Lactiplantibacillus plantarum, enhances their beneficial properties as regulators of the immune, digestive, and cardiovascular system. We investigated the effects of fermentation with Lactiplantibacillus plantarum on the antioxidant and immunomodulatory effects of Pushgay berries  in human umbilical vein endothelial cells (HUVECs) and macrophage cell line RAW264.7. Polyphenol content was assayed by Folin–Ciocalteu and HPLC-MS/MS analysis. The effects of berries solutions on cell viability or proliferation were assessed by WST8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium, monosodium salt and Lactate dehydrogenase (LDH) release, Trypan blue exclusion test, and Alamar blue assay. Antioxidant activity was evaluated by a cell-based chemiluminescent probe for the detection of intracellular H2O2 production in HUVECs. Heme oxygenase-1 (HO-1) expression levels were investigated by RT-qPCR. Glutathione reductase (GR), glutathione peroxidase (Gpx), superoxide dismutase (SOD), and catalase (CAT) activities, as markers of intracellular antioxidant defense, were evaluated by spectrophotometric analysis. The immunomodulatory activity was examined in RAW 264.7 by quantification of inducible nitric oxide synthase (iNOS) and Tumor Necrosis Factor—alpha (TNFα) by RT-qPCR. Data showed that fermentation of Pushgay berries (i) enhances the content of quercetin aglycone, and (ii) increases their intracellular antioxidant activity, as indicated by the reduction in H2O2-induced cell death and the decrease in H2O2-induced HO-1 gene expression in HUVECs treated for 24 h with fermented berries solution (10 µg/mL). Moreover, treatment with Pushgay berries for 72 h (10 µg/mL) promotes cells growth in RAW 264.7, and only fermented Pushgay berries increase the expression of iNOS in the same cell line. Taken together, our results show that LAB fermentation of Pushgay berries enhances their antioxidant and immunomodulatory properties. Accumulating evidence suggests that high consumption of natural antioxidants promotes health by reducing oxidative stress and, thus, the risk of developing cardiovascular diseases. Similarly, fermentation of natural compounds with lactic acid bacteria (LAB), such as  Oxidative stress is involved in the development of several diseases, including atherosclerosis, the major cause of many cardiovascular diseases (CVDs) . The molIn recent years, the potential role of natural bioactive compounds in preventing and treating disorders where oxidative stress is involved, such as cancers, metabolic diseases , neurodegenerative, and CVDs, has clearly emerged. Indeed, since natural products exert beneficial biological activities and possess pharmacological properties, they could play a valuable role in drug discovery ,6,7.Epidemiological and clinical studies have reported the beneficial properties of berries in terms of natural antioxidant capabilities and decreased risk of CVDs ,9. BerriVaccinium species from South America, such as bilberry or blueberry, on human health . The analytical method was developed and validated according to ICH guidelines to satisfy high analytical parameters in terms of accuracy and reproducibility. Limit of detection (LOD) and limit of quantification (LOQ) were determined by signal to noise ratio . Recovery and the matrix effect were evaluated for each compound before the sample analysis. Seven point calibration curves  were used for the quantification of each compound using standard solutions in the mobile phase (phase A:phase B 95:5). Internal standard hydroxytyrosol was used for calibration curves at a fixed concentration of 5 ng/mL. Then, 100 mg of each sample was extracted by sonication with 1 mL of 70% ethanol solution three times. The samples were centrifuged, the supernatants were pooled, and 10 ul of the sample was injected into the HPLC-MS.Liquid chromatography was performed using a 2690 Alliance system . Analytical separation was performed using Phenyl-Hexyl 1.7 µm, 150 mm × 2.1 mm i.d. . The mobile phase was constituted as follows: 15 mM ammonium acetate in water adjusted to pH 8.0 with ammonia (solvent A) and methanol 99.9% (solvent B). Chromatographic separation was achieved at a 0.15-mL/min flow rate under gradient elution conditions: 95% A for 5 min, 95–40% A from 5 to 15 min, 40–20% from 15 to 20 min, 20% A from 20 to 25 min, 20–95% A from 25 to 27 min, and 95% A from 27 to 35 min. All the changes in the mobile phase composition were linear. The analytical column was maintained at 30 °C. The column effluent was introduced into the ESI source, operating in positive ionization mode, connected to a triple quadruple mass spectrometer  operating in the multiple reaction monitoring (MRM) acquisition mode. Standard solutions of 3 anthocyanins were used to evaluate polyphenols profile of fermented and non-fermented Pushgay berries: malvidin-3-O-glucoside (MAL), quercetin-3-Oglucoside (QUE), and quercetin aglycone (QA). The most abundant signal for each compound in multiple reaction monitoring mode (MRM) was monitored for the quantification [2. HUVECs at passages 2 to 7 were actively proliferating (70–90% of confluency) when they were harvested and analyzed. HUVECs were plated on 1.5% gelatin-coated tissue culture dishes and maintained in phenol red-free basal medium M200 containing 2% FBS and EGM-2 at 37 °C with 5% CO2; for experiments, cells were used at passages 2 to 7, to avoid possible changes of cellular phenotype.RAW 264.7 cells (murine monocyte/macrophage cell line) were cultured in DMEM without phenol red containing 4 mM L-glutamine, 4500 mg/L glucose, 10% FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin at 37 °C with 5% COIn HUVECs, WST8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium, monosodium salt) was used to determine the cell viability. Indeed, in the presence of an electron mediator, WST8 is reduced to red formazan dye by dehydrogenases present in the cells. The amount of red dye generated is directly proportional to the number of living cells .4 cells/well HUVECs were seeded in a 96-well plate. After 80% confluence had been reached, cells were treated with different concentrations of berries solutions: fermented Pushgay (FP) and Pushgay (P) in complete culture medium for 24 h (range 0.25–100 µg/mL). The decrease in absorbance between the 24 h treatment (representing t1) and the control (representing t0) was monitored at 37 °C at 450 nm using an AMR-100 Microplate reader .For the experiments, 1.0 × 105 cells/well) overnight. Cells were incubated for 24 h with different concentrations of berries solutions (range 2.5–5 and 10 μg/mL in 0.1% DMSO). At the end of incubation, viability was determined with the Burker chamber utilizing the Trypan Blue exclusion test.To determine cell viability in RAW 264.7, cells in the logarithmic growth phase were harvested and seeded in six well-plates (3.0 × 104 cells/well) overnight. Subsequently, cells were grown at 37 °C, 5% CO2 for 24 h and treated with 10 μg/mL of each berry solution and further incubated for 24 and 72 h. At the end of incubation, cell number was determined with the Burker chamber after staining with Trypan Blue. The evaluation of the effects of berries on cell proliferation/metabolism in RAW 264.7 was investigated through the Trypan Blue and Alamar Blue assays. For Trypan Blue assay, RAW 264.7 cells, harvested in the logarithmic growth phase, were plated in triplicate in 24-well plates (3.0 × 104 cells/well) overnight. Next, cells were grown at 37 °C, 5% CO2 for 24 h and incubated with 10 μg/mL of each berry solution for 24 and 72 h before adding Alamar Blue solution (5%) in complete cell culture medium. Cells were incubated at 37 °C, 5% CO2 for 3 h. Next, 100 μL of the medium was transferred from each well to a 96-well plate. The reading was carried out with a spectrophotometer at 570 nm .For the Alamar Blue assay, RAW 264.7 cells in the logarithmic growth phase were harvested and plated in 24 wells in triplicate  release. The enzyme LDH catalyzes the conversion of lactate to pyruvate via NAD+ reduction to NADH; next the diaphorase reduces tetrazolium salt, oxidizing NADH, to a red formazan product that can be spectrophotometrically determined at 490 nm. LDH release from HUVECs treated for 24 h with berries solutions was monitored by collecting aliquots of the medium under the same experimental conditions performed above for cell viability assays, as previously reported . The dec5 cells/well) were seeded in six well-plates and incubated with 10 μg/mL of berries solutions, in the presence or absence of H2O2 (300 µM), for 24 h. RAW 264.7 cells (4.0 × 105 cells/well) were seeded in six well-plates and incubated with 10 μg/mL of berries solutions, in the presence or absence of IFN-γ 100 U/mL, for 24 h. Total RNA was extracted using the commercial kit RNeasy mini Kit . The concentration and purity of RNA were determined using the Nanodrop 1000 spectrophotometer (Thermo Fisher Scientific).HUVEC cells  was reverse transcribed in a volume of 25 μL using 250 units of SuperScript III reverse transcriptase  and 50 ng of random hexamers using the reaction conditions described in . Next, 2 formula , using RiNOS forward 5′-GGCAGACTGGATTTGGCTGG-3′, reverse 5′-CAACATCTCCTGGTGGAACAC-3′; TNFα forward 5′-ACTGAACTTCGGGGTGATCG-3′, reverse 5′-CCACTTGGTGGTTTGTGAGTG-3′.The intracellular antioxidant activity of the berries was estimated in HUVECs by using the chemiluminescent (CL) cell-based bioassay previously described .4 cells/well) were plated in a 96-well black microtiter with clear bottom and on the next day, cells were treated with serial dilutions of FP and P (range 0.25–25 µg/mL) for 24 h. Then, cell medium was removed and 100 μL of the H2O2-CL probe working solution  was added. After the incubation for 20 min at 37 °C, 100 µL of the oxidant agent menadione  was dispensed in each well to induce intracellular H2O2 production. CL emission signal was monitored for 40 min using a Luminoskan™ Ascent luminometric plate reader. The temperature was maintained at 37 °C during the measurement and the dose–response curve was obtained by plotting the CL signal versus the actual concentration of the menadione and fitting the experimental data to a straight line using the method of least squares.Briefly, two days before the experiment, HUVECs (1.0 × 106 cells/dish) were seeded in a 100 mm Petri dish and incubated with 10 μg/mL of berries solutions, in the presence or absence of H2O2 (300 µM), for 24 h. The lysates were obtained by scraping cells in a chilled buffer . The lysates were then incubated at +4 °C for 30 min and subsequently centrifuged at 10,000× g for 10 min. The protein concentration in the supernatant was determined according to Bradford method [HUVEC cells . A The total phenolic content was determined by the Folin–Ciocalteu reagent method obtaining a similar polyphenols concentration in fermented and non-fermented Pushgay berries (P: 2.38 ± 0.01 and FP: 2.95 ± 0.14 µg GAE/µg lyophilized berry) , in lineQuercetin glycoconjugates are the major flavonoid compounds in Pushgay berries ,25 and tIn this study, we performed a targeted/quantitative analysis of quercetin (both the aglycone and glycoconjugate forms) through HPLC-ESI MS/MS . In agreQuercetin aglycone significantly increases after fermentation, suggesting that LAB fermentation induces the hydrolysis of glucosides . QuercetHUVECs were treated for 24 h with FP or P solutions (between 0.25 and 25 µg/mL) to investigate their potential cytotoxicity. 2O2 production at 2.5, 10 and 25 μg/mL , while treatment with p does not have any significant effect. The IC50 of FP was evaluated by a dose–response curve, obtaining a value of 13.2 ± 0.6 μg/mL (A chemiluminescent (CL) cell-based bioassay was utilized to evaluate the intracellular antioxidant activity of FP or P solutions in HUVECs . Briefly.6 μg/mL C. 2O2 (300 µM) for 24 h, following treatment with FP and P solutions (10 μg/mL) for 24 h. As expected, treatment with H2O2 reduced cell viability of HUVECs and FP, but not P, significantly counteracts this effect  for 24 h before treatment with Hs effect .2O2. We found that FP strongly increases GR activity in HUVECs, both in the presence or absence of H2O2. No differences were observed for other enzymatic activity , glutathione peroxidase (Gpx), catalase (CAT), and superoxide dismutase (SOD) activities were assayed in HUVECs in the presence or absence of Hactivity . Treatments of RAW 264.7 with FP and P for 24 h at different concentrations (range 2.5–10 µg/mL) were not toxic . TherefoThe effects of berries on RAW 264.7 growth were evaluated by Alamar Blue staining after treatment of cells with FP and P solutions for 24 and 72 h. We found an increase in Alamar Blue staining after 72 h of treatment with P or FP, compared to control cells, but no effect was observed after 24 h of treatment A. Since To evaluate the immunomodulatory activity of berries solutions, the mRNA levels of inducible nitric oxide synthase (iNOS) and Tumor Necrosis Factor α (TNFα) were evaluated by qRT-PCR in RAW 264.7 treated with FP or P at 10 µg/mL for 24 h in the presence or absence of Interferon γ (IFNγ) (100 U/mL). We found that treatment with FP, but not P, leads to a significant raise in the levels of iNOS mRNA in the absence of IFN-γ A. TreatmLactiplantibacillus plantarum, may enhance the beneficial activity of phenolic compounds present in food, protecting endothelial cells from oxidative stress [The European vision of the Joint Programming Initiative “a healthy diet for a healthy life” (JPI HDHL) establishes that by 2030, all citizens will have the motivation, the ability, and the opportunity to consume a healthy diet from a variety of foods  to stay healthy and reduce the onset of food-related diseases. Epidemiological studies have shown that the intake of natural products, rich in antioxidants polyphenolic compounds, is related with a reduction in the risk of developing CVDs and cancer ,47. There stress  and modue stress . A variee stress . Variatie stress .Lactiplantibacillus plantarum. We report that treatment with Pushgay berries fermented with Lactiplantibacillus plantarum reduces oxidative stress induced by menadione and protects HUVECs against H2O2-induced cell toxicity more effectively than treatment with unfermented Pushgay berries. We also found that, after fermentation with Lactiplantibacillus plantarum, Pushgay berries promote proliferation and iNOS mRNA synthesis in mouse macrophages RAW 264.7.So far as we know, this is the first study that compares the antioxidant and immunostimulant properties of Pushgay berries before and after fermentation with 2O2-induced cell death [Lonicera caerulea berries have a higher antioxidant capacity than blueberries because they are enriched in anthocyanins, such as anidin, peonidin, and delphinidin, which are not present in other berries [L. caerulea may differ based on differences in the extraction approach [Our results are in contrast with others reporting that unfermented berries, due to their high levels of polyphenolic compounds, can reduce oxidative stress ,54,55, dll death , attenuall death . It is p berries ,60. Addiapproach . Polypheapproach . Cell cuapproach .2O2 production following menadione treatment [2O2, confirming that fermentation interferes with oxidative stress-induced cell damages. This is consistent with other studies that have shown that fermented blueberries have great scavenging ability against hydroxyl radicals [2O2-treated cells, pre-treated with FP, express a lower amount of HO-1 mRNA than cells treated with H2O2 in the absence of FP. Since HO-1 synthesis is a cell response against damages caused by oxidative stress [As described for other berries, FP gained the ability to interfere with Hreatment . Furtherreatment , FP berrradicals ,66,67. Fe stress , these dAlthough HO-1 induction promotes a protection against oxidative stress in various cell and animal models, high HO-1 levels may even sensitize the cell to oxidative stress, e.g., through the release of reactive iron . In addi2O2-induced damage by regulating the redox state of the cell, particularly increasing the activity of GR [2O2-induced oxidative damage by increasing GR activity [In order to gain, at least in part, insight into the mechanism underlying the protective activity of FP, we investigated the activity of well-known antioxidant enzymes that could be stimulated by berries treatment such as CAT, SOD, and enzymes involved in the glutathione (GSH) cycle, such as Gpx and GR . Our resty of GR , and pepactivity . Fruit-dactivity  and to tOur study also showed that treatment with FP modulates innate immunity, as indicated by increased RAW 264.7 macrophages proliferation. Furthermore, treatment of these cells with FP increased basal and IFN-γ-induced levels of iNOS.We found that Pushgay berries are enriched in quercetin and fermentation enhances their quercetin aglycone content, in line with previous data , and incEven if Hollman et al. suggested that quercetin glucoside has a higher bioavailability than the free form in ileostomized patients , it is pFinally, the contribution of phenolic acids in FP, which may exert an additive or synergistic effect with the quercetin aglycone within cells, must be carefully considered due to their greater bioavailability in comparison to larger phenolic molecules and their beneficial activities. More studies will be performed to clarify their possible beneficial effects in human cell models on vascular and immune system function.We report that fermentation strongly increases the content of quercetin aglycone  and promotes the antioxidant activity of Pushgay berries. One of the possible mechanisms underlying the antioxidant activity of FP could be the increased activity of GR, an enzyme responsible for the maintenance of GSH supply, which acts as an important player in controlling the cellular levels of ROS . Ferment


Morphine, one of the most efficacious analgesics, is effective in severe pain, especially in patients with concomitant painful cancers. The clinical use of morphine may be accompanied by increased immunosuppression, susceptibility to infection and postoperative tumor metastatic recurrence, and the specific mechanisms and clinical strategies to alleviate this suppression remain to be investigated. Expression of CD11b is closely associated with the macrophage phagocytosis of xenobiotic particles, bacteria or tumor cells. Here, we find that morphine at 0.1–10 nM levels inhibited CD11b expression and function on macrophages via a μ-opioid receptor (MOR)-dependent mechanism, thereby reducing macrophage phagocytosis of tumor cells, a process that can be reversed by thymopentin (TP5), a commonly used immune-enhancing adjuvant in clinical practice. By knocking down or overexpressing MOR on macrophages and using naloxone, an antagonist of the MOR receptor, and LA1, a molecule that promotes macrophage CD11b activation, we suggest that morphine may regulate macrophage phagocytosis by inhibiting the surface expression and function of macrophage CD11b through the membrane expression and activation of MOR. The CD47/SIRPα axis, which is engaged in macrophage-tumor immune escape, was not significantly affected by morphine. Notably, TP5, when combined with morphine, reversed the inhibition of macrophage phagocytosis by morphine through mechanisms that promote membrane expression of CD11b and modulate its downstream signaling . Thus, altered membrane expression and function of CD11b may mediate the inhibition of macrophage phagocytosis by therapeutic doses of morphine, and the reversal of this process by TP5 may provide an effective palliative option for clinical immunosuppression by morphine. Opioids such as morphine are widely used worldwide for clinical postoperative analgesia or other causes of severe pain. Clinical studies have found that the use of opioids  causes, in addition to some common side effects such as tolerance, addiction, respiratory depression, nausea, and constipation, an immunosuppressive effect that increases the risk of infection ,2. It isCD11b is a type 1 transmembrane glycoprotein of approximately 170 kDa in size that is highly expressed on myeloid cells  and regulates a broad range of immune responses, such as pathogen recognition, phagocytosis, and cell survival . CD11b fHere, we propose a possible mechanism of morphine-induced immunosuppression, namely that morphine reduces the expression and function of CD11b on macrophages via MOR, which inhibits the phagocytosis of macrophages and thus affects tumorigenesis, progression, and prognosis ,26. ThisTM Red 600, and co-cultured with macrophages for 24 h. The results were obtained by fluorescence intensity recorded by fluorescence microscopy photographs, and morphine at 10 nM significantly attenuated phagocytosis of A549 by macrophages compared to the controls  = 2.13 ± 0.16 nM, Opioid receptor agonists or antagonists affect tumor growth by modulating macrophage function ,32. To fp > 0.05, unpaired t-test, n = 3, p > 0.05, n = 3, unpaired t-test, p > 0.05, n = 3, one-way ANOVA followed by Dunnett’s multiple comparisons test, It has been shown that CD47, which is highly expressed on cancer cells, and regulatory protein α (SIRPα), a macrophage expressing receptor, form checkpoints for innate immunity, inhibiting macrophage-mediated phagocytosis and leading to immune escape of tumor cells . We conjp < 0.001, n = 4, one-way ANOVA followed by Dunnett’s multiple comparisons test, CD11b mediates multiple monocyte/macrophage responses in the immune inflammatory response, and it is tightly associated with phagocytosis of macrophages . Therefop < 0.01, n = 3, one-way ANOVA followed by Dunnett’s multiple comparisons test; 50 = 2.13 ± 0.16 nM) of phagocytosis was comparable to the inhibitory potency of morphine on CD11b expression , 41.3 ± 1.9% (8 μM LA1) vs. 38.8 ± 2.4% (Ctrl), respectively, TM Red 600 and co-cultured them with macrophages for 24 h. Macrophages pretreated with LA1 significantly restored phagocytosis of A549 cells compared with the morphine administration group alone (31.2 ± 4.1% (4 μM LA1 + 10 nM morphine) and 32.7 ± 4.7% (8 μM LA1 + 10 nM morphine) vs. 14.4 ± 3.7% (10 nM morphine), respectively, p < 0.01, n = 3, one-way ANOVA followed by Dunnett’s multiple comparisons test, p < 0.01, n = 3, one-way ANOVA followed by Dunnett’s multiple comparisons test, To further determine whether LA1 affects the phagocytosis of tumor cells by macrophages, we labeled cancer cells A549 with the fluorescent dye probe ProtonexThese results confirm that LA1 promotes macrophage CD11b activation and reverses morphine-mediated inhibition of macrophage phagocytosis.p < 0.001, n = 4, one-way ANOVA followed by Dunnett’s multiple comparisons test, Morphine at sub-nM to nM levels alone modulates macrophage CD11b function and reduces phagocytosis, while MOR is also expressed in macrophages, suggesting that morphine-mediated immunomodulation may be mediated through direct interaction with MOR on immune cells . First, 50 = 5.53 ± 3.70 nM, p < 0.001, n = 3, unpaired t-test, p < 0.001, n = 3, unpaired t-test, Then, morphine at concentrations of 0.01 nM~10 μM was applied to PMA-induced macrophages labeled with Anti-MOR antibody and FITC-anti-mouse IgG1 antibody, and flow cytometric results showed that morphine also significantly reduced the expression level of MOR on macrophage membranes , for validation . After pp < 0.001, NLX + morphine vs. morphine alone, n = 3, one-way ANOVA followed by Dunnett’s multiple comparisons. test, p < 0.01, n = 4, one-way ANOVA followed by Dunnett’s multiple comparisons test, Similarly, we tested whether NLX could also restore the morphine-induced decrease in phagocytic activity of macrophages at nM levels. The results of co-culture with macrophages and cancer cells A549 showed a significant increase in fluorescence intensity and phagocytosis ratio of macrophages towards cancer cell phagocytosis in response to NLX at 10 μM (38.9 ± 2.9%), and 20 μM (41.7 ± 1.1%) compared to morphine administration alone (16.6% ± 4.3%) and control (42.1 ± 2.3%)  is due to the sustained activation of MOR .It is also not well understood how morphine-mediated immunosuppression can be restored by combining it with other clinically used drugs. TP5 has very strong immunomodulatory activity and can significantly improve the cellular and humoral immune functions of the body, thus making it a potential drug for the treatment of primary or secondary immunodeficiency, tumors, and severe infections ,30. At tp < 0.05, n = 3, one-way ANOVA followed by Dunnett’s multiple comparisons test, p < 0.05, n = 4, one-way ANOVA followed by Dunnett’s multiple comparisons test, Indeed, TP5 administration alone significantly increased the mRNA expression of OPRM at concentrations of 50, 100 and 200 μM , 100 (49.2 ± 3.0%) and 200 μM (53.0 ± 5.3%) compared to control (41.6 ± 2.5%) , the coadministration of 50 μM, 100 μM or 200 μM TP5 and 10 nM morphine for 48 h significantly increased the expression of CD11b on macrophage membranes at 100 (32.4 ± 3.3%), and 200 μM (36.0 ± 3.9%) , 38.3 ± 8.0% (200 μM TP5 + 10 nM morphine), vs. 9.6 ± 1.4% , respectively, p < 0.01, vs. 200 μM TP5 + 10 nM morphine (34.0 ± 1.1%), n = 3, one-way ANOVA followed by Dunnett’s multiple comparisons test, Our previous study showed that TP5 can inhibit the stemness of colorectal cancer stem cells via nicotinic acetylcholine receptors (nAchRs) , and theTogether, these results suggest that TP5 reversed the inhibitory effect of morphine on macrophage phagocytosis, mainly by promoting membrane expression of CD11b and regulation of downstream signaling, a process in which nAchRs are also partially involved.Here, we find that morphine inhibited the phagocytic effect of macrophages on tumor cells in a system of macrophage and tumor cell co-culture, and that this inhibition was somewhat morphine concentration dependent (at nanomolar level). This inhibition of phagocytosis was not achieved by inhibiting the proliferative activity of macrophages as well as cancer cells A549, or the innate immunity CD47/SIRPα axis. Our results suggest that the inhibition of phagocytosis by morphine is due to its suppression of macrophage CD11b expression and function, and that the reduced phagocytic effect is consistent with a potency to suppress CD11b expression. In addition, LA1 can promote CD11b activation downstream to stimulate the expression of immune activation-related genes and inhibit immune suppression-related genes, thus reversing the immunosuppressive effects of morphine. Further studies with knockdown, antagonist inhibitors, and overexpression of MOR on macrophages suggest that morphine may inhibit phagocytosis of macrophages by reducing macrophage CD11b expression through MOR’s surface expression and activation. We found that in combination with morphine, TPF, which is an immune-enhancing adjuvant, reversed the inhibitory effect of morphine on macrophage phagocytosis and restored the macrophage phagocytic effect, mainly by promoting CD11b expression and activation of downstream signaling. Thus, CD11b and MOR jointly contribute to the immunosuppressive effects of morphine, with an association between them, and TP5 can regulate both, thereby reversing the immunosuppression of morphine.We suggest a possible mechanism for morphine-induced immunosuppression and decreased function, namely that morphine inhibits CD11b expression and function on macrophages via MOR activation, thereby suppressing macrophage phagocytosis. However, we do not know whether morphine only affects changes in CD11b expression during monocyte-to-macrophage conversion or also directly affects CD11b as a membrane receptor-mediated downstream immune-related signaling, which our current findings cannot distinguish. Additionally, whether there is a strong association between decreased membrane expression of MOR and decreased membrane expression of CD11b, and whether there is a direct interaction between the two, remains to be further investigated. The decrease in macrophage phagocytosis induced by nM levels of morphine may be due to two mechanisms: decreased expression of MOR and CD11b in the membrane, and activation of MOR receptors; it remains to be confirmed whether these two mechanisms interact or are causal. It is also not very clear whether the decreased membrane expression of MOR is caused by the sustained activation of MOR, which leads to a decreased surface expression of CD11b, or whether other factors mediate this process. Considering that the effect of LA1 activation does not fully restore morphine-suppressed CD11b surface expression, we speculate that there may be other mechanisms mediating this effect, which also need further investigation. This mode of action might not depend primarily on G-protein downstream signaling from the MOR receptors and could serve to modulate the immunosuppressive effects of morphine without affecting its analgesic effects.The relationship between prolonged opioid intake and tumor development in non-cancer chronic pain medication is not well understood, and there is growing evidence that patients in these settings are at risk for cancer . The comAnother point is that immune cells have been shown to secrete acetylcholine, dopamine, and gamma-aminobutyric acid (GABA), which mediate immune signaling and play important immunomodulatory roles ,46,47,48Another interesting question is whether the current results have the potential to be applied clinically to retain the analgesic effect of morphine while reducing its effect on immunosuppression. We have confirmed that there are no reports of TP5 affecting the clinical application of morphine analgesia. Only one study in an animal model found that TP5 treatment resulted in a slight decrease in sensitivity to painful stimuli in rats. TP5 enabled tumor-bearing animals to recover diminished behavioral activity and increased resistance to stressful stimuli and pain , suggestFinally, for the clinical use of TP5, clinical administration is mainly intramuscular or subcutaneous and can be 50 mg per day . PatientThe plasmid of pcDNA3.1-hMOR-WT was kindly gifted from Dr. Rui Wang. The siRNAs used in this study were purchased from Shanghai GenePharma .2 at 37 °C.TP5 was purchased from ChemBest Research Laboratories Ltd. . Leukadherin-1 (LA1) and D-Tubocurarine chloride pentahydrate (TUB) were purchased from MedChemExpress . Other compounds were purchased from Sigma. THP-1 cells were cultured in RPMI-1640 medium  supplemented with 20% fetal bovine serum , penicillin (100 IU/mL) and streptomycin . A549 cells were cultured in RPMI-1640 medium  supplemented with 10% fetal bovine serum , penicillin (100 IU/mL) and streptomycin . Cells were incubated in a humidified incubator containing 5% COTHP-1-differentiated macrophages were transfected  using 100 nM of siRNA against MOR (MOR siRNA) or non-silencing siRNA (Ctrl siRNA) . The medTHP1-differentiated macrophages were placed in 12-well plates and transfected with 1 μg pcDNA3.1-hMOR-WT or pcDNA3.1  . The medTHP-1 cells (5 × 105 cells/well) were seeded in 12-well plates and cultured with 200 ng/mL of phorbol-12-myristate-13-acetate  for 48 h to differentiate into macrophages . THP-1-dIn vitro phagocytosis was performed using THP-1 cells, and these cells differentiated into macrophages by incubation with PMA for 48 h. THP-1 cells (2 × 105 cells/well) were seeded in 24-well plates and treated with morphine for 48 h. A549 cells were labeled with fluorescent dye probe ProtonexTM Red 600  in the dark for 30 min. The fluorescence of Protonex™ Red dye increases sharply as the pH decreases from neutral to acidic according to the manufacturer’s protocol . Then, 2Total RNA was isolated and reverse transcribed using the EZ-press RNA purification kit  and PrimeScript RT kit , respectively, according to the manufacturer’s instructions, and qPCR was performed using TB Green Premix Ex Taq  . RelativCell viability was evaluated using the MTT assay kit  according to the manufacturer’s protocol . A549 cet-test, and differences between multiple groups were assessed by one-way ANOVA and Dunnett’s post hoc test. p less than 0.05 was considered statistically significant. Concentration-response relationships of CD11b and MOR were obtained by measuring fluorescence intensity in response to different concentrations of morphine, and all results that were used to generate a concentration-response relationship were from the same group. The data were fit to the Hill1 equation: I/I max = 1/[1 + (EC50/morphine) n], where I is the normalized fluorescence intensity at a given concentration of ligands, I max is the maximum normalized fluorescence intensity, EC50 is the morphine concentration producing half of the maximum fluorescence intensity, and n is the Hill1 coefficient.Data are expressed as mean ± SEM. All experiments were performed independently, at least 3 times. Statistical analyses were performed as described in each corresponding legend. Differences between two groups were assessed by unpaired two-sided Student’s


In addition to phytocannabinoids, cannabis contains terpenoids that are claimed to have a myriad of effects on the body. We tested a panel of five common cannabis terpenoids, myrcene, linalool, limonene, α-pinene and nerolidol, in two neuronal models, autaptic hippocampal neurons and dorsal root ganglion (DRG) neurons. Autaptic neurons express a form of cannabinoid CB1 receptor-dependent retrograde plasticity while DRGs express a variety of transient receptor potential (TRP) channels. Most terpenoids had little or no effect on neuronal cannabinoid signaling. The exception was nerolidol, which inhibited endocannabinoid signaling. Notably, this is not via inhibition of CB1 receptors but by inhibiting some aspect of 2-arachidonoylglycerol (2-AG) production/delivery; the mechanism does not involve reducing the activity of the 2-AG-synthesizing diacylglycerol lipases (DAGLs). Nerolidol was also the only terpenoid that activated a sustained calcium response in a small (7%) subpopulation of DRGs. In summary, we found that only one of five terpenoids tested had notable effects on cannabinoid signaling in two neuronal models. Our results suggest that a few terpenoids may indeed interact with some components of the cannabinoid signaling system and may therefore offer interesting insights upon further study. Δ9-THC primarily exerts its psychoactive effects via cannabinoid CB1 receptors hept-2-ene; nerolidol, cis/trans mixture of 3-Hydroxy-3,7,11-trimethyl-1,6,10-dodecatriene) were obtained from Sigma-Aldrich . Drugs were racemic mixtures of enantiomers if stereochemistry is not indicated. 2-AG was purchased from Cayman Chemical .Hippocampal culture preparation. All procedures used in this study were approved by the Animal Care Committee of Indiana University and conform to the Guidelines of the National Institutes of Health on the Care and Use of Animals. Mouse hippocampal neurons isolated from the CA1-CA3 region were cultured on microislands as described previously [eviously ,53. Neureviously . CultureElectrophysiology. When a single neuron is grown on a small island of permissive substrate, it forms synapses—or “autapses”—onto itself. All experiments were performed on isolated autaptic neurons. Whole cell voltage-clamp recordings from autaptic neurons were carried out at room temperature using an Axopatch 200A amplifier . The extracellular solution contained (in mM) 119 NaCl, 5 KCl, 2.5 CaCl2, 1.5 MgCl2, 30 glucose, and 20 HEPES. Continuous flow of solution through the bath chamber (~2 mL/min) ensured rapid drug application and clearance. Drugs were typically prepared as stocks, and then diluted into extracellular solution at their final concentration and used on the same day.2, 10 HEPES, 0.2 EGTA, 2 MgATP, and 0.5 LiGTP. Access resistance and holding current were monitored and only cells with both stable access resistance and holding current were included for data analysis. Conventional stimulus protocol: the membrane potential was held at –70 mV and excitatory postsynaptic currents (EPSCs) were evoked every 20 s by triggering an unclamped action current with a 1.0 ms depolarizing step. The resultant evoked waveform consisted of a brief stimulus artifact and a large downward spike representing inward sodium currents, followed by the slower EPSC. The size of the recorded EPSCs was calculated by integrating the evoked current to yield a charge value (in pC). Calculating the charge value in this manner yields an indirect measure of the amount of neurotransmitter released while minimizing the effects of cable distortion on currents generated far from the site of the recording electrode (the soma). Data were acquired at a sampling rate of 5 kHz.Recording pipettes of 1.8-3 MΩ were filled with (in mM) 121.5 KGluconate, 17.5 KCl, 9 NaCl, 1 MgClx-axis of DSE depolarization-response curves are log-scale seconds of the duration of the depolarization used to elicit DSE. Depolarization response curves are obtained to determine pharmacological properties of endogenous 2-AG signaling by depolarizing neurons for progressively longer durations . The data were fitted with a nonlinear regression , allowing calculation of an ED50, the effective dose or duration of depolarization at which a 50% inhibition is achieved. A statistically significant difference between these curves is defined as non-overlapping 95% confidence intervals of the ED50s. Values on graphs are presented as mean ± S.E.M.DSE stimuli: After establishing a 10–20 s 0.5 Hz baseline, DSE was evoked by depolarizing to 0 mV for 50 msec, 100 msec, 300 msec, 500 msec, 1 sec, 3 sec and 10 sec, followed in each case by resumption of a 0.5 Hz stimulus protocol for 20–80+ s, allowing EPSCs to recover to baseline values. This approach allowed us to determine the sensitivity of the synapses to DSE induction. To allow comparison, baseline values (prior to the DSE stimulus) are normalized to one. DSE inhibition values are presented as fractions of 1, i.e., a 50% inhibition from the baseline response is 0.50 ± standard error of the mean. The Enzchek lipase activity assay. Multiple 10 cm dishes of Chinese hamster ovary (CHO) cells were grown in parallel in Ham’s F12 medium. When cells reached ~70% confluence, one dish was transfected with DAGLα and mCherry plasmids using lipofectamine 2000  following the manufacturer’s instructions. The lipase activity of protein extracts of these cells was then compared with untransfected CHO cells on the next day using an Enzchek lipase substrate assay (Thermo Fisher). Protein was extracted from cells by treating them with a freshly prepared lysis buffer consisting of 50 mM HEPES, 100 mM NaCl, 5 mM CaCl2, 0.5% TritonX-100, pH 7.2. A Pierce protease inhibitor mini tablet (Thermo Fisher) was added to 25 mL of Lysis buffer. Cells were treated with 1mL ice-cold lysis buffer for 5 min and centrifuged for 5 min at 4 °C after which the supernatant was taken and maintained on ice for experiments the same day.t-test.Samples were tested on a 96 well plate using a Flexstation III . Wells received protein extract (50 µL); lysis buffer (50 µL); 3 µM nerolidol or vehicle (5 µL). Enzchek (20 µM in 5 µL) was added just before readings were taken . Samples were run in pairs or triplicates  in a given experiment. Experiments were repeated from separate cultures . Slopes from the linear phase of the time courses (rise time per minute divided by micrograms of protein per sample) were calculated to estimate lipase activity. Values for a given experiment were normalized to their vehicle control for that day and compared using an unpaired Student’s Gel-based competitive ABPP experiments. Mice were sacrificed, and brains removed, rinsed with lysis buffer , and placed in Dounce homogenizer with 2mL of cold lysis buffer and homogenized. Homogenates were maintained on ice for 15 min, then transferred to Eppendorf tubes and spun down . The supernatant was collected, and protein concentration determined using Bradford’s assay. Samples were used the same day. For gel-based competitive ABPP experiments, proteomes (1 mg/mL) were treated with drug or vehicle for 30 min, then treated with HT-01  in a 50 μL total reaction volume. After 30 min at 37 °C, the reactions were quenched with SDS-PAGE loading buffer . After separation by SDS-PAGE (10% acrylamide), samples were visualized by in-gel fluorescence scanning using a Typhoon 9500 fluorescent scanner. Band intensity was quantified using Fiji. To confirm the identity of the HT-01 labeled DAGLβ band, we tested whether signal of the predicted 74 kDa target band was blocked by the DAGLβ blocker KT109 [t-test.er KT109  (100 nM)Dorsal root ganglion cell culture. DRGs were harvested from P-0 through P-14 day-old rat pups following IACUC approval and following guidelines for the ethical care and use of laboratory animals. Rats were euthanized using isoflurane inhalation and cervical dislocation. DRGs were harvested using the protocol described by Sleigh et al. [2, 1X Glutamax , Penicillin/Streptomycin , and 10 mM HEPES  and the spinal cord was carefully removed and discarded. DRGs were pulled from the vertebrate and placed into a 15 mL conical centrifuge tube containing ice-cold Dissection Solution. DRGs were centrifuged at 100× g at 4 °C and the media was replaced with Dissection Solution containing 10 mg/mL collagenase type II . DRGs were incubated at 37 °C for 20 min. Trypsin/EDTA  was then added to a final concentration of 0.05% and the tissue was incubated a further 3 min. Tissue was centrifuged at 4 °C as above and washed three times in ice-cold DMEM  containing 10% fetal calf serum. DRGs were then triturated 30 times in 4 mL of this medium, centrifuged, and resuspended in 3 mL of cold culture medium , 2.5 mg/mL insulin, 5 mg/mL transferrin, 5 mg/mL nerve growth factor-β , 1X B27 , 1X Glutamax, 10% Fetal calf serum). Cells were then counted and plated at a concentration of ~5000 cells/cm2 on coverslips coated with poly-d lysine  and laminin . Cells were cultured at 37 °C and 5% CO2, with one half of the media changed every 3–4 days.h et al. . BrieflyCalcium imaging. DRGs were cultured for 2–10 days before the experiment. DRGs were treated with Fluo4-AM (5 µM) for 30 min at 37 °C, after which the cells were washed in extracellular solution (see electrophysiology) for 20 min to allow for de-esterification of Fluo4-AM. Fluorescence was then monitored on a Nikon TE200 inverted microscope with a 10× objective, a Hamamatsu Flash 4.0 camera and Nikon Elements AR software which controlled a SpectraX light engine for stimulation of fluorescence. Target DRGs were chosen before calcium imaging based on neuronal morphology using a brightfield image. This brightfield mask was then applied to the image series. Images were acquired every 30 s for 15 min and then analyzed using FIJI software with the 1-click ROI manager plugin [r plugin , to meas


Monoraphidium sp. CABeR41. The total tocopherol productivity of NRHC (Nitrate replete + 3% CO2) supplemented was (306.14 µg·L−1 d−1) which was nearly 2.5-fold higher compared to NRVLC (Nitrate replete + 0.03% CO2) (60.35 µg·L−1 d−1). The best tocopherol productivities were obtained in the NLHC (Nitrate limited + 3% CO2) supplemented cells (734.38 µg·L−1 d−1) accompanied by a significant increase in cell biomass (2.65-fold) and total lipids (6.25-fold). Further, global metabolomics using gas chromatography-mass spectrometry (GC-MS) was done in the defined conditions to elucidate the molecular mechanism during tocopherol accumulation. In the present study, the Monoraphidium sp. responded to nitrogen limitation by increase in nitrogen assimilation, with significant upregulation in gamma-Aminobutyric acid (GABA). Moreover, the tricarboxylic acid (TCA) cycle upregulation depicted increased availability of carbon skeletons and reducing power, which is leading to increased biomass yields along with the other biocommodities. In conclusion, our study depicts valorization of carbon dioxide as a cost-effective alternative for the enhancement of biomass along with tocopherols and other concomitant products like lipids and carotenoids in the indigenous strain Monoraphidium sp., as an industrial potential strain with relevance in nutraceuticals and pharmaceuticals.Tocopherols are the highly active form of the antioxidant molecules involved in scavenging of free radicals and protect the cell membranes from reactive oxygen species (ROS). In the present study, we focused on employing carbon supplementation with varying nitrate concentrations to enhance the total tocopherol yields in the native isolate  Ity acids . Moreoveoduction ,29. Furtoduction .v/v; 300 ppm) and high  CO2 supplementations in the newly isolated indigenous strain Monoraphidium sp. CABeR41. Based on the preliminary data analysis, the overall yield of tocopherols and lipids were induced during nutrient limited (NL) conditions in a well-correlated manner. Furthermore, CO2 supplementation drastically enhanced biomass along with other biocommodities such as lipids, carotenoids, and tocopherols. To illustrate the changes in the metabolic profiles of this microalga, we employed qualitative (untargeted) metabolomics to unveil the carbon flux within the essential metabolic pathways. Such findings may provide us new insights on the growth parameters required for improving biomass yields along with the industrially relevant HVABs, a roadmap towards cost-effective and sustainable microalgal biorefineries.In the present study, our focus was to improve tocopherol yields without compromising cell growth, i.e., biomass in response to varying nitrate concentrations subjected to very low : H3BO3—2.85, 0.045; MnCl2·0.4H2O—1.81, 0.014; ZnSO4·7H2O—0.22, 0.001; Na2MoO4·0.2H2O—0.39, 0.001; CuSO4·5H2O—0.08, 0.0008; Co(NO3)2·6H2O—0.05, 0.005. A total of ~106 cells mL−1 was inoculated at a logarithmic phase with an initial optical density at 750 nm wavelength (OD750) of 0.1. Growth was monitored by cell count using hemocytometer  and high carbon  with varying concentrations of nitrogen, i.e., nitrogen replete (NR), containing 1.5 g·L−1 of NaNO3, nitrogen limited (NL) with 0.5 g·L−1 of NaNO3, and nitrogen deplete (ND) with 0.0 g·L−1 of NaNO3, respectively. The cells were inoculated in BG-11 medium (as per conditions described earlier) within 500 mL Erlenmeyer flasks and cultivated for period of 10 days at 25 °C under 16:8 h light/dark photoperiods and light intensity of 150 µE m−2 s −1 with constant shaking at 150 rpm and the sampling was carried at intervals of 0, 2, 4, 6, 8, and 10 days.Further, these cells in BG-11 medium were subjected to supplementation of very low carbon  and HC  in presence of varying nitrate concentrations  is shown in −1) in inset. Our data analysis demonstrates that the biomass productivity of NRHC supplemented cells was significantly higher  (336.35 mg·L−1 d−1), i.e., increased by 4.9-fold with doubling time of 1.60 days than the control (NRVLC—56.93 mg·L−1 d−1 and doubling time of 2.82 days). Similar productivities were observed even in NLHc supplemented cells   as on the 10th day of cultivation . In such context, we hypothesize that the presence of additional carbon is diverting the flux towards the cell biomass, i.e., growth along with other biocommodities.The biomass yields  in NRHC remained higher at 0.8 on the 10th day of cultivation, which represents intact photoreaction centers and quinone pools that seems to be having better photochemical efficiency in comparison with all other conditions  0.7 until the eighth day but a slight decrease was observed at 0.65 on the 10th day, and this may be due to the non-availability of nitrate in medium  seems to be highest in NRHC supplemented cells whereas productivities of total carbohydrates (87.12 ± 7.0 mg·L−1 d−1) and total lipids (76.12 ± 13.4 mg·L−1 d−1) are highest in NLHC supplemented cells  has been used to detect intracellular lipids in  CABeR41 A. Cells he cells B.2−) formation. In this reaction, the DHE (low fluorescent) will be first oxidised to DHEox (high fluorescent) in the presence of O2− radical and the fluorescence was measured employing confocal laser scanning microscopy. During the non-cyclic electron transport chain (ETC), superoxide radical (O2−) is formed primarily in the thylakoid-localized PSII, as well as other cellular compartments. Then the superoxide radical (O2−) initiates lipid peroxidation chain reactions, which eventually destroy the chloroplast membrane integrity and cause cell death during the stress phenomenon. In the HC supplemeted cells demostrated higher DHEox fluorescence followed by NDVLC supplemented cells, which is due to drastic decrease in the quantum efficiency of photosystem II (Fv/Fm) along with the total chlorophyll content (The dihydroxy ethidium (DHE) fluorescent probes were used to visualise free radical  and δ-tocopherol (489.65 ± 4.53 µg·L−1 d−1) productivities, respectively, with the total tocopherol productivity at 734.38 ± 11.79 µg·L−1 d−1 (−1 d−1) was observed in NDHC supplemented cells but with impairment of the growth, the overall productivity was decreased.Tocopherols are the highly active form of antioxidant molecules involved in interacting with polyunsaturated acyl groups, scavenging of lipid peroxyl radicals, and quenching reactive oxygen species (ROS), thus protecting fatty acids from lipid peroxidation during the stress phenomenon. In the present study, we have quantified two major isoforms of tocopherols in the native isolate ·L−1 d−1 . A drastMonoraphidium sp. Our data analysis  in comparison with the NRVLC supplemented cells (1.54 ± 0.08 mg·g−1 DCW). However, the primary carotenoids such as α-carotene and β-carotene yields improved in NRHC condition along with a significant increase in zeaxanthin content (1.43 ± 0.07 mg·g−1 DCW)   and the g−1 DCW) .Monoraphidium sp. are illustrated as HC condition followed by NLHC and other carbon supplemented cells  after filtering the raw data which includes major metabolites namely amino acids, sugars, organic acids, fatty acids, alcohols, antioxidants, and sterols.HC in comparsion with other conditions  metabolites like malic and fumaric acid are increased, while succinic acid and 2-ketoglutaric acid were decreased. Further, fatty acids like palmitoleic acid, α-linolenic acid, linoleic acid, and steric acid were increased significantly in NLHC supplemented cells. In addition, the other essential metabolites namely phytol and tocopherols were also found to be upregulated (Monoraphidium sp. was illustrated in (A) NDHC and (B) NLHC.Heat map, VIP score, and dot-plot graphs illustrate the overall changes in the metabolome profiles in the NLnditions . A decreegulated . Also, i2, but content of lipids and HVABs appears to be lower than expected  and high carbon  subjected to varying nitrate concentrations as nitrogen replete , nitrogen limited  and nitrate deplete . The biomass productivity of 3%CO2 (NRHC) supplemented cells was significantly higher (336.35 mg·L−1 d−1) with reduced doubling time (1.60 days) compared to the control (NRVLC) on the 10th day of cultivation (HC supplemented cells demonstrated higher biomass productivity (316.63 mg·L−1 d−1) with reduced doubling time (1.54 days). Despite having 0.5 g·L−1 of the nitrate than the NRHC, the NLHC displayed no apparent phenotypic changes under 3% CO2 (v/v), showing that high CO2 supplementation acts as a major contributor in the growth enhancement. Studies in Chlorella saccharophila and Microchloropsis gaditana also demonstrated that the growth was significantly elevated in 3% CO2 supplementation with reduced doubling times [HC supplemented cells with lower biomass productivities of 48.19 mg·L−1 d−1. Further, significant increase in lipid content was observed in NDHC & NDVLC supplemented cells  and ETRII in NDHC condition demonstrating cellular stress and damaged reaction centres, thus depicting drastic decline in photosynthetic activity. Zao et al. [Porphyridium cruentum subjected to nitrogen deplete condition where the photochemical efficiency of PSII decreased along with photoinhibition, indicating the phenomenon of nutrient stress. Further, NLHC supplemented cells depicted slight decrease in (vF/mF) and ETRII due to non-availability of nitrate in medium on the 10th day of cultivation  formation, which is a major damaging ROS in green photosynthetic organisms [HC supplemented cells demonstrate higher DHEox fluorescence followed by NDVLC and NLHC conditions, suggesting higher accumulation of ROS radicals  are produced in the thylakoid membrane when the absorption of light by chlorophylls exceeds the capacity for energy consumption by photosynthetic apparatus. They play a key role in cell defense, cell signaling, and apoptosis [HC give rise to the highest level of total tocopherol content (2743.03 ± 7.21 µg.g−1 DCW) followed by NLHC (1879.38 ± 137.51 µg.g−1 DCW) and NDVLC (1393.16 ± 32.2 µg.g−1 DCW) conditions, respectively.Furthermore, the DHE fluorescent probes were used to visualize superoxide  without compromising growth. Studies has demonstrated that the tocopherols help in protecting the oxidative modification of D1 protein in Arabidopsis sp. Further, the tocopherol-deficient mutant (vte1) D1 protein was modified to either tyrosine hydroperoxide or dihydroxyphenylalanine, suggesting the role of tocopherols in protecting PSII from oxidative damage [npq1 mutant of A. thaliana, accumulates more α-tocopherol in early leaves when exposed to high light [Arabidopsis vte1 mutant, which is tocopherol-deficient, suggests their functional interaction against lipid peroxidation and photooxidative stress [VLC, NDHC, and NLHC conditions wherein decrease of zeaxanthin content was observed with improved total tocopherol yields. Moreover, total antioxidant activity estimated by TAC, FRAP, and DPPH assays showed higher antioxidant efficiency in NRHC followed by NLHC and NRVLC conditions, respectively  metabolomics was done to identify molecular changes in the Monoraphidium sp. CABeR41 in excess carbon and nitrogen limitation conditions, utilizing a feedbackward omics approach to elucidate metabolic pathways involved in tocopherol biosynthesis. A total number of nearly 50 metabolites were analyzed and normalized with ribitol as an internal standard [In addition, both total tocopherols and lipids are induced to accumulate under stress conditions like ND2 > 0.90  was obsestandard ,70.HC condition, the metabolites such as glycine was downregulated than the NRVLC cells, which depicts lower photorespiration activity in presence of 3% CO2 (v/v) supplementation. Glycine is components of the photorespiratory glycolate cycle in algae, and its overexpression is used to assess photorespiratory activity [2 and CO2 depicts the rate of carbon absorption, photosynthetic efficiency along with reduced quotient and net photosynthesis [HC condition. Trehalose, a non-reducing disaccharide, recognized to be a stabilizing agent for membranes stability from damage aids in the preservation of cellular integrity [Isochrysis galbana during nutrient limited condition [P. tricornutum and Isochrysis zhangjiangensis during early stages of nitrogen deficiency, ensuring nitrate assimilation, and providing required nitrogen for protein synthesis [In NLactivity . Competiynthesis . In addintegrity . Moreoventegrity . Furtherondition . Glutamiondition ,76,77. Iynthesis ,79.HC supplemented cells. GABA, a non-protein amino acid, quickly accumulates in photosynthetic organisms in response to biotic and abiotic stress and governs endogenous signaling and cell development [HC conditions, protein and amino acid accumulation was triggered in order to maintain crucial metabolic activities for fast development. Interestingly, another metabolites myo-inositol was upregulated (1.41-fold) in NLHC condition, an important component of structural lipids, serves as a phosphate reserve and involved in cell development [myo-inositol (500 mg L−1) to Dunaliella salina increased biomass up to 1.34-fold with a rise in lipid productivity and similar comparable results were also reported in Monoraphidium sp. QLY-1 [Also, significant increase in gamma-aminobutyric acid (GABA) (2.75-fold), a glutamate derivative was observed in the NLelopment . Increaselopment . An exogp. QLY-1 ,83.Schizochytrium sp. B4D1 [Monoraphidium sp. CABeR41, revealed that lipid synthesis rises in NLHC, which strongly co-related with our metabolomic profiling. Another TCA cycle intermediate, fumaric acid was found to be upregulated (6.15-fold), involved in decarboxylation of metabolites acting as carbon sink similar to starch which metabolizes to release energy and carbon skeleton for the production of other compounds [pgm1 mutant of Arabidopsis sp. which lacks starch, accumulated more fumarate which appears to act as both a transient carbon sink for photosynthate and a pH regulator in nitrate absorption [Meanwhile, malic acid an intermediate in tricarboxylic acid (TCA) cycle, was also significantly upregulated (1.24-fold), which catalyzes the oxidative decarboxylation of malate to pyruvate by converting NADP to NADPH, thus providing a supply of NADPH for fatty acid biosynthesis . In a resp. B4D1 . Also, tompounds ,87. A stsorption . Therefosorption .HC supplemented cells. Tocopherols are synthesized by the combination of two major pathways: the MEP (methylerytrithol phosphate pathway) route and the shikimate pathway. The primary structure of tocopherol incorporates polar chromanol head group attached with hydrophobic prenyl side chain synthesized from the farnesyl diphosphate (FPP). The chromanol ring is provided by the shikimate route, whereas the hydrophobic prenyl tail is provided by the MEP pathway [HC, thus specifying the carbon flux towards tocopherol biosynthesis. Apart from de novo synthesis, tocopherols are synthesized from chlorophyll degradation, where released free phytol is phosphorylated to phytylmonophosphate (phytyl-P) and phytyl-PP by phytol kinase (VTE5) and is fed into biosynthesis of tocopherols, resulting in upregulation of tocopherol during cellular stress [Monoraphidium sp. CABeR41.Moreover, downregulation of lactic acid (1.06-fold) signifies the shift of carbon flux towards HVABs. The phenomenon is also supported by elevated tocopherol by nearly 3.25-folds in NL pathway ,91. Morer stress . Thus, oMonoraphidium sp. CABeR41 when subjected to NLHC condition demonstrates a concomitant increase in cell biomass (316.63 ± 15.47 mg·L−1 d−1), total lipids (76.12 ± 13.39 mg·L−1 d−1) and total tocopherols (734.38 ± 11.79 µg L−1 d−1), respectively. Furthermore, such stratergy demonstrates the cost-effective method for accumulating increased levels of total tocopherols and lipids employing carbon supplementation with varying nitrate concentrations leading to multi-fold enhancement of biocommodities without compromising growth. Also, illustrates the relevance of carbon and nitrogen (C:N) ratio which plays a significant role in achieving higher biomass productivities. In near future, our focus is to employ multi-omics approach to unveil the regulatory metabolic pathways involved in the tocopherol biosynthesis in these photosynthetic cell factories. Perhaps the study is a pivotal laboratory model for scale-up using cost-effective substrates. Such an approach will aim towards fulfilment of sustainable development goals (SDGs) involved in the innovation and cost-effective industrial algal biorefineries providing a better sustainability process with biorenewables, paving a new perspective for the improvement of the bioeconomy.In conclusion, the new indigenous microalgae


Kinesin family member 2A (KIF2A) is reported as an oncogene and a potential biomarker for progression and prognosis in several cancers such as cervical, ovarian, and gastric. However, its clinical value in basal-like breast cancer (BLBC) is unclear. This study aims to evaluate KIF2A expression and its correlation with clinical features and survival rates in BLBC patients.KIF2A mRNA and protein expressions in tumor and adjacent tissues from 89 BLBC patients are assessed by reverse transcription-quantitative polymerase chain reaction and immunohistochemistry assays, respectively.p < 0.001) and mRNA expressions (p < 0.001) were higher in tumor than in adjacent tissue. Besides, tumor KIF2A protein expression was positively correlated with N (p = 0.028) and TNM (p = 0.014) stages; meanwhile, tumor KIF2A mRNA expression was positively correlated with N stage (p = 0.046), TNM stage (p = 0.006), and tumor size (p = 0.043). Additionally, both tumor KIF2A protein (p = 0.035) and mRNA (p = 0.039) high expressions were correlated with worse disease-free survival (DFS) but not with overall survival (both p > 0.05). Moreover, tumor KIF2A protein expression was higher in relapsed patients than in non-relapsed patients within 3 years (p = 0.015) and 5 years (p = 0.031), whereas no difference was found between the dead and survivors within 3 years (p = 0.057) or 5 years (p = 0.107). Lastly, after adjustment, tumor KIF2A mRNA high exhibited a trend that correlated with DFS but without statistical significance (p = 0.051).Both KIF2A protein (KIF2A correlates with more frequent lymph node metastasis and worse DFS in BLBC patients, shedding light on its potency as a biomarker for BLBC. Breast cancer is the most common malignancy in females, with an estimated morbidity of 2 million and a mortality of 0.6 million worldwide in the year 2018 . As the Kinesin family member 2A (KIF2A), an M-type nonmotile microtubule depolymerase of the Kinesin-13 family , 13, serThis study aims to detect KIF2A protein and mRNA expressions in the BLBC tissue and its adjacent non-cancerous tissue by immunohistochemistry (IHC) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), respectively, so as to evaluate the correlation of KIF2A with clinical characteristics, disease-free survival (DFS), and OS in BLBC patients.After the approval of the Ethics Committee of The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, this study retrospectively collected the clinical data and the fresh-frozen specimens of 89 BLBC patients who underwent surgery in the hospital between January 2016 and December 2019. The patients were screened from the database according to the following criteria: (i) histopathological diagnosis of BLBC; (ii) resection; (iii) fresh-frozen tumor and adjacent tissues; (iv) the availability of clinicopathologic data and survival data. However, patients with the following conditions were not included in the study: (i) due to improper preservation, the specimens of patients were unable to be used for IHC assay and RT-qPCR assay; (ii) those who had a history of other primary tumors or malignant disease at diagnosis. Signed informed consents were obtained from patients or their relatives.We reviewed the database of the hospital and collected the relevant clinicopathological data and treatment information of patients. For survival analysis, follow-up data of patients were collected for the calculation of DFS and OS. Meanwhile, the tumor and adjacent tissues of patients were obtained from the specimen library, all of which were stored in liquid nitrogen and made available for RT-qPCR assay and IHC assay.−ΔΔCt method with β-actin as an internal reference. The forward primer for KIF2A was 5′-GCCTTTGATGACTCAGCTCC-3′, and the reverse primer for KIF2A was 5′-TTCCTGAAAAGTCACCACCC-3′. The primers used in qPCR were designed in accordance with the previous study , followed by reverse transcription into cDNA using the ReverTra Ace qPCR RT Kit . Subsequently, qPCR was conducted by SYBR Green Realtime PCR Master Mix  to quantify the KIF2A mRNA expression, which was calculated by the 2us study . The higThe KIF2A protein expression in the specimens was examined by IHC assay. The IHC staining procedures were implemented as in previous studies , 25. KIFt-test, Wilcoxon signed-rank test, independent sample t-test, or Wilcoxon rank-sum test. Association analysis was determined by using Spearman's rank correlation test. DFS and OS were analyzed using the Kaplan–Meier method and the log-rank test. Cox's proportional hazards model method regression was performed to analyze the factors affecting DFS and OS. Data analysis was carried out with SPSS 26.0 . Graphs were constructed by using GraphPad Prism 7.01 . A value p < 0.05 indicated statistical significance.The difference comparison was checked by using the paired Table 1).The mean age of the BLBC patients was 56.3 ± 11.1 years. Besides, the number of BLBC patients with good differentiation, intermediate differentiation, and poor differentiation was 17 (19.1%), 38 (42.7%), and 34 (38.2%), respectively, and the median [interquartile range (IQR)] value of tumor size was 2.5 (1.5–3.5) cm in the BLBC patients. In terms of T stage, the number of BLBC patients with T1, T2, and T3 was 35 (39.3%), 45 (50.6%), and 9 (10.1%), respectively. In regard to the N stage, there were 37 (41.6%), 22 (24.7%), and 30 (33.7%) BLBC patients with N0, N1, and N2 stages, respectively. With respect to the TNM stage, the number of BLBC patients with stage I, IIA, IIB, and IIIA was 17 (19.1%), 33 (37.1%), 7 (7.9%), and 32 (36.0%), respectively. Furthermore, the number of BLBC patients receiving neoadjuvant chemotherapy and adjuvant chemotherapy was 50 (56.2%) and 71 (79.8%), respectively  (p < 0.001) and KIF2A mRNA expression [median (IQR) value: 2.521 (2.004–3.769) vs. 1.031 (0.716–1.263)] (p < 0.001) were higher in tumor than in adjacent tissue .The stained photographs of KIF2A protein expression in tumor and adjacent tissues of BLBC patients are shown in p = 0.028) and TNM stage (p = 0.014). Also, tumor KIF2A mRNA expression was positively correlated with N stage (p = 0.046), TNM stage (p = 0.006), and tumor size (p = 0.043). However, no correlation of tumor KIF2A IHC score or tumor KIF2A mRNA expression with other clinical characteristics was observed (Table 2).In BLBC patients, tumor KIF2A IHC score was positively correlated with both N stage (p = 0.035) , and a tumor KIF2A IHC score was higher in patients who relapsed within 3 years (p = 0.015) or 5 years (p = 0.031) than in those who did not relapse within 3 years or 5 years, respectively . However, no correlation of tumor KIF2A protein expression with accumulating OS was found in BLBC patients (p = 0.105) . Also, no difference in the tumor KIF2A IHC score between BLBC deaths and the survival rate within 3 years (p = 0.057) or 5 years (p = 0.107) was found .In BLBC patients, a high tumor KIF2A protein expression was correlated with worse accumulating DFS (p = 0.039) . Besides, tumor KIF2A mRNA expression was higher in patients who relapsed within 3 years than in those who did not relapse within 3 years (p = 0.050), while no difference of tumor KIF2A mRNA expression was found between patients who relapsed within 5 years and those who did not relapse within 5 years (p = 0.117) . In addition, there was no correlation of tumor KIF2A mRNA expression with accumulating OS in BLBC patients (p = 0.136) . Besides, there was no difference of tumor KIF2A mRNA expression between BLBC deaths and survivors within 3 years (p = 0.122) or 5 years (p = 0.100) .In BLBC patients, a high tumor KIF2A mRNA expression was correlated with worse accumulating DFS  [95% confidential interval (CI): 2.347 (1.033–5.336)], tumor KIF2A mRNA high , T stage , and N stage  were factors related to 0020 worse DFS. From the forward stepwise multivariate Cox's regression analysis, T stage was an independent factor for a shortened DFS , while tumor KIF2A mRNA high showed a similar trend but without statistical significance  (Table 3).Cox's proportional hazards regression analyses were performed to evaluate factors affecting DFS and OS, and the findings are displayed in p = 0.011, HR (95% CI): 2.684 (1.253–5.752)], N stage , TNM stage , and neoadjuvant chemotherapy  were factors for worse OS, but neither tumor KIF2A protein high (p = 0.117) nor tumor KIF2A mRNA high (p = 0.145) were factors. From the forward stepwise multivariate Cox's regression analysis, N stage was an independent factor for a shortened OS , whereas adjuvant chemotherapy was an independent factor for a prolonged OS .In regard to OS, T stage [The major findings are outlined as follows: (1) KIF2A was overexpressed in tumor than in the adjacent tissue of BLBC patients. (2) Tumor KIF2A expression was correlated with advanced N and TNM stages in BLBC patients. (3) Tumor KIF2A high expression was correlated with worse DFS but not with OS in BLBC patients.in vitro; furthermore, it is upregulated in tumor tissues of patients with these cancers (Kinesin superfamily proteins (KIFs) are microtubule-dependent motor proteins that provide strength to the intracellular transportation of membranous organelles and macromolecules, as well as cell division , 27. KIF cancers , 20, 21. cancers , 21, as  cancers , leadingKIF2A is clinically correlated with poor clinicopathological features in multiple cancers , 28, 29.In terms of survival, KIF2A is associated with poor DFS or OS in patients with hepatocellular, cervical, lung, and colorectal cancers , 20, 29.It could not be denied that there were a few limitations in this study. Firstly, the small sample size led to low statistical power, and a further study with a larger sample size is now proposed to be conducted. Secondly, the potential mechanisms underlying the oncogenic effects of KIF2A in BLBC are required to be clarified in the future. Thirdly, there might exist several confounding factors such as treatment after relapse, which influenced the findings in this study. Fourthly, the clinical value of KIF2A in other types of breast cancer could be investigated in the future.Conclusively, KIF2A is highly expressed in tumor than in adjacent tissue; also, tumor KIF2A high expression correlates with more frequent lymph node metastasis and worse DFS in BLBC patients. The above discoveries shed light on the potency of KIF2A as a biomarker for BLBC.


All steers underwent a TM depletion period for a minimum of 42-d prior to the administration experimental treatments. Treatments included a negative control (CON) in which cattle received no additional TM supplementation or TM supplementation treatments in which cattle received added Co, Cu, I, Mn, Se, or Zn from inorganic TM sources at 2016 Nutrient Requirements of Beef Cattle (NASEM) requirement levels (1X), at 2 times NASEM requirements (2X), or at 4 times NASEM requirements (4X). Selenium was included at 0.1, 0.2, and 0.3 mg/kg for 1X, 2X, and 4X respectively, based on federal law. There was no difference in overall BW, average daily gain (ADG), dry matter intake (DMI), or gain to feed (G:F) due to TM supplementation (CON vs. SUPP P ≥ 0.47). There was no difference in hot carcass weight, rib eye area, fat thickness, dressing percentage, marbling score, or USDA Yield Grade due to TM supplementation (CON vs. SUPP P ≥ 0.30). One steer was chosen at random from each pen to be evaluated for serum and liver TM status and antibody concentrations to common respiratory viruses. There was a treatment × day interaction for serum Co and liver Cu and Se (P < 0.0001). Serum Co was greatest for the 4X treatment from d 28 through harvest. Liver Cu was greatest for the 2X and 4X treatments from d 56 through harvest. Liver Se was greatest for 2X and 4X from d 28 through harvest. Serum Zn was greatest for the 4X treatment (P = 0.02). There was an effect of day on liver Co, Fe, Mn, Mo, and Zn (P ≤ 0.0001) and serum Cu, Mn, Mo, Se, and Zn (P ≤ 0.002). Individual TM concentrations differed over time; however, none were ever considered deficient or toxic based on published reference ranges. There was an effect of time on bovine viral diarrhea virus Type 1A, bovine herpesvirus type 1, and bovine parainfluenza 3 virus antibody concentrations (P ≤ 0.0001). Supplementation of TM above NASEM requirements did not affect overall cattle performance, carcass characteristics, or antibody concentrations, but did affect the storage and circulation of certain TM.The objective of this experiment was to determine the effects of increasing trace mineral (TM) supplementation on finishing cattle performance, carcass characteristics, TM balance, and antibody concentrations. Commercial Angus steers ( Combined and individual trace mineral (TM) supplementation of Cobalt (Co), Copper (Cu), Iron (Fe), Iodine (I), Manganese (Mn), Selenium (Se), and Zinc (Zn) has produced variable results in feedlot cattle. Research indicates that supplying the 7 TM with published requirements does not consistently enhance performance or carcass characteristics . TypicalAll procedures were approved by the Institutional Animal Care and Use Committee at Oklahoma State University This experiment used a total of 240 crossbred Angus steers that were received in 2 shipments from central South Dakota. While cattle were received 5 d apart, treatment allocation was staggered by 7 d to accommodate logistical challenges due to the time required to obtain body weights (BW) and biological samples and to keep sampling intervals consistent among groups.In mid-February 2020, 164 crossbred Angus steers (BW = 297 ± 31.2 kg) were transported on 2 trucks approximately 948 km from Burke, SD to the Willard Sparks Beef Research Center (WSBRC) in Stillwater, OK. Of these initial 164 steers, 140 were initially processed upon arrival to be allocated to the experiment. The arrival processing procedures for the 140 steers included collection of individual BW and application of individual identification tags. Cattle were then held in a pen overnight with ad libitum access to prairie hay and water. The initial 140 processed cattle were deemed Group 1 (G1).The remaining 24 head from the first shipment were placed in a feedlot pen and were fed ad libitum prairie hay and water until the arrival of the second shipment of cattle. Five days later, 81 additional steers  were transported on 1 truck approximately 1,328 km from Mobridge, South Dakota to the WSBRC. These 81 steers were combined with the remaining 24 cattle from the first shipment for processing and deemed Group 2 (G2). Five steers  from G2 were removed prior to treatment allocation. The remaining 100 animals from G2 were individually weighed, tagged, and held in a pen overnight with ad libitum access to prairie hay and water.n = 140; G2 n = 100) were processed the day following collection of arrival BW . Steers were blocked by BW within group and randomly allocated to pens within block. Processing procedures were similar to that described by n = 13) that arrived as bulls were evenly distributed between treatments . Steers were housed in forty-eight 4.57 × 13.24 m partially covered dirt feedlot pens. Cattle housed in 2 adjacent pens shared a 76-L concrete water fountain . Cattle were revaccinated for viral and bacterial respiratory pathogens on d 0 . Cattle were reimplanted on d 28 .Cattle from both groups . Within block, cattle were randomly assigned to each pen and experimental TM treatments were randomly assigned to pens within each block. A total of 48 pens were used for this experiment, with 12 pens per treatment and 5 cattle per pen  to allow cattle to acclimate to the feedlot environment . After bDuring the 28-d transitioning period, all cattle received only the negative control (CON) supplement (no added TM) that was formulated to meet macro mineral deficiencies present from the basal finishing diet. As a result, cattle on all experimental treatments received no TM supplementation from the time of arrival until d 0 when experimental TM treatments were initiated. This resulted in a period of depletion where cattle were not receiving any TM supplementation for at least 42 d  prior to experimental treatment administration.−1·d−1).All treatment supplements contained 0.65% Ca, 0.30% NaCl, 4,715 IU/kg of Vitamin A, 25.1 IU/kg of Vitamin E, 142 IU/kg of Vitamin D, 30 g/ton of monensin , and 9 g/ton of tylosin . On a DM basis, finishing diets consisted of 7% hay, 15% Sweet Bran, 65% rolled corn, 5% liquid supplement, 1% urea, and 5% pelleted vitamin and treatment supplement . Twenty-Feeding procedures were similar to those described by Diet samples were collected twice weekly, and DM was calculated after samples were dried in a forced air oven at 60 °C for 48 h. A monthly composite was created after DM was calculated and stored in a freezer until nutrient analysis could be completed. Feed refusals were weighed back before feeding on d 0, 14, 28, 56, 84, 112, 140, and 168 or if excessive orts remained in the bunk, refusal samples were dried to determine DM content and were subtracted from DM delivered in order to calculate dry matter intake (DMI).CON) in which cattle received no supplemental TM. This was the same supplement feed to all cattle during the depletion period, a treatment designed to meet 1X; 15.0 mg Co/kg, 10.0 mg Cu/kg, 50.0 mg Fe/kg, 0.50 mg I/kg, 20.0 mg Mn/kg, 0.10 mg Se/kg, and 30.0 mg Zn/kg; DM basis), a 2 times 2X), and 4 times 4X) treatment. Exceptions to the 1X, 2X, and 4X experimental treatment structure were Fe and Se. Iron was kept constant at 50 mg/kg (DM basis) for 1X, 2X, and 4X treatments based on consulting feedlot nutritionist survey recommendations (3), copper sulfate (CuSO4), ferrous sulfate (FeSO4), ethylenediamine dihydriodide (EDDI), manganese oxide (MnO), sodium selenite (Na2SeO3), and zinc sulfate (ZnSO4).Dietary TM treatments were modeled from the ndations . SeleniuIndividual BW were recorded for all steers on d 0,14, 28, 56, 84, 112, and prior to shipping for harvest. Body weights were measured before feeding at approximately 0400 h with no withdrawal from feed or water. All BW were adjusted using a calculated 4% shrink (BW × 0.96). Average daily gain (ADG) was calculated by dividing shrunk individual BW gain in kg by days on feed (DOF) for each period. Pen ADG was calculated as the average of individual ADG for each steer in the pen for that period. Dry matter intake was calculated from total DM feed delivery for the pen for that period divided by days on feed (DOF) and number of steers in the pen for each period (head days). Gain to feed (G:F) ratio was calculated by dividing pen ADG by pen average daily DMI for each period.m concentration and the m) = 0.077 (shrunk BW)0.75.The data from the 8 steers that died or were removed from the experiment were excluded from all analyses (deads out data). Since feed intake was measured on a pen basis not on an individual animal basis, feed intake data were corrected by removing the average daily DMI for each steer removed from the pen until the respective steer ceased gaining BW. From the time the steer ceased gaining BW until the steer was physically removed from the pen and simultaneously the experiment, maintenance DMI were estimated and removed using the diet NEA subset of steers  was used to obtain blood and liver biopsy samples prior and subsequent to administration of TM supplementation treatments to examine the impact of the TM treatments on TM status and antibody concentrations. All blood samples were collected via the jugular venipuncture and stored on ice prior to harvesting serum. On d 0, 14, 28, 56, and 7 d prior to shipping to harvest, two 6-mL blood samples were collected into a tube containing silicone  for TM analysis. On d 0, 14, 28, and 56, two 6 mL blood samples were collected into a tube containing a positive gel barrier and silicone glass powder  for antibody concentration analysis. Blood was allowed to clot for an average of 2 h before centrifuging. All blood tubes were centrifuged at 1,294 × g for 30 min at 4 °C . Serum collected for TM analysis was collected and shipped overnight to the Texas A & M Veterinary Medical Diagnostic Laboratory (TVMDL) for further analysis. Serum collected for antibody concentration analysis was stored in a −80 °C freezer until analysis. Samples were analyzed for bovine viral diarrhea virus (BVDV) types 1A, 1B, and 2, bovine herpesvirus type 1 (BHV-1), and bovine parainfluenza 3 (PI-3) virus. All serum samples for antibody concentration analysis were sent to TVMDL for analysis after the completion of the experiment.On d 0, 28, 56, and 7 d prior to harvest, a 100-mg liver sample was collected using the procedure described by The biopsy site was surgically scrubbed 3 times with a commercially-available iodine scrub  followed by rinsing with a 70% isopropyl alcohol solution. After the third scrub and rinse, a commercial iodine solution was sprayed on the injection sites prior to obtaining a biopsy. After ensuring the biopsy sites were thoroughly anesthetized , a scalpel was utilized to make an approximately 1-cm stab incision for the insertion of the biopsy needle. The incision was made between the 11th and 12th ribs approximately 25 cm lateral to the vertebrae, and a 2.1 mm × 15.2 cm 14-gauge Tru-cut biopsy needle was inserted and then directed cranially and ventrally toward the opposite elbow. The biopsy needle was advanced through the peritoneum, the diaphragm, and then into the liver to obtain an approximately 100-mg sample of liver tissue. The incision site was closed with veterinary glue to prevent infection and sprayed again with iodine. Animals were observed twice daily for 7 d after the procedure. The 100-mg liver sample was transferred to a 2 mL centrifuge tube and stored on ice. Liver samples were shipped overnight to TVMDL for TM analysis.Cattle were shipped approximately 116 km to a commercial abattoir in Arkansas City, Kansas in 3 harvest groups. The 5 heaviest blocks were shipped on d 126 of the experiment, the 4 middle blocks were shipped on d 140, and the 3 lightest blocks were shipped on d 156. Carcass data were collected by abattoir trained personnel and via carcass camera imaging because of COVID-19 protocols.For all rations, a single 400-g sample from the middle of the feed batch was collected from the mixer twice weekly. A 400-g sample of the dietary TM supplements were collected twice weekly. Within each month, the twice weekly finishing diet and TM supplement samples were individually composited and stored until analysis. Monthly composite samples of finishing diets and TM supplements, as well as composites of receiving and transition diets, were ground through a 2mm screen , and were composited for each diet and TM supplement. Approximately 200 g of each diet and supplement composite were sent in duplicate to a commercial laboratory for proximate and TM analysis .3, 0.25 mL HCl, and a glass encased stir bar. One mL of H2O2 was added. The sample was then stirred for approximately 10 min and transferred to an automated microwave digester . The sample was then transferred to a tube and brought to 50 mL with tissue sample diluent (2% HNO3 and 1 µg/mL ISTD), inverted, and assayed. Aliquots of serum were diluted 1:10 with a serum diluent . Samples were then vortexed and assayed. All samples were analyzed on a PerkinElmer NexION 350x ICP-MS using Syngistix 1.1 software.Liver tissue samples and serum samples were analyzed for Co, Cu, Fe, Mn, Mo, Se, and Zn concentrations using inductively coupled plasma mass spectrometry (ICP-MS) at TVMDL . In brief, samples were weighed and combined with 5 mL HNO2 atmosphere. After 72 to 96 h of incubation, depending on virus; samples were analyzed. A serum dilution was considered positive if there is a 100% reduction in the amount of virus in the serum test wells compared to that present in the positive control wells. The highest dilution of serum resulting in complete neutralization of virus is the end point titer for that serum. All titers are reported as the final serum dilution prior to the addition of the cell culture suspension. Titers are reported as the reciprocal of the dilution.Serum samples were analyzed for antibody concentrations specific to BRD, specifically BVDV types 1A, 1B, and 2, infectious bovine rhinotracheitis (IBR) specifically bovine herpes virus 1 (BVH-1) titers, and parainfluenza 3 (PI-3) using the serum neutralization test in the virology section at the TVMDL . In brief, a 2-fold serial dilution was performed for all serum. Diluted samples were challenged with aliquots of specific stock virus. Samples are agitated and incubated at 37 ± 1 °C, in a humidified 5 ± 1% COn = 48). All performance and carcass characteristic data were analyzed using the MIXED procedure of SAS 9.4  with treatment as a fixed effect and block as a random effect. There was no effect of treatment in the overall model for any performance or carcass parameters (P ≥ 0.12), so predetermined contrasts were evaluated including CON vs. TM supplement (SUPP) and the linear and quadratic effects of TM supplementation. Due to unequal spacing in dietary treatments, contrast coefficients were determined using the IML procedure of SAS.This experiment was organized in a randomized complete block design and blocked by BW within group. For all performance measurements, pen served as the experiential unit . Analysis for Co liver, Cu serum and liver, Se serum concentrations and BVDV Type 1B, 2, and BHV-1 antibody titers utilized an unstructured covariance structure. Analysis for Co serum, Fe liver, Mn serum, and Se liver concentrations utilized a heterogeneous autoregressive (1) covariance structure. Day was included as a repeated measure with pen as the subject. All data from steers removed from the experiment were excluded from statistical analysis. Significance was determined when Experimental diets were formulated to be identical except for the TM composition of the treatment supplements. Proximate analysis of diets excluding TM is presented in P ≥ 0.18) or ADG (CON vs. SUPP P ≥ 0.11) due to TM supplementation. Furthermore, there was no linear or quadratic trend in BW  or ADG among treatments . There was no difference in DMI during from d 0 to d 13 of the experiment due to TM supplementation (CON vs. SUPP P = 0.39). Interestingly, from d 14 to d 27, DMI for cattle consuming the CON treatment was less than (P = 0.04) cattle fed 1X, 2X, and 4X. Over the next several intervals, cattle fed the CON diet had average DMI that did not differ from that of the other 3 TM treatments (P ≥ 0.58). During the last 28 d of the experiment, when a beta agonist was included in the diet, cattle consuming the CON diet tended (P = 0.08) to have decreased DMI than cattle consuming the 1X, 2X, or 4X diets. There was no difference in overall DMI between cattle consuming the CON diet and cattle consuming the 1X, 2X, or 4X diets (P = 0.99). While TM supplemented cattle were also more efficient during the final 28 d, when a beta agonist was included (P = 0.03), compared with CON cattle, there was no difference in G:F for any other interval (P ≥ 0.31) or overall (P = 0.48) for cattle offered the CON diet compared with cattle offered the 1X, 2X, or 4X diets.The effects of supplemental TM on steer performance and DMI are presented in Previous research involving individual TM supplementation has been variable . VariatiP = 0.11), a tendency for an increase in DMI (P = 0.08), and an increase in G:F (P = 0.03) for cattle receiving TM supplementation. Previous research has been conducted regarding the synergistic effects of TM and beta agonists. Results from the current experiment indicate that the addition of TM at or above With the exception of the results observed during the beta agonist period, the present experiment is in agreement with the majority of previous research, where TM supplementation above published requirements had no effect on overall feedlot cattle performance.P ≥ 0.30). There were also no linear or quadratic trends for any carcass characteristics .Carcass data are presented in These results are similar to those published by Trace mineral concentrations were observed from a subset of 48 steers (1 steer per pen/12 steers per treatment). Trace mineral concentrations are reported on a wet weight basis.P = 0.31) or effect of treatment on Co concentrations (P = 0.11). There was an effect of day on Co concentrations in the liver (P < 0.0001) where concentrations for all treatments decreased on d 0 to d 28, increased on d 56, and then remained relatively constant until harvest. Cobalt concentrations in the liver are displayed in P < 0.0001) as shown in P < 0.0001) serum Co concentrations on d 28 through harvest compared to cattle on the CON, 1X, and 2X treatments, which did not differ (P ≥ 0.17) from d 28 through harvest. There was a treatment × day interaction for Co concentrations in serum (P < 0.0001) for Cu concentrations in the liver as shown in P = 0.57). However, concentrations for cattle on the 1X, 2X, and 4X treatments increased steadily over the course of the experiment while cattle on the CON treatment maintained near baseline liver Cu concentrations through harvest. Liver Cu concentrations differed (P ≤ 0.04) between all 4 treatments on d 28 with 4X having the greatest concentrations, followed by 2X, 1X, and then the CON. Liver Cu concentrations were not different for cattle on the 4X and 2X treatments from d 56 through harvest (P ≥ 0.20). However, liver Cu concentrations were greater for 4X and 2X than the 1X or CON treatments and liver Cu concentrations were greater for 1X treatment than CON from d 56 through harvest (P ≤ 0.001). Cattle on all experimental treatments exhibited concentrations that were considered adequate (CON and 1X) or high (2X and 4X) for beef cattle (4 reported similar findings. 4 fed to Angus steers and reported a treatment × time interaction in liver Cu concentrations. Liver Cu concentrations were similar across treatments on d 0 and increased over time. Liver Cu concentrations were greater at harvest for steers fed 20 mg/kg of Cu than cattle consuming 10 mg/kg of Cu (There was a treatment × day interaction (f cattle . Howeverf cattle . Previoukg of Cu . Other rkg of Cu . Liver Ckg of Cu .P = 0.35) or effect of treatment (P = 0.11) for serum Cu concentrations as shown in P < 0.0001). Copper concentrations remained stable from d 0 to d 14. From d 14 to 28 serum Cu concentrations increased, followed by a decrease in serum concentrations from d 28 to d 56. Serum Cu concentrations increased for all treatments from d 56 through harvest. Although serum Cu concentrations fluctuated over time, cattle maintained concentrations within a narrow margin. According to There was no treatment × day interaction  or effect of treatment (P = 0.59) for concentrations for Fe in the liver, as shown in P < 0.0001). Concentrations for all for treatments were similar on d 0. On d 28, liver Fe concentrations remained stable. However, on d 56 liver Fe concentrations increased and continued to increase through harvest. As expected, there was not a treatment × day interaction (P ≥ 0.13) as shown in There was no treatment × day interaction or effect of treatment or day on Fe serum concentrations (P = 0.74) or effect of treatment (P = 0.39) for Mn concentrations in the liver as shown in P < 0.0001). Liver Mn concentrations did not differ on d 0. On d 28, liver Mn concentrations increased, then slightly decreased on d 56. Liver Mn concentrations then increased through harvest. All liver Mn concentrations were within normal ranges indicating that Mn was neither deficient in the basal diet nor toxic in the 4X diet (4. There was no treatment × day interaction ( 4X diet . In an e diet 4. . Due to P = 0.45) or treatment effect (P = 0.82) for serum concentrations of Mn as shown in P < 0.0001). Serum Mn concentrations decreased from d 0 through d 28 and then stabilized through harvest. There was no treatment × day interaction (P = 0.19) or effect of treatment (P = 0.12) on Mo concentrations in the liver as shown in P < 0.0001). Liver Mo concentrations did not differ on d 0. However, Mo liver concentrations varied on d 28, tended to increase on d 56, and then plateaued through harvest. The present experiment did not provide supplemental Mo, so it was expected that regardless of treatment, Mo liver concentration would not differ. The changes in liver Mo concentrations over time could reflect use of Mo within the body. As Mo is an important antagonist for other TM, changes in Mo concentrations over time could influence the metabolism of other TM. There was no treatment × day interaction (P = 0.13) or effect of treatment (P = 0.75) for serum concentrations of Mo as shown in P = 0.0019). Serum Mo concentrations decreased from d 0 through d 14. Serum Mo concentrations slightly increased on d 28 and then stabilized through harvest. To the authors knowledge, there are no published studies evaluating TM supplementation on serum Mo. However, There was no treatment × day interaction (P < 0.0001) as shown in P ≥ 0.11). However, liver Se concentrations of cattle on the 2X and 4X treatments were greater (P ≤ 0.05) on d 28 than cattle on the CON and 1X treatments, which did not differ (P = 0.37). Liver Se concentrations of 2X cattle did not differ from 1X cattle on d 56 (P = 0.19). Cattle offered the 1X, 2X, and 4X diets had greater liver Se concentrations than CON cattle on d 56 and at harvest (P ≤ 0.05). There was a treatment × day interaction for Se concentrations in the liver (P = 0.99) or effect of treatment (P = 0.32) for Se concentrations in serum as shown in P < 0.0001). Serum Se concentrations exhibited a decrease from d 0 through d 14 and then an increase on d 28. Serum Se concentrations remained stable through harvest. In classical research in sheep, serum Se increased as dietary Se increased (There was no treatment × day interaction (ncreased . In geneP = 0.91) or effect of treatment (P = 0.47) for Zn concentrations in the liver as shown in P < 0.0001). Liver Zn concentrations decreased from d 0 through d 28. Liver Zn concentrations then increased for all treatments through d 56 and continued to increase through harvest. There was no treatment × day interaction (P = 0.33). However, there was an effect of treatment (P = 0.02) and day (P < 0.0001) on serum Zn concentrations as shown in P = 0.001). There was no treatment × day interaction for serum Zn concentrations (P = 0.34) or effect of treatment (P = 0.89) for BVDV Type 1A antibody concentrations as shown in P = 0.03). Antibody concentrations for BVDV Type 1A increased from d 0 to d 14 then gradually decreased over the remainder of the sampling period. There was no treatment × day interaction, effect of treatment, or effect of day on BVDV Type 1B or BVDV Type 2 antibody concentrations (P ≥ 0.44) as shown in There was no treatment × day interaction (P = 0.34) or effect of treatment (P = 0.77) for BHV-1 concentrations. However, there was an effect of day (P < 0.0001) as shown in There was no treatment × day interaction (P = 0.93) or effect of treatment (P = 0.22) for PI-3 antibody concentrations as shown in P < 0.0001). Antibody concentrations were similar on d 0. Similar to BVDV Type 1A and BHV-1, antibody concentrations for PI-3 increased from d 0 to d 14, then gradually decreased over the remainder of the sampling period. There was no treatment × day interaction (Antibody concentration data from the current experiment and previous research report varying results. Cattle in the current experiment were considered low to moderate risk . VaccinaTrace mineral supplementation had no effect on overall cattle performance or carcass characteristics in the current experiment. Antibody concentrations for common respiratory viruses were also not affected by supplemental TM inclusion. The supplementation of TM did have an impact on serum Co concentrations, liver Cu concentrations, liver Se concentrations, and serum Zn concentrations, where increased dietary inclusion of TM increased the previously mentioned TM concentrations over time. There are several reasons why TM supplementation may not have effected overall cattle performance, carcass characteristics, and antibody titers in the current experiment. All cattle were receiving sufficient TM concentrations from the basal diet, with the exception of Cu, during the 42-d depletion period without additional TM supplementation. As such, it is probable that additional supplementation would not enhance performance or carcass characteristics, due to the presence of sufficient levels of most TM in the basal diet. It should also be noted that cattle were considered to be low to moderate risk upon arrival and were acclimated to the feedlot environment during the depletion period prior to the allocation of dietary treatments. As such, there was not an initial depression in DMI that could result in the potential for decreased TM consumption from the basal diet at the beginning of the experiment. Based on the overall performance, carcass characteristics, and antibody concentrations observed in this experiment, the supplementation of TM beyond the