Rosa roxburghii fruit were used as research objects to study the effects of different concentrations of benzothiazole (BTH) treatment on quality parameters, reactive oxygen species (ROS) metabolism, and the phenylpropanoid pathway during storage at 4°C for 14days. Results showed that BTH effectively delayed senescence with lower decay incidence, weight loss, and lipid peroxidation level and maintained the quality with higher contents of total soluble solid (TSS) content, titratable acidity (TA) in R. roxburghii fruit. Moreover, BTH increased hydrogen peroxide (H2O2) content, superoxide anion (O2•−) production rate, and the activities and expression of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione (GSH) reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), and peroxidase (POD), and the contents of GSH and ascorbic acid (AsA), but reduced the oxidized GSH (GSSG) content. In addition, the activities and gene expression of phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL) and the concentrations of flavonoids, total phenols, and lignin were significantly elevated by BTH. These findings imply that BTH can delay senescence and maintain the quality of R. roxburghii fruit by modulating ROS metabolism and the phenylpropanoid pathway under low-temperature conditions. Rosa roxburghii (R. roxburghii), a perennial deciduous shrub from the Rosaceae family, is widely distributed in southwest China. The vitamin C and superoxide dismutase (SOD) contents of R. roxburghii fruit are particularly rich and exhibit considerable promotion prospects in the market  metabolism remain few.e market . Howeverely rare . To dateThe structural analogue of salicylic acid, benzothiazole (BTH) has confirmed to induce the production and improvement of disease resistance in various plants . BTH canReactive oxygen species have been confirmed to act directly on pathogens and function as a defensive gene signals in plant defense responses . HoweverR. roxburghii remain unclear.The phenylpropanoid pathway is a unique secondary metabolism in plants, and it is important in the development of plant disease resistance. Many major antibacterial substances, such as lignin, flavonoids, and phenolic compounds are directly or indirectly generated by the phenylpropanoid pathway . The buiR. roxburghii fruit during low-temperature storage, (2) the ROS metabolism of R. roxburghii fruit during low-temperature storage, and (3) the phenylpropanoid pathway of R. roxburghii fruit. That is, this research intended to evaluate the potential of BTH in enhancing the postharvest quality and delaying the senescence of R. roxburghii fruit under low-temperature conditions.The current study aimed to investigate the effects of BTH treatment on the following: (1) the quality including: weight loss rate, decay incidence, malondialdehyde (MDA), cell membrane permeability, titratable acidity (TA), soluble solids (TSS), and AsA content of R. roxburghii fruit harvested at 85days after full blossom (about 70% maturity) without mechanical damage were obtained from an orchard planted with the AsA–GSH cycle comprises R. roxburghii in Anshun, Guizhou Province, China. Sigma-Aldrich provided support for BTH.−1 of BTH dissolving with 0.1mlL−1 Tween-80 for 10min, with distilled water as the control. All the processed fruit were air-dried at ambient temperature (22°C) before being stored at 4°C for 14days. Each treatment consisted of three replicates, and each replicate comprised 80 fruits.Undamaged fruit were selected and washed three times with distilled water. All the selected fruit were immersed in 25, 50, 100, and 200mgLOn days 0, 2, 4, 6, 8, 10, 12, and 14 after BTH treatment, tissues were detached from 3 to 6mm below the skin and around the equator of a fruit. Consequently, the collected samples were frozen in liquid nitrogen and immediately stored at −80°C.−1 fresh weight (FW). The optimal concentration of BTH was determined on the basis of the weight loss rate and AsA content.The weighing method used for evaluation and measurement was based on the following formula: weight loss rate/%=[/quality before storage]×100. The AsA content was assayed by applying the method of Decay index is divided into six levels. Level 0: no decay and mold; Level 1: small brown spots, no obvious decay area; Level 2: decay area <1/4 of the fruit surface area; Level 3: 1/4<decay area<1/3 of the fruit surface area; Level 4: 1/3<decay area<1/2 of the fruit surface area; and Level 5: decay area>1/2 of the fruit surface area. The decay incidence (%) was calculated according to the following equation: ∑ (number of fruits×decay index)/×100.−1 of sodium hydroxide standard solution. The method of CF. Cell membrane integrity was estimated using the following formula: cell membrane integrity (%)=[1−(CI/CF)]×100. The study of −1 FW.Pulp tissues (2.0g) from 10 fruits were homogenized. The supernatant was dropped onto a TF-65 handheld refractometer. Total soluble solid (TSS) content was expressed in %. Then, acid–base titration was performed with 0.1molL2•−. The rate of O2•− production was measured using an mmol g−1 min−1 FW scale. H2O2 content was determined in accordance with the method of g for 20min at 4°C. Thereafter, 1ml of supernatant with 200μl of 20% titanium tetrachloride dissolved in concentrated hydrochloric acid  was used in the reaction. The titanium–hydroperoxide complex precipitate was filtered three times in cold acetone before being dissolved in 3ml of 1M sulfuric acid and centrifuged for 10min at 4°C at 9,000×g. H2O2 content was measured using an absorbance of 410nm and expressed in μmol g−1 FW. GSH content was assayed by applying the method of −1 FW. GSSG contents were determined using GSSG Detection Assay Kit  and were expressed as μmol g−1 FW.The method of All the extractions were performed at 4°C. Liquid nitrogen was used to grind frozen tissues (1.0g) into powder, which was subsequently extracted with the following solutions:−1 of polyvinyl polypyrrolidone (PVPP). SOD activity was determined using the method described by −1 protein, where U=50% inhibition of NBT. CAT activity was determined using the method described by −1 H2O2 and 0.3ml of crude enzyme extract were used in the reaction. The absorbance of the supernatant was then measured at 240nm.SOD and CAT: 3.0ml of phosphate buffer  with 5.0mM of dithiothreitol (DTT) and 10gL2O2, and 0.2ml of enzyme extract were used in the assay mixture. APX activity was measured in units of U mg−1 protein, i.e., 1U=0.01 Δ340 min−1. DHAR activities were measured using the method of −1 protein, where 1U=0.01A290 min−1.APX and DHAR: 3.0ml of phosphate buffer  with 1.0mM of EDTA. APX activity was determined as described by −1 protein.GSH reductase: 0.1M of phosphate buffer . The GR-dependent oxidation of NADPH was used to determine GR activity at 340nm by using the method of −1 of PVPP, and 5mM of β-mercaptoethanol). MDHAR activities were measured using the method of −1 protein, where 1U=0.01A340 min−1.MDHAR: 0.04M of phosphate buffer . POD activity was assayed in accordance with the method of −1 protein, where 1U=0.01 Δ470 min−1.Peroxidase (POD): 0.05M of phosphate buffer . PAL activity was determined using the method of −1 protein. Here, U=0.01 ΔOD290 min−1.Phenylalanine ammonia lyase (PAL): 5ml of sodium borate buffer  of PVPP, and 10% (v/v) glycerin. C4H activity was determined in accordance with the method of Cinnamate 4-hydroxylase (C4H): 5ml of 50mM Tris–HCl buffer (pH 8.9) with 4mM of MgCl2, 0.15ml of 1M coenzyme A (CoA), 0.15ml of 0.8mM adenosine triphosphate, and 0.15ml of 2mM p-cumaric acid. Activity was measured in U·mg−1 protein by using U=0.01 ΔOD333 min−1.4-Coumarate-CoA ligase (4CL): 5ml of 0.2M Tris–HCl buffer (pH 7.5) with 25% glycerin and 0.1M of DTT. The activity of 4CL was assessed using the technique of −1 FW.Total phenolic, flavonoid, and lignin contents were extracted and measured using the method described by RNA was extracted from tissues (1.0g) by using an RNeasy Plant Mini Kit  following meticulous procedures. First-strand complementary DNA (cDNA) was synthesized from 1μg of total RNA by utilizing a Reverse-iT™ 1st Strand Synthesis Kit  and strictly following the manufacturer’s instructions.−ΔΔt formula was used to calculate the target gene expression and the actin gene was used as reference. Three duplicates of real-time PCR were conducted for each sample. Primers were indicated in A real-time quantitative PCR experiment was conducted by adding 10μl of SYBR™ Green qPCR Master Mix, 1μl of template cDNA, and 1μl of 10μM forward and reverse primers to each well with a 20μl reaction volume. An ABI PRISM™ 7,000 Sequence Detection System was used for measurements (Applied Biosystems). Cycling conditions were 95°C for 10min, followed by 40cycles of 15s at 95°C and 40s at 60°C. The 2p<0.05).By using SPSS 19.0, all data on enzyme activities and substance contents were evaluated using one-way ANOVA of least significant difference ; National Natural Science Foundation of China (32002103), and Guangdong Basic and Applied Basic Research Foundation (2020A1515110092); and Major Research Project of Innovation Group of Guizhou Provincial Department of Education (2018017).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Collectively, this study identifies new aspects of the metabolic reprogramming that occur in distinct macrophage polarization states. The channeling of exogenous fatty acids into particular lipid synthetic pathways may contribute to the sensitivity/resistance of macrophage subsets to the inflammatory effects of increased environmental fatty acid levels.Adipose-tissue-resident macrophages (ATMs) maintain metabolic homeostasis but also contribute to obesity-induced adipose tissue inflammation and metabolic dysfunction. Central to these contrasting effects of ATMs on metabolic homeostasis is the interaction of macrophages with fatty acids. Fatty acid levels are increased within adipose tissue in various pathological and physiological conditions, but appear to initiate inflammatory responses only upon interaction with particular macrophage subsets within obese adipose tissue. The molecular basis underlying these divergent outcomes is likely due to phenotypic differences between ATM subsets, although how macrophage polarization state influences the metabolism of exogenous fatty acids is relatively unknown. Herein, using stable isotope-labeled and nonlabeled fatty acids in combination with mass spectrometry lipidomics, we show marked differences in the utilization of exogenous fatty acids within inflammatory macrophages (M1 macrophages) and macrophages involved in tissue homeostasis (M2 macrophages). Specifically, the accumulation of exogenous fatty acids within triacylglycerols and cholesterol esters is significantly higher in M1 macrophages, while there is an increased enrichment of exogenous fatty acids within glycerophospholipids, ether lipids, and sphingolipids in M2 macrophages. Finally, we show that functionally distinct ATM populations  Macrophages are cells of the innate immune system with critical roles in the elimination of infectious microorganisms, the resolution of tissue damage, and the maintenance of tissue and organismal homeostasis . MacrophOne of the many tissue-specific signals that macrophages are exposed to is altered nutrient levels. This is exemplified in the adipose tissue and liver, where resident macrophages are exposed to marked fluctuations in fatty acid levels that result from physiological challenges such as fasting , 6 and c2), proinflammatory cytokines) and are antimicrobial  and then performed mass-spectrometry-based targeted lipidomic profiling. In the absence of exogenously supplied fatty acids, with the exception of those FAs and other lipids present in serum, we observed that M1 macrophages have substantially higher levels of TGs, diacylglycerols (DGs), and cholesterol esters (CEs) (B–D), consistent with the results of previous studies . In contrast, M1 macrophages had a higher proportion of TGs containing unsaturated fatty acids, in particular TGs containing polyunsaturated fatty acids (PUFAs), i.e., TGs containing six to ten double bonds within their constituent fatty acyl chains. These differences are the result of a substantial increase in TG levels in M1 macrophages, which, although occurring across all TG species, are most marked in PUFA-containing TGs. E–I provide examples of the changes in TGs containing fatty acids of varying saturation status. Previous work would indicate that the increase in TG levels in M1 macrophages is likely due to increased fatty acid supply via de novo synthesis as well as an increase in the expression of multiple enzymes in the TG synthesis pathway (de novo fatty acid synthesis during M1 macrophage polarization by inhibiting either acetyl-CoA carboxylase (TOFA) or the mitochondrial pyruvate transporter (UK5099) completely prevented the increase in TG species (J–N). Of note, PUFAs cannot be synthesized de novo; therefore, in the case of increasing PUFA-containing TG species, the PUFA likely derives from serum via extracellular uptake or potentially from phospholipid remodeling. Indeed, we observed lower levels of numerous PUFA-containing phospholipid species in M1 compared with M2 macrophages , consistent with the possibility that the breakdown of PUFA-containing glycerophospholipids (GPLs) may supply PUFAs that are subsequently used in TG synthesis.To determine how macrophage phenotypic state influences the metabolism of exogenous fatty acids, we firstly generated bone-marrow-derived M0 macrophages (unpolarized), which were subsequently polarized to the M1 (LPS + IFNy for 24 h) or M2 (IL-4 for 24 h) state A and thers (CEs) , B–D, co studies , 27, 28. studies . Specifi pathway , 28. Ind species , J–N. Ofversus M2) influenced the metabolism of the major fatty acid species that are released following adipocyte lipolysis. Thus, we exposed M1 and M2 macrophages to exogenously added palmitic acid , oleic acid , arachidonic acid , and docosahexaenoic acid . Mass-spectrometry-based targeted lipidomic profiling was used to determine the endogenous lipid species that were altered following treatment with exogenous fatty acids. Macrophage polarization state had a marked impact on the fate of exogenous fatty acids. Strikingly, the levels of numerous TG (A–E), DG (F–H), and CE species (I–L) were higher in M1 macrophages following Pal, Ol, and AA treatment. Notably, the fate of exogenous DHA was distinct from Pal and Ol. Specifically, while Pal and Ol markedly increased the levels of TG species containing a 16:0 or 18:1 fatty acyl chain (A and B), DHA treatment had relatively minor effects on the levels of DHA-containing TG species (D and E). In contrast, while exogenous Pal and Ol led to only minor increases in 16:0- and 18:1-containing CEs (I and J), 22:6-containing CEs were markedly increased following DHA treatment (L). Exogenous AA increased both 20:4-containing TG and CE species (C and K). These results demonstrate that in addition to having higher basal levels of neutral lipids, M1 macrophages accumulate significantly more TG and CE than M2 macrophages following treatment with exogenous fatty acids. They also demonstrate that the fate of exogenous fatty acids within M1 macrophages is dependent upon the saturation status/acyl chain length, with saturated and monounsaturated fatty acids (16:0 and 18:1) preferentially accumulating within TGs and PUFA preferentially accumulating within CEs.Within the adipose tissue, adipocytes are constantly undergoing lipolysis, releasing fatty acids that can be sensed by resident macrophages. We aimed to explore how macrophage polarization state , DG , F–H, an species , I–L weryl chain , A and B species , D and Ening CEs , I and Jreatment L. Exogen species , C and Ki.e., fatty acids in which the terminal methyl group is replaced by an alkyne) followed by conjugation via copper-catalyzed azide-alkyne cycloaddition to an azide-Alexa488 conjugate to enable the assessment of fatty acid uptake by flow cytometry. M2 macrophages had a significantly higher uptake of alkyne-oleate and alkyne-palmitate compared with M1 macrophages  phosphate ester head group. Treatment with exogenous fatty acids increased the levels of multiple GPL species from different classes, including phosphatidylcholine (PC) (A–H), phosphatidylethanolamine (PE) (I–N), phosphatidylinositol (PI) (O–R), phosphatidylglycerol (PG) (S), and phosphatidylserine lipid species (T). As expected, the GPL species altered were generally those containing acyl chains that corresponded to the specific exogenously added fatty acid; e.g., PE 18:1/18:1 was increased only following exposure to oleic acid. Treatment with exogenous fatty acids increased the abundance of multiple PC and PE species in both M1 and M2 macrophages (A–N). Interestingly, while treatment with Pal and Ol resulted in similar increases in PE and PC species containing 16:0 and 18:1 acyl chains, the increase in PC and PE species containing 20:4 and 22:6 acyl chains following treatment with exogenous AA and DHA was significantly greater in M2 relative to M1 macrophages (A–N). Similarly, the increase in numerous PI species was significantly greater in M2 macrophages (O–R).We hypothesized that exogenous fatty acids are incorporated into alternate lipid biosynthetic pathways in M2 macrophages, thereby reducing their esterification within TGs and CEs. GPLs are the major constituents of cell membranes, with important structural and biochemical roles. GPLs consist of a glycerol backbone with ester-linked fatty acyl chains and a variable (ine (PC) , A–H, phine (PE) , I–N, phtol (PI) , O–R, phrol (PG) S, and ph species T. As exprophages , A–N. Inrophages , A–N. Sirophages , O–R.via an ether or vinyl-ether bond at the Sn1 position of glycerol. Like their diacyl counterparts, ether GPLs are abundant membrane lipids, but their altered chemical linkage endows ether lipids, and in particular vinyl-ether lipids , with unique biophysical and biochemical properties ) and both vinyl-ether PCs ) and PEs ). Where exogenous fatty acids increased the level of a specific ether/vinyl-ether GPL, these increases were generally significantly greater in M2 macrophages . Consistent with the data presented above, d9-oleate was enriched within TGs, DGs, and CEs at substantially greater levels in M1 relative to M2 macrophages (A–F). Of note, d9-oleate enrichment within TGs, DGs, and CEs in M1 macrophages was observable after as little as 5 min of treatment and was markedly increased after only 30 min. This contrasts with M2 macrophages in which detectable increases in TGs, DGs, and CEs were only apparent after 30 and 240 min. A marked d9-oleate enrichment within 18:1 acylcarnitine was also observed in M1 macrophages, which was significantly lower in M2 macrophages (G). The enrichment of d9-oleate within ether and vinyl-ether GPLs was significantly higher in M2 macrophages (H–N). Owing to the specific fragmentation of PE plasmalogens in our lipidomics profiling conditions  or Sn1 alkenyl positions (J–N), indicating that exogenous fatty acids can be both reduced to fatty alcohols to serve as substrates for the peroxisomal synthesis of 1-alkyl-DHAP as well as serve as substrates for acyl/alkyl-G3P-acyltransferase, which catalyzes the addition of an acyl chain to the Sn2 position of 1-alkyl-G3P  . As expected, d9-oleate was also enriched within major 18:1-containing PC and PE GPLs (O and P), which tended to be higher in M2 macrophages. The results from this time course analysis were confirmed in an independent experiment performed in M1 and M2 macrophages treated with d9-oleate for 4 h (A–P).A limitation to measuring the abundance of intracellular lipid species using conventional MS-lipidomics with the goal of determining the fate of exogenous fatty acids is that the levels observed reflect both endogenous and exogenous lipid sources. Therefore, to gain greater insight specifically into the fate of exogenous fatty acids within M1 and M2 macrophages, we utilized stable-isotope labeled fatty acid tracers. To gain insight into the rate of exogenous fatty acid incorporation into various endogenous lipid pools, we treated M1 and M2 macrophages for 5, 30, and 240 min with deuterated oleate  . Interesposition , L–N. As PE GPLs , O and P for 4 h , A–P.Fig3-palmitate) within specific lipid species in M1 and M2 macrophages. As expected, d3-palmitate was enriched in lipid species from multiple lipid classes. Consistent with the results described above using either d9-oleate or nonlabeled fatty acids, the enrichment of d3-palmitate within TG and CE species was significantly greater in M1 macrophages (A–C). Enrichment of d3-palmitate in DGs was unaffected by macrophage polarization state (D and E). In contrast, the enrichment of d3-palmitate within various diacyl, ether, and vinyl-ether GPLs was significantly greater in M2 macrophages (F–L). Collectively, these results demonstrate that M2 macrophages have a propensity to channel exogenous fatty acids into glycerophospholipid synthetic pathways. This effect likely contributes to the reduced accumulation of TGs and CEs observed in M2 macrophages.Next, we assessed the enrichment of deuterated palmitate  . Strikingly, d3-palmitate enrichment within the d18:1 sphingoid base was markedly higher in M2 macrophages (A–D). With regard to the incorporation of d3-palmitate within the ceramide acyl chain, a marked increase in Cer(d18:1/16:0-d3) was observed in both M1 and M2 macrophages, and this was slightly, although significantly, higher in M2 macrophages (E). Furthermore, while very little d3-palmitate enrichment within the acyl chains of Cer(d18:1/20:0-d3) and Cer(d18:1/24:0-d3) was observed in M1 macrophages, significant d3-palmitate enrichment into these ceramide species was observed in M2 macrophages (F and G), indicating active fatty acid elongation, e.g., from 16:0 to 18:0, 20:0, 22:0, and 24:0. With the exception of SM 42:1, we did not observe d3-palmitate enrichment across multiple measured sphingomyelin species (H); likely reflecting the relatively short incubation time of macrophages with d3-palmitate (4 h). With regard to SM 42:1, while d3-palmitate enrichment was observed in both M1 and M2 macrophages, this was significantly higher in M2 macrophages (H). These findings using d3-palmitate are consistent with results obtained using non-stable-isotope-labeled palmitate. Specifically, several d18:1 sphingoid base-containing ceramide species were increased following palmitate treatment, effects that were significantly greater in M2 relative to M1 macrophages . Very few sphingomyelin species were increased following palmitate treatment; however, among those sphingomyelin species that were increased following palmitate treatment, these increases were typically significantly greater in M2 macrophages (M–P). Collectively, these results demonstrate an increased entry of exogenous palmitate into the de novo ceramide synthetic pathway in M2 macrophages, which, similar to the case for increased glycerophospholipid synthesis, may contribute to the reduced accumulation of TGs and CEs observed in M2 macrophages.Because palmitate is the preferred substrate in the formation of the sphingoid base that forms the backbone of ceramides and other sphingolipids , we were length) . Accordi pathway , A–D. Strophages , A–D. Wirophages E. Furtherophages , F and G species H; likelyrophages H. These rophages , I–L. Verophages , M–P. Coin vivo have altered lipid compositions. CD9+ ATMs reside within crown-like structures in adipose tissue and display an M1-like phenotype, being proinflammatory and lipid-laden  and performed mass spectrometry lipidomics on the isolated cell populations. Principal component analysis (PCA) differentiated CD9+ and Ly6C+ macrophages, indicative of a difference in global lipid composition between the two cell types (A). Somewhat unexpectedly, we observed increased total TG levels in the M2-like Ly6C+ macrophages (B). Interestingly, there was a marked difference in the composition of the TG pool, with M1-like CD9+ macrophages having an increased proportion of PUFA-containing TG species, and Ly6C+ macrophages having an increased proportion of saturated and monounsaturated fatty acid–containing TG species (C). This finding is consistent with our data showing an increased proportion of PUFA-containing TG species in M1 macrophages , CD9+ macrophages had higher levels of diacyl PC and lower levels of ether-PC relative to Ly6C+ macrophages (E and F). Differences in PE composition and PS were also observed, while the proportion of PI was unaltered (G–K). Finally, we also noted a marked difference in the proportions of major sphingolipid classes, ceramide, hexosyl ceramide, and sphingomyelin, between CD9+ and Ly6C+ macrophages (L–N), indicative of differences in sphingolipid metabolism between cell types. These observations suggest that even though they are resident within the same adipose tissue depot, and have been fed the same HF diet, distinct ATM populations have alterations in their lipid metabolism.Finally, we wanted to investigate whether functionally distinct populations of ATM id-laden , while Lipogenic . Therefoll types A. Somewhrophages B. Intere species C. This frophages  and S1. ll types D, CD9+ mrophages , E and Fnaltered , G–K. Firophages , L–N, inSince the discovery of macrophage recruitment into adipose tissue in response to stimuli such as fasting , cold 3, and obein vitro model of macrophage polarization is an oversimplification of the far more nuanced effector phenotypes present in tissue-resident macrophages in vivo, it nonetheless provides an important conceptual framework with which to understand macrophage biology. Indeed, recent studies using this in vitro model to study the phenotypes associated with distinct macrophage polarization states have revealed new biological insights into macrophage metabolism with important in vivo consequences  4 by LPS induces TG and CE synthesis , 28. Sev2 . PUFAs s2 (2 . In the 2 (2 .via the diacylglycerol acyltransferase (DGAT)-mediated esterification of excess free fatty acids into TGs  an Ly6C+ population that is uniformly distributed throughout the adipose tissue, is not lipid-laden, and has a gene expression profile that would be expected to facilitate the maintenance of normal adipose tissue physiology , conditions that increase adipose tissue lipolysis and therefore fatty acid levels within the extracellular adipose tissue environment, are not inflammatory and instead express markers associated with M2 polarization , 32. Usiysiology . We hypoysiology . Insteadysiology . This inde novo sphingolipid synthesis pathway  CD9+ and Ly6C+ macrophages have distinct lipid profiles, whether the metabolism of exogenous fatty acids is likewise altered between CD9+ and Ly6C+ macrophages will require future investigations, e.g., comparing how the cellular lipid content of CD9+ and Ly6C+ macrophages differ in response to stimuli such as fasting or cold exposure.In the final part of this work, we assessed the lipid composition of functionally distinct CD9tro work . SpecifiIn conclusion, our results add significant new knowledge with regard to how lipid metabolism is reprogrammed by macrophage polarization. In particular, we show that macrophage polarization state markedly alters the channeling of exogenous fatty acids into endogenous lipid pools. We propose that these differences, in addition to previously described alterations in lipid metabolism between M1 and M2 macrophages such as differences in fatty acid oxidation capacity, may contribute to the sensitivity, in the case of M1 macrophages, and the resistance, in the case of M2 macrophages, of ATM to lipotoxicity and inflammation.In vitro experiments—In all experiments, bone-marrow-derived macrophages (BMDMs) were generated from wild-type, male C57Bl6/J mice aged 4 to 8 weeks old. Mice were bred and housed at the Alfred Research Alliance Animal Facility and were maintained on a 12:12 h light:dark cycle. All procedures complied with the national guidelines for the care and use of laboratory mice and were approved by an institutional animal ethics committee (AMREP AEC). In vivo experiments—11-week-old C57Bl6/J mice were placed on a HFD  for 10 weeks. After 10 weeks of HFD, mice were humanely culled and the epididymal adipose tissue collected; mice were not fasted prior to being culled. These procedures complied with the national guidelines for the care and use of laboratory mice and were approved by the institutional animal ethics committee of the University of Melbourne.2 asphyxiation, femurs and tibias were removed, cleaned, and bone marrow (BM) flushed from the bones with RPMI-1640 media containing GLUTAMAX . BM was centrifuged  and resuspended in RPMI media containing 5% Fetal Bovine Serum  before cells were counted on an automated analyzer (Sysmex XS-1000i). BM cells (1 million/ml) were cultured in macrophage differentiation media , and 20 ng/ml macrophage-colony stimulating factor ) in a T75 flask. Following an overnight incubation , nonadherent cells were plated in 24-well tissue culture plates (Jet-Biofil) at a density of ∼4 × 105 cells per well in 1 ml of macrophage differentiation media. After 3 days, an additional 1 ml of macrophage differentiation media was added. BM cells were considered to be fully differentiated into macrophages following 7 days of culture. To induce polarization to the M1 and M2 states, BMDMs were treated with 100 ng/ml ultrapure lipopolysaccharide  and 20 ng/ml interferon-y  for M1 polarization, or 20 ng/ml IL-4 (PeproTech) for M2 polarization, in macrophage differentiation media for 24 h. De novo lipogenesis was inhibited by pretreating M0 (nonpolarized) macrophages with either an inhibitor of acetyl-CoA carboxylase  or the mitochondrial pyruvate carrier  for 1 h. M0 BMDMs were subsequently polarized to either the M1 or M2 states or remained as M0 macrophages. TOFA and UK5099 were present in the media during the 24 h M1 polarization. Unlabeled FAs  were initially solubilized in ethanol before being added to filtered  RPMI media containing 5% FBS and 2% w/v bovine serum albumin  to give a final concentration of 200 μM. For experiments using stable-isotope labeled fatty acids, d9-oleate (Avanti Polar Lipids # 861809) and d3-palmitate (Sigma-Aldrich #615951), deuterated FAs were likewise initially solubilized in ethanol before being added to RPMI media containing 5% FBS and 0.2% w/v BSA to give a final concentration of 20 μM. FA mixtures were gently rocked for an hour at 37 °C to aid conjugation of the fatty acids to BSA. Once prepared, FAs or vehicle controls (BSA + ethanol) were added to polarized BMDM, after which BMDMs were washed (×2) with cold PBS and cells stored at −80 °C until further processing (Lipidomics and Western blotting).Following COBMDMs were scraped, collected in 200 μl of ice-cold PBS, sonicated , and protein concentrations determined  to allow for data normalization during analysis. Samples were then dried overnight in a Speedvac (Thermo Scientific) in preparation for lipid extraction. Lipids were extracted using a single-phase chloroform/methanol extraction as described previously with modification for cultured cells , 46.Liquid chromatography–tandem mass spectrometry (LC-MS/MS) was performed according to previously published methods, with slight modification for cultured cells , 46, 47.9-oleate, or d3-palmitate for the durations indicated within specific figure legends and were subsequently extracted as described above. Mass spectrometry transitions were modified to detect trace incorporation of deuterated fatty acids into lipids in a targeted manner based on lipid species of interest. Lipid species expected to incorporate d9-oleate had their precursor mass offset by 9 Da, while the product ions were offset depending on lipid class fragmentation patterns. In instances where double incorporation was possible, the precursor mass was also offset by 18 Da. This was additionally performed for d3-palmitate but with 3 and 6 Da for single and double incorporation, respectively. The corresponding nonlabeled species were also measured. Data was analyzed using Agilent MassHunter 10.0. Percent enrichment was calculated as: Area of the labeled species/(Area of labeled species + Area of non-labeled species) ×100.Cells were treated with either BSA, d3. Membranes were then washed (TBST) for 30 min, incubated with secondary antibody (Anti-rabbit IgG (H + L)-HRP Conjugate, Bio-Rad #1706515; 1:2000 in TBST +2.5% BSA) for 1 h at room temperature, and then washed (TBST) for a further 30 min before being visualized using a chemiluminescent substrate (mixture of Thermo Fisher Scientific chemiluminescent substrates – #34095 and #34577) on a ChemiDoc XRS system (Bio-Rad). Where required, membranes were stripped  to allow for reprobing with a different primary antibody.To confirm that BMDMs were effectively polarized into the M1 and M2 states, BMDMs were analyzed by Western blot. Briefly, after being washed (×2) with ice cold PBS, 100 μl of 2× Laemmli buffer containing 200 μM dithiothreitol was directly added to wells, and cells were scraped and collected before being incubated at 95 °C for 10 min. Samples were resolved by SDS polyacrylamide gel electrophoresis and then transferred to 0.2 μm nitrocellulose membranes (Bio-Rad) by wet transfer at 4 °C. Membranes were washed with Tris-buffered saline containing Tween 20 (TBST) for ∼30 min before being blocked in TBST containing 5% skim milk powder for 1 h, and finally washed in TBST, all conducted at room temperature. Membranes were incubated overnight at 4 °C on a rotary shaker with antibodies  made up in TBST containing 2.5% (wt/vol) BSA and 0.01% (wt/vol) NaNin vitro and in vivo  in that the fatty acid used is very minimally modified . Posttreatment, alkyne-FAs can then be visualized using reporter-tagged azide-conjugates, e.g., azide-AlexaFluor488. Studies to date have shown that alkyne fatty acids are metabolized in a similar manner to fatty acids, making them more physiologically representative of their native counterparts compared with fatty acids directly conjugated to a fluorophore.Copper-catalyzed azide-alkyne cycloaddition (“click”) chemistry-based approaches have been used to visualize fatty acids  in vivo . Using c2. After which, sterile PBS supplemented with 10% FBS was added and any remaining adherent cells were dislodged by gentle, repeated pipetting before being collected in Eppendorf tubes, and centrifuged . Next, cells were resuspended in Fixation and Permeabilization solution  and incubated for 20 min on ice. The process was stopped with Perm/Wash buffer , and cells were spun  and then washed . Next, a cocktail of Click-iT reaction reagents  was prepared according to the manufacturer’s protocol  and added to cells . Controls that were treated identically except for the absence of CuSO4 in the Click-iT cocktail, and which are therefore unable to undergo the click reaction, were included to account for background fluorescence. FACS buffer was then added to wash the cells, samples were spun  and then resuspended in an antibody cocktail containing macrophage-specific markers  and a viability dye  and incubated on ice, in the dark, for 30 min. Cells were washed and transferred to FACS tubes for analysis using an LSRII Fortessa (BD Biosciences) flow cytometer with FACSDiva software. The gating strategy used to identify macrophages and to quantify the mean fluorescence intensity of the alkyne-FA/azide-AlexaFluor488 conjugate is shown in Alkyne-palmitate  and alkyne-oleate  were initially solubilized in absolute ethanol and then added to RPMI containing 2% FBS and 0.2% w/v BSA to attain the final desired concentrations shown in . Mixture+ and Ly6C+ cells were gated essentially as described previously (+ cells—viable (Ghost Dye negative), single cells were initially gated, from which CD45+ cells were gated, from which CD11b+ Ly6C− were gated, from which F4/80+ CD64+ cell were gated, from which CD9+ cells were gated for sorting; for Ly6C cells—viable (Ghost Dye negative), single cells were initially gated, from which CD45+ cells were gated, from which CD9− cells were gated, from which CD11b+ Ly6C+ cells were gated for sorting. We were able to sort ∼70,000 to 250,000 CD9+ macrophages and ∼5000 to 60,000 Ly6C+ macrophages. After cells were sorted, they were washed once with PBS, and the cell pellets stored at −80 °C in 100 μl PBS. Prior to lipidomic analysis, samples were lyophilized in a Savant SpeedVac (Thermo Scientific) and subsequently reconstituted in milliQ water. In this experiment, samples were extracted using the butanol:methanol method, as we have described previously  to investigate TG compositional changes, we normalized each TG species to the sum total triglyceride concentration, (2) to investigate phospholipid changes between cells types, we examined each lipid class total relative to the total glycerophospholipid pool. This was done similarly for each sphingolipid class and the total sphingolipid pool. While we present some of the data from this work in Adipose tissue was placed in a 5 ml tube with 2 ml liberase  and finely minced. Minced adipose tissue was moved to a small Petri dish and then incubated in a rotary shaker for 45 min at 37 °C. Next, 10 ml of cold RPMI was added to the Petri dish, and this RPMI/minced adipose tissue mixture subsequently filtered (250 μm) and transferred to a 15 ml falcon tube. Samples were then spun , the floating adipocytes and supernatant removed, 1 ml of red blood cell lysis buffer added for 5 min, the reaction stopped with the addition of ∼5 ml of FACS buffer, and samples spun again . After removing the supernatant, samples were resuspended in 1 ml FACS buffer and cells counted (Sysmex XS-1000i). Cells were then incubated with the following antibodies (1:400 dilution): CD45-BUV395 , Ly6C-PerCP (Cat# 45-5932-82), CD11b-APC , CD64-BV650 , F4/80-FITC , CD9-PE , and a viability dye  for 30 min, on ice, in the dark. After 30 min, ∼1 ml of FACS buffer was added, cells spun (2800 rpm for 3 min), and the cell pellet resuspended in ∼300 μl of FACS buffer and filtered (40 μm) prior to sorting on a BD FACSAria Fusion flow cytometer at the AMREP flow cytometry core facility (Burnet Institute). CD9eviously : for CD9eviously , and prot test, one-way analysis of variance (ANOVA), or two-way ANOVA; the specific statistical tests used are indicated in the figure legends. PCA was performed in R (v4.0.5) using the FactoMineR and factoextra packages. Data was log-transformed prior to PCA. In the TG species heatmap in Figure 6C, the TG species shown are those that were significantly different (unpaired t test) after performing a Benjamini–Hochberg false discovery rate correction set at 10%. One biological replicate in the M1 BSA group (from the experiment in which the four different FAs were used) was excluded in all analysis as it was a clear technical artefact. In the in vivo experiment, two mice were excluded from the final analysis due to low cell numbers obtained from the sort (<2000 Ly6C+ cells). A p-value of less than 0.05 was used to define statistical significance, although more precise p-values are indicated in the figures. All data are shown as the mean ± S.D.Data were analyzed by unpaired Graeme.lancaster@baker.edu.au) and Andrew J. Murphy (Andrew.murphy@baker.edu.au) upon request.All data are contained within the manuscript and supporting information or are available from the authors: Graeme I. Lancaster (This article contains The authors declare that they have no conflicts of interest with the contents of this article.


G. applanatum is a valuable medicinal mushroom that has been widely used as a folk medicine for the treatment and prevention of various diseases. In this study, we obtained crude extract from G. applanatum mushroom with a subcritical water extraction method; cell viability assay was carried out and the crude extract showed an antiproliferative effect in HeLa cells with IC50 of 1.55 ± 0.01 mg/mL; however, it did not show any sign of toxicity in HaCaT. Protein expression was detected by Western blot, stability of IκBα and downregulation of NFκB, IKKα, IKKβ, p-NFκB-65(Ser 536) and p-IKKα/β(Ser 176/180), suggesting loss of survival in a dose-dependent manner. RT-qPCR revealed RNA/mRNA expression; fold changes of gene expression in Apaf-1, caspase-3, cytochrome-c, caspase-9, Bax and Bak were increased, which implies apoptosis, and NFκB was decreased in a dose-dependent manner. DNA fragmentation was seen in the treatment groups as compared to the control group using gel electrophoresis. Identification and quantification of compounds were carried out by GC–MS and HPLC, respectively; 2(5H)furanone with IC50 of 1.99 ± 0.01 μg/mL could be the responsible anticancer compound. In conclusion, these findings suggest the potential use of the crude extract of G. applanatum as a natural source with anticancer activity against cervical cancer.Cancer is currently one of the foremost health challenges and a leading cause of death worldwide. Cervical cancer is caused by cofactors, including oral contraceptive use, smoking, multiparity, and HIV infection. One of the major and considerable etiologies is the persistent infection of the oncogenic human papilloma virus.  Cancer is currently one of the foremost health challenges and a leading cause of death worldwide. According to the World Health Organization (WHO), cancer was responsible for approximately 9.6 million deaths in 2018. Environment , certain viral infections , and diets  are the main etiologies in the occurrence of cancer .Cervical cancer is characterized as an invasive  and primG. applanatum is one of the wild mushroom species which attaches and grows on the wood; it is a valuable medicinal mushroom that has been extensively utilized as a folk medicine for the treatment and prevention of various diseases  × 100. This experiment was carried out in triplicate.To determine the cytotoxicity of the GAMCE, the control, and experimental group cells were seeded in a 96-well plate with a density of 1 × 105 cells/mL) was prepared, then 5 mL was inoculated on each 100 mm cell culture dish incubated at 37 °C with 5% CO2 for 24 h. The media were then changed, and the control group (no treatment) and the experiment group were treated with 1.4, 1.5, 1.6, and 1.7 mg/mL of GAMCE and incubated at 37 °C with 5% CO2 for 12 h. Subsequently, the media was removed, HeLa cells were washed, scraped and collected with cold PBS, transferred into 15 mL conical tubes. Following by centrifugation with 1400 RPM for 5 min. After centrifugation cells are lysed by the addition of 35 µL cell lysis buffer . Incubated for 10 min on ice, lysates were collected and clarified by centrifugation at 14,000 rpm for 20 min at 4 °C. Protein quantification was carried out by using Albumin bovine 2 mg standard  and Bradford reagent for ELISA microplate reader at 595 nm. Aliquots of whole-cell lysates were formed on 12% SDS-PAGE and bound to the nitrocellulose membrane. The membranes were blocked with 5% skim milk in PBST (PBS buffer and 0.5% Tween-20). After blocking non-specific sites, the membranes were probed with primary antibodies  and then washed in PBST three times, followed by incubation for 1 h with anti-rabbit IgG or anti-mouse IgG as secondary antibodies . The blots were then washed in PBST and visualized by an enhanced chemiluminescent detection solution  and imaging system . Finally, Band density of the blotted image was measured by using Adobe photoshop CS6.A total of 25 mL of MEM constituted HeLa cells suspension (adjusted: 1 × 105 cells/mL) was prepared, then 5 mL was inoculated on each 100mm cell culture dish and incubated at 37 °C with 5% CO2 for 24 h. The media were then changed, and the control group (no treatment) and the experiment group were treated with 1.5 and 1.6 mg/mL of GAMCE and incubated at 37 °C with 5% CO2 for 12 h. Next, the media was removed and then 1 mL cold PBS was added in each dish, the control with no treatment and GAMCE (1.5 and 1.6 mg/mL) treated cells were scraped and collected into separated 1.5 mL-eppendorf tubes. Centrifuged at 8000 RPM for 10 min at 4 °C. RNA was extracted using (RNeasy® Mini Kit (50); QIAGEN, Hilden, Germany) with protocol.15 mL of MEM constituted HeLa cells suspension , and kept in PCR machine , incubated at  1-cycle. Finally kept at 4 °C.TM 96; Bioneer, Korea) with a qPCR condition at (95 °C for 5 min) 1-cycle, then at  45-cycles. The relative quantification result was determined by 2−∆∆CT method [RT-qPCR was carried out to observe the relative fold change of genes expression in HeLa cells. This was conducted by mixing 7 μL distilled water, primer  (1 μL foT method .5 cells/mL) was prepared, then 5 mL was inoculated on each 100mm cell culture dish and incubated at 37 °C with 5% CO2 for 24 h. After 24 h, the media were changed, and the control and experimental groups treated with GAMCE concentrations of 1500 and 1600 µg/mL incubated for 24 h. Next, the media were removed and then 1 mL cold PBS was added in each dish, cells were scraped and collected into separated 1.5 mL-eppendorf tubes. Genomic DNA was extracted from the control and experiment groups using a kit  with protocol. Finally, DNA fragmentation was checked using 1.5% agarose gel electrophoresis with 100 V run for 30 min followed by an imaging system .15 mL of MEM constituted HeLa cells suspension (adjusted: 1 × 10Identification of compounds was carried out by using gas chromatography–mass spectrometry (GC–MS)  and Column (DB-5MS Ultra (30 × 0.25 × 0.25)). The sample was dissolved in water, diluted with methanol, and the precipitated portion was centrifuged and analyzed as a supernatant.Slightly modified quantitative HPLC analysis was performed in accordance with the protocol discussed by Pérez et al. 1996). HPLC fit out with Luna 5μ C18(2) 100A New Column 250 × 4.6 mm was operated. UV detector with a wavelength of 280 nm and an injection volume of 20 μL were set. The mobile phase used to separate furanone consists of two eluents: 0.2 M sodium acetate/acetic acid, pH 4 (solvent A), and methanol (solvent B). Chromatographic conditions were 0–11 min, isocratic 13% B; 11–26 min, gradient 13–23% B; 26–30 min, isocratic 23% B; and 30–33 min, gradient 23–80% B (cleaning process) [96. HPLC t-test using Microsoft Excel where the mean value of control group was individually compared with the mean value of every treated group. p-value was used as statistical significant.All data are presented and shown as Mean ± SE. Statistical analysis was conducted with ANOVA and 50 of GAMCE for HeLa was 1.55 ± 0.01 mg/mL (H)furanone in the 1 g of GAMCE; accordingly, the compound 2(5H)furanone with IC50 of 1.99 ± 0.01 μg/mL available in the crude extract with IC50 of 1.55 ± 0.01 mg/mL, could be responsible for the antiproliferative activity.Crude extraction was carried out by subcritical water extraction method and 820 mg of freeze-dried GAMCE was obtained and collected from 20 g of sample powder input. Cell viability was carried out on HeLa and HaCaT cell lines in vitro; the results obtained with IC01 mg/mL  when com01 mg/mL  compared01 mg/mL . Western01 mg/mL  was perf01 mg/mL  in the c01 mg/mL . GC–MS a01 mg/mL  for crosG. applanatum mushroom as a natural ingredient. The crude extract derived from the G. applanatum own anticancer activity effect in HeLa cells was observed by killing 50% HeLa cells at a 1.55 ± 0.01 mg/mL concentration level in vitro. This result showed the anti-cervical cancer potency of the crude extract of G. applanatum in HeLa cells.The aim of this research is to help and serve humanity in discovering suitable and effective therapeutic sources against cervical cancer. Crude extracts of natural sources are able to demonstrate numerous biological activities, including anticancer activity, and research reports are increasing. Specifically, this study focuses on treating and curing cervical cancer by using crude extract of Apoptosis is a special type of cell death program that can trigger either the extrinsic or intrinsic apoptotic pathway . NFκB isThe interaction between anti-apoptotic and pro-apoptotic members can control the integrity of mitochondria and take a crucial part in regulating the release of mitochondrial cytochrome-c . AdditioApoptosis in HeLa is characterized biochemically by the cleavage of chromosomal DNA into oligonucleosomal-sized pieces after the treatment with the crude extract. Apoptotic stimuli can cause cells to die without causing DNA degradation, while DNA fragmentation might hasten the process. Later phases of the apoptotic process in HeLa resulted in DNA fragmentation. Furthermore, the caspase family predominantly mediates apoptotic signal pathways, which finally result in DNA fragmentation. However, both caspase-dependent and caspase-independent apoptotic DNA fragmentation may have the possibility of occurring in HeLa after the treatment. The DNA ladder gel electrophoresis method was performed in this investigation , and it H)-furanone, 2(5H)-furanone, and 3(2H)-furanone [H)-furanone (H)-furanone is a commonly used conduit for many drugs that have a variety of bio-activities, such as antifungal, antibacterial, and anti-inflammatory. Many 2(5H)-furanone derivatives have also been reported as antitumor agents in nature [H)-furanone derivative has shown cytotoxicity effect on MCF-7 breast cancer cells, and cytotoxicity against A549 cells has been observed [H)-furanone-piperazine shows effective cytotoxicity against HeLa and SMMC-7721 human hepatocellular carcinoma cells [H) showed to promote apoptosis and cell cycle arrest in HepG2 (liver) and A549 (lung) human neoplastic cells, mediated by mitochondrial extrinsic, or death receptor pathways [H)-furanone derivatives show effective cytotoxicity on A549 and SK-OV-3 cell lines [H)-furanone derivatives show acceptable antiproliferative activity against cancer cell lines, including C6 (rat glioma cell), EC-1 , MDA-MB-231 (estrogen receptor-negative human breast cell), MCF-7 (estrogen receptor-positive human breast cell), HepG2 (human hepatoma cell), and CNE-1  [H)furanone in cancer chemotherapy. The anticancer action of G. applanatum may be attributed to the 2(5H)furanone ingredient in the crude extract; however, further research is needed to make the estimation more reliable and tangible.Furanone is a five-membered heteroaromatic ring containing oxygen atoms, which is of great significance in pharmaceuticals. The presence of this bio-active compound in natural and artificial origin has played an essential role in the design and development of novel drugs. These days, the synthesis of furanone derivatives and the exploration of their medicinal effects have been some of the main concerns of researchers . Furanonfuranone . 2(5H)-ffuranone  units arn nature ,39. Accon nature , 2(5H)-fobserved . Chiral ma cells . In vitrll lines . Bis-2(5ma cell) . The resma cell) . These rG. applanatum mushroom. These significant outcomes lead us to conclude that the crude extract of G. applanatum mushroom has natural therapeutic potency in the treatment for cervical cancer: by launching intrinsic apoptotic suicidal signals, by suppressing DNA survival and proliferation transcription factors, by causing DNA fragmentation, and by ceasing the growth and the metastatic prospect of the cervical cancer cells.In this study, there are apparent and objective changes observed on HeLa cells’ viability, gene expression, protein expression, and DNA fragmentation test results after treating the cells with the crude extract of Rather than this preliminary report, additional research is recommended to observe the application, route, dose, pharmacodynamics, pharmacokinetics, and other properties of the crude extract and specific isolated compounds, as well as to implement anticancer therapy products as a drug or supplement in the commercial field.


Pichia pastoris GS115, generating a recombinant P. pastoris GS115 strain expressing the feIFN-ω’. After induction, we found that the expression level of the feIFN-ω’ was two times more than that of feIFN-ω (p < 0.01). Subsequently, the feIFN-ω’ was purified and modified with polyethylene glycol, and its antiviral efficacy was evaluated in vitro and in vivo, using vesicular stomatitis virus (VSV) and FPV as model virus. Our results clearly demonstrated that the feIFN-ω’ had significant antiviral activities on both homologous and heterologous animal cells in vitro. Importantly, the feIFN-ω’ can effectively promote the expression of antiviral proteins IFIT3, ISG15, Mx1, and ISG56, and further enhance host defense to eliminate FPV infection in vivo, suggesting a potential candidate for the development of therapeutic agent against feline viral diseases.Feline viral diseases, such as feline panleukopenia, feline infectious peritonitis, and feline coronaviral enteritis, seriously endanger the health of cats, and restrict the development of pet industry. Meanwhile, there is a current lack of effective vaccines to protect against feline viral diseases. Thus, effective therapeutic agents are highly desirable. Interferons (IFNs) are important mediators of the antiviral host defense in animals, particularly type I IFNs. In this study, a novel feline IFN omega (feIFN-ω) gene was extracted from the cat stimulated with feline parvovirus (FPV) combined with poly(I:C), and following codon optimization encoding the feIFN-ω, the desired gene (feIFN-ω’) fragment was inserted into plasmid pPICZαA, and transformed into  Interferon (IFN) is an antiviral protein found from influenza virus-infected chicken embryos in 1957, and subsequent studies have demonstrated that human and animal cells also can produce IFNs, which are considered as the main natural immune barrier for the host against viral infections . IFNs arNowadays, it is more and more common to keep companion animals, especially pet cats. However, cat viral diseases, such as feline infectious peritonitis caused by feline infectious peritonitis virus (FIPV), feline panleukopenia caused by feline parvovirus (FPV), and cat enteritis caused by feline enteric coronavirus seriously endanger the health of cats and the development of the pet cat industry . In addiPichia pastoris strain, and after being modified with polyethylene glycol, the antiviral efficacy of the feIFN-ω’ was evaluated in vitro and in vivo.In this study, in order to develop a potent antiviral agent for the treatment of cat viral diseases, a gene encoding a novel feline IFN-ω (feIFN-ω) was obtained from the peripheral blood of the cat stimulated with FPV combined with poly(I:C). Following codon optimization of the gene encoding feIFN-ω, the desired feIFN-ω’ was produced by a recombinant n = 20) were purchased from a pet market in China. Animal experiments were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and were approved by the Ethical Committee for Animal Experiments of Zhejiang A&F University (ZAFUAC2021021), China. Vesicular stomatitis virus (VSV) and feline parvovirus (FPV) were kept in our laboratory. Pichia pastoris strain GS115 (P. pastoris GS115) kept in our laboratory was cultured in Yeast-extract Peptone Dextrose (YPD) medium  at 30°C. Madin–Darby bovine kidney (MDBK) cells, Madin–Darby canine kidney (MDCK) cells, and feline kidney (F81) cells were cultured in Dulbecco’s Modified Eagle Medium, DMEM  supplemented with 10% fetal bovine serum, FBS  in a 5% CO2 incubator at 37°C. The plasmid pPICZαA kept in our laboratory was used to construct recombinant P. pastoris expressing feline IFN-ω.Healthy 4-month-old Dragon Li cats (3 TCID50) and 500 µL of 1.0 mg/mL poly(I:C)  through subcutaneous injection route for three consecutive days. On day 10 post-stimulation, the peripheral blood of the cat was collected, and the total RNA was extracted by Total RNA Isolation kit  followed by reverse transcription, generating first-strand cDNA. Next, using the cDNA as template, a gene encoding feIFN-ω was obtained by PCR amplification with the primer pair listed in The cat was co-stimulated by 500 µL of FPV  was synthesized , which was harbored in a recombinant plasmid pMD-ω’.In this study, we aimed to produce the feIFN-ω by yeast to test its antiviral efficacy BamH I and Kpn I , and the gene fragments of interest were purified, followed by ligation with T4 DNA ligase , generating the recombinant plasmid pPICZαA-ω (or pPICZαA-ω’). Next, the recombinant plasmid pPICZαA-ω (or pPICZαA-ω’) was linearized by AVRII enzyme digestion , and then the linearized pPICZαA-ω (or pPICZαA-ω’) was electroporated into the P. pastoris GS115 competent cells, followed by screening positive clones on YPD plates supplemented with 100 µg/mL of Zeocin , generating the recombinant strains named GS115-pPICZαA-ω or GS115-pPICZαA-ω’. In order to produce the feline interferon, the recombinant strain GS115-pPICZαA-ω (or GS115-pPICZαA-ω’) was grown overnight to an OD600 of approximately 1.5 in BMGY medium . After centrifugation, the cells pellets were transferred into BMMY medium containing 1.5% YNB, biotin, and 0.05% methanol , and cultured for 120 h. Sterile-filtered methanol was supplemented every 24 h to maintain induction condition. The expression level of the feIFN-ω in GS115-pPICZαA-ω (and GS115-pPICZαA-ω’) and its culture supernatants was analyzed by 12% SDS-PAGE. After that, the feIFN-ω’ was purified by Ni2+ affinity chromatography column, and PEGylation was conducted in sodium borate buffer with the feIFN-ω’ and 40 kDa mPEG-2-N-hydroxysuccinimide, and then the polyethylene glycol-modified feIFN-ω’ was purified by a Q-Sepharose column [The recombinant plasmid pMD-ω containing the feIFN-ω gene (or pMD-ω’ containing the optimized feIFN-ω’ gene) and vector pPICZαA were, respectively, digested by e column .in vitro using VSV and FPV as model viruses. The supernatants from induced GS115-pPICZαA-ω’ were serially 10-fold diluted in DMEM supplemented with 5% FBS, and were then added into a 96-well plate (8 replicates per dilution) followed by addition of MDBK cells to a concentration of 5 × 104 cells/well. Then, the MDBK cells were incubated at 37°C in a 5% CO2 incubator for 24 h. After discarding the supernatants, the MDBK cells were infected with VSV (or FPV) at an MOI of 1.0. When the cytopathic effect (CPE) reached 100% in virus control group (untreated with feIFN-ω’), the cells were stained with 0.1% (w/v) of crystal violet, and incubated at 37°C for 30 min. After elution with 50% ethanol-0.1% acetic acid, absorbance was measured at 595 nm. In parallel, the supernatants from GS115-pPICZαA were used as negative control. Subsequently, the antiviral activity of the feIFN-ω’ purified by Ni2+ affinity chromatography column at a concentration of 0.001 ng ~ 10 ng was determined by MCIA, using FPV as model virus and INTERCAT IFN  as feline IFN positive control. The test was repeated five times. Furthermore, the species-specific antiviral activity was also determined by the ability of the feIFN-ω’ to inhibit the CPE of VSV on F81 cells, MDCK cells, and MDBK cells. In parallel, the INTERCAT IFN was used as feline IFN control. The assay was repeated three times (8 replicates each sample).The antiviral activity of the recombinant feIFN-ω’ was determined by microdose cytopathogenic effect inhibition assay (MCIA) in vivo, eighteen 4-month-old Dragon Li cats were randomly divided into three groups: FPV infection group , feIFN-ω’ treatment group , and normal control group . On days 5 and 15 after challenge, three cats were randomly selected from each group, respectively, and the viral loads of the FPV in blood, kidney, liver, spleen, intestine, and feces of the cats were determined using a SYBR Green I-based real-time quantitative RT-PCR (RT-qPCR) assay with the primer pair targeting the FPV VP2 gene  values, and Tukey’s multiple comparison tests and one-way analysis of variance (ANOVA) were used to analyze the differences among groups by GraphPad Prism V8.0 software.http://www.sbg.bio.ic.ac.uk), and result demonstrated that the 3D structure of the feIFN-ω conformed to the 3D structural characteristics of type I IFN (After being stimulated by FPV combined with poly(I:C), a gene of approximately 612 bp encoding feIFN-ω was amplified by RT-PCR from the peripheral blood of the cat , and phype I IFN .Figure P. pastoris, the gene sequences encoding the feIFN-ω were optimized according to the codon usage bias of P. pastoris (p < 0.01) (In order to achieve highly efficient expression of the feIFN-ω by pastoris . Subsequpastoris  and strapastoris . By cont < 0.01) .Figure In order to evaluate the biological activity of the recombinant GS115-expressed feIFN-ω’, we first tested the antiviral activity of the supernatants of the recombinant strain GS115-pPICZαA-ω’ induced with methanol, using VSV and FPV as model viruses. As shown in in vivo, the cats were infected with FPV followed by the feIFN-ω’ therapy. Our results showed that after the feIFN-ω’ treatment, virus loads in blood, kidney, liver, spleen, intestine, and feces of the cats in the feIFN-ω’ treatment group significantly decreased on days 10 after feIFN-ω’ treatment, indicating effective antiviral activity of the recombinant feIFN-ω’ in vivo  and FPV infection group (p < 0.05), indicating that the feIFN-ω’ can effectively promote the expression of antiviral proteins. Furthermore, we also determined the mRNA transcript levels of cytokines IL-1β, TNF-α, IL-4, IL-6, IL-10, and IL-12 in the blood samples of the cats from each group by RT-qPCR, and results showed that compared to the FPV infection group, the levels of these cytokines significantly decreased (p < 0.05 or p < 0.01) in the feIFN-ω’ treatment, but still significantly higher (p < 0.05) than those in the mock control group  expression system to produce the feIFN-ω in this study, and our result showed that the feIFN-ω can be effectively expressed by P. pastoris and secreted into supernatants.Protein expression system includes prokaryotic and eukaryotic systems. Although prokaryotic expression system is most commonly used , the actity loss ,32. Yeasity loss , and canity loss . TherefoP. pastoris is related to the conformity of expression system, selection of vectors, expression conditions, and other factors [P. pastoris, the gene sequences encoding the feIFN-ω were subjected to optimization according to the codon usage bias for P. pastoris, such as using high-frequency dominant codon, reducing the GC content, adjusting AT-rich regions, etc. [P. pastoris could significantly improve protein expression of the interest.Importantly, the efficient expression of exogenous protein in  factors ,36. More factors . Therefons, etc. . With thin vitro and in vivo. In vitro, we first analyzed the antiviral activity of the supernatants secreted by GS115-pPICZαA-ω’, and results showed that the supernatants displayed effective antiviral activity with a dose-dependence, and significant reduction in CPE on the MDBK cells infected by VSV and FPV was observed, while not observed in negative control group. Then, we purified the recombinant feIFN-ω’ and further tested its antiviral activity, and our data showed that the purified feIFN-ω’ also displayed effective antiviral activity, and significant reduction in the FPV-induced CPE can be observed. By contrast, the antiviral effect of the feIFN-ω’ obtained in this study was better than that of INTERCAT IFN. Utilizing a standard VSV assay, we evaluated the antiviral effect of the feIFN-ω’ on F81 cells (feline), MDBK cells (bovine), and MDCK cells (canine) using the INTERCAT IFN as positive control, and our data showed that the feIFN-ω’ showed highly efficient antiviral effect against VSV on homologous and heterologous animal cells. However, the INTERCAT IFN only showed effective antiviral effect against VSV on homologous animal cells, but not on MDBK and MDCK, which was consistent with previous report [Subsequently, we systematically assessed the antiviral activity of the feIFN-ω’ s report .in vivo experiments to evaluate the antiviral effect of the recombinant feIFN-ω’, in order to enhance the stability of the feIFN-ω’ and extend its half-life in vivo, the feIFN-ω’ was modified with polyethylene glycol. And then, the animal experiment was carried out. The cats were infected with feline parvovirus (FPV), followed by feIFN-ω’ treatment, using FPV-infected cats without interferon treatment as control. We found that the viral loads in blood, kidney, liver, spleen, intestine, and feces of the FPV-infected cats with the feIFN-ω’ treatment (feIFN-ω’ treatment group) significantly decreased, and the virus could be effectively eliminated detected by IHC assay, indicating that the FPV replication was effectively inhibited. However, the viral loads in FPV-infected cats without feIFN-ω’ treatment (FPV infection group) continued to increase. FPV infection can cause panleukopenia in cats. So, we determined the total WBC levels in the cats from mock control group, FPV infection group, and feIFN-ω’ treatment group. Our data showed that the total WBC counts significantly decreased after virus infection, and with the feIFN-ω’ treatment, the total WBC counts in feIFN-ω’ treatment group were gradually returned to normal, but not in FPV infection group, indicating effective therapeutic effect of the feIFN-ω’. To further explore the antiviral effect of the feIFN-ω’, the mRNA levels and protein levels of the antiviral proteins Mx1, ISG15, ISG56, and IFIT3 in the blood of the cats from each group were determined, and from these results, we clearly see that the feIFN-ω’ treatment can significantly promote the expression of antiviral proteins Mx1, ISG15, ISG56, and IFIT3, which would further exert their antiviral functions to inhibit the replication of FPV. Many studies have also demonstrated that interferon ω could stimulate the expression of the antiviral proteins ISGs [p < 0.05 or p < 0.01) in the cats from the feIFN-ω’ treatment group, compared with the FPV infection group. These findings are consistent with an overall reduction of pro-inflammatory pathways in cats treated with IFN [Before performing ins ISGs ,41. In awith IFN .P. pastoris, and achieved highly efficient expression of the feIFN-ω from a recombinant P. pastoris. The antiviral efficacy of the feIFN-ω was subsequently evaluated, and our results clearly demonstrated that the feIFN-ω displayed effective antiviral activity against VSV and FPV, suggesting a promising therapeutic agent for viral diseases in cats.A global overview of the gene clone, codon optimization, expression, and antiviral effect evaluation of a novel feIFN-ω is given in


This overview article will fully explore the phase space of boron–hydrogen chemistry in order to discuss parameters that optimize these materials as solid electrolytes for battery applications. Key properties for effective solid-state electrolytes, including ionic conduction, electrochemical window, high energy density, and resistance to dendrite formation, are also discussed. Because of their open structures (for closo-boranes) leading to rapid ionic conduction, and their ability to undergo phase transition between low conductivity and high conductivity phases, borohydrides deserve a focused discussion and further experimental efforts. One challenge that remains is the low electrochemical stability of borohydrides. This overview article highlights current knowledge and additionally recommends a path towards further computational and experimental research efforts.The goal of this article is to highlight crucial breakthroughs in solid-state ionic conduction in borohydrides for battery applications. Borohydrides, M Solid-state electrolytes, which have high ionic conduction, will reduce the need for organic liquid electrolytes and will, in turn, have a large impact on further growth of lithium ion battery technologies [Recent advances in battery technologies have permitted the development of rechargeable lithium ion batteries with high energy density, high power density, durability, long term cycle life, and low loss in capacity with cycling. The intent of this article is to provide inspiration to undertake research to advance “beyond lithium battery concepts”, specifically towards borohydride solid-state electrolytes. However, today lithium ion batteries are dominating the market. Lithium ion batteries boast specific power densities of ~500 W/kg and specific energy densities of up to 200 kWh/kg  . These lnologies . Materia4, LiNH2, and LiAlH4 systems (and their mixtures), providing a prospectus on improving room temperature ionic conduction [This overview article work seeks to provide discussion germane to materials based in the borohydride family for solid-state electrolyte applications. The development of borohydrides as a class of materials for use as solid electrolytes could lead to innovations in low temperature fuel cells and safer ion conducting batteries. This class of materials has not been fully explored experimentally or computationally for use as solid-state electrolytes. A review by Matsuo and Orimo published in 2011 describes fast ionic conduction in LiBHnduction . Neverthnduction . That co+BH4, closo-M2+B10H10, closo-M+B12H12, and planar-M6+B6H6 (with M = cations) can form ionic compounds with cations such as Li+, K+, Na+, Ca2+, and Mg2+. Anion structural renderings are shown in 10H102− and -B12H12−) structures have open channels for ionic conduction [The attractiveness of borohydrides is that they form in various molecular structures. Boron–hydrogen chemistry is versatile (forming bridge and cyclic structures), and this could lead to improved tunability in cationic conduction. For example, nido- Mnduction .There are several technical challenges remaining for lithium ion batteries  and, in particular, for solid-state batteries. Among those are increasing ionic conduction, broadening the electrochemical stability window, alleviating dendrite formation, and achieving high energy density. 4)2 and LiBH4 mixtures as electrolytes in operational batteries and showed reasonably high full cell electric potential and cycle life. Work using Mg(BH4)2 and LiBH4 was followed by use of carboranes, e.g., (C2BH10H11)2Mg, in diglyme and tetraglyme solvents as electrolytes [4 with 0.5 M NaBH4 in THF solution. Microstructure changes are indeed apparent before  [Contemporaneous with work done using borohydrides inside of solvents, borohydrides in the closo-borane phases were being considered as superionic conductors for rapid ion motion through their open channel pore structures ,23,24,25 in 1959 , there h in 1959 ,29. Only in 1959 ,32,33,34tructure . Experimredicted . Furtherredicted  demonstragnitude . Other sterials) ,18,19,354−) has a room temperature ionic conductivity of ~10−7 S/cm [2, to affect ionic conduction in borohydrides. For example, MgBH4 having an ionic conductivity of 10−8 S/cm realizes an increase in ionic conduction to 10−5 S/cm with the addition of an -NH2 adduct [Ionic conduction is considered the most important variable for solid electrolytes, and hence there have been many material developments for improved ionic conductivity ,36,37,380−7 S/cm . Doping 0−7 S/cm ,41. Work2 adduct .4)2 with an adduct of -NH2 reaches ionic conductivity levels of 10−6 S/cm at 150 °C [3(PO4)2, which has an ionic conductivity of 10−6 S/cm at temperatures in excess of 400 °C. Another factor that is well known to affect ionic conduction in LiBH4 is crystallographic phase. When measured above the orthorhombic to hexagonal phase transition (occurring at 130 °C), a higher ionic conduction is realized in the elevated temperature hexagonal phase [4 is 10−3 S/cm [3(BH4)(BO3), formed by decomposing LiBH4, Ca(BH4)2 in the presence of carbon, has demonstrated a Li+ conduction of 10−5 S/cm at room temperature [3 molecular unit is reported to define the transport pathway for the lithium ion [2+, Eu2+, and Na+ have all demonstrated improved Li+ conductivity when added to LiCa3BH4BO3. The larger-sized divalent substitutions, Sr2+ and Eu2+, expand the lattice to enhance transport. The monovalent Na+ substituting onto Ca2+ O-sites also causes a lattice expansion when there is excess Li+ [The conductivity for Mg results in increased ionic conduction over the nido- BH4− units. In fact, closo-boranes realize ionic conductivities, which are five orders of magnitude higher than nido- structures. Other work has shown that rapid cationic conduction (3 × 10−2 S/cm) occurs in the LiB10H10 and NaB10H10 phases [12H12 closo-borane phases [nHn−) could readily be synthesized following the resin-based ion exchange methods as described in the thesis by Blake [nHn purchased from Boron Specialties, LLC. Some borohydrides, such as borane, diborane, and pentaborane, are known to be toxic and pose environmental safety hazards and so safe handling must be considered. These are primarily in stoichiometries x < 5 in BxHy  and are liquid or gaseous at room temperature, toxic, and highly reactive with oxygen in air.As noted earlier in this paper, the open structure of the closo-boranes  [2S and Li3PS4, are stable up to 2.3 V to 2.5 V vs. Li metal, while LiH is stable down to 0 V [12H12) [xHy product species cannot be easily removed and must be considered. In practical applications, the electrochemical windows could be far from those predicted computationally. This is an important avenue of experimental research that should be undertaken. Moreover, research which considers interlayered compounds comprised of several borohydrides as the solid electrolyte could serve the objective of increasing electrochemical window of the entire layer while isolating product phases into close proximity to one another for overcoming diffusion barriers to forming starting compounds upon reverse cycling.The electrochemical stability window is the electric potential range over which the material is electrochemically stable. One drawback of borohydrides is their narrow electrochemical windows ,44. Figuulations . Lu et au et al.  also pro species . The ele [12H12) . It will4 is 10−3 S/cm in the high temperature hexagonal phase [4− unit with temperature on ramp up and ramp down at a slow heating and cooling rate of 5 °C/min  transition from a ν3 splitting to a single broad peak at elevated temperature, which is consistent with a phase transition to the hexagonal phase (which occurs at 130 °C) [4−  [4 has a marked effect on room temperature ionic conduction, measured at 10−3 S/cm [4− unit (determined by techniques such as Raman spectroscopy), and ionic conductivity (using impedance spectroscopy) should be explored for both nanostructured LiBH4 and the LiBH4 having additives such as lithium halides incorporated in the materials processing. Vibrational spectroscopy and other crystallographic and structural measurement techniques should be, ideally, partnered with ionic conductivity measurements in order to gain “fine-structure” insights that could lead to novel approaches to improved properties.The experimental data are determined by the most widely used approach for measuring ionic conductivity: impedance spectroscopy. Impedance spectroscopy is able to distinguish localized, non-localized, and space charge effects ,47,48. Mal phase . To exam5 °C/min . Gomes,  130 °C) . Upon co 130 °C) . Additio 130 °C) . Additiol phase) . Nanostr0−3 S/cm . The rel2B12H12 phase. Results suggest that the high rotational mobility of the in -B12H122− anionic structures aids in the high ionic mobility of Na+ [12H122− anions plays a correlated role in cationic conduction [4) in -B12H122− structures is possible [Other advanced characterization techniques, such as quasi-elastic neutron scattering (QENS), which can deliver both structural information (such as H-H distances), diffusivities, and mechanism of motion , should be considered when studying ionic mobility in borohydrides. Impedance spectroscopy was used to study superionic conduction in the Nay of Na+ . Furthernduction ,51. In apossible .In work documenting battery development by Scrosati, the timeline growth in lithium ion battery sales in various market sectors  between 2000 and 2015 demonstrates the enormous potential of the technology to penetrate further into new markets . There i


The gut is able to “sense” nutrients and release gut hormones to regulate digestive processes. Accordingly, various gastrointestinal cell types possess transient receptor potential channels, cation channels involved in somatosensation, thermoregulation and the sensing of pungent and spicy substances. Recent research shows that both channels are expressed in enteroendocrine cell types responsible for the release of gut peptide hormones such as Cholecystokinin (CCK) and Glucagon-like Peptide-1 (GLP-1). A large array of herbal compounds, used in pig nutrition mostly for their antibacterial and antioxidant properties, are able to activate these channels. Cinnamaldehyde, occurring in the bark of cinnamon trees, acts as an agonist of Transient Receptor Potential Ankyrin 1 (TRPA1)-channel. Capsaicin, the active component of chili peppers, acts as an agonist of Transient Receptor Potential Vanilloid 1 (TRPV1)-channel. This study explored the ability of both compounds to stimulate the release of CCK and GLP-1, and whether this response was mediated by TRP channels, using an ex-vivo intestinal segment model. Results showed mainly the potential of cinnamaldehyde to interact with TRPA1 and trigger CCK and GLP-1 release, while yet being dependent on the location in the small intestine.Cinnamaldehyde and capsaicin have been reported to exert effects on the gastric function, mediated by the interaction with transient receptor potential ankyrin channel 1 (TRPA1) and transient receptor potential vanilloid channel 1 (TRPV1), respectively. This study examined whether these compounds could trigger the release of cholecystokinin (CCK) and/or glucagon-like peptide 1 (GLP-1) in the pig’s gut in a porcine ex-vivo intestinal segment model. Furthermore, it was verified whether this response was mediated by TRPA1 or TRPV1 by using the channel’s antagonist. These gut peptides play a key role in the “intestinal brake”, a feedback mechanism that influences the function of proximal parts of the gut. Structural analogues of cinnamaldehyde were screened as well, to explore structure-dependent activation. Results showed a significant effect of capsaicin on GLP-1 release in the proximal small intestine, TRPV1 independent. TRPA1 showed to be strongly activated by cinnamaldehyde, both in proximal and distal small intestine, evidenced by the release of CCK and GLP-1, respectively. Out of all structural derivates, cinnamaldehyde showed the highest affinity for TRPA1, which elucidates the importance of the α,β-unsaturated aldehyde moiety. In conclusion, cinnamaldehyde as a TRPA1 agonist, is a promising candidate to modulate gastric function, by activating intestinal brake mechanisms. Only treatments in the second incubation step were changed: The KRB/HEPES buffer was then replaced by a pre-warmed KRB/HEPES buffer  without glucose but containing one of treatments  or a control  and incubated for an additional 1 h at 37 °C, O2/CO2 (95%/5%). All treatments were tested in each animal in triplicate, using a different 24-well plate per animal.The second and third experiment were conducted in accordance with the ethical standards and recommendations for the accommodation and care of laboratory animals, covered by the European Directive 2010/63/EU on the protection of animals used for scientific purposes and the Belgian Royal Decree KB29.05.13 on the use of animals for experimental studies. No ethical approval was required for this trial according to local legislation as animals were kept under farm practices without interventions causing harm equivalent to, or higher than, that caused by the introduction of a needle in accordance with good veterinary practice, and because animals were killed solely for the use of their organs or tissues (2010/63/EU). Six 45-day-old (three weeks post weaning) piglets (Topigs Norsvin × German Pietrain) with a mean body weight (BW) of 12.21 ± 1.32 kg were selected from a commercial herd  and housed in groups of two per pen with full slated floors in the experimental facility of the Department of Animal Sciences and Aquatic Ecology of Ghent University . The stable had a conventional ventilation scheme, ambient temperature at 24 °C, and an 18 light/6 dark schedule. Piglets had ad libitum access to water and a commercial starter diet. Piglets were humanely sacrificed by inducing electronarcosis, followed by exsanguination. To investigate the effect of cinnamaldehyde on CCK and GLP-1 release in the distal SI, GIT-tissue segments from duodenum  and ileum , were collected within 10 min. Tissue was stored in ice cold KRB/HEPES buffer bubbled with OGLP-1 and CCK hormone levels were analysed using commercially available ELISA kits, according to the manufacturer’s instructions. Concentrations of CCK-58 were analysed using a Porcine Cholecystokinin ELISA kit from MyBioSource, San Diego, CA, USA (#MBS264395). Inter-assay coefficient of variation for the CCK kit was 7.5% and intra-assay coefficient of variation was 3.2%. GLP-1 release was measured using a Glucagon-Like Peptide-1  ELISA kit from Millipore, Billerica, MA, USA (EZGLPT1-36K). Inter-assay coefficient of variation for the GLP-1 kit was 7.8% and intra-assay coefficient of variation was 2.1%. When necessary, samples were diluted in assay buffer in order to obtain values within the detection range of this kit. Both hormone levels were measured on a TECAN INFINITE M NANO Microplate Reader.Viability of tissues was monitored as previously described, using a Roche LDH reagent kit PLUS  according to the manufacturer’s instructions.Six 38-day-old piglets  with a mean body weight (BW) of 9.05 ± 0.42 kg were selected from a herd  and housed in a group of two per pen with full slated floors. The stable had a conventional ventilation scheme, ambient temperature at 24 °C, and an 18 light/6 dark schedule. Piglets had ad libitum access to water and a standard starter diet. Piglets were humanely sacrificed by inducing electronarcosis followed by exsanguination.−1) or a control  and incubated for an additional 1 h at 37 °C, O2/CO2 (95%/5%). All treatments were tested in each animal in triplicate, using a different 24-well plate per animal.To explore which structural features of cinnamaldehyde are important for the activation of TRPA1 in the duodenum of piglets, the effect on CCK release of cinnamaldehyde next to four structural derivates was investigated in the duodenum of piglets. As such, 2-methoxy cinnamaldehyde (CIN2M), 4-methoxycinnamaldehyde (CIN4M) , hydrocinnamaldehyde (HCIN)  and cinnamic acid (CINA)  were tested next to cinnamaldehyde and a control (KRB). GIT-tissue segments from duodenum  were collected within 10 min and processed as previously described. Only treatments in the second incubation step were changed: The KRB/HEPES buffer was then replaced by a pre-warmed KRB/HEPES buffer  without glucose but containing one of the treatments . Regarding experiment 1 and experiment 3, data were analysed using a one-way ANOVA according to the GLM procedure of SAS with a linear model that included effects of treatment (fixed) and pig (random). Regarding experiment 2, statistical analysis was performed using two-way ANOVA with the GLM procedure of SAS with a 2-level full factorial design: treatment, location in GIT, and interaction, using pig as a random factor. Multiple comparisons were performed by a Tukey test. Data were expressed as means and LDH leakage for samples was on average 8.9 ± 0.2% for KRB, 9.0 ± 0.7% for CIN, 9.9 ± 0.2 for CAP, 9.0 ± 0.6 for CIN-, and 9.3 ± 0.2 for CAP-. Following the criteria stated by Voortman et al. (2012)  and Ripk−1 and 15.9 ± 11.1 pg mL−1, respectively). p = 0.008) on CCK release. Compared to the control treatment only CIN (94.4 ± 11.8 pg mL−1) stimulated CCK secretion (p < 0.05). CAP (35.6 ± 12.6 pg mL−1), CIN- (44.34 ± 11.8 pg mL−1) and CAP- (47.04 ± 10.6 pg mL−1) did not change CCK release as compared to the control, and no differences between these treatments were observed. Compared to CIN, CIN-  CCK levels were almost 47% lower (p = 0.05), and at the same level as the basal CCK secretion. p = 0.007) on GLP-1 release. Compared to the control treatment (KRB), CIN (27.8 ± 12.4 pg mL−1), CAP (29.3 ± 9.5 pg mL−1), CIN- (27.9 ± 11.6 pg mL−1) and CAP- (30.3 ± 13.2 pg mL−1) significantly (p < 0.05) stimulated GLP-1 secretion resulting in concentrations that were almost two times higher as compared to control levels. No differences were observed between incubations with pure compounds  and with their respective antagonist .The basal secretion of CCK in duodenal tissue (KRB treatments) was higher than GLP-1 secretion  upregulated the expression of TRPA1 (p < 0.05) compared to KRB. The addition of 20 µmol L−1 capsaicin (CAP) did not alter TRPA1 mRNA expression (p = 0.091) towards higher mRNA expression in tissue incubated with 20 µmol L−1 capsaicin (CAP) as compared to control (KRB)  was determined. Addition of 200 µmol Lpression a. In theol (KRB) b. Incubaol (KRB) c,d.LDH leakage for duodenal samples was on average 8.4 ± 0.3% for KRB, 7.2 ± 0.3% for CIN and 8.2 ± 0.7 CIN-. Ileum samples showed an LDH leakage of 8.7 ± 0.4% for KRB, 8.4 ± 0.3% for CIN and 8.5 ± 0.3 CIN-. All intestinal samples were within the limit of LDH leakage and treatment did not have a toxic effect.p < 0.001) and the interaction (p < 0.001) have an effect. Compared to KRB, CIN potently stimulated CCK release , in duodenal tissue (−1). No differences were observed between control (KRB) and treatments  in the ileum regarding CCK release. Basal CCK secretion (KRB) in duodenum and ileum was around the same level (p = 0.995). p < 0.001), and the interaction (p < 0.001) have an effect. Regarding GLP-1 in duodenal tissue (−1) stimulated release compared to control . This response was not blocked by incubating together with the antagonist . In ileal tissue, cinnamaldehyde evoked a higher response  compared to control . This response was mostly blocked by incubating together with the antagonist . Basal GLP−1 secretion (KRB) in ileum was higher compared to basal secretion in the duodenum (p < 0.05).l tissue . This rel tissue , cinnamap < 0.001) on CCK release. Compared to the control treatment , all treatments , stimulated CCK release in duodenal tissue. Incubation with cinnamaldehyde (CIN) stimulated CCK release more potently compared to the derivates (p > 0.05); however, no differences were observed between incubation with derivates .Our study confirmed the hypothesis that cinnamaldehyde and capsaicin have an effect on GLP-1 and CCK release. As expected, basal CCK secretion in the proximal SI was higher compared to basal GLP-1 secretion. Voortman et al. (2012), observed a clear pattern of CCK, GLP-1, GLP-2 and PYY release along the porcine intestinal tract. CCK levels were predominantly released in the proximal small intestine from I cells. In contrast, tissue levels of GLP-1, known to be predominantly released from L cells in the ileum, are higher in the distal small intestine . CinnamaCapsaicin did not trigger CCK release from duodenal tissue cultures. This pungent compound is known to stimulate TRPV1, which is expressed in the duodenum of the pig . Since w−1 to 510 mg kg−1 feed) resulted in higher TRPV1 mRNA abundance in both duodenum and ileum [Cinnamaldehyde upregulated TRPA1 mRNA expression, which highlights the molecule’s ability to activate this ion channel. TRPV1 gene expression shows a trend for a higher expression after treatment with capsaicin. In a study performed by Toschi et al. (2020), feeding 28-day-old piglets for 14 days with different doses of thymol  and distal  exposure of nutrients on gastrointestinal motility. A study performed by Maljaars et al. (2007) showed that gastric emptying of a liquid meal was significantly delayed after ileal fat perfusion compared to an equicaloric duodenal infusion in man . In dogsResults obtained from the third experiment highlighted which structural features are important to stimulate CCK release. TRPA1 contains ankyrin repeat motifs in the intracellular N-terminal moiety which possess cysteine and lysine residues, essential for activation by reactive agonists. TRPA1 could be activated by covalent binding of electrophiles to these cysteine residues, since the nucleophilic mercapto-group of cysteines is able to attack the α,β-unsaturated bond of cinnamaldehyde, via Michael addition .To study the precise mechanism of covalent modification with unsaturated carbonyl-containing compounds, Sadofsky et al. (2011) examined a range of compounds which can undergo both conjugate and/or direct addition . ResultsIn conclusion, capsaicin stimulates GLP-1 release, but TRPV1 seemed not to be involved. It was demonstrated that cinnamaldehyde potentiated CCK release in the duodenum and GLP-1 release in the ileum by activation of TRPA1. Moreover, our findings elucidate that this compound, and not its structural derivatives missing the α,β-unsaturated aldehyde moiety, is a promising candidate to modulate gastric function in the pig, improving digestion.