Optimizing Jojoba Oil Methanolysis and Unveiling the Immunomodulatory Potential of cis-13-Docosenol Fatty Alcohol: A Circular Biorefinery Perspective

Optimizing Jojoba Oil Methanolysis and Unveiling the Immunomodulatory Potential of cis-13-Docosenol Fatty Alcohol: A Circular Biorefinery Perspective | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Optimizing Jojoba Oil Methanolysis and Unveiling the Immunomodulatory Potential of cis-13-Docosenol Fatty Alcohol: A Circular Biorefinery Perspective Laura Mendoza, Marcos Sánchez, Jorge Mario Marchetti, María Montoya This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4750304/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract To ensure the sustainability of Jojoba oil production, research and development must prioritize the adoption of environmentally friendly extraction processes. Firstly, optimal conditions for extracting Jojoba oil were predicted marking a significant step towards realizing its economic potential. Molar ratio, temperature and catalysts concentration were taken into consideration to achieve optimal production. Secondly, interactions of cis-13-docosenol (C13D), a key component of Jojoba oil, with innate immune cells were analysed. By meticulously examining the interactions between C13D and critical elements of the innate immune system, including monocytes, macrophages, and dendritic cells (DC), we aim to uncover the immunomodulatory properties of this compound. In experiments with THP-1 cells and DC, low doses of C13D were found to elevate pro-inflammatory cytokines TNF-α, IL-6, and IL-1β to levels comparable to those induced by LPS. Furthermore, modulation of T cell stimulation by monocyte-derived dendritic cells (MoDC) previously treated with C13D resulted in increased T cell proliferation, likely due to the enhanced activation of surface markers. This detailed exploration into the effects of C13D on innate immune cells not only deepens our understanding of Jojoba oil's therapeutic potential but also establishes a foundation for future advancements in immunology and biotechnology. Physical sciences/Chemistry/Chemical biology Biological sciences/Chemical biology Biological sciences/Immunology Physical sciences/Chemistry jojobyl alcohols 13-docosenol adjuvants jojoba oil Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION There is an increasing global demand for both circularity and carbon neutrality. The use of biomass and waste biomass to produce new bio-based chemicals is rising and will continue to do so in the coming decades. This trend is driven by societal and political pressure to replace petroleum-based chemicals with natural bio-based alternatives 12 . Various types of biomasses can serve as substrates for the production of different chemicals. For instance, algae can be utilized to produce succinic acid 3 , lignocellulose and cellulose to produce wood-based derivatives (like vanillin) 4 or waste oils can be upgraded into bio-based fuels, jet fuel, alcohol based and biodiesel, among others 5 6 . Although many new bio-based chemicals are being developed, there is limited research on the production of bio-based components with potential medical applications. One example is biomass-based fibre materials designed for use as antibacterial agents 7 , antioxidant, anti-fungal as well as for cosmetics 8 . The jojoba plant, botanically classified as a desert-like perennial shrub from the Buxaceae family 9 , is native to the Sonoran Desert. It is typically found in the US, Israel, Mexico, Argentina, India, and South Africa due to its unique characteristics and resistance to extreme weather conditions 9 10 , particularly salinity, drought, and heat 11 . From a chemical perspective, jojoba oil consists of a mixture of high molecular weight esters extracted from the jojoba plant. The composition of jojoba oil is distinctly different from that of other vegetable oils due to the absence of a glycerol backbone in its chemical structure. The most common method for obtaining jojoba oil from jojoba seeds involves a combination of mechanical pressing and hexane extraction to maximize the overall yield of the process, which is slightly higher than 50% of the total seed. Furthermore, jojoba oil can be converted into biodiesel and jojobyl alcohols through methanolysis transesterification. The long-chain alcohols produced in this process include 11-eicosenol, 13-docosenol (C13D), and 15-tetracosenol, which are classified as high-value-added products. This reaction is illustrated in Fig. 1 . Importantly, jojoba oil has been shown to have certain pharmaceutical applications 12 . Similar fatty alcohols derived from very long-chain aliphatic alcohols have been reported to inhibit the production of pro-inflammatory cytokines in LPS-stimulated macrophages, indicating potential anti-inflammatory effects of long-chain fatty alcohols 13 . Other studies investigating eicosenoid compounds have revealed their ability to enhance the production of pro-inflammatory cytokines, such as TNF-α and IL-1β, while decreasing levels of the anti-inflammatory cytokine IL-10 in THP1-derived macrophages 14 . The functionality of C13D against enveloped viruses is still unknown. However, very similar compounds have been studied in various cell types, including THP-1 cells, PMA-differentiated THP-1 cells, macrophages, and monocyte-derived dendritic cells (MoDCs) 13 , 14 , 15 . Depending on their nature, some compounds exhibit anti-inflammatory effects, whereas others are capable of stimulating immune cells. Biorefineries are processing facilities that convert biomass into value-added products such as biofuels, biochemicals, bioenergy/biopower, and other biomaterials with potential medical applications. In this study, the production optimization for valorizing jojoba oil into fatty alcohols was carried out using a response surface methodology (RSM) approach. Bio-methanol and calcium oxide were employed in a completely green process. The molar ratio, temperature, and catalyst concentration were optimized to achieve the highest production efficiency. Among the jojoba alcohols produced, C13D was investigated for its potential to modulate immune cells. This approach is illustrated in the graphical abstract. The results from our study revealed the predicted optimal conditions for jojoba oil production and suggested a possible new application of C13D as an immunomodulator in innate immune system cells. This biorefinery approach focuses on the production of high-value products, such as fatty alcohols, while also generating fatty acid methyl esters as a biofuel co-product. MATERIALS AND METHODS Materials: Jojoba oil was supplied by Jojoba Israel (Israel). Gas chromatography (GC) standards were purchased from Sigma Aldrich. The catalyst was produced by the calcination of mussel shells (Mytilus galloprovincialis species) from Galicia (Spain). C13D was purchased from Cymit Quimica S.L. and was used in a liquid state at 37°C in all experiments. The purity of C13D is > 98.0% (GC), as indicated in its datasheet ( https://cymitquimica.com/es/productos/3B-D2174/629-98-1/cis-13-docosenol/ ). Reaction steps and optimization The experimental data referenced was previously published 16 . This existing dataset was examined from a kinetics standpoint; however, its optimization and the influence of the variables under investigation were not addressed. To address this gap, this study conducted a statistical analysis of data from all 27 experiments to elucidate the individual effects of each variable and their quadratic combinations. StatGraphics Software was employed for this analysis. The selected model was quadratic, with the primary factors being the three individual variables: molar ratio (defined as the moles of alcohol relative to the moles of oil in the process), temperature, and catalyst amount (expressed as the weight percentage of catalyst relative to the quantity of oil). The reaction was conducted in a 250 mL reactor where the appropriate reactants were premixed, and the desired temperature was reached prior to the initiation of the reaction. Temperature was maintained constant using a Proportional-Integral-Derivative (PID) controller. At predetermined intervals, samples were withdrawn and prepared for gas chromatography (GC) analysis. The analyses were performed on a Hewlett-Packard GC model 5890 connected to an integrator. Samples were analyzed using a Flame Ionization Detector (FID) set at a detector temperature of 320°C. Further details of the methodology can be found elsewhere 17 . THP1 cell culture and differentiation THP-1 cells (TIB-202) were cultured in 24-well plates at a seeding density of 1 x 10 6 cells/ml in Roswell Park Memorial Institute (RPMI) medium 1640 with addition of 1% penicillin/streptomycin and 5% Fetal Bovine Serum (FBS) (Sigma-Aldrich, Dorset, UK). Cells were treated with different doses of C13D (0,3 − 1–3–9 µl/ml) either alone or in combination with LPS (100 ng/ml) for 24h at 37˚C and 100% humidity in 5% CO 2 . THP-1 cells were cultured in 6-well plates at a concentration of 2 x 10 5 cells/ml and stimulated with 25 ng/ml of phorbol 12-myristate 13-acetate (PMA, 25 ng/ml final concentration, Sigma-Aldrich, Dorset, UK) to differentiate into macrophage-like cells. Cells were then incubated for 48 h, after which the PMA-containing media was replaced with fresh media and the cells rested for an additional 24 h prior to C13D treatment, as explained above. In this case, LPS concentration was 10 ng/ml. Viability from C13D treated cells was measured by MTS assay, based on the reduction of MTS tetrazolium compound by viable mammalian cells to generate a coloured formazan dye that is soluble in cell culture media and quantified by measuring at 490–500 nm absorbance. For that procedure, cells were seeded in 96 well plates with C13D and LPS treatments and subsequently, MTS reagent was added to cell culture media and incubated for 1-2h in standard culture conditions. After that, the plate was shaken briefly and optical density measured at 490 nm. Enzyme-Linked Immunosorbent Assay (ELISA) Cytokines such as TNF-α, IL-1β, IL-6 and IL-10 were measured in the supernatants of the cell cultures after 24h using commercially available Human DuoSet ELISA kits (R&D Systems – DY210, DY201, DY206, DY217B) in accordance with the supplier’s protocol. Absorbance was converted to pg/ml according to the standard curve generated with a five-parameter logistic curve fit. Primary human cells Buffy coats from healthy blood donors were collected from the blood bank “Centro de Transfusión” in Comunidad de Madrid. MoDC differentiation was achieved as previously described 18 . This procedure was approved by CSIC Ethical Committee. Briefly, PBMC were depleted using mouse anti-human CD14 microbeads (Miltenyi Biotec) as indicated by the manufacturer. To generate MoDCs, CD14 + cells were incubated following Miltenyi Biotec´s protocol ( https://www.miltenyibiotec.com/ES-en/applications/all-protocols/generation-of-mo-dcs.html ). After 7 days, cells were harvested and seeded in plates for stimulation. For CD3 + isolation, PBMC were counted and labelled with CFSE (5 µM; Invitrogen; Thermo Fisher) for 20 min at 37˚C and straightaway incubated with anti-human CD3 (PE/Cyanine7 anti-human CD3 Antibody; clon SK7; isotype: Mouse IgG1, k; BioLegend) for 30 min at 4˚C in the dark. Cells were sorted using BD FACSAria Fusion Flow Cytometer, separating CD3 + cells with a standard purity around 95%. Flow cytometry immunophenotyping For MoDC, 2 x 10 5 cells were stained at a final volume of 50 µl. First, cells were resuspended in 25 µl of PBS supplemented with 0,5% BSA and 0,1% NaN 3 (FACS buffer) mixed with 25 µl of anti-goat serum (Merck Millipore) and incubated for 20 min on ice. After the blocking step, an antibody cocktail was prepared with a total volume of 50 µL per sample, containing all antibodies from the panel at the final concentrations shown in Table 1 , and diluted in FACS buffer. Fifty microliters of the antibody mix were added to each sample and incubated for 30 minutes at 4°C in the dark. Following extensive washing with 100 µL PBS and centrifugation (1100 rpm, 5 minutes, 4°C), the pellets were resuspended in 200 µL PBS and analyzed using a Cytek Aurora Spectral Flow Cytometer. Table 1 Antibodies used for immunophenotyping. PE, Phycoerythrin; APC, Allophycocyanin; APC R700, Allophycocyanin R700. Conjugated Marker Supplier Clone Catalogue number Concentration (µl/Test) PE CD80 BD Bioscience 2D10.4 566992 2.5 APC CD40 BD Bioscience 5C3 555591 10 APC R700 CD86 BD Bioscience 2331 (FUN-1) 565149 2.5 eFluor450 HLA-DR II Thermo Fisher L243 48-9952-42 2.5 MLR assay For mixed lymphocyte reaction (MLR), allogeneic CFSE- labelled T-cells (10 6 cells/ml) were co-cultured with MoDC (2 x 10 5 cells/ml) in 96-well round-bottom plates (Corning, Corning, NY) at a stimulator: responder ratio of 1:5. Unstimulated CFSE-labelled T cells served as a negative control. After 6 days, cells were stained with PE/Cyanine7-conjugated anti-CD3 (at 1:100 dilution, PE/Cyanine7 anti-human CD3 Antibody; clon SK7; isotype: Mouse IgG1, k; BioLegend) antibody and analysed in CytoFLEX S Cytometer. T-cell proliferation analysis was performed using FlowJo software proliferation tool (Becton Dickinson, Franklin Lakes, NJ). Data analysis Statistical analyses were performed using GraphPad PRISM 5. P-values were determined using two-way ANOVA and Bonferroni test correction was applied. Unless otherwise stated, data are shown as the mean of at least three biological replicates. Significant differences in the figures were indicated as: *, p < 0.05; **, < 0.01; ***, p < 0.001, ****, p < 0.0001. RESULTS Optimization using individual variables Molar ratio (MR), temperature (Temp), and catalyst concentration (Cat) are identified as the main variables for extracting jojoba oil. Consequently, these three variables were initially analyzed individually. Each of these main variables alone has a significant effect on the model under study, as shown in the Pareto chart in Fig. 2 . All three variables (MR(A), Temp(B), Cat(C)) have a positive effect, while the quadratic terms of each variable with itself (AA, BB, CC) have a significantly smaller negative effect. Figure 2 Based on these results, the effect of each individual variable was studied while keeping the remaining operational conditions constant. Figure 3 A shows changes in jojoba oil yield at different Temp. An increase from 45 to 65°C improves the reaction yield from 79.6–90.3%. Similarly, the effect of changing the amount of alcohol in the reaction medium was analyzed to understand how the ratio among the different feedstocks influences the process yield. Figure 3 B shows that an increase in the MR, and therefore an increase in the amount of alcohol, improves the reaction yield. There is a significant increase when changing the molar ratio from 6:1 to 9:1 (jojoba oil yield increases from 59.9–90.3%), while the increase is much less significant when MR increases from 9:1 to 12:1 (yield increases up to 93%). This smaller increase could be related to the reaction approaching equilibrium, where higher amounts of alcohol have a significantly reduced effect. The third variable studied was Cat. For this purpose, all other operational variables were kept constant while the catalyst amount was increased from 6 to 10 wt.%. Figure 3 C shows this effect. Increasing the catalyst concentration increased the reaction yield, with a more significant impact observed between 6 to 8 wt.% compared to changes from 8 to 10 wt.%. This effect could be due to an excess of catalysts that cannot be utilized effectively due to the limited amount of feedstock. Additionally, a higher Cat increases the reaction rate, leading to the reaction reaching equilibrium sooner. Consequently, higher Cat for a fixed time could result in the reaction being closer to equilibrium, leading to smaller yield increments compared to lower catalyst concentrations. Optimization by combined effect Besides the individual effect of each variable, it is important to understand the combined effect of multiple variables. Here, the effect of two variables at a time is presented. Figure 3 D shows the combined effect of temperature and molar ratio on the reaction yield. An increase in both variables significantly increases the reaction yield while keeping other variables constant. This result aligns with the findings presented in Fig. 2 , where these two variables showed a positive effect both individually and in combination. In Fig. 3 E, the combined effect of reaction Temp and Cat on jojoba oil yield is presented. A higher Cat and a higher Temp do not result in the optimal yield. Instead, the highest Cat is necessary, but the reaction Temp should be close to 55 ºC for the best effect on reaction yield. Figure 3F shows the combined effect of MR and Cat on the reaction yield. These two variables, as well as their interaction, have a positive effect. Therefore, an increase in both variables leads to an increase in jojoba oil yield. As shown in Fig. 3 F, the highest yield is achieved when both MR and Cat reached their maximum levels. Figure 3 Using all 27 experiments presented elsewhere 17 , a quadratic model was applied to understand the individual effects of all the variables, their quadratic effects, and their combined quadratic effects. This analysis yielded the following equation: Where T: temperature [°C], M: molar ratio, and C: catalysts amount [wt.%] Using Eq. 1, a comparison between the experimental and predicted yields was conducted. Figure 4 presents this comparison for all the data collectively. The regression was robust, with a coefficient of regression of 0.8192, indicating that the model fits the data quite accurately. Figure 4 Finally, an optimization analysis was performed using the presented data and the developed model. The optimal operational conditions and the corresponding theoretical yields are described and presented in Table 2 . Table 2 Predicted optimal conditions. Temp [°C] MR Cat. [%] Yield [%] Predicted 60.87 12:1 9.79 94.87 Having developed the optimal conditions to generate jojoba oil fatty alcohols, we wonder whether one of the main jojoba oil component, C13D, exhibited an ability to interact with cells from the immune system, paving the way of new applications of Jojoba oil after its optimization. Cytokine secretion in C13D treated THP-1 and PMA-differentiated THP-1 cells Monocytes and macrophages have crucial and diverse roles in the regulation of the innate immune system and act as antigen presenting cells in adaptive immunity 19 . They also initiate inflammation against invading antigens and antigen presentation 20 . Therefore, THP-1 cell line of monocytes were initially used to measure whether C13D was toxic in our tissue culture conditions. As described in Supplementary Fig. 1, concentrations ranging from 0.3 to 9 µl/ml did not significantly affect cell viability in our experiments. Therefore, subsequent experiments were conducted within this concentration range. Previous studies have utilized LPS as a stimulatory factor to analyze cytokine expression following activation in these cells 19 . In this study, LPS served as a positive control for stimulation when C13D was added to THP-1 cells at various doses (Fig. 5 , A-D) and to PMA-stimulated THP-1 cells (Fig. 5 , E-H). Generally, cytokine secretion was higher in PMA-differentiated THP-1 cells (Fig. 5 , E-H) compared to non-PMA-treated THP-1 cells (Fig. 5 , A-D) when assessing IL-1β, TNF-α, IL-6, and IL-10. C13D induced IL-6 secretion in THP-1 cells at doses of 1 and 3 µl/ml (Fig. 5 C). In contrast, in PMA-differentiated THP-1 cells, the highest doses significantly increased IL-1β and TNF-α secretion (Fig. 5 E, F) without affecting IL-6 and IL-10 secretion (Fig. 5 G, H). When C13D was combined with LPS in THP-1 cells, cytokine secretion exhibited a distinct pattern. For both IL-1β and IL-6, higher doses of C13D correlated with lower cytokine release (Fig. 5 A, C). IL-10 was not detected in the secretion (Fig. 5 D), and TNF-α levels varied depending on the combined dose (Fig. 5 B). In PMA-differentiated THP-1 cells, a similar pattern was observed for IL-1β, IL-6, and IL-10 secretion, with decreasing levels as the dose of C13D increased (Fig. 5 E, G, and H). However, TNF-α secretion increased with C13D doses up to 3 µl/ml (Fig. 5 F). Figure 5 Cytokine secretion in C13D treated MoDC MoDCs are highly valuable for in vitro studies to simulate the behavior of DC under specific conditions. Hence, increasing concentrations of C13D were added in the presence or absence of LPS (Fig. 6 ). C13D significantly enhanced secretion of the pro-inflammatory cytokine TNF-α at all doses tested and the anti-inflammatory cytokine IL-10, but the latter effect was observed only at the highest concentration used (Figs. 6 B, D). It did not affect the release of IL-1β or IL-6 (Figs. 6 A, C). However, upon LPS activation, TNF-α secretion decreased at doses of 3 and 9 µl/ml (Fig. 6 B), while IL-6 secretion remained unaffected (Fig. 6 C). IL-1β secretion significantly increased at lower concentrations of C13D (Fig. 6 A). The pattern for IL-10 secretion appeared to be dose-dependent, similar to PMA-differentiated THP-1 cells (Fig. 5 H), with decreased secretion as the C13D dose increased (Fig. 6 D). MoDC surface markers’ levels after C13D treatment MoDCs remain immature after 7 days of differentiation from monocytes. Increasing the expression of markers such as CD80, CD86, CD40, or MHC-II on the surface of DCs is considered the gold standard for measuring activation in these cells (ref. https://doi.org/10.3109/10520295.2015.1017536 ). Therefore, to assess MoDC maturation induced by C13D treatment, surface expression was measured using specific monoclonal antibodies for each of these markers by flow cytometry (Fig. 7 A-D). Levels of CD80 or CD40 remained unchanged in the presence of C13D compared to untreated cells (data not shown). MHC-II levels were already high, making further activation difficult to detect (data not shown). Surprisingly, after 24 hours of C13D treatment, CD86 levels increased in a dose-dependent manner, as shown in Fig. 7 E. Although not reaching the same levels as LPS, such increase is significantly visible compared to untreated cells. Effect of C13D on T cell proliferation Functional maturation of DCs is commonly assessed by their ability to stimulate allogeneic T cells using mixed lymphocyte reaction (MLR) assays 21 . DCs are potent inducers of lymphocyte activation in allogeneic MLR due to their expression of several surface costimulatory molecules, such as CD86, as shown in Fig. 7 A-E. High levels of MHC expression and the presentation of costimulatory molecules make DCs particularly effective in eliciting a robust T cell response 22 . Therefore, an MLR assay was employed to investigate the impact of C13D-treated MoDCs on their capacity to induce proliferation of naïve T lymphocytes (Fig. 7 F-L). After 6 days of co-culture, the data indicated no significant proliferation of T cells alone (Fig. 7 F), and only a slight percentage of proliferation was observed in the presence of untreated MoDCs (Fig. 7 G), suggesting minimal activation in the absence of stimuli. Interestingly, C13D-treated MoDCs induced approximately 37% proliferation in the T CD3 + population at a concentration of 0.3 µl/ml (Fig. 7 I). Notably, under our experimental conditions, LPS-treated MoDCs induced similar levels of proliferation, reaching up to approximately 39.9% (Fig. 7 H). Although the level of proliferation was not as pronounced at other doses of C13D as it was at 0.3 µl/ml, MoDCs treated with any dose of C13D significantly induced T cell proliferation compared to untreated MoDCs (Fig. 7 J-L). Again, it was interesting to observe a dose dependent effect, the higher the concentration of C13D, the lower percentage of T cell proliferation induced (Fig. 7 M). DISCUSSION Among emerging biomass sources, non-edible oils are receiving increasing attention due to their minimal competition with food resources. Additionally, to avoid competition with arable land, there is significant interest in non-edible oils that can be cultivated in non-arable areas. Jojoba oil, for instance, is a drought-resistant shrub which thrives in arid conditions. This makes it well-suited for cultivation in desert non-arable areas worldwide due to its unique resistance to extreme weather conditions 9 . The cultivation of jojoba plants holds great promise for the development of desert areas. As jojoba cultivation requires minimal water and can withstand harsh environmental conditions, it presents a sustainable and economically viable option for transforming barren desert lands into productive agricultural spaces. Establishing jojoba plantations contributes not only to local economic growth but also to environmental conservation by preventing desertification and enhancing soil stability. This dual benefit of economic prosperity and ecological sustainability positions jojoba cultivation as a key driver for development in desert regions, offering a unique solution to harness the untapped potential of arid landscapes. However, while jojoba oil holds significant economic potential, its extraction process requires optimization to ensure sustainability. Currently, the cultivation and extraction of jojoba oil can be resource-intensive, necessitating a careful balance between economic gains and environmental impact. In response to the global need for a circular economy and waste valorization, several studies have explored the use of renewable alcohols, such as methanol, ethanol, and butanol, in conjunction with bio- and waste-based catalysts like mussel shells and eggshells. Catarino et al. 23 studied the use of calcium diglyceroxide from scallop shells with promising results, they achieved a yield of 96% towards fatty acid methyl esters within 2 h. of reaction at a temperature of 65°C with 5 wt% of catalyst and a molar ratio of 12:1. Similar work was conducted by Dias et al. 24 , where lime was used as a calcium source and different configurations of CaO were tested. They found that the presence of glycerol in a CaO and methanol solution exhibited kinetic behavior without an induction period, as previously reported. Sánchez et al. 25 studied the transesterification of jojoba oil using renewable calcium oxide catalysts and methanol. The results showed a reaction conversion of 95% under the conditions of a reaction temperature of 65°C, a molar ratio of 12:1, and 8 wt.% catalyst. In addition, Sanchez et al. 25 presented a kinetics model and a reaction pathway for such process. Furthermore, Sánchez et al. 26 improved the reaction conditions by using a high-pressure reactor, allowing the process to reach a final conversion of 96.3 %. This hih conversion was achieved with a reaction time of 5 h with similar operational conditions as in their previous work 25 . Likewise, Avhad et al. 17 studied the butanolysis of jojoba oil in the presence of mussel shell naturally derived catalyst (CaO based). The authors have found the highest conversion of 96.11 % when the reation temperature is 85°C with a molar ratio of 10:1 and 12 wt% catalyst. The transesterification reaction produces jojoba alcohols (11-eicosenol, C13D, and 15-tetracosenol) in a mixture of 36–40%, 43–49%, and 9–10%, respectively 27 . Once the Jojoba oil is being produced, its transformation into biodiesel and jojobyl alcohols can be achieve using different reactive pathways. Singh et al 28 studied the supercritical methanolysis of jojoba oil using a response surface methodology (RSM) approach. The authors found the highest yield of 95 %at a temp of 287°C, 123 bar of pressure and 30:1 molar ratio within 23 minutes of reaction. Similarly, the work by Ravikumar et al. 29 used methanol for the transesterification of jojoba oil, in this case the authors used a basic homogeneous catalyst (potassium hydroxide) achieving 90% yield after 25 minutes at a reaction temperature of 50 ºC. Similar research was carried out by Buoaid et al. 30 where jojoba was transesterified with methanol in the presence of potassium hydroxide with a yield of 83 % whe working at 25°C with a catalyst concentration of 1.35 wt%. Abdulrhman et al. 16 studied the transesterification of jojoba oil with methanol but using sodium hydroxide. The authors found the highest yield of 70 % for a15-minute reaction with a molar ratio of 16:1 and catalyst amount of 1.5 wt%. Aiming to make jojoba oil production sustainable, this work has sought to define the predicted optimal conditions for temperature, molar ratio, and catalyst amount, which are shown in Table 2 . Our results showed an optimal yield of 94.87% achieved when temperature was 60.87°C, molar ratio of alcohol to oil of 12:1, catalyst amount of 9.79 wt.% and reaction time of 10 h with constant steering of 300 rpm. As presented in Fig. 3 , the increase in yield observed with rising temperature can be attributed to the endothermic nature of the reaction. A higher reaction temperature results in a higher final yield. Similar results were obtained by Mohadesi et el. 31 and Bargole et al. 32 where both used waste cooking oil as the sources in the presence of calcium-based catalysts. The optimal yield of 97 % ad 95 % rspectively were obtained for reaction temperatures of 54.97°C and 64.8°C respectively. In fact, the results obtained by Sulaiman et al. 33 in their study on the transesterification reaction of waste cooking oil using CaO-doped catalysts in the presence of methanol were similar. They observed that increasing the molar ratio resulted in higher yields. However, a further increase in the molar ratio tended to stabilize the yield, possibly indicating that excessively high values led to decreased yields due to the dissolution of catalysts in the reaction medium. Figure 3 in this study illustrates the impact of temperature and molar ratio on the reaction yield. Increasing both variables significantly enhance the reaction yield when other variables are held constant. This finding corroborates the results depicted in Fig. 2 , where these two variables individually and in combination showed a positive effect. Moreover, this observation aligns with the findings of Mohadesi et al. 31 , who investigated the combined effect of temperature and molar ratio on the transesterification of waste cooking oil. In both studies, higher temperatures and molar ratios led to increased yields. The influence of reaction temperature and catalyst amount on jojoba oil yield is depicted in Fig. 3 . It was observed that the highest catalyst concentration was necessary, while the optimal reaction temperature for maximizing yield was around 55°C. This indicates that a higher catalyst amount and temperature do not necessarily yield the best possible outcome. Foroutan et al. 34 observed a similar relationship between temperature and catalyst amount in their study on biodiesel production from edible oils using CaO-based catalysts. They reported a maximum yield of 98.83% at a reaction temperature of 65°C and a catalyst amount of 4%, which was not the highest amount tested. Between 43–49% of jojoba alcohols produced by transesterification is C13D, also known as erucyl alcohol. C13D possesses multiple applications, including its characterization as an antiviral agent effective against lipid-enveloped viruses such as herpes simplex virus (HSV) and respiratory syncytial virus (RSV). Based such old experiments, it is generally believed that 1-docosanol inhibit viral replication by disrupting viral membranes 35 , although its mechanism of action has yet to be precisely defined. It has also been used as an emulsifier and surfactant in various cosmetic and pharmaceutical formulations as well as it is used in skincare products for its moisturizing and conditioning properties 36 , 37 . It is also used in industrial applications such as lubricants and coatings due to its lubricating and film-forming properties 38 , 39 . However, there are no studies focusing on possible C13D interaction with cells from the immune system. Thus, after establishing optimal conditions for producing jojoba oil fatty alcohols, we were investigating whether one of its components, C13D, interacts with immune system cells. This exploration could potentially open up new applications for jojoba oil following its optimization. The functionality of C13D against enveloped viruses is still unknown, although very similar compounds have been studied on different cell types such as THP-1 cells, PMA-differentiated THP-1 cells, macrophages and monocyte derived dendritic cells (MoDC) 40 , 41 , 42 , 43 , 44 . Immune cells are quite valuable candidates to study immune processes and responses. Mononuclear phagocytes, monocytes, and macrophages have crucial and diverse roles in the regulation of the innate immune system and they act as antigen presenting cells 45 . The stablished cell line THP-1 is widely applied to mimic monocytes in cell culture models 46 and they are widely used as model for primary human macrophages. This is because following differentiation using phorbol 12-myristate 13-acetate (PMA), THP-1 cells acquire a macrophage-like phenotype 21 , which help understanding this cell type behaviour under different conditions. Dendritic cells (DCs) are professional antigen presenting cells, they can be found in practically all tissues, where they detect imbalances and process antigens for presenting to T lymphocytes (T cells). DCs serve as the bridge between the innate and adaptive immune systems and are critical in the initiation of primary immune responses 19 20 22 . Functionally and phenotypically, MoDCs are believed to be typical immature DCs, characterized by their low expression of major histocompatibility complex class II (MHC-II) and costimulatory molecules. Immature MoDCs can be subsequently matured after treatment with compounds known to induce DC maturation, such as LPS (Lipopolysaccharide), TNF-α, or IFN-γ 47 . DCs are not plentiful in peripheral blood, in fact, circulating blood DCs constitute from 0.1–1.0% of peripheral blood mononuclear cells. Thus, moDCs have been the most widely used model to investigate human DC biology and function 48 49 . Cytokines are secreted from a variety of cells such as lymphocytes, macrophages, monocytes, DC, etc. They participate in the immune response and have an important function as mediators associated with the communication network of the immune system 50 51 . Cytokines are responsible for the regulation of the maturation, growth and capacity of reacting quickly of immune cells 52 51 . Generally speaking, cytokines can be pro-inflammatory (IL-1β, IL‐6, IL‐8, IL‐12, IFN‐γ, and TNF‐α) or anti-inflammatory cytokines (L‐4, IL‐6, IL‐10, IL‐11, IL‐13, and TGF‐β) 51 , although some cytokines exert both functions depending on the concentration and the environmental milieu. Pro-inflammatory cytokines can go into the systemic circulation and produce immune cell activation 13 whereas anti-inflammatory cytokines are immunoregulatory molecules which inhibit the excess inflammatory response of the pro-inflammatory ones 15 . Depending on their nature, some chemical compounds present anti-inflammatory effects whereas other are able to stimulate immune cells by, for example, making them produce high levels of pro-inflammatory cytokines. Some studies reveal that long chain fatty alcohols can reduce NO 2 amount, pro-inflammatory cytokines and inflammatory mediators produced by macrophages in a dose-dependent manner 13 15 . Other studies suggest an immune stimulating capacity of fatty alcohols due to their capacity of increasing TNF-α and IL-1β production, which are pro-inflammatory cytokines, and decreasing IL-10 level, which is an anti-inflammatory cytokine, by PMA-differentiated THP-1 cells 14 . This groundbreaking study marks the first exploration of C13D within the context of cells belonging to the innate immune system, such as monocytes, macrophages, or DC (Fig. 8 ). When those cells were treated with different C13D doses in presence or absence of LPS different responses were shown. Low doses of C13D were able to increase TNF-α, IL-6 or IL-1β pro-inflammatory cytokines to similar levels as LPS in MoDC. Also, increased T cell proliferation was observed when C13D-activated MoDC were present, possible due to a higher expression of surface activation markers like CD86. By delving into the interactions between C13D and these crucial components of the immune system, we aimed to uncover novel insights into potential immunomodulatory effects and therapeutic applications, shedding light on a previously unexplored aspect of cellular immunity. This pioneering research holds the potential to broaden our understanding of C13D's impact on immune responses, opening avenues for innovative therapeutic strategies and contributing to advancements in the field of immunology. Conclusions The pursuit of optimizing jojoba oil extraction underscores a commitment to both economic prosperity and environmental stewardship, paving the way for a more sustainable and responsible industry in arid regions. Our study firstly focused on optimizing jojoba oil production, revealing that all operational variables evaluated positively influenced jojoba oil yield. However, the interaction between temperature and catalyst concentration showed a distinct effect. Optimal operational temperature was found to be in the mid-range of the tested domain, outperforming other scenarios. In summary, the variables ranked in decreasing order of relevance were: molar ratio > temperature > catalyst concentration. The fit between experimental and predicted data was robust, with a regression value of approximately 0.82. After establishing optimal conditions for producing jojoba oil fatty alcohols, we explored whether one of its components, C13D, could interact with immune system cells. This investigation aimed to uncover new applications for jojoba oil following optimization. Our exploration into the effects of cis-13-docosenol (C13D) on innate immune cells revealed that low doses of C13D could elevate pro-inflammatory cytokines TNF-α, IL-6, and IL-1β to levels comparable to those induced by LPS in monocytes and DCs. Moreover, modulation of T cell stimulation by MoDCs previously treated with C13D enhanced T cell proliferation, likely through increased activation of surface markers. Our findings establish optimal conditions for jojoba oil extraction, a critical step in maximizing its economic viability. This research adopts a bio-refinery approach, focusing on the immunomodulatory activity of C13D within the context of innate immune cells, contributing to a comprehensive understanding of its potential applications in sustainable biorefinery practices. Declarations Acknowledgments: JMM would like to thank NMBU for their financial support. MM would like to thank CSIC's Global Health Platform (PTI+ Salud Global) for their support. Permissions: We have the approval from the CSIC Ethical committee for the experiments using cells from human peripheral blood from healthy donors. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Authors' contributions Conceptualization: Marcos Sanchez, Jorge Marchetti, María Montoya. Methodology: Marcos Sanchez, Jorge Marchetti, María Montoya. Formal analysis and investigation: Marcos Sanchez, Jorge Marchetti, Laura Mendoza, Maria Montoya. Writing - original draft preparation: Laura Mendoza, Marcos Sanchez. Writing - review and editing: Jorge Marchetti, María Montoya Funding acquisition: Jorge Marchetti, María Montoya Resources: Marcos Sanchez, Jorge Marchetti, María Montoya Supervision: Jorge Marchetti, María Montoya Funding This research work was partially funded by the European Commission – NextGenerationEU (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI+ Salud Global) (COVID-19-117 and SGL2103015), Spanish Ministry of Science project (PID2021-123399OB-I00) and by the Norwegian University of Life Science. Availability of data and materials The data is available upon request. Dra. María Montoya should be contacted if any request of the data from this study. References Cho, E. J., Trinh, L. T. P., Song, Y., Lee, Y. G. & Bae, H. J. Bioconversion of biomass waste into high value chemicals. Bioresource Technology vol. 298 Preprint at https://doi.org/10.1016/j.biortech.2019.122386 (2020). Kircher, M. The transition to a bio-economy: Emerging from the oil age. Biofuels, Bioproducts and Biorefining 6, 369–375 (2012). Knoshaug, E. P. et al. Demonstration of parallel algal processing: Production of renewable diesel blendstock and a high-value chemical intermediate. Green Chemistry 20, 457–468 (2018). Maeda, M. et al. Vanillin production from native softwood lignin in the presence of tetrabutylammonium ion. Journal of Wood Science 64, 810–815 (2018). Hvidsten, I. B. & Marchetti, J. M. Securing liquid biofuel by upgrading waste fish oil via renewable and sustainable catalytic technology. Fuel 333, (2023). Bouaid, A., Acherki, H., Bonilla, M. H. & Marchetti, J. M. Enzymatic ethanolysis of high free fatty acid jatropha oil using Eversa Transform. Energy Advances 159–168 (2022) doi: 10.1039/d1ya00057h . Li, D., Wang, Y., Huang, W. & Gong, H. Biomass-derived fiber materials for biomedical applications. Frontiers in Materials vol. 10 Preprint at https://doi.org/10.3389/fmats.2023.1058050 (2023). Gad, H. A. et al. Jojoba Oil: An Updated Comprehensive Review on Chemistry, Pharmaceutical Uses, and Toxicity. (2021) doi: 10.3390/polym . Sánchez, M., Avhad, M. R., Marchetti, J. M., Martínez, M. & Aracil, J. Jojoba oil: A state of the art review and future prospects. Energy Conversion and Management vol. 129 293–304 Preprint at https://doi.org/10.1016/j.enconman.2016.10.038 (2016). Yang, L., Takase, M., Zhang, M., Zhao, T. & Wu, X. Potential non-edible oil feedstock for biodiesel production in Africa: A survey. Renewable and Sustainable Energy Reviews vol. 38 461–477 Preprint at https://doi.org/10.1016/j.rser.2014.06.002 (2014). sony. COM PAR AT IVE STUDY OF ALK AL INE AND SAL INE STRESSES ON TWO OIL-PRODUCING PL ANT S A f a f. J. Exp. Biol. (Bot.) 10, 13–25 (2014). Habashy, R. R., Abdel-Naim, A. B., Khalifa, A. E. & Al-Azizi, M. M. Anti-inflammatory effects of jojoba liquid wax in experimental models. Pharmacol Res 51, 95–105 (2005). Fernández-Arche, A. et al. Long-chain fatty alcohols from pomace olive oil modulate the release of proinflammatory mediators. Journal of Nutritional Biochemistry 20, 155–162 (2009). Alqarni, A. M. et al. Metabolomic profiling of the immune stimulatory effect of eicosenoids on PMA-differentiated THP-1 cells. Vaccines (Basel) 7, (2019). Montserrat-De La Paz, S., García-Giménez, M. D., Ángel-Martín, M., Pérez-Camino, M. C. & Fernández Arche, A. Long-chain fatty alcohols from evening primrose oil inhibit the inflammatory response in murine peritoneal macrophages. J Ethnopharmacol 151, 131–136 (2014). Abdulrahman, A., Ali, A. & Alfazazi, A. Synthesis and Process Parameter Optimization of Biodiesel from Jojoba Oil Using Response Surface Methodology. Arab J Sci Eng 46, 6609–6617 (2021). Avhad, M. R. et al. Renewable production of value-added jojobyl alcohols and biodiesel using a naturally-derived heterogeneous green catalyst. Fuel 179, 332–338 (2016). Crisci, E. et al. In vivo tracking and immunological properties of pulsed porcine monocyte-derived dendritic cells. Mol Immunol 63, 343–354 (2015). Patente, T. A. et al. Human dendritic cells: Their heterogeneity and clinical application potential in cancer immunotherapy. Front Immunol 10, (2019). O’Keeffe, M., Mok, W. H. & Radford, K. J. Human dendritic cell subsets and function in health and disease. Cellular and Molecular Life Sciences vol. 72 4309–4325 Preprint at https://doi.org/10.1007/s00018-015-2005-0 (2015). Lund, M. E., To, J., O’Brien, B. A. & Donnelly, S. The choice of phorbol 12-myristate 13-acetate differentiation protocol influences the response of THP-1 macrophages to a pro-inflammatory stimulus. J Immunol Methods 430, 64–70 (2016). Gao, Y. et al. Single-Cell Analysis Reveals the Heterogeneity of Monocyte-Derived and Peripheral Type-2 Conventional Dendritic Cells. The Journal of Immunology 207, 837–848 (2021). Catarino, M., Martins, S., Soares Dias, A. P., Costa Pereira, M. F. & Gomes, J. Calcium diglyceroxide as a catalyst for biodiesel production. J Environ Chem Eng 7, (2019). Soares Dias, A. P., Puna, J., Gomes, J., Neiva Correia, M. J. & Bordado, J. Biodiesel production over lime. Catalytic contributions of bulk phases and surface Ca species formed during reaction. Renew Energy 99, 622–630 (2016). Sánchez, M., Marchetti, J. M., El Boulifi, N., Aracil, J. & Martínez, M. Kinetics of Jojoba oil methanolysis using a waste from fish industry as catalyst. Chemical Engineering Journal 262, 640–647 (2015). Sánchez, M., Avhad, M. R., Marchetti, J. M., Martínez, M. & Aracil, J. Enhancement of the jojobyl alcohols and biodiesel production using a renewable catalyst in a pressurized reactor. Energy Convers Manag 126, 1047–1053 (2016). Aracil J, Martínez M, El-Boulifi N, Sánchez M & Serrano M. Proceso integrado de obtención de alcoholes monoinsaturados, biodiesel y productos biodegradables a partir de aceite de Jojoba. (2013). Singh, N. K., Singh, Y. & Sharma, A. Optimization of biodiesel synthesis from Jojoba oil via supercritical methanol: A response surface methodology approach coupled with genetic algorithm. Biomass Bioenergy 156, (2022). Ravikumar Solomon, G. et al. Evaluation of the transesterification process and physio-chemical properties of jojoba biodiesel. International Journal of Innovative Technology and Exploring Engineering 9, 2553–2557 (2019). Bouaid, A., Bajo, L., Martinez, M. & Aracil, J. Optimization of biodiesel production from jojoba oil. Process Safety and Environmental Protection 85, 378–382 (2007). Mohadesi, M., Aghel, B., Gouran, A. & Razmehgir, M. H. Transesterification of waste cooking oil using Clay/CaO as a solid base catalyst. Energy 242, (2022). Bargole, S. S., Singh, P. K., George, S. & Saharan, V. K. Valorisation of low fatty acid content waste cooking oil into biodiesel through transesterification using a basic heterogeneous calcium-based catalyst. Biomass Bioenergy 146, (2021). Sulaiman, N. F., Ramly, N. I., Abd Mubin, M. H. & Lee, S. L. Transition metal oxide (NiO, CuO, ZnO)-doped calcium oxide catalysts derived from eggshells for the transesterification of refined waste cooking oil. RSC Adv 11, 21781–21795 (2021). Foroutan, R., Mohammadi, R., Razeghi, J. & Ramavandi, B. Biodiesel production from edible oils using algal biochar/CaO/K2CO3 as a heterogeneous and recyclable catalyst. Renew Energy 168, 1207–1216 (2021). Katz, D. H., Marcellettit, J. F., Khalilt, M. H., Popet, L. E. & Katzt, L. R. Antiviral Activity of 1-Docosanol, an Inhibitor of Lipid-Enveloped Viruses Including Herpes Simplex (Viral Inhibition/Long-Chain Alcohols) . Proc. Natl. Acad. Sci. USA vol. 88 (1991). Leung, D. T. & Sacks, S. L. Docosanol: A topical antiviral for herpes labialis. Expert Opin Pharmacother 5, 2567–2571 (2004). Khalil, M. H., Marcelletti, J. F., Katz, L. R., Katz, D. H. & Pope, L. E. Topical application of docosanol-or stearic acid-containing creams reduces severity of phenol burn wounds in mice. Contact Dermatitis 43, 79–81 (2000). Ottone, C., Bernal, C., Serna, N., Illanes, A. & Wilson, L. Enhanced long-chain fatty alcohol oxidation by immobilization of alcohol dehydrogenase from S. cerevisiae. Appl Microbiol Biotechnol 102, 237–247 (2018). Callau, M., Sow-Kébé, K., Jenkins, N. & Fameau, A. L. Effect of the ratio between fatty alcohol and fatty acid on foaming properties of whipped oleogels. Food Chem 333, (2020). Smith, N. C., Christian, S. L., Taylor, R. G., Santander, J. & Rise, M. L. Immune modulatory properties of 6-gingerol and resveratrol in Atlantic salmon macrophages. Mol Immunol 95, 10–19 (2018). Lee, T. Y., Lee, K. C., Chen, S. Y. & Chang, H. H. 6-Gingerol inhibits ROS and iNOS through the suppression of PKC-α and NF-κB pathways in lipopolysaccharide-stimulated mouse macrophages. Biochem Biophys Res Commun 382, 134–139 (2009). Guimarães, M. R., Leite, F. R. M., Spolidorio, L. C., Kirkwood, K. L. & Rossa, C. Curcumin abrogates LPS-induced proinflammatory cytokines in RAW 264.7 macrophages. Evidence for novel mechanisms involving SOCS-1, -3 and p38 MAPK. Arch Oral Biol 58, 1309–1317 (2013). Qureshi, A. A. et al. Inhibition of nitric oxide and inflammatory cytokines in LPS-stimulated murine macrophages by resveratrol, a potent proteasome inhibitor. Lipids Health Dis 11, (2012). Mu, M. M. et al. The Inhibitory Action of Quercetin on Lipopolysaccharide-Induced Nitric Oxide Production in RAW 264.7 Macrophage Cells . Journal of Endotoxin Research vol. 7 (2001). Ginhoux, F. & Jung, S. Monocytes and macrophages: Developmental pathways and tissue homeostasis. Nature Reviews Immunology vol. 14 392–404 Preprint at https://doi.org/10.1038/nri3671 (2014). Schildberger, A., Rossmanith, E., Eichhorn, T., Strassl, K. & Weber, V. Monocytes, peripheral blood mononuclear cells, and THP-1 cells exhibit different cytokine expression patterns following stimulation with lipopolysaccharide. Mediators Inflamm 2013, (2013). Dudek, A. M., Martin, S., Garg, A. D. & Agostinis, P. Immature, semi-mature, and fully mature dendritic cells: Toward a DC-cancer cells interface that augments anticancer immunity. Frontiers in Immunology vol. 4 Preprint at https://doi.org/10.3389/fimmu.2013.00438 (2013). Romani, N. et al. Proliferating Dendritic Cell Progenitors in Human Blood . Inaba, K. et al. Identification of Proliferating Dendritic Cell Precursors in Mouse Blood . O’Shea, J. J. & Murray, P. J. Cytokine Signaling Modules in Inflammatory Responses. Immunity vol. 28 477–487 Preprint at https://doi.org/10.1016/j.immuni.2008.03.002 (2008). Liu, C. et al. Cytokines: From Clinical Significance to Quantification. Advanced Science vol. 8 Preprint at https://doi.org/10.1002/advs.202004433 (2021). Burska, A., Boissinot, M. & Ponchel, F. Cytokines as biomarkers in rheumatoid arthritis. Mediators of Inflammation vol. 2014 Preprint at https://doi.org/10.1155/2014/545493 (2014). Additional Declarations No competing interests reported. Supplementary Files SppFig.Mendozaetal.1.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4750304","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":344696618,"identity":"ab79f076-5a86-48a5-9001-bd62e060f0ee","order_by":0,"name":"Laura Mendoza","email":"","orcid":"","institution":"Margarita Salas Center for Biological Research (CIB), CSIC","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Mendoza","suffix":""},{"id":344696619,"identity":"d5f45917-9c9a-47da-805c-be15d66db563","order_by":1,"name":"Marcos Sánchez","email":"","orcid":"","institution":"Fundación Centro Tecnológico de 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14:08:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4750304/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4750304/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":63306330,"identity":"fac521b9-54d8-411b-ac0d-79b008bc7c94","added_by":"auto","created_at":"2024-08-26 17:35:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":38220,"visible":true,"origin":"","legend":"\u003cp\u003eScheme of the methanolysis of jojoba oil using CaO as catalyst.\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4750304/v1/1ceddbbf4884b7195c6eff6f.png"},{"id":63306329,"identity":"42966aa1-de44-4e20-b318-8da8225cce25","added_by":"auto","created_at":"2024-08-26 17:35:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":9006,"visible":true,"origin":"","legend":"\u003cp\u003ePareto Chart illustrating the contribution of various factors to the yield.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4750304/v1/a00b9e68a7e51e54a5736077.png"},{"id":63306331,"identity":"b627faf1-2d3a-4623-a704-a88d3403d8f3","added_by":"auto","created_at":"2024-08-26 17:35:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":418449,"visible":true,"origin":"","legend":"\u003cp\u003eYield variation as a function of all the different variables tested. a) Effect of temperature (T) in the reaction yield, b) variation of the reaction yield due to modifications on the molar ratio (M), c) effect of the catalysts amount (C) in the production yield, d) combine effect of temperature and molar ratio on the reaction yield, e) effect of temperature and catalyst on the yield of the reactions, and f) is the combine effect of molar ratio and catalysts on the yield.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4750304/v1/6f83116e0f3ec888240c531a.png"},{"id":63306337,"identity":"2c37e5f2-08b4-4f25-a43d-228e347b1714","added_by":"auto","created_at":"2024-08-26 17:35:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":29660,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the experimental values and those predicted by the model to assure the accuracy of the model.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4750304/v1/d1afdcf134d8c19324dbef8a.png"},{"id":63306740,"identity":"dfdb2d4c-9e02-4bec-a3f3-53d4c1672dc5","added_by":"auto","created_at":"2024-08-26 17:43:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":133958,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytokines in THP-1 and PMA-differentiated THP-1 cells.\u003c/strong\u003e A – D show cytokine levels released by THP-1 cells when treated with C13D in presence (squared bars) or absence of LPS (solid bars). E – H show cytokine levels released by PMA-differentiated THP-1 cells when treated with C13Din the presence or absence of LPS. C13D doses used were: 0,3µl/ml; 1µl/ml; 3µl/ml and 9µl/ml. This is a representative experiment of 3 replicas with similar results. Significant differences are indicated as: *, p \u0026lt;0.05; **, \u0026lt;0.01; ***, p\u0026lt;0.001, ****, p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4750304/v1/b45a25060bf78f1d2366e32e.png"},{"id":63306739,"identity":"468f9b49-cc0b-4a15-87fd-626362636083","added_by":"auto","created_at":"2024-08-26 17:43:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":61045,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytokines in monocyte derived dendritic cells (MoDCs).\u003c/strong\u003e A – D show the release of pro and anti-inflamatory cytokines by MoDC after being treated with C13D in presence (squared bars) or absence of LPS (solid bars). C13D doses used were: 0,3µl/ml; 1µl/ml; 3µl/ml and 9µl/ml. This is a representative experiment of 3 replicas with similar results. Significant differences are indicated as: *, p \u0026lt;0.05; **, \u0026lt;0.01; ***, p\u0026lt;0.001, ****, p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4750304/v1/cc4a7b12523bcfba26ffe800.png"},{"id":63306335,"identity":"227367e2-c716-4561-ac46-f0c5726fcfab","added_by":"auto","created_at":"2024-08-26 17:35:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":231945,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMoDC immunophenotyping (A – E).\u003c/strong\u003e Spectral Flow cytometry analysis showing CD86 expression in untreated DC (UT), LPS-treated MoDC in comparison with MoDC treated with different C13D doses: (A) 0,3µl/ml; (B) 1µl/ml; (C) 3µl/ml and (D) 9µl/ml. Barr graph showed statistical differences between samples (E). \u003cstrong\u003eMixed Lymphocyte Reaction (MLR) (F – M).\u003c/strong\u003e (F) T cells unstimulated; (G) T cells + untreated MoDC; (H) T cells + LPS-treated MoDC; (I) T cells + 0,3µl/ml C13D-treated-MoDC; (J) T cells + 1µl/ml C13D-treated-MoDC; (K) T cells + 3µl/ml C13D-treated-MoDC; (L) T cells + 9µl/ml C13D-treated-MoDC; (M) Bar graph showing significant differences. This is a representative experiment of 2 independent experiment with similar results. Significant differences are indicated as: *, p \u0026lt;0.05; **, \u0026lt;0.01; ***, p\u0026lt;0.001, ****, p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4750304/v1/bc906d6faa20291dc369b516.png"},{"id":63307186,"identity":"a3ca25d7-8bfd-4db9-a0fb-1aa78b3b48e1","added_by":"auto","created_at":"2024-08-26 17:51:52","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":397173,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical\u003cstrong\u003e \u003c/strong\u003esummary.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4750304/v1/19ff9bb3aa4fb0e3f60ab750.png"},{"id":68231428,"identity":"53d65e2b-ee61-4d6f-9bcc-516563fef0c4","added_by":"auto","created_at":"2024-11-05 06:03:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1834507,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4750304/v1/1f139558-48f1-4d07-94b0-1a34e1c20a91.pdf"},{"id":63306336,"identity":"5247bd82-14aa-4fdc-b5b2-17de0ed41cd0","added_by":"auto","created_at":"2024-08-26 17:35:52","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":51502,"visible":true,"origin":"","legend":"","description":"","filename":"SppFig.Mendozaetal.1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4750304/v1/20f836e6860f00a00a9073b7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Optimizing Jojoba Oil Methanolysis and Unveiling the Immunomodulatory Potential of cis-13-Docosenol Fatty Alcohol: A Circular Biorefinery Perspective","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThere is an increasing global demand for both circularity and carbon neutrality. The use of biomass and waste biomass to produce new bio-based chemicals is rising and will continue to do so in the coming decades. This trend is driven by societal and political pressure to replace petroleum-based chemicals with natural bio-based alternatives\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Various types of biomasses can serve as substrates for the production of different chemicals. For instance, algae can be utilized to produce succinic acid\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, lignocellulose and cellulose to produce wood-based derivatives (like vanillin)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e or waste oils can be upgraded into bio-based fuels, jet fuel, alcohol based and biodiesel, among others\u003csup\u003e5 6\u003c/sup\u003e. Although many new bio-based chemicals are being developed, there is limited research on the production of bio-based components with potential medical applications. One example is biomass-based fibre materials designed for use as antibacterial agents\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, antioxidant, anti-fungal as well as for cosmetics\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe jojoba plant, botanically classified as a desert-like perennial shrub from the Buxaceae family \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, is native to the Sonoran Desert. It is typically found in the US, Israel, Mexico, Argentina, India, and South Africa due to its unique characteristics and resistance to extreme weather conditions \u003csup\u003e9 10\u003c/sup\u003e, particularly salinity, drought, and heat\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. From a chemical perspective, jojoba oil consists of a mixture of high molecular weight esters extracted from the jojoba plant. The composition of jojoba oil is distinctly different from that of other vegetable oils due to the absence of a glycerol backbone in its chemical structure. The most common method for obtaining jojoba oil from jojoba seeds involves a combination of mechanical pressing and hexane extraction to maximize the overall yield of the process, which is slightly higher than 50% of the total seed. Furthermore, jojoba oil can be converted into biodiesel and jojobyl alcohols through methanolysis transesterification. The long-chain alcohols produced in this process include 11-eicosenol, 13-docosenol (C13D), and 15-tetracosenol, which are classified as high-value-added products. This reaction is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImportantly, jojoba oil has been shown to have certain pharmaceutical applications \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Similar fatty alcohols derived from very long-chain aliphatic alcohols have been reported to inhibit the production of pro-inflammatory cytokines in LPS-stimulated macrophages, indicating potential anti-inflammatory effects of long-chain fatty alcohols\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Other studies investigating eicosenoid compounds have revealed their ability to enhance the production of pro-inflammatory cytokines, such as TNF-α and IL-1β, while decreasing levels of the anti-inflammatory cytokine IL-10 in THP1-derived macrophages\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. The functionality of C13D against enveloped viruses is still unknown. However, very similar compounds have been studied in various cell types, including THP-1 cells, PMA-differentiated THP-1 cells, macrophages, and monocyte-derived dendritic cells (MoDCs)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Depending on their nature, some compounds exhibit anti-inflammatory effects, whereas others are capable of stimulating immune cells.\u003c/p\u003e \u003cp\u003eBiorefineries are processing facilities that convert biomass into value-added products such as biofuels, biochemicals, bioenergy/biopower, and other biomaterials with potential medical applications. In this study, the production optimization for valorizing jojoba oil into fatty alcohols was carried out using a response surface methodology (RSM) approach. Bio-methanol and calcium oxide were employed in a completely green process. The molar ratio, temperature, and catalyst concentration were optimized to achieve the highest production efficiency. Among the jojoba alcohols produced, C13D was investigated for its potential to modulate immune cells. This approach is illustrated in the graphical abstract. The results from our study revealed the predicted optimal conditions for jojoba oil production and suggested a possible new application of C13D as an immunomodulator in innate immune system cells. This biorefinery approach focuses on the production of high-value products, such as fatty alcohols, while also generating fatty acid methyl esters as a biofuel co-product.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials:\u003c/h2\u003e \u003cp\u003eJojoba oil was supplied by Jojoba Israel (Israel). Gas chromatography (GC) standards were purchased from Sigma Aldrich. The catalyst was produced by the calcination of mussel shells (Mytilus galloprovincialis species) from Galicia (Spain). C13D was purchased from Cymit Quimica S.L. and was used in a liquid state at 37\u0026deg;C in all experiments. The purity of C13D is \u0026gt;\u0026thinsp;98.0% (GC), as indicated in its datasheet (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cymitquimica.com/es/productos/3B-D2174/629-98-1/cis-13-docosenol/\u003c/span\u003e\u003cspan address=\"https://cymitquimica.com/es/productos/3B-D2174/629-98-1/cis-13-docosenol/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eReaction steps and optimization\u003c/h2\u003e \u003cp\u003eThe experimental data referenced was previously published\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. This existing dataset was examined from a kinetics standpoint; however, its optimization and the influence of the variables under investigation were not addressed. To address this gap, this study conducted a statistical analysis of data from all 27 experiments to elucidate the individual effects of each variable and their quadratic combinations. StatGraphics Software was employed for this analysis. The selected model was quadratic, with the primary factors being the three individual variables: molar ratio (defined as the moles of alcohol relative to the moles of oil in the process), temperature, and catalyst amount (expressed as the weight percentage of catalyst relative to the quantity of oil).\u003c/p\u003e \u003cp\u003eThe reaction was conducted in a 250 mL reactor where the appropriate reactants were premixed, and the desired temperature was reached prior to the initiation of the reaction. Temperature was maintained constant using a Proportional-Integral-Derivative (PID) controller. At predetermined intervals, samples were withdrawn and prepared for gas chromatography (GC) analysis. The analyses were performed on a Hewlett-Packard GC model 5890 connected to an integrator. Samples were analyzed using a Flame Ionization Detector (FID) set at a detector temperature of 320\u0026deg;C. Further details of the methodology can be found elsewhere \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eTHP1 cell culture and differentiation\u003c/h2\u003e \u003cp\u003eTHP-1 cells (TIB-202) were cultured in 24-well plates at a seeding density of 1 x 10\u003csup\u003e6\u003c/sup\u003e cells/ml in Roswell Park Memorial Institute (RPMI) medium 1640 with addition of 1% penicillin/streptomycin and 5% Fetal Bovine Serum (FBS) (Sigma-Aldrich, Dorset, UK). Cells were treated with different doses of C13D (0,3\u0026thinsp;\u0026minus;\u0026thinsp;1\u0026ndash;3\u0026ndash;9 \u0026micro;l/ml) either alone or in combination with LPS (100 ng/ml) for 24h at 37˚C and 100% humidity in 5% CO\u003csub\u003e2\u003c/sub\u003e. THP-1 cells were cultured in 6-well plates at a concentration of 2 x 10\u003csup\u003e5\u003c/sup\u003e cells/ml and stimulated with 25 ng/ml of phorbol 12-myristate 13-acetate (PMA, 25 ng/ml final concentration, Sigma-Aldrich, Dorset, UK) to differentiate into macrophage-like cells. Cells were then incubated for 48 h, after which the PMA-containing media was replaced with fresh media and the cells rested for an additional 24 h prior to C13D treatment, as explained above. In this case, LPS concentration was 10 ng/ml.\u003c/p\u003e \u003cp\u003eViability from C13D treated cells was measured by MTS assay, based on the reduction of MTS tetrazolium compound by viable mammalian cells to generate a coloured formazan dye that is soluble in cell culture media and quantified by measuring at 490\u0026ndash;500 nm absorbance. For that procedure, cells were seeded in 96 well plates with C13D and LPS treatments and subsequently, MTS reagent was added to cell culture media and incubated for 1-2h in standard culture conditions. After that, the plate was shaken briefly and optical density measured at 490 nm.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-Linked Immunosorbent Assay (ELISA)\u003c/h2\u003e \u003cp\u003eCytokines such as TNF-α, IL-1β, IL-6 and IL-10 were measured in the supernatants of the cell cultures after 24h using commercially available Human DuoSet ELISA kits (R\u0026amp;D Systems \u0026ndash; DY210, DY201, DY206, DY217B) in accordance with the supplier\u0026rsquo;s protocol. Absorbance was converted to pg/ml according to the standard curve generated with a five-parameter logistic curve fit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003ePrimary human cells\u003c/h2\u003e \u003cp\u003eBuffy coats from healthy blood donors were collected from the blood bank \u0026ldquo;Centro de Transfusi\u0026oacute;n\u0026rdquo; in Comunidad de Madrid. MoDC differentiation was achieved as previously described \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. This procedure was approved by CSIC Ethical Committee. Briefly, PBMC were depleted using mouse anti-human CD14 microbeads (Miltenyi Biotec) as indicated by the manufacturer. To generate MoDCs, CD14\u003csup\u003e+\u003c/sup\u003e cells were incubated following Miltenyi Biotec\u0026acute;s protocol (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.miltenyibiotec.com/ES-en/applications/all-protocols/generation-of-mo-dcs.html\u003c/span\u003e\u003cspan address=\"https://www.miltenyibiotec.com/ES-en/applications/all-protocols/generation-of-mo-dcs.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). After 7 days, cells were harvested and seeded in plates for stimulation.\u003c/p\u003e \u003cp\u003eFor CD3\u0026thinsp;+\u0026thinsp;isolation, PBMC were counted and labelled with CFSE (5 \u0026micro;M; Invitrogen; Thermo Fisher) for 20 min at 37˚C and straightaway incubated with anti-human CD3 (PE/Cyanine7 anti-human CD3 Antibody; clon SK7; isotype: Mouse IgG1, k; BioLegend) for 30 min at 4˚C in the dark. Cells were sorted using BD FACSAria Fusion Flow Cytometer, separating CD3\u0026thinsp;+\u0026thinsp;cells with a standard purity around 95%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry immunophenotyping\u003c/h2\u003e \u003cp\u003eFor MoDC, 2 x 10\u003csup\u003e5\u003c/sup\u003e cells were stained at a final volume of 50 \u0026micro;l. First, cells were resuspended in 25 \u0026micro;l of PBS supplemented with 0,5% BSA and 0,1% NaN\u003csub\u003e3\u003c/sub\u003e (FACS buffer) mixed with 25 \u0026micro;l of anti-goat serum (Merck Millipore) and incubated for 20 min on ice. After the blocking step, an antibody cocktail was prepared with a total volume of 50 \u0026micro;L per sample, containing all antibodies from the panel at the final concentrations shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, and diluted in FACS buffer. Fifty microliters of the antibody mix were added to each sample and incubated for 30 minutes at 4\u0026deg;C in the dark. Following extensive washing with 100 \u0026micro;L PBS and centrifugation (1100 rpm, 5 minutes, 4\u0026deg;C), the pellets were resuspended in 200 \u0026micro;L PBS and analyzed using a Cytek Aurora Spectral Flow Cytometer.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntibodies used for immunophenotyping. PE, Phycoerythrin; APC, Allophycocyanin; APC R700, Allophycocyanin R700.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eConjugated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMarker\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSupplier\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eClone\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCatalogue number\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003cp\u003e(\u0026micro;l/Test)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCD80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBD Bioscience\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2D10.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e566992\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAPC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCD40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBD Bioscience\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5C3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e555591\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAPC R700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCD86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBD Bioscience\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2331 (FUN-1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e565149\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eeFluor450\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHLA-DR II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThermo Fisher\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eL243\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e48-9952-42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMLR assay\u003c/h2\u003e \u003cp\u003eFor mixed lymphocyte reaction (MLR), allogeneic CFSE- labelled T-cells (10\u003csup\u003e6\u003c/sup\u003e cells/ml) were co-cultured with MoDC (2 x 10\u003csup\u003e5\u003c/sup\u003e cells/ml) in 96-well round-bottom plates (Corning, Corning, NY) at a stimulator: responder ratio of 1:5. Unstimulated CFSE-labelled T cells served as a negative control. After 6 days, cells were stained with PE/Cyanine7-conjugated anti-CD3 (at 1:100 dilution, PE/Cyanine7 anti-human CD3 Antibody; clon SK7; isotype: Mouse IgG1, k; BioLegend) antibody and analysed in CytoFLEX S Cytometer. T-cell proliferation analysis was performed using FlowJo software proliferation tool (Becton Dickinson, Franklin Lakes, NJ).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad PRISM 5. P-values were determined using two-way ANOVA and Bonferroni test correction was applied. Unless otherwise stated, data are shown as the mean of at least three biological replicates. Significant differences in the figures were indicated as: *, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **, \u0026lt;\u0026thinsp;0.01; ***, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, ****, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003eOptimization using individual variables\u003c/h2\u003e\n \u003cp\u003eMolar ratio (MR), temperature (Temp), and catalyst concentration (Cat) are identified as the main variables for extracting jojoba oil. Consequently, these three variables were initially analyzed individually. Each of these main variables alone has a significant effect on the model under study, as shown in the Pareto chart in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. All three variables (MR(A), Temp(B), Cat(C)) have a positive effect, while the quadratic terms of each variable with itself (AA, BB, CC) have a significantly smaller negative effect.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eBased on these results, the effect of each individual variable was studied while keeping the remaining operational conditions constant. Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA shows changes in jojoba oil yield at different Temp. An increase from 45 to 65°C improves the reaction yield from 79.6–90.3%. Similarly, the effect of changing the amount of alcohol in the reaction medium was analyzed to understand how the ratio among the different feedstocks influences the process yield. Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB shows that an increase in the MR, and therefore an increase in the amount of alcohol, improves the reaction yield. There is a significant increase when changing the molar ratio from 6:1 to 9:1 (jojoba oil yield increases from 59.9–90.3%), while the increase is much less significant when MR increases from 9:1 to 12:1 (yield increases up to 93%). This smaller increase could be related to the reaction approaching equilibrium, where higher amounts of alcohol have a significantly reduced effect.\u003c/p\u003e\n \u003cp\u003eThe third variable studied was Cat. For this purpose, all other operational variables were kept constant while the catalyst amount was increased from 6 to 10 wt.%. Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC shows this effect. Increasing the catalyst concentration increased the reaction yield, with a more significant impact observed between 6 to 8 wt.% compared to changes from 8 to 10 wt.%. This effect could be due to an excess of catalysts that cannot be utilized effectively due to the limited amount of feedstock. Additionally, a higher Cat increases the reaction rate, leading to the reaction reaching equilibrium sooner. Consequently, higher Cat for a fixed time could result in the reaction being closer to equilibrium, leading to smaller yield increments compared to lower catalyst concentrations.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003eOptimization by combined effect\u003c/h2\u003e\n \u003cp\u003eBesides the individual effect of each variable, it is important to understand the combined effect of multiple variables. Here, the effect of two variables at a time is presented. Figure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD shows the combined effect of temperature and molar ratio on the reaction yield. An increase in both variables significantly increases the reaction yield while keeping other variables constant. This result aligns with the findings presented in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, where these two variables showed a positive effect both individually and in combination.\u003c/p\u003e\n \u003cp\u003eIn Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE, the combined effect of reaction Temp and Cat on jojoba oil yield is presented. A higher Cat and a higher Temp do not result in the optimal yield. Instead, the highest Cat is necessary, but the reaction Temp should be close to 55 ºC for the best effect on reaction yield.\u003c/p\u003e\n \u003cp\u003eFigure 3F shows the combined effect of MR and Cat on the reaction yield. These two variables, as well as their interaction, have a positive effect. Therefore, an increase in both variables leads to an increase in jojoba oil yield. As shown in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF, the highest yield is achieved when both MR and Cat reached their maximum levels.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eUsing all 27 experiments presented elsewhere \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, a quadratic model was applied to understand the individual effects of all the variables, their quadratic effects, and their combined quadratic effects. This analysis yielded the following equation:\u003c/p\u003e\n \u003cdiv id=\"Equa\" class=\"Equation\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\u003cimg 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\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003eWhere T: temperature [°C], M: molar ratio, and C: catalysts amount [wt.%]\u003c/p\u003e\n \u003cp\u003eUsing Eq. 1, a comparison between the experimental and predicted yields was conducted. Figure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e presents this comparison for all the data collectively. The regression was robust, with a coefficient of regression of 0.8192, indicating that the model fits the data quite accurately.\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e\u003c/p\u003e\n \u003cp\u003eFinally, an optimization analysis was performed using the presented data and the developed model. The optimal operational conditions and the corresponding theoretical yields are described and presented in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePredicted optimal conditions.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTemp [°C]\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMR\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCat. [%]\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eYield [%]\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePredicted\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12:1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94.87\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eHaving developed the optimal conditions to generate jojoba oil fatty alcohols, we wonder whether one of the main jojoba oil component, C13D, exhibited an ability to interact with cells from the immune system, paving the way of new applications of Jojoba oil after its optimization.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eCytokine secretion in C13D treated THP-1 and PMA-differentiated THP-1 cells\u003c/h2\u003e\n \u003cp\u003eMonocytes and macrophages have crucial and diverse roles in the regulation of the innate immune system and act as antigen presenting cells in adaptive immunity \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. They also initiate inflammation against invading antigens and antigen presentation \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Therefore, THP-1 cell line of monocytes were initially used to measure whether C13D was toxic in our tissue culture conditions. As described in Supplementary Fig.\u0026nbsp;1, concentrations ranging from 0.3 to 9 µl/ml did not significantly affect cell viability in our experiments. Therefore, subsequent experiments were conducted within this concentration range.\u003c/p\u003e\n \u003cp\u003ePrevious studies have utilized LPS as a stimulatory factor to analyze cytokine expression following activation in these cells \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. In this study, LPS served as a positive control for stimulation when C13D was added to THP-1 cells at various doses (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, A-D) and to PMA-stimulated THP-1 cells (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, E-H). Generally, cytokine secretion was higher in PMA-differentiated THP-1 cells (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, E-H) compared to non-PMA-treated THP-1 cells (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, A-D) when assessing IL-1β, TNF-α, IL-6, and IL-10. C13D induced IL-6 secretion in THP-1 cells at doses of 1 and 3 µl/ml (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). In contrast, in PMA-differentiated THP-1 cells, the highest doses significantly increased IL-1β and TNF-α secretion (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE, F) without affecting IL-6 and IL-10 secretion (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG, H).\u003c/p\u003e\n \u003cp\u003eWhen C13D was combined with LPS in THP-1 cells, cytokine secretion exhibited a distinct pattern. For both IL-1β and IL-6, higher doses of C13D correlated with lower cytokine release (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA, C). IL-10 was not detected in the secretion (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD), and TNF-α levels varied depending on the combined dose (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). In PMA-differentiated THP-1 cells, a similar pattern was observed for IL-1β, IL-6, and IL-10 secretion, with decreasing levels as the dose of C13D increased (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE, G, and H). However, TNF-α secretion increased with C13D doses up to 3 µl/ml (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF).\u003c/p\u003e\n \u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eCytokine secretion in C13D treated MoDC\u003c/h2\u003e\n \u003cp\u003eMoDCs are highly valuable for in vitro studies to simulate the behavior of DC under specific conditions. Hence, increasing concentrations of C13D were added in the presence or absence of LPS (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). C13D significantly enhanced secretion of the pro-inflammatory cytokine TNF-α at all doses tested and the anti-inflammatory cytokine IL-10, but the latter effect was observed only at the highest concentration used (Figs. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB, D). It did not affect the release of IL-1β or IL-6 (Figs. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA, C). However, upon LPS activation, TNF-α secretion decreased at doses of 3 and 9 µl/ml (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB), while IL-6 secretion remained unaffected (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC). IL-1β secretion significantly increased at lower concentrations of C13D (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). The pattern for IL-10 secretion appeared to be dose-dependent, similar to PMA-differentiated THP-1 cells (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eH), with decreased secretion as the C13D dose increased (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eMoDC surface markers’ levels after C13D treatment\u003c/h2\u003e\n \u003cp\u003eMoDCs remain immature after 7 days of differentiation from monocytes. Increasing the expression of markers such as CD80, CD86, CD40, or MHC-II on the surface of DCs is considered the gold standard for measuring activation in these cells (ref. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3109/10520295.2015.1017536\u003c/span\u003e\u003c/span\u003e). Therefore, to assess MoDC maturation induced by C13D treatment, surface expression was measured using specific monoclonal antibodies for each of these markers by flow cytometry (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA-D). Levels of CD80 or CD40 remained unchanged in the presence of C13D compared to untreated cells (data not shown). MHC-II levels were already high, making further activation difficult to detect (data not shown). Surprisingly, after 24 hours of C13D treatment, CD86 levels increased in a dose-dependent manner, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eE. Although not reaching the same levels as LPS, such increase is significantly visible compared to untreated cells.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect of C13D on T cell proliferation\u003c/h2\u003e\n \u003cp\u003eFunctional maturation of DCs is commonly assessed by their ability to stimulate allogeneic T cells using mixed lymphocyte reaction (MLR) assays \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. DCs are potent inducers of lymphocyte activation in allogeneic MLR due to their expression of several surface costimulatory molecules, such as CD86, as shown in Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eA-E. High levels of MHC expression and the presentation of costimulatory molecules make DCs particularly effective in eliciting a robust T cell response \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Therefore, an MLR assay was employed to investigate the impact of C13D-treated MoDCs on their capacity to induce proliferation of naïve T lymphocytes (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eF-L). After 6 days of co-culture, the data indicated no significant proliferation of T cells alone (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eF), and only a slight percentage of proliferation was observed in the presence of untreated MoDCs (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eG), suggesting minimal activation in the absence of stimuli. Interestingly, C13D-treated MoDCs induced approximately 37% proliferation in the T CD3 + population at a concentration of 0.3 µl/ml (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eI). Notably, under our experimental conditions, LPS-treated MoDCs induced similar levels of proliferation, reaching up to approximately 39.9% (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eH). Although the level of proliferation was not as pronounced at other doses of C13D as it was at 0.3 µl/ml, MoDCs treated with any dose of C13D significantly induced T cell proliferation compared to untreated MoDCs (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eJ-L). Again, it was interesting to observe a dose dependent effect, the higher the concentration of C13D, the lower percentage of T cell proliferation induced (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003eM).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAmong emerging biomass sources, non-edible oils are receiving increasing attention due to their minimal competition with food resources. Additionally, to avoid competition with arable land, there is significant interest in non-edible oils that can be cultivated in non-arable areas. Jojoba oil, for instance, is a drought-resistant shrub which thrives in arid conditions. This makes it well-suited for cultivation in desert non-arable areas worldwide due to its unique resistance to extreme weather conditions\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The cultivation of jojoba plants holds great promise for the development of desert areas. As jojoba cultivation requires minimal water and can withstand harsh environmental conditions, it presents a sustainable and economically viable option for transforming barren desert lands into productive agricultural spaces. Establishing jojoba plantations contributes not only to local economic growth but also to environmental conservation by preventing desertification and enhancing soil stability. This dual benefit of economic prosperity and ecological sustainability positions jojoba cultivation as a key driver for development in desert regions, offering a unique solution to harness the untapped potential of arid landscapes. However, while jojoba oil holds significant economic potential, its extraction process requires optimization to ensure sustainability. Currently, the cultivation and extraction of jojoba oil can be resource-intensive, necessitating a careful balance between economic gains and environmental impact. In response to the global need for a circular economy and waste valorization, several studies have explored the use of renewable alcohols, such as methanol, ethanol, and butanol, in conjunction with bio- and waste-based catalysts like mussel shells and eggshells. Catarino et al.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e studied the use of calcium diglyceroxide from scallop shells with promising results, they achieved a yield of 96% towards fatty acid methyl esters within 2 h. of reaction at a temperature of 65\u0026deg;C with 5 wt% of catalyst and a molar ratio of 12:1. Similar work was conducted by Dias et al. \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, where lime was used as a calcium source and different configurations of CaO were tested. They found that the presence of glycerol in a CaO and methanol solution exhibited kinetic behavior without an induction period, as previously reported. S\u0026aacute;nchez et al. \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e studied the transesterification of jojoba oil using renewable calcium oxide catalysts and methanol. The results showed a reaction conversion of 95% under the conditions of a reaction temperature of 65\u0026deg;C, a molar ratio of 12:1, and 8 wt.% catalyst. In addition, Sanchez et al. \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e presented a kinetics model and a reaction pathway for such process. Furthermore, S\u0026aacute;nchez et al. \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e improved the reaction conditions by using a high-pressure reactor, allowing the process to reach a final conversion of 96.3 %. This hih conversion was achieved with a reaction time of 5 h with similar operational conditions as in their previous work \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Likewise, Avhad et al. \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e studied the butanolysis of jojoba oil in the presence of mussel shell naturally derived catalyst (CaO based). The authors have found the highest conversion of 96.11 % when the reation temperature is 85\u0026deg;C with a molar ratio of 10:1 and 12 wt% catalyst. The transesterification reaction produces jojoba alcohols (11-eicosenol, C13D, and 15-tetracosenol) in a mixture of 36\u0026ndash;40%, 43\u0026ndash;49%, and 9\u0026ndash;10%, respectively\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOnce the Jojoba oil is being produced, its transformation into biodiesel and jojobyl alcohols can be achieve using different reactive pathways. Singh et al \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e studied the supercritical methanolysis of jojoba oil using a response surface methodology (RSM) approach. The authors found the highest yield of 95 %at a temp of 287\u0026deg;C, 123 bar of pressure and 30:1 molar ratio within 23 minutes of reaction. Similarly, the work by Ravikumar et al. \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e used methanol for the transesterification of jojoba oil, in this case the authors used a basic homogeneous catalyst (potassium hydroxide) achieving 90% yield after 25 minutes at a reaction temperature of 50 \u0026ordm;C. Similar research was carried out by Buoaid et al. \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e where jojoba was transesterified with methanol in the presence of potassium hydroxide with a yield of 83 % whe working at 25\u0026deg;C with a catalyst concentration of 1.35 wt%. Abdulrhman et al. \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e studied the transesterification of jojoba oil with methanol but using sodium hydroxide. The authors found the highest yield of 70 % for a15-minute reaction with a molar ratio of 16:1 and catalyst amount of 1.5 wt%.\u003c/p\u003e \u003cp\u003eAiming to make jojoba oil production sustainable, this work has sought to define the predicted optimal conditions for temperature, molar ratio, and catalyst amount, which are shown in Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Our results showed an optimal yield of 94.87% achieved when temperature was 60.87\u0026deg;C, molar ratio of alcohol to oil of 12:1, catalyst amount of 9.79 wt.% and reaction time of 10 h with constant steering of 300 rpm.\u003c/p\u003e \u003cp\u003eAs presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the increase in yield observed with rising temperature can be attributed to the endothermic nature of the reaction. A higher reaction temperature results in a higher final yield. Similar results were obtained by Mohadesi et el. \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e and Bargole et al. \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e where both used waste cooking oil as the sources in the presence of calcium-based catalysts. The optimal yield of 97 % ad 95 % rspectively were obtained for reaction temperatures of 54.97\u0026deg;C and 64.8\u0026deg;C respectively. In fact, the results obtained by Sulaiman et al. \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e in their study on the transesterification reaction of waste cooking oil using CaO-doped catalysts in the presence of methanol were similar. They observed that increasing the molar ratio resulted in higher yields. However, a further increase in the molar ratio tended to stabilize the yield, possibly indicating that excessively high values led to decreased yields due to the dissolution of catalysts in the reaction medium. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e in this study illustrates the impact of temperature and molar ratio on the reaction yield. Increasing both variables significantly enhance the reaction yield when other variables are held constant. This finding corroborates the results depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, where these two variables individually and in combination showed a positive effect. Moreover, this observation aligns with the findings of Mohadesi et al. \u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, who investigated the combined effect of temperature and molar ratio on the transesterification of waste cooking oil. In both studies, higher temperatures and molar ratios led to increased yields.\u003c/p\u003e \u003cp\u003eThe influence of reaction temperature and catalyst amount on jojoba oil yield is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. It was observed that the highest catalyst concentration was necessary, while the optimal reaction temperature for maximizing yield was around 55\u0026deg;C. This indicates that a higher catalyst amount and temperature do not necessarily yield the best possible outcome. Foroutan et al. \u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e observed a similar relationship between temperature and catalyst amount in their study on biodiesel production from edible oils using CaO-based catalysts. They reported a maximum yield of 98.83% at a reaction temperature of 65\u0026deg;C and a catalyst amount of 4%, which was not the highest amount tested.\u003c/p\u003e \u003cp\u003eBetween 43\u0026ndash;49% of jojoba alcohols produced by transesterification is C13D, also known as erucyl alcohol. C13D possesses multiple applications, including its characterization as an antiviral agent effective against lipid-enveloped viruses such as herpes simplex virus (HSV) and respiratory syncytial virus (RSV). Based such old experiments, it is generally believed that 1-docosanol inhibit viral replication by disrupting viral membranes\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e, although its mechanism of action has yet to be precisely defined. It has also been used as an emulsifier and surfactant in various cosmetic and pharmaceutical formulations as well as it is used in skincare products for its moisturizing and conditioning properties\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. It is also used in industrial applications such as lubricants and coatings due to its lubricating and film-forming properties\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. However, there are no studies focusing on possible C13D interaction with cells from the immune system. Thus, after establishing optimal conditions for producing jojoba oil fatty alcohols, we were investigating whether one of its components, C13D, interacts with immune system cells. This exploration could potentially open up new applications for jojoba oil following its optimization.\u003c/p\u003e \u003cp\u003eThe functionality of C13D against enveloped viruses is still unknown, although very similar compounds have been studied on different cell types such as THP-1 cells, PMA-differentiated THP-1 cells, macrophages and monocyte derived dendritic cells (MoDC)\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Immune cells are quite valuable candidates to study immune processes and responses. Mononuclear phagocytes, monocytes, and macrophages have crucial and diverse roles in the regulation of the innate immune system and they act as antigen presenting cells \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. The stablished cell line THP-1 is widely applied to mimic monocytes in cell culture models \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e and they are widely used as model for primary human macrophages. This is because following differentiation using phorbol 12-myristate 13-acetate (PMA), THP-1 cells acquire a macrophage-like phenotype \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, which help understanding this cell type behaviour under different conditions. Dendritic cells (DCs) are professional antigen presenting cells, they can be found in practically all tissues, where they detect imbalances and process antigens for presenting to T lymphocytes (T cells). DCs serve as the bridge between the innate and adaptive immune systems and are critical in the initiation of primary immune responses \u003csup\u003e19 20 22\u003c/sup\u003e. Functionally and phenotypically, MoDCs are believed to be typical immature DCs, characterized by their low expression of major histocompatibility complex class II (MHC-II) and costimulatory molecules. Immature MoDCs can be subsequently matured after treatment with compounds known to induce DC maturation, such as LPS (Lipopolysaccharide), TNF-α, or IFN-γ \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. DCs are not plentiful in peripheral blood, in fact, circulating blood DCs constitute from 0.1\u0026ndash;1.0% of peripheral blood mononuclear cells. Thus, moDCs have been the most widely used model to investigate human DC biology and function \u003csup\u003e48 49\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCytokines are secreted from a variety of cells such as lymphocytes, macrophages, monocytes, DC, etc. They participate in the immune response and have an important function as mediators associated with the communication network of the immune system \u003csup\u003e50 51\u003c/sup\u003e. Cytokines are responsible for the regulation of the maturation, growth and capacity of reacting quickly of immune cells \u003csup\u003e52 51\u003c/sup\u003e. Generally speaking, cytokines can be pro-inflammatory (IL-1β, IL‐6, IL‐8, IL‐12, IFN‐γ, and TNF‐α) or anti-inflammatory cytokines (L‐4, IL‐6, IL‐10, IL‐11, IL‐13, and TGF‐β) \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, although some cytokines exert both functions depending on the concentration and the environmental milieu. Pro-inflammatory cytokines can go into the systemic circulation and produce immune cell activation \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e whereas anti-inflammatory cytokines are immunoregulatory molecules which inhibit the excess inflammatory response of the pro-inflammatory ones \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDepending on their nature, some chemical compounds present anti-inflammatory effects whereas other are able to stimulate immune cells by, for example, making them produce high levels of pro-inflammatory cytokines. Some studies reveal that long chain fatty alcohols can reduce NO\u003csub\u003e2\u003c/sub\u003e amount, pro-inflammatory cytokines and inflammatory mediators produced by macrophages in a dose-dependent manner \u003csup\u003e13 15\u003c/sup\u003e. Other studies suggest an immune stimulating capacity of fatty alcohols due to their capacity of increasing TNF-α and IL-1β production, which are pro-inflammatory cytokines, and decreasing IL-10 level, which is an anti-inflammatory cytokine, by PMA-differentiated THP-1 cells \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis groundbreaking study marks the first exploration of C13D within the context of cells belonging to the innate immune system, such as monocytes, macrophages, or DC (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). When those cells were treated with different C13D doses in presence or absence of LPS different responses were shown. Low doses of C13D were able to increase TNF-α, IL-6 or IL-1β pro-inflammatory cytokines to similar levels as LPS in MoDC. Also, increased T cell proliferation was observed when C13D-activated MoDC were present, possible due to a higher expression of surface activation markers like CD86. By delving into the interactions between C13D and these crucial components of the immune system, we aimed to uncover novel insights into potential immunomodulatory effects and therapeutic applications, shedding light on a previously unexplored aspect of cellular immunity. This pioneering research holds the potential to broaden our understanding of C13D's impact on immune responses, opening avenues for innovative therapeutic strategies and contributing to advancements in the field of immunology.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe pursuit of optimizing jojoba oil extraction underscores a commitment to both economic prosperity and environmental stewardship, paving the way for a more sustainable and responsible industry in arid regions. Our study firstly focused on optimizing jojoba oil production, revealing that all operational variables evaluated positively influenced jojoba oil yield. However, the interaction between temperature and catalyst concentration showed a distinct effect. Optimal operational temperature was found to be in the mid-range of the tested domain, outperforming other scenarios. In summary, the variables ranked in decreasing order of relevance were: molar ratio\u0026thinsp;\u0026gt;\u0026thinsp;temperature\u0026thinsp;\u0026gt;\u0026thinsp;catalyst concentration. The fit between experimental and predicted data was robust, with a regression value of approximately 0.82.\u003c/p\u003e \u003cp\u003eAfter establishing optimal conditions for producing jojoba oil fatty alcohols, we explored whether one of its components, C13D, could interact with immune system cells. This investigation aimed to uncover new applications for jojoba oil following optimization. Our exploration into the effects of cis-13-docosenol (C13D) on innate immune cells revealed that low doses of C13D could elevate pro-inflammatory cytokines TNF-α, IL-6, and IL-1β to levels comparable to those induced by LPS in monocytes and DCs. Moreover, modulation of T cell stimulation by MoDCs previously treated with C13D enhanced T cell proliferation, likely through increased activation of surface markers.\u003c/p\u003e \u003cp\u003eOur findings establish optimal conditions for jojoba oil extraction, a critical step in maximizing its economic viability. This research adopts a bio-refinery approach, focusing on the immunomodulatory activity of C13D within the context of innate immune cells, contributing to a comprehensive understanding of its potential applications in sustainable biorefinery practices.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJMM would like to thank NMBU for their financial support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMM would like to thank\u0026nbsp;CSIC\u0026apos;s Global Health Platform (PTI+ Salud Global)\u0026nbsp;for their support.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePermissions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;We have the approval from the CSIC Ethical committee for the experiments using cells from human peripheral blood from healthy donors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: Marcos Sanchez, Jorge Marchetti, Mar\u0026iacute;a Montoya.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMethodology: Marcos Sanchez, Jorge Marchetti, Mar\u0026iacute;a Montoya.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFormal analysis and investigation: Marcos Sanchez, Jorge Marchetti, Laura Mendoza, Maria Montoya.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWriting - original draft preparation: Laura Mendoza, Marcos Sanchez.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWriting - review and editing: Jorge Marchetti, Mar\u0026iacute;a Montoya \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding acquisition: Jorge Marchetti, Mar\u0026iacute;a Montoya\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResources: Marcos Sanchez, Jorge Marchetti, Mar\u0026iacute;a Montoya\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSupervision: Jorge Marchetti, Mar\u0026iacute;a Montoya\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research work was partially funded by the European Commission \u0026ndash; NextGenerationEU (Regulation EU 2020/2094), through CSIC\u0026apos;s Global Health Platform (PTI+ Salud Global) (COVID-19-117 and SGL2103015), Spanish Ministry of Science project (PID2021-123399OB-I00) and by the\u0026nbsp;Norwegian University of Life Science.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data is available upon request. Dra. Mar\u0026iacute;a Montoya should be contacted if any request of the data from this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCho, E. J., Trinh, L. T. P., Song, Y., Lee, Y. G. \u0026amp; Bae, H. J. Bioconversion of biomass waste into high value chemicals. \u003cem\u003eBioresource Technology\u003c/em\u003e vol. 298 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.biortech.2019.122386\u003c/span\u003e\u003cspan address=\"10.1016/j.biortech.2019.122386\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKircher, M. The transition to a bio-economy: Emerging from the oil age. Biofuels, Bioproducts and Biorefining 6, 369\u0026ndash;375 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKnoshaug, E. P. \u003cem\u003eet al.\u003c/em\u003e Demonstration of parallel algal processing: Production of renewable diesel blendstock and a high-value chemical intermediate. Green Chemistry 20, 457\u0026ndash;468 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaeda, M. \u003cem\u003eet al.\u003c/em\u003e Vanillin production from native softwood lignin in the presence of tetrabutylammonium ion. Journal of Wood Science 64, 810\u0026ndash;815 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHvidsten, I. B. \u0026amp; Marchetti, J. M. Securing liquid biofuel by upgrading waste fish oil via renewable and sustainable catalytic technology. Fuel 333, (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBouaid, A., Acherki, H., Bonilla, M. H. \u0026amp; Marchetti, J. M. Enzymatic ethanolysis of high free fatty acid jatropha oil using Eversa Transform. Energy Advances 159\u0026ndash;168 (2022) doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1039/d1ya00057h\u003c/span\u003e\u003cspan address=\"10.1039/d1ya00057h\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, D., Wang, Y., Huang, W. \u0026amp; Gong, H. Biomass-derived fiber materials for biomedical applications. \u003cem\u003eFrontiers in Materials\u003c/em\u003e vol. 10 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmats.2023.1058050\u003c/span\u003e\u003cspan address=\"10.3389/fmats.2023.1058050\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGad, H. A. \u003cem\u003eet al.\u003c/em\u003e Jojoba Oil: An Updated Comprehensive Review on Chemistry, Pharmaceutical Uses, and Toxicity. (2021) doi:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/polym\u003c/span\u003e\u003cspan address=\"10.3390/polym\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS\u0026aacute;nchez, M., Avhad, M. R., Marchetti, J. M., Mart\u0026iacute;nez, M. \u0026amp; Aracil, J. Jojoba oil: A state of the art review and future prospects. \u003cem\u003eEnergy Conversion and Management\u003c/em\u003e vol. 129 293\u0026ndash;304 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.enconman.2016.10.038\u003c/span\u003e\u003cspan address=\"10.1016/j.enconman.2016.10.038\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, L., Takase, M., Zhang, M., Zhao, T. \u0026amp; Wu, X. Potential non-edible oil feedstock for biodiesel production in Africa: A survey. \u003cem\u003eRenewable and Sustainable Energy Reviews\u003c/em\u003e vol. 38 461\u0026ndash;477 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.rser.2014.06.002\u003c/span\u003e\u003cspan address=\"10.1016/j.rser.2014.06.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003esony. COM PAR AT IVE STUDY OF ALK AL INE AND SAL INE STRESSES ON TWO OIL-PRODUCING PL ANT S A f a f. J. Exp. Biol. (Bot.) 10, 13\u0026ndash;25 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHabashy, R. R., Abdel-Naim, A. B., Khalifa, A. E. \u0026amp; Al-Azizi, M. M. Anti-inflammatory effects of jojoba liquid wax in experimental models. Pharmacol Res 51, 95\u0026ndash;105 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFern\u0026aacute;ndez-Arche, A. \u003cem\u003eet al.\u003c/em\u003e Long-chain fatty alcohols from pomace olive oil modulate the release of proinflammatory mediators. Journal of Nutritional Biochemistry 20, 155\u0026ndash;162 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlqarni, A. M. \u003cem\u003eet al.\u003c/em\u003e Metabolomic profiling of the immune stimulatory effect of eicosenoids on PMA-differentiated THP-1 cells. Vaccines (Basel) 7, (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontserrat-De La Paz, S., Garc\u0026iacute;a-Gim\u0026eacute;nez, M. D., \u0026Aacute;ngel-Mart\u0026iacute;n, M., P\u0026eacute;rez-Camino, M. C. \u0026amp; Fern\u0026aacute;ndez Arche, A. Long-chain fatty alcohols from evening primrose oil inhibit the inflammatory response in murine peritoneal macrophages. J Ethnopharmacol 151, 131\u0026ndash;136 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdulrahman, A., Ali, A. \u0026amp; Alfazazi, A. Synthesis and Process Parameter Optimization of Biodiesel from Jojoba Oil Using Response Surface Methodology. Arab J Sci Eng 46, 6609\u0026ndash;6617 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAvhad, M. R. \u003cem\u003eet al.\u003c/em\u003e Renewable production of value-added jojobyl alcohols and biodiesel using a naturally-derived heterogeneous green catalyst. Fuel 179, 332\u0026ndash;338 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrisci, E. \u003cem\u003eet al.\u003c/em\u003e In vivo tracking and immunological properties of pulsed porcine monocyte-derived dendritic cells. Mol Immunol 63, 343\u0026ndash;354 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatente, T. A. \u003cem\u003eet al.\u003c/em\u003e Human dendritic cells: Their heterogeneity and clinical application potential in cancer immunotherapy. Front Immunol 10, (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO\u0026rsquo;Keeffe, M., Mok, W. H. \u0026amp; Radford, K. J. Human dendritic cell subsets and function in health and disease. \u003cem\u003eCellular and Molecular Life Sciences\u003c/em\u003e vol. 72 4309\u0026ndash;4325 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00018-015-2005-0\u003c/span\u003e\u003cspan address=\"10.1007/s00018-015-2005-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLund, M. E., To, J., O\u0026rsquo;Brien, B. A. \u0026amp; Donnelly, S. The choice of phorbol 12-myristate 13-acetate differentiation protocol influences the response of THP-1 macrophages to a pro-inflammatory stimulus. J Immunol Methods 430, 64\u0026ndash;70 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao, Y. \u003cem\u003eet al.\u003c/em\u003e Single-Cell Analysis Reveals the Heterogeneity of Monocyte-Derived and Peripheral Type-2 Conventional Dendritic Cells. The Journal of Immunology 207, 837\u0026ndash;848 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCatarino, M., Martins, S., Soares Dias, A. P., Costa Pereira, M. F. \u0026amp; Gomes, J. Calcium diglyceroxide as a catalyst for biodiesel production. J Environ Chem Eng 7, (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoares Dias, A. P., Puna, J., Gomes, J., Neiva Correia, M. J. \u0026amp; Bordado, J. Biodiesel production over lime. Catalytic contributions of bulk phases and surface Ca species formed during reaction. Renew Energy 99, 622\u0026ndash;630 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS\u0026aacute;nchez, M., Marchetti, J. M., El Boulifi, N., Aracil, J. \u0026amp; Mart\u0026iacute;nez, M. Kinetics of Jojoba oil methanolysis using a waste from fish industry as catalyst. Chemical Engineering Journal 262, 640\u0026ndash;647 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eS\u0026aacute;nchez, M., Avhad, M. R., Marchetti, J. M., Mart\u0026iacute;nez, M. \u0026amp; Aracil, J. Enhancement of the jojobyl alcohols and biodiesel production using a renewable catalyst in a pressurized reactor. Energy Convers Manag 126, 1047\u0026ndash;1053 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAracil J, Mart\u0026iacute;nez M, El-Boulifi N, S\u0026aacute;nchez M \u0026amp; Serrano M. Proceso integrado de obtenci\u0026oacute;n de alcoholes monoinsaturados, biodiesel y productos biodegradables a partir de aceite de Jojoba. (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh, N. K., Singh, Y. \u0026amp; Sharma, A. Optimization of biodiesel synthesis from Jojoba oil via supercritical methanol: A response surface methodology approach coupled with genetic algorithm. Biomass Bioenergy 156, (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRavikumar Solomon, G. \u003cem\u003eet al.\u003c/em\u003e Evaluation of the transesterification process and physio-chemical properties of jojoba biodiesel. International Journal of Innovative Technology and Exploring Engineering 9, 2553\u0026ndash;2557 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBouaid, A., Bajo, L., Martinez, M. \u0026amp; Aracil, J. Optimization of biodiesel production from jojoba oil. Process Safety and Environmental Protection 85, 378\u0026ndash;382 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohadesi, M., Aghel, B., Gouran, A. \u0026amp; Razmehgir, M. H. Transesterification of waste cooking oil using Clay/CaO as a solid base catalyst. Energy 242, (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBargole, S. S., Singh, P. K., George, S. \u0026amp; Saharan, V. K. Valorisation of low fatty acid content waste cooking oil into biodiesel through transesterification using a basic heterogeneous calcium-based catalyst. Biomass Bioenergy 146, (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSulaiman, N. F., Ramly, N. I., Abd Mubin, M. H. \u0026amp; Lee, S. L. Transition metal oxide (NiO, CuO, ZnO)-doped calcium oxide catalysts derived from eggshells for the transesterification of refined waste cooking oil. RSC Adv 11, 21781\u0026ndash;21795 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eForoutan, R., Mohammadi, R., Razeghi, J. \u0026amp; Ramavandi, B. Biodiesel production from edible oils using algal biochar/CaO/K2CO3 as a heterogeneous and recyclable catalyst. Renew Energy 168, 1207\u0026ndash;1216 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKatz, D. H., Marcellettit, J. F., Khalilt, M. H., Popet, L. E. \u0026amp; Katzt, L. R. \u003cem\u003eAntiviral Activity of 1-Docosanol, an Inhibitor of Lipid-Enveloped Viruses Including Herpes Simplex (Viral Inhibition/Long-Chain Alcohols)\u003c/em\u003e. \u003cem\u003eProc. Natl. Acad. Sci. USA\u003c/em\u003e vol. 88 (1991).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeung, D. T. \u0026amp; Sacks, S. L. Docosanol: A topical antiviral for herpes labialis. Expert Opin Pharmacother 5, 2567\u0026ndash;2571 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhalil, M. H., Marcelletti, J. F., Katz, L. R., Katz, D. H. \u0026amp; Pope, L. E. Topical application of docosanol-or stearic acid-containing creams reduces severity of phenol burn wounds in mice. Contact Dermatitis 43, 79\u0026ndash;81 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOttone, C., Bernal, C., Serna, N., Illanes, A. \u0026amp; Wilson, L. Enhanced long-chain fatty alcohol oxidation by immobilization of alcohol dehydrogenase from S. cerevisiae. Appl Microbiol Biotechnol 102, 237\u0026ndash;247 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCallau, M., Sow-K\u0026eacute;b\u0026eacute;, K., Jenkins, N. \u0026amp; Fameau, A. L. Effect of the ratio between fatty alcohol and fatty acid on foaming properties of whipped oleogels. Food Chem 333, (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmith, N. C., Christian, S. L., Taylor, R. G., Santander, J. \u0026amp; Rise, M. L. Immune modulatory properties of 6-gingerol and resveratrol in Atlantic salmon macrophages. Mol Immunol 95, 10\u0026ndash;19 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee, T. Y., Lee, K. C., Chen, S. Y. \u0026amp; Chang, H. H. 6-Gingerol inhibits ROS and iNOS through the suppression of PKC-α and NF-κB pathways in lipopolysaccharide-stimulated mouse macrophages. Biochem Biophys Res Commun 382, 134\u0026ndash;139 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuimar\u0026atilde;es, M. R., Leite, F. R. M., Spolidorio, L. C., Kirkwood, K. L. \u0026amp; Rossa, C. Curcumin abrogates LPS-induced proinflammatory cytokines in RAW 264.7 macrophages. Evidence for novel mechanisms involving SOCS-1, -3 and p38 MAPK. Arch Oral Biol 58, 1309\u0026ndash;1317 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQureshi, A. A. \u003cem\u003eet al.\u003c/em\u003e Inhibition of nitric oxide and inflammatory cytokines in LPS-stimulated murine macrophages by resveratrol, a potent proteasome inhibitor. Lipids Health Dis 11, (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMu, M. M. \u003cem\u003eet al. The Inhibitory Action of Quercetin on Lipopolysaccharide-Induced Nitric Oxide Production in RAW 264.7 Macrophage Cells\u003c/em\u003e. Journal of Endotoxin Research vol. 7 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGinhoux, F. \u0026amp; Jung, S. Monocytes and macrophages: Developmental pathways and tissue homeostasis. \u003cem\u003eNature Reviews Immunology\u003c/em\u003e vol. 14 392\u0026ndash;404 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nri3671\u003c/span\u003e\u003cspan address=\"10.1038/nri3671\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchildberger, A., Rossmanith, E., Eichhorn, T., Strassl, K. \u0026amp; Weber, V. Monocytes, peripheral blood mononuclear cells, and THP-1 cells exhibit different cytokine expression patterns following stimulation with lipopolysaccharide. \u003cem\u003eMediators Inflamm\u003c/em\u003e 2013, (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDudek, A. M., Martin, S., Garg, A. D. \u0026amp; Agostinis, P. Immature, semi-mature, and fully mature dendritic cells: Toward a DC-cancer cells interface that augments anticancer immunity. \u003cem\u003eFrontiers in Immunology\u003c/em\u003e vol. 4 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fimmu.2013.00438\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2013.00438\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRomani, N. \u003cem\u003eet al. Proliferating Dendritic Cell Progenitors in Human Blood\u003c/em\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eInaba, K. \u003cem\u003eet al. Identification of Proliferating Dendritic Cell Precursors in Mouse Blood\u003c/em\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO\u0026rsquo;Shea, J. J. \u0026amp; Murray, P. J. Cytokine Signaling Modules in Inflammatory Responses. \u003cem\u003eImmunity\u003c/em\u003e vol. 28 477\u0026ndash;487 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.immuni.2008.03.002\u003c/span\u003e\u003cspan address=\"10.1016/j.immuni.2008.03.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, C. \u003cem\u003eet al.\u003c/em\u003e Cytokines: From Clinical Significance to Quantification. \u003cem\u003eAdvanced Science\u003c/em\u003e vol. 8 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/advs.202004433\u003c/span\u003e\u003cspan address=\"10.1002/advs.202004433\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurska, A., Boissinot, M. \u0026amp; Ponchel, F. Cytokines as biomarkers in rheumatoid arthritis. \u003cem\u003eMediators of Inflammation\u003c/em\u003e vol. 2014 Preprint at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1155/2014/545493\u003c/span\u003e\u003cspan address=\"10.1155/2014/545493\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2014).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"jojobyl alcohols, 13-docosenol, adjuvants, jojoba oil","lastPublishedDoi":"10.21203/rs.3.rs-4750304/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4750304/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTo ensure the sustainability of Jojoba oil production, research and development must prioritize the adoption of environmentally friendly extraction processes. Firstly, optimal conditions for extracting Jojoba oil were predicted marking a significant step towards realizing its economic potential. Molar ratio, temperature and catalysts concentration were taken into consideration to achieve optimal production. Secondly, interactions of cis-13-docosenol (C13D), a key component of Jojoba oil, with innate immune cells were analysed. By meticulously examining the interactions between C13D and critical elements of the innate immune system, including monocytes, macrophages, and dendritic cells (DC), we aim to uncover the immunomodulatory properties of this compound. In experiments with THP-1 cells and DC, low doses of C13D were found to elevate pro-inflammatory cytokines TNF-α, IL-6, and IL-1β to levels comparable to those induced by LPS. Furthermore, modulation of T cell stimulation by monocyte-derived dendritic cells (MoDC) previously treated with C13D resulted in increased T cell proliferation, likely due to the enhanced activation of surface markers. This detailed exploration into the effects of C13D on innate immune cells not only deepens our understanding of Jojoba oil's therapeutic potential but also establishes a foundation for future advancements in immunology and biotechnology.\u003c/p\u003e","manuscriptTitle":"Optimizing Jojoba Oil Methanolysis and Unveiling the Immunomodulatory Potential of cis-13-Docosenol Fatty Alcohol: A Circular Biorefinery Perspective","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-26 17:35:47","doi":"10.21203/rs.3.rs-4750304/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"14c39750-96eb-4de0-8f22-a3c4604d96ec","owner":[],"postedDate":"August 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":36532924,"name":"Physical sciences/Chemistry/Chemical biology"},{"id":36532925,"name":"Biological sciences/Chemical biology"},{"id":36532926,"name":"Biological sciences/Immunology"},{"id":36532927,"name":"Physical sciences/Chemistry"}],"tags":[],"updatedAt":"2024-11-05T05:54:06+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-26 17:35:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4750304","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4750304","identity":"rs-4750304","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}