Here, we show that mouse embryonic fibroblasts (MEFs) lacking Rab8 have severely impaired adipocyte differentiation in vivo and ex vivo. Immunofluorescent localization and biochemical analyses of Rab8a-deficient, Rab8b-deficient, and Rab8a and Rab8b double-deficient MEFs revealed that Rab8 controls the Lrp6 vesicular compartment, clearance of basal signalosome, traffic of frizzled two receptor, and thereby a proper attenuation of Wnt signaling in differentiating MEFs. Upon induction of adipogenesis program, Rab8a- and Rab8b-deficient MEFs exhibited severely defective lipid-droplet formation and abnormal cilia morphology, despite overall intact cilia growth and ciliary cargo transport. Our results suggest that intracellular Rab8 traffic regulates induction of adipogenesis via proper positioning of Wnt receptors for signaling control in mesenchymal cells.Differentiation of mesenchymal stem cells into adipocyte requires coordination of external stimuli and depends upon the functionality of the primary cilium. The Rab8 small GTPases are regulators of intracellular transport of membrane-bound structural and signaling cargo. However, the physiological contribution of the intrinsic trafficking network controlled by Rab8 to mesenchymal tissue differentiation has not been fully defined  Adipocytes are differentiated mesenchymal cells that store excess food energy as fat in organelles known as lipid droplets. Metabolic disorders such as obesity disrupt lipid homeostasis and adipogenesis by inducing hypertrophy of existing adipocytes or by increasing proliferation to form new adipocytes to cope with excess fat , 2. Adipin vivo or in primary MEFs.A hallmark of adipocyte maturation is the formation, trafficking, and fusion of lipid droplets, all processes linked to the Rab family of small GTPases . ApproxiAdipocyte differentiation may be coordinated by Wnt, Hedgehog, bone morphogenetic protein, and hormone (namely insulin and leptin) signaling , 4, 5, 6Primary cilia, or nonmotile cilia, are microtubule structures at the plasma membrane made up of an axoneme arranged in 9 + 0 formation and a basal body , 25, 26.Wnt proteins are secreted extracellular growth factors that bind the coreceptors LRP5/6 and Fzd , 38. UpoWhether Rab8-mediated membrane transport affects mesenchymal cell differentiation remains incompletely defined. We show here that MEFs deficient in Rab8a, Rab8b, or both proteins exhibit a severe impairment in activating the adipogenesis program. Mechanistically, Rab8 deficiency alters the intracellular Lrp6 vesicular compartment, Fzd2 membrane trafficking, and Wnt signaling.−/− MEFs. Using the TopFlash luciferase reporter as a readout for Wnt/β-catenin transcriptional activity, Rab8a−/− MEFs displayed increased sensitivity to Wnt3a stimulation . WT MEFs exhibited a 4.9-fold Wnt3a-induced TopFlash activity, whereas Rab8a−/− MEFs exhibited a 30-fold induction of reporter activity.We first investigated the role of Rab8 on induction and activation of Wnt/β-catenin signaling components in Rab8a−/− MEFs, the abundance of pLRP6  was approximately 14-fold of that in WT MEFs (B and C). Upon Wnt3a treatment, pLRP6 levels progressively increased over 4 h after stimulation in both WT and Rab8a−/− MEFs .Wnt engagement with surface receptors on ligand-receiving cells elicits rapid formation of LRP6 signalosomes , which a WT MEFs , B and Ce.g., Axin and Dvl) and promote inactivation of the destruction complex  but not Axin1 and phosphorylated β-catenin.Wnt-induced Lrp6 signalosomes recruit key pathway components ( complex . The obs−/− MEFs exhibited 9-fold increase in TopFlash activity in response to CHIR99021 compared with WT MEFs (E). We then overexpressed a constitutively active β-catenin (β-cat-ΔN) construct lacking the GSK3β phosphorylation sites (−/− MEFs (F). These data suggested that the enhanced Wnt signaling in Rab8a−/− MEFs occurred upstream of β-catenin, possibly at the cell surface and endocytic compartment.These data suggested that loss of Rab8a in MEFs appeared to elevate Lrp6 signalosome activity that was accompanied by an enhanced intracellular signaling response. To examine at what molecular level Rab8a deficiency enhanced the signaling cascade, GSK3-mediated signaling inhibition was first assessed by using CHIR99021, the GSK3 inhibitor . Rab8a−/ WT MEFs E. We theon sites  and foun−/− MEFs F. These A). Rab8b KD efficacy was verified by Western blot even after multiple passages . We first examined Lrp6 intracellular localization in WT and Rab8-deficient MEFs in the presence or absence of exogenous Wnt ligands, by transiently expressing a GFP–Lrp6. In serum-starved WT MEFs treated by the vehicle, vesicular Lrp6 was rare, with approximately two Lrp6+ puncta per cell on average. Upon Wnt3a addition for 15 min, vesicular Lrp6 increased by approximately 9-fold (C and D). In contrast, vehicle-treated Rab8a−/−, Rab8bKD, and Rab8a−/−;Rab8bKD MEFs had significantly increased vesicular Lrp6 compartments compared with WTs, and the total numbers of Lrp6+ puncta did not increase further upon Wnt3a addition (C and D).The two mammalian Rab8 isoforms, Rab8a and Rab8b, share 83% amino acid sequence homology and both participate in apical cargo transport . To examFig. 2E, data represent three independent experiments). In Wnt3a-stimulated WT MEFs, Lrp6 was detected in fractions 6 to 11, while pLrp6 became detectable in fractions 8 to 11, where Gsk3, endocytic markers, and caveolin were found to be cosedimented (A).The above results suggested a changed basal Lrp6 compartmentalization in Rab8-deficient MEFs. To biochemically examine Lrp6 intracellular distribution, we performed sucrose gradient sedimentation on lysates of MEFs upon 4 h of Wnt3a or vehicle treatments, essentially following a previous study . In vehiFig. 2dimented A.−/− MEFs showed notably higher basal pLrp6 in fractions 9 to 11 (F), where Dvl2, Rab7, Rab9, and caveolin were cosedimented (B). Wnt stimulation increased pLrp6 abundance and intracellular domains in Rab8a−/− MEFs (F). In addition, Wnt-induced sedimentations of Axin1, Rab7, and caveolin were altered in Rab8a−/− MEFs compared with WT MEFs (C), suggesting that ablation of Rab8a vesicular compartment affected the membrane trafficking network in response to exogenous Wnt ligand stimulation.Vehicle-treated Rab8a 9 to 11 F, where dimented B. Wnt st−/− MEFs F. In add WT MEFs C, suggesKD MEFs reached a similar but less robust effect on pLrp6, as Rab8a−/− MEFs (G). Owing to the detection of basal pLrp6 in these MEFs, we examined localization of Lrp6 puncta in starved cells and found that in addition to intracellular puncta, there were significantly increased numbers of peripheral Lrp6+ puncta in Rab8-deficient MEFs . These results collectively suggested that there was an altered Lrp6 vesicular compartment that potentially contributed to a sensitized MEF response to Wnt ligands.Examination of Rab8b−/− MEFs G. Owing in vitro to induce growth arrest (A). Adipocyte differentiation was confirmed by the formation of lipid droplets detected by BODIPY staining (B). Consistent with previous studies (A).To examine whether Rab8 is expressed during MEF differentiation to adipocyte, WT MEFs were first grown to confluency h arrest , followeh arrest , 60, 61.h arrest A. Adipocstaining B. Consis studies , 63, 64, studies A.Figure −/− MEFs contained small intracellular lipid droplets that were of a similar size to those of WT MEFs (C and D). To test if Rab8a deficiency might impact MEFs’ response to adipogenic stimuli, we treated Rab8a−/− MEFs with the same induction cocktail. Fluorescence staining did not detect the formation of large lipid droplets in Rab8a−/− MEFs after treatment with the differentiation protocol (E and F). The average diameter of WT droplets increased by over 20-fold upon differentiation, while the droplets in Rab8a−/− MEFs increased by less than 2-fold .Before adipogenic induction, Rab8a WT MEFs , C and Dprotocol , E and FG and H, data represent three independent experiments). In contrast, induced Rab8a−/− MEFs exhibited elevated abundances of both total β-catenin and Tcf1 (G and H). These data demonstrated that the impaired Rab8−/− MEF differentiation was accompanied by aberrantly elevated Wnt signaling, an observation consistent with the above biochemical analysis . Although there was the presence of subcutaneous cells marked by aP2  in Rab8a−/− mice, these cells did not appear to contain the characteristic lipid droplets that were densely packed as the aP2 subcutaneous adipocytes in WT mice (B and D). Similar observations for aP2-labeled cells were made in anatomically matched adipocyte tissues adjacent to skeletal muscles (E and F). Together, these results suggested that Rab8a is necessary for adipose tissue development in vivo and ex vivo.Examination of the subcutaneous adipose tissues in newborn (P0) Rab8aate pups , A and C WT mice , B and D muscles , E and FKD MEFs exhibited an overall similar Lrp6 profile as Rab8a−/− MEFs. Thus, we next examined whether Rab8bKD MEFs had a similar adipogenic defect. Similar to Rab8a−/− MEFs, the differentiation of Rab8bKD MEFs was also impaired: there was a prolonged delay, taking approximately 13 days, to start the formation of significantly smaller lipid droplets than WT cultures .Rab8b−/−;Rab8bKD MEFs. Rab8a−/−;Rab8bKD MEFs, upon induction by the same protocol, failed to attach to glass coverslips around day 5 and 6 of the differentiation protocol. To ameliorate this attachment issue, we coated glass coverslips with 0.1% gelatin. After 2 weeks of induction, Rab8a−/−;Rab8bKD MEFs only formed scattered, small lipid droplets, with a significantly decreased total BODIPY-stained lipid area compared with Rab8a−/− or Rab8bKD MEFs (G). However, the diameter or the size of lipid droplets in Rab8a−/−;Rab8bKD cultures did not differ from those in Rab8a−/− or Rab8bKD MEFs (F). Owing to defects in primary cilia, MEFs lacking Kif3a failed to undergo adipogenesis , which also displayed a delayed formation and fewer lipid droplets (E). These data suggested a defective activation of the adipogenic program in Rab8-deficient MEFs, which was further supported by significantly reduced transcripts of adipocyte differentiation, including Fabp4 and Glut4 in these Rab8a−/−;Rab8bKD MEFs compared with WT MEFs (H). PPAR-γ expression was only slightly reduced in Rab8a−/−;Rab8bKD MEFs.To further test if there remained some redundancy between Rab8a and Rab8b, we tested adipogenic capacity of Rab8abKD MEFs G. HowevebKD MEFs F. Owing ogenesis , so we u−/− MEFs , F and Gdroplets E. These  WT MEFs H. PPAR-γE–G), in disrupting MEF differentiation into adipocytes. As Rab8 has been implicated in ciliary cargo transport and cilia development . Before serum starvation (0 h), Rab8a−/− MEFs showed the lowest percentage (62%) of ciliated cells, followed by Rab8a−/−;Rab8bKD MEFs (71%), Rab8bKD MEFs (76%), and WT MEFs (82%). As expected, primary cilia were not observed in Kif3a−/− MEFs (A and B).To induce primary cilia formation, MEFs were grown as confluent monolayers for 48 h, serum-starved for 0, 2, 24, or 48 h and then immunofluorescently stained for acetylated tubulin (a cilium marker) and γ-tubulin  at each time point A. Before−/− MEFs , A and B−/−;Rab8bKD MEFs exhibited the shortest primary cilia before serum starvation, followed by Rab8a−/− or Rab8bKD MEFs, whereas WT MEFs possessed the longest primary cilia (C). After serum starvation, the percentage of ciliated cells and the cilium length increased in WT and the three Rab8-deficient MEF cell lines (C). After 24 h of serum starvation, 92.8% of WT MEFs, 76% of Rab8a−/− MEFs, 85.4% of Rab8bKD MEFs, and 88.0% of Rab8a−/−;Rab8bKD MEFs were ciliated. After prolonged serum starvation (48 h), all MEF cell lines retained primary cilia . Kif3a−/− MEFs remained nonciliated at all time points examined (A and B).Rab8ary cilia C. After ll lines C. After examined , A and B−/− and Rab8a−/−;Rab8bKD MEFs contained multiple basal bodies and aberrant cilia . Interestingly, in some Rab8a−/−;Rab8bKD MEFs, multiple cilia grew from a single basal body, while some other Rab8a−/−;Rab8bKD cells contained two sets of basal body and cilia . These changes were rarely observed in WT or Rab8bKD MEFs. Thus, although the overall growth of a primary cilium may not require Rab8, the loss of both Rab8a and Rab8b did appear to affect basal body duplication and the morphological outcome of primary ciliogenesis.Although the absence of either single or double Rab8 did not appear to prevent the formation of the primary cilium, a fraction of Rab8ant cilia D, inset.−/−, Rab8bKD, and Rab8a−/−;Rab8bKD MEFs (A). Likewise, BBS1, a subunit of the Bardet–Biedl Syndrome protein complex shown to interact with Rab8, was also localized to primary cilia in WT and Rab8-deficient MEFs (B). Thus, ciliary transport of Smo and BBS1 was not affected by Rab8 deficiency in MEFs.Although Rab8 deficiency did not prevent the formation of the primary cilium in our analysis, literature suggested that there might be abnormal transport of certain ciliary cargos in the absence of Rab8 . We firsbKD MEFs A. Likewient MEFs B. Thus, C and E). After 24 h of starvation, patches of Fzd2, at the base of primary cilia, was detected in 88.7% of WT cells and numerous small Fzd2-stained puncta (C and E). Approximately 50% of Rab8a−/− MEFs also displayed Fzd2 membrane patches localized to primary cilia and that was increased to 80% after 24 h of serum starvation. In addition, Rab8a−/− MEFs contained more fluorescent Fzd2 puncta even before serum starvation, and these puncta increased after 24 h of serum starvation . Although Rab8a−/− and Rab8bKD MEFs had approximately equal numbers of Fzd2 patches at the base of cilia, in Rab8bKD MEFs, the cilium-associated patches appeared relatively small and Rab8bKD cells lacked puncta either before or after starvation (C). Rab8a−/−;Rab8bKD MEFs were found to have fewer cilium-associated Fzd2 patches before starvation, and this deficiency became significant after 24 h of starvation (D and E). The formation of Fzd2 puncta in Rab8a−/−;Rab8bKD cells was not impaired (D). Kif3a−/− MEFs, while lacking primary cilia, still contained Fzd2 membrane patches and puncta (D).Planar cell polarity effectors are trafficked to the base of primary cilia through a mechanism dependent upon Fuzzy recruitment of Dishevelled to Rab8 vesicles , 29, 67.d puncta , C and Earvation C. Rab8a−arvation , D and Eimpaired D. Kif3a−d puncta D.F). Interestingly, we observed the induction of a truncated Fzd2 that was prominent in Rab8a−/− and Rab8a−/−;Rab8bKD MEFs, and to a lesser extent in Rab8bKD , suggesting that lack of Rab8 led to a possible post-translationally modified Fzd2. Rab8a−/− and Rab8a−/−;Rab8bKD MEFs also showed a concomitant induction of Tcf1 that was not seen in WT or Rab8bKD cells . Together, these data collectively suggested that Rab8 plays a critical role in regulating the membrane positioning of Wnt signaling components and proper MEF differentiation in response to adipogenic signals.The above data indicated abnormal Fzd2 trafficking in the absence of Rab8 during adipogenic induction. We examined Fzd2 protein in various MEFs by Western blots before and after adipogenic induction. Full-length Fzd2 appeared in all cell lines before and after adipogenic induction  . Future studies will elucidate the precise molecular mechanisms of such regulation.We also observe an interesting localization of Fzd2 to a disk of membrane at the base of the primary cilium. In  cleaved , 81, 82.−/−;Rab8bKD MEFs exhibited the strongest defect in adipogenesis, to an extent similar to Kif3a−/−cells that had both impaired ciliogenesis and adipogenic potential  for viral concentration. The viral pellet was resuspended with 200 μl of DMEM and aliquoted for later usage. For Rab8b KD, MEFs were infected with diluted lentivirus stock  for 5 h in DMEM in the presence of polybrene (8 μg/ml) and then incubated with complete DMEM containing 10% FBS for another 24 h. Puromycin (3 μg/ml) was added into the culture medium for selection. KD efficiency and the maintenance of KD were confirmed by Western blotting.Rab8b-specific lentiviral shRNA construct targeting against the 3'UTR of mouse Rab8b was constructed by inserting the annealed complementary oligonucleotides (5'-CCGGGCCAAGAACTAACAGAACTTTCCATGGAAAGTTCTGTTAGTTCTTGGCTTTTTG-3' and 5'-AATTCAAAAAGCCAAGAACTAACAGAACTTTCCATGGAAAGTTCTGTTAGTTCTTGGC-3') into pLK0.1 lentiviral vector (Addgene) between AgeI and EcoRI sites. For viral packaging, this lentiviral vector along with pVSV-G was transfected into GP2-293 cells (Clontech) using Lipofectamine 2000 (Invitrogen). After 48 h, the supernatant was collected and subjected to ultracentrifugation . 24 h after transfection, 100% confluent cells were serum-starved for 8 h and treated with 50 ng/ml Wnt3a  dissolved in PBS or 20-μM CHIR99021 (Stemgent) for 16 h. For β-catenin ΔN group, 100% confluent cells were serum-starved for 24 h. Cells were then lysed, and the luciferase assay was carried out by using the Dual-Luciferase Reporter Assay Kit (Promega) and the GloMax-Multi Detection System (Promega) according to the manufacturer's instruction. pcDNA3 ΔN47 β-catenin was from Addgene   at 4 °C. The lysates were incubated on ice for 15 min and spun down in 4 °C, 17,000g for 15 min. The supernatant was collected and analyzed for the protein concentration. After adding lithium dodecyl sulfate and 50-mM DTT, the samples were incubated at 70 °C for 10 min. Samples were loaded on SDS-PAGE gels, transferred to polyvinylidene difluoride membranes, and blocked in 5% skim milk in Tris-buffered saline with 0.1% Tween 20 for 1 h at room temperature (RT). The membrane was incubated with primary antibodies: LRP6 , p-LRP6 (S1490) , Axin1 , Dvl2 , Dvl3 , β-catenin , p-β-catenin , GSK3β , p-GSK3β , β-actin , TCF-1 , Caveolin-1 , Rab5 , Rab7 , Rab9 , Rab8 , Rab11a , Fzd2 , Fabp4 , Glut4 , Erk1/2 , and pErk1/2  at 4 °C overnight in a dilution of 1:1000 unless indicated. After washing with 1x Tris-buffered saline with 0.1% Tween 20, the membranes were incubated in the secondary antibody  for 1 to 2 h at RT and developed in ECL detection reagents .After starvation for 16 to 24 h, WT and Rab8a −/− MEFs cells were treated with vehicle or Wnt3a (100 ng/ml) and harvested at 0, 2, and 4 h. MEF cells were washed with ice-cold PBS and lysed in the lysis buffer  for 4 h before harvest. Cells were harvested in Hank’s Balanced Salt Buffer on ice, pelleted, and lysed for 20 min in an extraction buffer which contains 30-mM Tris (pH 7.4), 140-mM sodium chloride, 1% Triton X-100, 25-mM sodium fluoride, 3-mM sodium orthovanadate, 2-mM PMSF, and protease inhibitor cocktail tablet (Roche). The lysates were centrifuged, and the supernatant was layered on top of a 15 to 40% sucrose gradient 30-mM Tris (pH 7.4), 140-mM sodium chloride, 0.02% Triton X-100, 25-mM sodium fluoride, 3-mM sodium orthovanadate, and protease inhibitors. Ultracentrifugation was performed in a Beckman SW55Ti rotor at 45,200 rpm for 4 h at 4 °C. After centrifugation, fractions were collected from the bottom of the tube by a peristaltic pump and analyzed by SDS-PAGE and immunoblot.This method is according to the previous study . BrieflyFor BODIPY 493/503 staining, the powder was dissolved in 100% ethanol to a final concentration of 1 mg/ml. In the dark, the differentiated cells plated on coverslips were fixed in 4% PFA for 10 min, washed in 1x high-quality PBS twice, and incubated in 0.1% Triton X-100 for 10 min at RT. Cells were then incubated in BODIPY 493/503 (1:1000 of stock in 1x PBS) for 30 min at RT and counterstained with TO-PRO-3 (1:500 in 1x PBS) for 15 min at RT. Coverslips were dried and mounted onto slides using the ProLong Anti-fade reagent (Thermo Fisher Scientific) and sealed with clear nail polish. Imaging was performed using a Zeiss LSM 510 Confocal Microscope.For ciliogenesis, cells were plated in 6-well plates containing coverslips. All cells were grown and maintained in overconfluency for 48 h and then incubated in a serum-free medium for 0, 2, 24, and 48 h, respectively. On the day of staining, cells were fixed in methanol at −20 °C for 5 min. Permeabilization occurred in 0.1% Triton X-100 in PBS for 10 min at RT. Cells were blocked in 10% goat serum in PBS for 30 min at RT and then incubated with primary antibodies: acetylated α-tubulin , γ-tubulin  in 10% goat serum in PBS, at 4 °C overnight. On the second day after washing with PBS, cells were incubated with the fluorescently conjugated secondary antibody  in the dark for 1 h at RT. After washing in PBS, cells were counterstained with TO-PRO-3  in PBS for 15 min at RT. Coverslips were dried and mounted onto slides with the ProLong Anti-fade reagent  and sealed with clear nail polish. Imaging was performed using the Zeiss LSM 510 confocal microscope.Immunostaining was performed as described previously , 43, 45.For LPR6 immunofluorescence detection, cells were transiently transfected with pCS2+LRP6 GFP . At 100%Uninduced MEFs were harvested at 90% confluency. Induced MEFs were harvested 48 h after the start of induction. RNA extraction was performed by using the RNeasy kit (QIAGEN) according to the manufacturer's instruction. Quantitative PCR analysis was described previously , 84. Prit test, one-way ANOVA, or two-way ANOVA. Significance was accepted at p < 0.05. Calculations and graphs were generated using GraphPad Prism (7.04).All results represent three or more independent experiments unless stated otherwise. Western blots, dimension of lipid droplets, and the cilia length were measured by ImageJ . For each cell line, ciliated cells were counted manually against total cells from six low-magnification confocal images. The lipid-droplet number and size were quantified using particle analysis in ImageJ. Data represented the means ± SEM from independent experiments. Statistical analysis was performed by Student’s All data are contained within the article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


C1QTNF6 (CTRP6), a member of the CTRP family, has recently been implied to play a role in the tumorigenesis of for a variety of cancer types. However, the role of C1QTNF6 in oral squamous cell carcinoma (OSCC) and its potential molecular remains unclear.C1QTNF6 expression was detected by qRT-PCR and western blot analysis. Lentiviral vectors were constructed to knockdown C1QTNF6 in CaL27 and SCC-9 human OSCC cell lines. Cell viability, cell cycle and cell apoptosis analyses were performed by MTT assay, PI/Annexin V staining, and flow cytometry. The effect of C1QTNF6 knockdown on in vivo tumorigenicity of OSCC cells in vivo was evaluated using nude mouse xenograft tumor model. Downstream signaling mechanisms were identified by microarray and Ingenuity Pathway Analysis.Immunohistochemistry of OSCC tissue and data from TCGA demonstrate that C1QTNF6 was overexpressed in OSCC tissues, and that cellular proliferation was significantly decreased after C1QTNF6 was knockdown in CaL27 and SCC-9 cell lines. Knockdown of C1QTNF6 also resulted in cell cycle arrest at the G2/M phase and enhanced cell apoptosis in in CaL27 and SCC-9 cell lines. Furthermore, knockdown of C1QTNF6 in Cal-27 cells inhibited tumor growth of OSCC in vivo. Microarray analysis revealed that C1QTNF6 silencing resulted in significant alterations of gene expression, with the Acute Phase Response signaling pathway significantly activated following C1QTNF6 silencing.These results suggest that C1QTNF6 plays an important role in promoting OSCC tumorigenesis, which indicates that C1QTNF6 may comprise a promising therapeutic target for OSCC treatment.The online version contains supplementary material available at 10.1186/s12935-021-02377-x. Oral squamous cell carcinoma (OSCC) is the most common epithelial malignancy in the oral cavity, accounting for almost 90% of oral malignancies . Despite−/− mice, C1QTNF6 was demonstrated to serve as a novel regulator of the complement alternative pathway with potential to treat rheumatoid arthritis in clinical studies [The C1q/tumor necrosis factor‑related protein family (CTRPs/C1QTNF), including 16 members, contains four domains: a N-terminal signal peptide, a collagenous-like domain, a short variable region, and a C-terminal globular domain . CTRPs a studies . However studies , 19. In  studies . Consist studies . In part studies . This evIn this study, we analyzed The Cancer Genome Atlas (TCGA) and found that C1QTNF6 might be a potential tumor-associated regulator involved in the carcinogenesis of OSCC. Furthermore, we confirmed the high expression of C1QTNF6 in OSCC tissues by immunohistochemical analysis and positive expression in OSCC cell lines by RT-PCR. We performed C1QTNF6 knockdown in OSCC cell lines and xenografted tumors to explore the pathological relevance between C1QTNF6 expression and OSCC, including its potential role in the proliferation and apoptosis of OSCC cells in vitro and tumor growth in vivo. We also performed signal pathway analysis to explore the possible molecular mechanisms underlying the regulatory function of C1QTNF6 in OSCC by Ingenuity Pathway Analysis (IPA). The data suggest that the Acute Phase Response signal pathway might play a role in C1QTNF6-mediated promotion of OSCC tumorigenesis.2 at 37 °C.CAL27 and SCC-9 head and neck squamous cell carcinoma cell lines were cultured in DMEM  supplemented with 10% fetal calf serum , 100 μg/ml streptomycin , and 100 U/ml penicillin . Human immortalized oral epithelial cell (HioEC) were cultured in a defined keratinocyte-SFM medium . Cells were maintained in a fully humidified atmosphere containing 5% COhttps://cancergenome.nih.gov/). For head and neck squamous cell carcinoma (HNSCC), there were 528 samples with available data in the TCGA database, including 520 RNAseqV2 samples, and 40 pairs with sample data and pathological information. Our expression profile analysis was based on these 40 paired samples of RNAseqV2 data. The Trimmed Mean of M-values (TMM) method was applied for data standardization. To avoid errors caused by inappropriate sample grouping, the BCV  was observed for quality control.For expression analysis, we selected RNAseq and RNAseqV2 paired sample data. The raw sequencing data and pathological information were downloaded from TCGA database  in expression were calculated, and differentially expressed genes with log2|FC|≥ 1.0  were considered to be significant. The remaining genes were filtered out. Additionally, several other criteria were adopted to filter genes: reported functional and clinical relation in HNSCC; multiple transmembrane proteins; those for which gene annotation was not clear (such as the open reading frame); and those reported in more than 100 articles in PubMed. The final gene list was randomly concentrated, and C1QTNF6 was selected for further experimental research.5 cells per well) and incubated at 37 °C with 5% CO2 until they reached ~ 70% confluence. Then, lentivirus was added at MOI 1. After 72 h, cells were observed under a fluorescence microscope and harvested for subsequent experiments.C1QTNF6-targeting short hairpin RNA (shRNA) oligonucleotide sequences (TGTGTGAGATCCCTATGGT) were designed, synthesized and cloned into the pGV115-GFP vector by GeneChem Corporation . Scrambled shRNA (TCTCCGAACGTGTCACGT) was also inserted into the pGV115-GFP vector and used as the negative control. Lentivirus transfection was performed according to the manufacturer’s recommended protocol. CAL27 and SCC-9 cells were seeded into a six-well plate  according to the manufacturer’s instructions. Reverse transcription was performed to synthesize cDNAs using M-MLV reverse transcriptase (Promega) and OligodT primers . C1QTNF6 mRNA expression was detected by quantitative real-time PCR using SYBR Master Mixture  on a Real-Time PCR machine TP800 (Takara). The cycling parameters were: 95 °C for a 30 s hot start followed by 45 cycles of 95 °C for 5 s and 60 °C for 30 s. Primers used were as follows: GAPDH forward: 5′-TGACTTCAACAGCGACACCCA-3′, GAPDH reverse: 5′-CACCCTGTTGCTGTAGCCAAA-3′, C1QTNF6 forward: 5′-GAAAGGGTCTTTGTGAACCTTGA-3′, and C1QTNF6 reverse: 5′-CTGCGCGTACAGGATGACAG-3′. The relative mRNA expression was calculated using the 2Proteins were extracted, and the protein concentration was adjusted to 2 μg/μL and stored at − 80 °C for later use. Total protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes at 4 °C (300 mA for 150 min). The PVDF membranes were blocked in 5% skim milk in TBST for 1 h and incubated overnight with C1QTNF6 , ID1 , JAK2 , DDIT3 , MK167 , or BBC3  at 4 °C. After three washes in TBST, the membranes were incubated with secondary antibody coupled to HRP  before visualization. β-actin  was used as reference.OSCC tissues and normal tissues were immunostained for C1QTNF6 expression using a C1QTNF6 antibody  at a 1:100 dilution for 1 h according to the supplier’s instructions. The secondary antibody (goat anti-rabbit IgG) was added for 1 h at 37 °C. Images were obtained at the optical facility. Immunohistochemical staining of C1QTNF6 was evaluated semi-quantitatively by percentage of staining cells and staining intensity. The intensity score was defined as follows: 0, no appreciable staining; 1, weak intensity; 2, moderate intensity; 3, strong intensity; 4, very strong intensity. The staining percentage score was based on the proportion of positively stained cells (0–100%).Lentivirus-transfected cells were seeded into 96-well plates. Cell density was 2,000 cells/well in incubator with 37˚C and 5% CO2. Starting from the second day after laying the board, Celigo will test the reading board once a day for 3–5 consecutive days. By adjusting the input parameters of Analysis Settings, the number of cells with green fluorescence in each scanning orifice was accurately calculated. The data were statistically plotted and the cell proliferation curve for 5 days was plotted. Normalization process of celigo experiment is as follows: the cell count values of each group at each time point were calculated and compared with those of the group on the first day, so as to obtain the proliferation multiple of the cells in this group at each time point. According to the proliferation multiple and points in time, the growth curve based on cell proliferation multiple was drawn.Lentivirus-transfected cells were seeded in 6-cm dishes. When the cells achieved approximately 80% confluence, they were trypsinized, washed twice in PBS, and fixed with pre-chilled 70% ethanol for at least 1 h at 4 °C. Then the cells were washed with PBS and stained with PI mixture (40 × PI stock (2 mg/ml), 100 × RNase stock (10 mg/ml) and 1 × PBS buffer at a dilution of 25:10:1,000) for 45 min at 37 °C. The cell cycle results were measured on a Guava easyCyte HT , and all experiments were performed in triplicate.MTT assay was used to measure the cell viability. Briefly, Lentivirus-transfected cells (2000 cells/well) were seeded in five 96-well plates for test last 5 days. MTT  solution (20 μl) was added to a final concentration of 5 mg/ml, and the cells were incubated for 4 h. The medium was then removed, and 150 μl of dimethylsulfoxide (DMSO)  was added and incubated for 30 min at room temperature. The absorbance of the samples was measured at 490 nm using a microplatereader .Cell apoptosis analysis was measured using the eBioscience™ Annexin V Apoptosis Detection Kit APC according to the manufacturer’s instructions. Briefly, Lentivirus-transfected cells were trypsinized, washed twice in pre-chilled D-hank’s, and resuspended in binding buffer. Then 5 μl of annexin V-APC was added to 100 μl of cell suspension and incubated at room temperature in the dark for 15 min. Samples were analyzed on a Guava easyCyte HT .7) were inoculated into the right side of the axillary of nude mice subcutaneously. The mice were observed every 5 days. The length and width of tumors were measured using calipers, and the volume of tumors was calculated using the equation (L × W2)/2. On day 35, the animals were sacrificed, and the tumors were removed and weighed. Luciferase activity in tumor tissue was detected by IVIS LuminaXR system .Four-week-old female BALB/c nude mice were purchased from the Shanghai Institute for Biological Sciences . Twenty BALB/c nude mice were randomly divided into two groups (n = 10/group). CaL-27 cells were transfected with either C1QTNF6-shRNA or Ctrl-shRNA lentiviral vector labeled with luciferase. Lentivirus-transfected cells  tool to examine biological functions and disease correlations as well as functional relationships between genes and gene networks.Next, we used the Ingenuity Pathways Analysis . Data were expressed as the mean ± standard deviation (SD) and the statistical significance between two groups was assessed using To determine whether C1QTNF6 may have a role in OSCC, we downloaded and analyzed data from 40 paired cancer and normal tissues from The Cancer Genome Atlas (TCGA). The results suggest that C1QTNF6 tends to be more highly expressed in OSCC, indicating that it could potentially be a tumor-associated regulator involved in the carcinogenesis of OSCC. As additional verification, we collected 13 paired OSCC and normal tissues and observed the levels of C1QTNF6 by qRT-PCR. Compared with adjacent normal tissues, the expression of C1QTNF6 was higher in the OSCC tissues . The efficiency of the C1QTNF6 knockdown was confirmed by qRT-PCR and western blotting. C1QTNF6 mRNA levels were significantly reduced in both cell lines after C1QTNF6-shRNA transfection, with approximately 60–80% knockdown efficiency , including 943 upregulated genes and 685 downregulated genes. The heat map shows hierarchical clustering analysis of differentially expressed genes, which revealed that OSCC cells from three samples shared similar gene expression patterns after C1QTNF6 knockdown , the Acute Phase Response was the top-activated signaling pathway following C1QTNF6 silencing. This signaling pathway is an important component of anticancer responses among multicellular organisms . The acu. DDIT3 (DNA-damage inducible transcript 3) promotes ovarian cell apoptosis in mice via ER stress activation, and knockdown of DDIT3 suppresses cell apoptosis [BH3-only protein BBC3 (BCL-2-binding component 3) belongs to the Bcl-2 family and is a strongly proapoptotic gene that is subject to transcriptional regulation by multiple cell death-signaling pathways . It has poptosis . DDIT3 ipoptosis . Over-expoptosis . High leThere is a balance between the proliferation and apoptosis of tumor cells, and the regulation of this balance determines the development and progression of the tumor. Based on our findings, we speculate that C1QTNF6 knockdown leads to complex changes in OSCC, which produces an anti-cancer effect through acute phase response both in vivo and in vitro. Microarray analysis suggests that changes in the expression of target genes, including ID1, BBC3, and DDIT3, could cause cell cycle arrest and promote apoptosis of OSCC. Our study provides a possible molecular mechanism underlying C1QTNF6-mediated promotion of OSCC tumorigenesis, which needs to be further studied, especially cell cycle factors, apoptosis proteins, and ER stress. Our findings not only provide a novel target gene for OSCC therapy but also add new evidence supporting the relationship between C1QTNF6 and tumorigenesis  and parp (B) related with apotosis in SCC-9 cells. *P<0.05, **P<0.01, ***P<0.001.Additional file 4. The total amount of fluorescence expression of the C1QTNF6-shRNA group and Ctrl-shRNA group.


Vaccinia virus (VACV) belongs to the genus Orthopoxvirus of the family Poxviridae. There are four different forms of infectious virus particles: intracellular mature virus (IMV), intracellular en-veloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). The F13 protein occupies the inner side of the CEV- and EEV-membranes and the outer side of the IEV-membranes. It plays an important role in wrapping progress and EEV production. We constructed a human single-chain fragment variable (scFv) library with a diversity of ≥4 × 108 independent colonies using peripheral blood from four vaccinated donors. One anti-F13 scFv was isolated and characterised after three rounds of panning. In Western blotting assays, the scFv 3E2 reacted with the recombinant F13VACV protein with a reduction of binding under denatured and reduced conditions. Two antigenic binding sites (139-GSIHTIKTLGVYSDY-153 and 169-AFNSAKNSWLNL-188) of scFv 3E2 were mapped using a cellulose membrane encompassing 372 15-mere peptides with 12 overlaps covering the whole F13 protein. No neutralisation capa-bilities were observed either in the presence or absence of complement. In conclusion, the con-struction of recombinant immunoglobulin libraries is a promising strategy to isolate specific scFvs to enable the study of the host-pathogen interaction. Orthopoxvirus (OPXV) of the family Poxviridae [Vaccinia virus (VACV), a member of the genus xviridae , was usexviridae . While vxviridae ,6,7,8,9.xviridae . TherefoThere are four different types of infectious virus particles: intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV) ,12. The Although smallpox has been eradicated, there is a rising interest in neutralising antibodies as well as antiviral drugs because of the fear of bioterrorism ,36. The VACV scFv antibody retrieved from a human immunoglobulin library isolated from an OPXV vaccine. The specificity, binding affinity and virus neutralisation capacities of the F13 scFv were compared to that of a monoclonal antibody.In this study, we constructed an anti-F13The permanent monkey kidney cell line MA-104 cultured in minimum essential medium (MEM) and supplemented with 7% foetal calf serum was used to propagate the VACV strains Elstree and Munich 1 . InfectiVirus preparations were purified and concentrated by sucrose gradient centrifugation as described previously ,41. The VACV [g. Protein contents of all antibodies preparations were determined according to the method of Lowry et al. [For this study, the A27-specific murine mAb anti-VACV 5B4/2F2 (epitope #1A) ,41 and tVACV ,41, usedy et al. .®  according to the manufacturer’s instructions as described before [6)  as directed by the manufacturers. To construct the scFv library, RT-PCR was performed using total RNA of at least 107 cells per volunteer as described before [Four human volunteers were immunised via scarification with Dryvaxd before . 20–28 dd before .BamHI and HindIII were introduced to both ends of the primers. The reserve primer “F13 ” 5′-GAT CAA GCT TTT AAA TTT TTA ACG ATT TAC-3′, and the following forward primers were used to amplify the full-size F13 protein and the truncated fragment #1: “F13 forward ”: 5′-GAT CGG ATC CAT GTG GCC ATT TGC ATC GG-3′, “fragment #1 ”: 5′-GAT CGG ATC CAG AAT CCT ATA GGT GGA GTG-3′. The PCR reactions were set as follows: initial denaturation at 95 °C/10 min, 35 cycles of 94 °C/1 min, primer annealing at 50.6 °C/1 min and 72 °C/2 min, and a final extension step of 72 °C/10 min.For the amplification of the F13L gene and its truncated sub-fragment, primers were selected by using a published sequence (GenBank accession number: NC_006998.1). The restriction enzymes E. coli. Ligation and transformation were performed according to the manufacturer. Plasmids of positive colonies were isolated from 5 mL LB media using the MiniPrep Kit . The genes were sequenced with an ABI Prism 3100 Analyzer . Sequences were analysed using the DNAStar program  and BLAST [The PCR products were ligated into the pSC-A-amp/kan PCR cloning vector  and transformed into chemically competent nd BLAST .® TOP10 Electrocomp™ E. coli.  and were grown in LB media with 1 mM Ampicillin at 37 °C until OD600 nm = 0.6. The expression was induced with 1 mM IPTG at 37 °C for 5 h while shaking at 200 rpm . Cells were pelleted (4500 rpm/20 min) and resuspended in TBS buffer containing 7.7 mM Tris pH 7.5/150 mM NaCl before sonicated (100% on ice/ 20 min). Cell debris was removed by centrifugation (2100 rpm/15 min). The lysate was pelleted at 7000 rpm for 1 h and resuspended in suspension buffer containing 12.5% phosphate buffer/1% 2 M imidazole stock/23% of 43.5% glycerol stock at 4 °C overnight. Afterwards, the lysates were mixed with an equal volume of 16 M urea stock and incubated by gently shaking at 4 °C for 1 h. Lysates were clarified at 17,000× g at 4 °C for 45 min. Supernatants were compounded with Ni-NTA agarose  and incubated under gently shaking at 4 °C overnight. The mixtures were loaded onto 5 mL Ni-NTA columns . The purifications were done according to the manufacturer instructions by using a binding buffer pH 8.0 containing 12.5% phosphate buffer/1% 2 M imidazole stock/23% of 43.5% glycerol stock/50% 16 M urea stock and an elution buffer containing 12.5% phosphate buffer/5% 2 M imidazole stock/23% of 43.5% glycerol stock/50% 16M urea stock. After overnight dialysis against PBS, the protein concentrations were determined by Lowry protein assay as described previously [Plasmids were ligated into the expression vectors pQE80L and pQE81L , electroporated into One Shot eviously .To get specific anti-F13 scFv antibodies, the constructed human anti-OPXV-scFv phage library was panned three times, applying purified recombinant F13 protein of VACV Munich 1. The exact screening procedure was performed as previously described .E. coli HB2151 pre-cultures were transferred into 100 µL 2 × TYG (0.1%)-A and incubated at 30 °C for 4 h. The expression was induced by the addition of IPTG to a final concentration of 2 mM dissolved in 50 µL 2 × TY-A by gentle shaking at 30 °C overnight . The cells were centrifuged  and the supernatants were applied in an ELISA for pre-screening as described before [2 server [To produce antibody fragments without pIII fusion, 0.5 µL of the d before . The posd before ,48 and I2 server  and VMD Production of the scFv yielding the highest ELISA value was scaled up to one litre and the antibody was purified as described before . FinallyFor Western blotting analyses, 5 µg/slot of gradient purified VACV Elstree, gradient purified VACV Munich1, purified recombinant A27 protein and of the purified recombinant F13 protein were fractionated by vertical 12% sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis  and subsVACV, 5 µg/mL A27VACV, and with 2 µg/mL BSA. After blocking, antibodies were added in two-fold serial dilutions starting with a concentration of 200 µg/mL. Incubation was performed at 37 °C for 3 h. After washing with PBS five times, the detection of the second antibody occurred either with goat pAb to E tag (HRP) (1:2000), anti-mouse IgG (whole molecule) or anti-rat IgG (whole molecule) peroxidase conjugate developed in goat (1:2000) at 37 °C for 1 h. After washing with PBS ten times, the developing solution composed of tetramethylbenzidine (TMB) was added. The reaction development was stopped using hydrochloric acid. The photometric reading  was performed at 450 nm. The binding affinity was calculated from the average adsorption of three independent assays using the Michaelis–Menten behaviour [Microtiter plates were coated with either of 2 µg/mL VACV Elstree, 2 µg/mL VACV Munich1, 15 µg/mL F13ehaviour  using GrA total of 372 amino acids representing whole F13 protein were synthesised directly on derivatised cellulose membranes in the form of 15-mere peptides with 12 aa overlaps. The synthesis of derivatised cellulose membranes was performed as described before . The proA confluent monolayer of vero cells was cultivated on glass coverslips placed in 24-well tissue culture plates. Then, 100 pfu (100 µL/well) of VACV Munich1 was added to each well. After incubation at 37 °C for 3 h, the virus mixture was replaced with a fresh medium containing 2.5% FCS. Cells were incubated at 37 °C for about 18 h, followed by removing the medium. Cells were fixed on glass coverslips with 4% paraformaldehyde in PBS at room temperature for 20 min. After that, 0.2% Triton X-100 in PBS was added and incubated at room temperature for 5 min to permeabilise the cells. After three washing-steps with PBS, cells were blocked with PBS supplemented with 10% FCS (200 µL/well) at room temperature for 30 min. Subsequently, the blocking solution was removed and 100 µg/mL of the primary Abs diluted in PBS supplemented with 10% FCS (250 µL/well) were incubated at 4 °C for 2 h, followed by further 30 min at room temperature. After three washing-steps with PBS, both the Hoechst’ reagent  to stain the cell nuclei and the secondary Abs  diluted in PBS containing 10% FCS (250 µL/well)) were added and incubated at room temperature for 1 h. After three washing steps with PBS, the glass coverslips were mounted in Mowiol/DABCO. Fluorescence was examined on a confocal laser scanning microscope  equipped with a Plan-Apochromat 63×/1.40 Oil DIC objective.To assess the neutralisation abilities of selected antibodies, a confluent monolayer of vero cells was grown in 24-well tissue culture plates. The PRT was performed as described before . PlaquesE. coli HB2151 colonies were isolated to produce soluble antibodies in a microtiter well format. No specific binding of scFv was observed after the first and second selection rounds. The third selection round revealed one clone with an adsorption three times over the background designated as 3E2. The scFv 3E2 (Acc. No.: MW520862) was sequenced and classified to the human VH3/D2/JH3-VK3/JK4 families . The binding affinities were calculated according to Michaelis–Menten  Figure .m) of 10.97 ng/mL and a maximal velocity (vmax) of 1.25 were recorded against the F13 protein  starting at aa 197 with a total length of 532 bp was constructed A to coveThe epitope mapping was verified by Western blotting analysis, whereby the mAbs 15B6 and 5B4/2F2, as well as an anti-his tag Ab, were used as controls . The scFThe VACV proteins F13 and A27 have both functions in virion wrapping. To investigate a believed interaction between them, a confocal study was done by infecting a confluent monolayer of Vero cells with 100 pfu of VACV Munich1 for 3 h. After fixing and permeabilisation of the cells, the Abs were incubated. Fluorescence was examined under a confocal laser scanning microscope. Penetrating the cell through permeabilisation was recognised for the following: scFv 3E2 A, the mAThe classical plaque reduction test (PRT) was performed in triplicates with a starting concentration of 200 µg/mL of the antibodies. The VACV-Elstree was neither neutralised by the scFv 3E2 A nor by VACV scFv, which was isolated from a human anti-OPXV-immunoglobulin library by phage display. The epitope of the scFv 3E2 was assigned to the 37 kDa F13 protein, which is the most abundant membrane protein of the EEV [In this study, we characterised a purified human anti-F13 the EEV ,29 and l the EEV . F13 pla the EEV ,57. In W the EEV ,57. ThesClostridium difficile [For epitope mapping, a cellulose membrane, spanning 372 15-mere peptides with 12 overlapping amino acids covering the whole F13 protein was used. The scFv 3E2 reacted with two epitopes differently located on the F13 primary structure (139-GSIHTIKTLGVYSDY-153 and 169-AFNSAKNSWLNL-188). Another study shows that one antibody detected two almost identical epitopes of ifficile . Howeverifficile . Other difficile . For theifficile  were eviifficile . Althougifficile ,64,65,66ifficile . Howeverifficile ,68. In c


Apoptosis of vascular smooth muscle cells (VSMCs) is closely related to the pathogenesis of cardiovascular diseases, and oxidative stress is an important cause of VSMCs’ death. Inhibiting VSMCs apoptosis is an effective preventive strategy in slowing down the development of cardiovascular disease, especially for atherosclerosis. In this study, we found that oxidation resistance protein 1 (OXR1), a crucial participator for responding to oxidative stress, could modulate the expression of p53, the key regulator of cell apoptosis. Our results revealed that oxidative stress promoted VSMCs apoptosis by overexpression of the OXR1-p53 axis, and 6-shogaol (6S), a major biologically active compound in ginger, could effectively attenuate cell death by preventing the upregulated expression of the OXR1-p53 axis. Quantitative proteomics analysis revealed that the degradation of p53 mediated by OXR1 might be related to the enhanced assembly of SCF ubiquitin ligase complexes, which is reported to closely relate to the modification of ubiquitination or neddylation and subsequent degradation of p53. Nowadays, cardiovascular disease remains the major cause of morbidity and mortality worldwide . Increas2O2) exposure (Oxidation resistance protein 1 (OXR1), a crucial participator for the early response to oxidative stress , has beeexposure , but thevia the activation and inactivation of specific genes  and exhibits important pharmacological effects, including anti-oxidative, anti-inflammatory, neuroprotective, and anti-apoptosis  is a major biologically active compound present in ginger  was dissolved in DMSO with a high concentration of 100 mM and stored at −80°C as a stock solution. Hydrogen peroxide  and 6S was diluted freshly in a growth medium immediately before use. Cells were treated with H2O2 or 6S following recovery in fresh medium for the indicated time.The Rat arterial vascular smooth muscle cell (VSMC) was obtained from the Cell Bank of Chinese Academy of Sciences  and cultured in Dulbecco’s modified Eagle medium (DMEM), including 4.5% glucose  and supplemented with 10% fetal bovine serum , 100 IU/ml penicillin and 100 μg/ml streptomycin at 37°C with 5% CO2. The optical density (OD) of each well was determined under 450 nm wavelength with the Varioskan Flash .Cell Counting Kit-8  was employed to determine the cell viability of VSMCs. VSMCs were seeded in 96-well plates and grown to an 80% confluence in complete medium. Then, VSMCs were performed with a series of treatments, replaced with fresh medium containing 10% CCK-8 solution, and incubated for 3 h at 37°C with 5% CO2O2 for 2 h after incubation with 6S  for 24 h. Then, the SOD activity, MDA content, and LDH leakage were measured by commercial chemical assay kits  according to the manufacturer’s instructions.The VSMCs were exposed to 1 mM H2O2 for 2 h after pre-treated with different concentrations of 6S for 24 h.The OXR1 siRNA was synthesized by GenePharma  and transfected the VSMCs using siRNA-mate-plus following the manufacturer’s recommendations. The sense sequence for the OXR1 siRNA was 5′-GGAGAUAAAGCUACAGGAATT-3′, and the antisense sequence was 5′- UUCCUGUAGCUUUAUCUCCTT-3′. The sense sequence of the negative-control siRNA was 5′-UUCUCCGAACGUGUCACGUTT-3′, and the antisense sequence was 5′-ACGUGACACGUUCGGAGAATT-3′. After 24 h transfection, the cells were exposed to 1 mM HThe RIPA lysis buffer  was used to lyse the cell pellets and then collected the supernatants and determined the protein concentrations by BCA protein assay kit . The protein extracts were separated by SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane , and incubated with specific antibodies of OXR1 , p53 , p21 , Bax , cleaved caspase-3 , and GAPDH  at dilution of 1:500 incubated at 4°C overnight after being blocked with 5% non-fat dry milk in Tris-buffered saline containing 1% Tween-20 (TBST) at room temperature for 2 h. Then, the membranes were incubated with secondary antibody at room temperature for 2 h and exposed to the enhanced chemiluminescence-plus reagents according to the manufacturer’s protocol after extensive washing. Finally, the protein bands were detected using Bio-Rad Image Studio and normalized against GAPDH.p < 0.05 was considered statistically significant.All the data were presented as the mean ± standard deviation (SD) and analyzed by one-way analysis of variance with Tukey’s multiple comparison tests in GraphPad Prism 5.0 software. 2O2 injury, and 6S treatment (8 μM) groups were precipitated with five volumes of ice-cold acetone/ethanol/acetic acid (v/v/v = 50/50/0.1) at −20°C overnight. The precipitated protein was collected by centrifuging at 15,000 g for 30 min and washing with acetone and 75% ethanol in turn. Then, the protein sample was re-dissolved with 8 M urea/100 mM TEAB (pH 8.0) solution, and the protein concentration was determined by BCA assay. After reduction by 10 mM DTT at 60°C for 1 h and alkylation by 20 mM IAA in the darkness at room temperature for 30 min, the protein was eight times diluted with 100 mM TEAB buffer and digested by trypsin ) overnight at 37°C.The protein extracts of control, H2O2 injury, and 6S treatment (8 μM) groups were labeled with light (L), medium (M), and heavy (H) stable isotope dimethyl labels, and the equal amounts of protein digests from control and siRNA group were labeled with light (L) and medium (M) stable isotope dimethyl labels. The labeling reactions were performed by adding light labeling reagents (5 mM CH2O and 5 mM NaCNBH3), medium labeling reagents (5 mM CD2O and 5 mM NaCNBH3), and heavy labeling reagents (5 mM 13CD2O and 5 mM NaCNBD3) at 25°C for 1 h, respectively. 10% (v/v) ammonia solution was added to quench the labeling reactions, and then 10% (v/v) formic acid was added to further quench the reactions and acidify the samples. Then, the labeled samples were mixed for further usage. For phosphopeptides enrichment, the sample was mixed with equal volume of 80% ACN/6% TFA solution containing Ti4+-IMAC microspheres. After shaking for 30 min, Ti4+-IMAC microspheres were collected by centrifugation and rinsed with 50% ACN/6% TFA/200 mM NaCl and 30% ACN/0.1% TFA, respectively. Finally, the phosphopeptides were eluted with 10% (v/v) ammonia solution and lyophilized for analysis by LC-MS/MS.Stable isotope dimethyl labeling and phosphopeptides enrichment were performed in solution according to The samples were analyzed by LTQ Orbitrap Velos  with Accela 600 HPLC system  as described previously  with minRattus norvegicus, 8,133 sequences). Cysteine carboxamidomethylation was set as a static modification, and methionine oxidation, acetylation of protein N-term, and phosphorylation (STY) were set as variable modifications with up to two missing cleavages of trypsin allowed. Precursor ion mass tolerances were 20 ppm, and fragment ion mass tolerance was 0.8 Da. The false discovery rates (FDR) for peptide, protein, and protein phosphorylation sites were all set as < 1%, and a minimum length of six amino acids was used for peptides identification. Triplets were selected as the quantification mode with the dimethyl Lys 0 and N-term 0 as light labels, dimethyl Lys 4 and N-term 4 as medium labels, and dimethyl Lys 8 and N-term 8 as heavy labels. All other parameters are the default setting in MaxQuant. After filtering by the FDR ≤ 1%, only phosphorylation sites with location probability ≥0.75 and score difference ≥5 were considered as highly reliable results and used for further investigation. Finally, the results were imported into the MaxQuant associated software suite Perseus (The obtained raw files were searched using MaxQuant (version 1.3.0.5)  with a r Perseus  for furtThe mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD015127.2O2 is widely used as a rapid and sensitive oxidative stress-induced apoptotic model  were significantly increased upon H2O2 treatment, indicating that H2O2 successfully induced VSMCs apoptosis. Fortunately, their expression levels were attenuated by pre-treatment with 6S in a dose-dependent manner  was performed on localized sites  that were observed in a minimum of two replicates. As a result, a total of 282 sites (16.3% of the 1729 sites) belonging to 175 phosphoproteins changed significantly upon oxidative stress , which suppress p53 activity and induce its proteasome-dependent degradation by ubiquitinating it. On the contrary, p53 is stabilized and activated in response to stresses because of the posttranslational modification changes on p53 or/and its negative regulators, which disrupt the interaction between them . We obsen of p53 . Acetylan of p53 . Besidesn of p53 . Those oAfter demonstrating the possibility of p53 activation upon oxidative stress, we next investigated whether p53 participated in the protection process of 6S in VSMCs. Therefore, we looked at the phosphoproteomic difference occurring with 6S pre-treatment. A total of 449 sites (26.0% of the 1725 sites) belonging to 250 phosphoproteins displayed different changes upon oxidative stress  significantly contributes to the pathogenesis of cardiovascular diseases, including inflammation, hypertension, atherosclerosis, and even myocardial infarction . Recentl6S is the major bioactive compound present in ginger and possesses serious pharmacological activities, such as neuroprotective, anti-oxidative, anti-inflammatory, anti-diabetic, anti-tumor, and anti-platelet aggregation . 6S has 2O2-induced VSMCs apoptosis by inhibiting the activation of p53 and p53-mediated cell cycle arrest and apoptosis pathways. However, the modulation mechanism of 6S on p53 expression/activation in VSMCs is still unclear.As one of the most important PTMs, phosphorylation plays crucial roles in almost all cellular processes, including the regulation of apoptosis. To discover the action mechanism by which 6S protects VSMCs from oxidative stress-induced apoptosis, mass spectrometry- (MS-) based quantitative phosphoproteomic analysis had demonstrated the crucial role of p53 in oxidative stress-induced apoptosis and the protective effect of 6S. It has been well established that overexpression of p53 leads to growth inhibition/apoptosis in VSMCs, while suppression of p53 activation abrogates the p53-induced apoptosis . To date2O2 in a dose-dependent manner, suggesting that 6S protected VSMCs apoptosis by inhibiting the overexpression of the OXR1-p53 axis.OXR1 has always been considered a crucial participator for the early response to oxidative stress  and an iAt present, the regulation mechanism of p53 expression by OXR1 is completely unknown. In order to preliminarily find out the potential mechanisms, an MS-based quantitative proteomic analysis was applied to compare the protein expression changes after OXR1 silencing in VSMCs, and SKP1 was observed to change most notably with a 12.9-fold upregulation. SKP1 is an essential adaptor protein linking CUL1 and a series of F-box proteins to form the SCF complexes, which act as multi-protein E3 ubiquitin ligase complexes that promote ubiquitination and degradation of a large number of regulatory proteins involved in diverse processes . Accumulvia enhanced assembly of SCF complexes, and, by extension, protect VSMCs against apoptosis. Therefore, 6S could be of therapeutic use in preventing VSMC damage-related cardiovascular diseases.In summary, our results showed that 6S protected VSMCs against oxidative stress-induced apoptosis by regulating the OXR1-p53 axis. We had verified that the expression level of p53 was regulated by OXR1. In VSMCs, oxidative stress significantly promoted the expression of OXR1, resulting in the stabilization and activation of p53, which promoted VSMCs apoptosis. 6S could effectively prevent the upregulated expression of OXR1, which might lead to promoted ubiquitination or neddylation and subsequent degradation of p53


The biotransformation products were extracted, purified and isolated using silica gel column chromatography and semi-preparative HPLC. Spectroscopic methods including LC-HRMS, GC–MS, FT-IR, 1D and 2D NMR were used to elucidate the structure of the artemisinin metabolites.1H NMR spectroscopy was further used to study the time-course biotransformation. The antiplasmodial activity (IC50) of the biotransformation products of 1 against intraerythrocytic cultures of Plasmodium falciparum were determined using bioluminescence assays. A filamentous fungus Aspergillus niger CICC 2487 was found to possess the best efficiency to convert artemisinin (1) to a novel derivative, 4-methoxy-9,10-dimethyloctahydrofuro--isochromen-11(4H)-one (2) via ring rearrangement and further degradation, along with three known derivatives, compound (3), deoxyartemisinin (4) and 3-hydroxy-deoxyartemisinin (5). Kinetic study of the biotransformation of artemisinin indicated the formation of artemisinin G as a key intermediate which could be hydrolyzed and methylated to form the new compound 2. Our study shows that the anti-plasmodial potency of compounds 2, 3, 4 and 5 were ablated compared to 1, which attributed to the loss of the unique peroxide bridge in artemisinin (1). This is the first report of microbial degradation and ring rearrangement of artemisinin with subsequent hydrolysis and methoxylation by A.niger.Artemisinin is a component part of current frontline medicines for the treatment of malaria. The aim of this study is to make analogues of artemisinin using microbial transformation and evaluate their in vitro antimalarial activity. A panel of microorganisms were screened for biotransformation of artemisinin (Aspergillus niger CICC 2487 was found to be efficient for biotransformation of artemisinin• A novel and unusual artemisinin derivative was isolated and elucidated• peroxide bridge in artemisinin is crucial for its high antimalarial potency• The The pathway of biotransformation involves the formation of artemisinin G as a key intermediate• The online version contains supplementary material available at 10.1007/s00253-022-11888-0. Artemislaria Tu . Recent laria Tu . This th1) was transformed to various metabolites via deoxygenation (reduction)  was purchased from Macklin Co., Ltd (China) which was used for biotransformation and bioactivity studies. HPLC grade formic acid, acetonitrile, hexane and ethyl acetate were purchased from Merck . The other chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd. The thin layer chromatography (TLC) plate was obtained from Qingdao Haiyang Chemical Group Co., People’s Republic of China.Aspergillus niger CICC 2487, Bacillus subtilis CICC 10157, Streptococcus thermophiles CICC 6038 and Phanerochaete chrysosporium CICC 40719 were purchased from the China General Microbiological Culture Collection Center. Trichoderma reesei ATCC 56765 and Lactococcus lactis ATCC 11454 were purchased from the American Type Culture Collection. Saccharomyces cerevisiae is from Mauripan Co.A. niger was maintained on potato-dextrose agar plates at 4 ℃ and freshly sub-cultured before the transformation experiment. Fungal mycelia from agar slope cultures were transferred into 2-L Erlenmeyer flasks containing 1 L of potato dextrose liquid (PDL) medium, and incubated for 3 days at 180 rpm and 28 ℃ in a rotary shaker.The fungal strain Fungal mycelia were moved into 100-mL shaker flasks containing 40 mL of different types of culture media depending on the microbial strains . After 2A. niger mycelia were transferred into 100-mL Erlenmeyer flasks containing 40 mL of PDL medium for 3 days of incubation. The fermentation broth from the above Erlenmeyer flask was transferred into 2-L Erlenmeyer flasks containing 1 L of PDL medium with the same culture conditions. 20 mL of artemisinin (100 mg/mL in acetone) was added into each of the above flask with 1L-fermentation broth to achieve a final concentration of 2 mg/mL. The control was kept without addition of artemisinin. Substrate controls consisted of sterile medium containing the same amount of artemisinin and incubated under the same conditions. A total of 4 g of artemisinin was transformed with 2 L of fermentation broth. The cultures were maintained under the same conditions for 14 days. Later on, all fermentation broth was combined and the sediment was separated by centrifugation at 8500 g for 20 min. The supernatant (about 2 L) was extracted three times with ethyl acetate. The extract was evaporated under reduced pressure to afford a residue for further analysis and purification.A. niger. The re-dissolved transformation product was analyzed using LC-HRMS . The resolving power was 17,500 for full-scan and mass accuracy for 5 ppm. Chromatographic separation was achieved on an Agilent zorbax SB18 column . The LC–MS experiments were performed using a mobile phase A of 0.1% formic acid and a mobile phase B of acetonitrile containing 0.1% formic acid at a flow rate of 0.7 ml/min. The high-performance liquid chromatography (HPLC) gradient system for metabolites identification started with 30% A and linearly increased to 80% A in 5 min.Liquid chromatography and high-resolution mass spectrometry (LC-HRMS) were applied for detecting the biotransformation products of artemisinin by the culture supernatant of 2 , compound 3 , a mixture of compound 1 and 4 , and compound 5  were isolated from the extract of A. niger.The ethyl acetate extract (2.0 g) consisting of artemisinin transformation products was added to 4 mL of methanol, the precipitated solid was removed and identified as the untransformed artemisinin. The solution phase was evaporated and submitted for silica gel chromatography  eluting with hexane with increasing ethyl acetate (from 5–100%). 15 fractions were collected and the solvents were evaporated. The sub-fractions were further purified by semi-preparative high-performance liquid chromatography (HPLC) using Agilent 1220 LC (USA) system. The mobile phase A consisted of water and solvent B was methanol. The mobile phase calibration rose from 20% by 80% (A + B) over a period of 25 min to 100% B and kept at 100% for 10 min at a flow rate of 4 mL/min at 215 nm on semi-preparative HPLC column . Four compounds: compound A.niger. 100 mg of artemisinin in 2 mL acetone was also transferred to a flask containing only medium and incubated for 336 h. Cell culture of A. niger without addition of 1 was used as control. The transformation flasks were incubated and the extraction of transformation products at 48, 120, 192, 264 and 336 h in two replicates was performed as described above. The transformation products were identified by 1H NMR spectroscopy (400 MHz Bruker AVANCE NEO instrument) in CDCl3 in comparison with the 1H NMR data of 1–5 obtained before and that of 7 as reported . Bruker TopSpin 4.1.3 software or ACD/Labs 10 Freeware  was used to analyze the NMR spectra.1D and 2D NMR spectra of compounds 1–2 μL of the isolated compounds dissolved in ethyl acetate was injected into gas chromatography mass spectrometry (GC–MS) system consisting of an Agilent 7890 coupled to Agilent MS model 5975C MSD . The GC started from150 °C increasing to 300 °C at the rate of 10 °C/min, which was then kept at 300 °C for 4 min with a constant helium pressure (10 psi). The mass spectra data were acquired in the scan mode in the m/z range 40–1000.2, 3, 5, and a mixture of 1 and 4 were dissolved in methanol and injected to a LTQ Orbitrap mass spectrometer system  to determine their high-resolution molecular masses and their fragment ions (MS/MS) using  a positive or negative electrospray ion source.The purified compounds −1 using an attenuated total reflection (ATR) mode. The powder of isolated artemisinin derivatives were pressed against the ATR wafer.FT-IR instrument  was used to determine the functional groups of isolated compounds. FT-IR spectra were recorded from 4000 to 800 cm2: white powder. GC-EI-MS, m/z (%): 223.1 (1), 195.0 (1), 166.1 (80), 151.1 (92), 137.0 (100), 98.0 (40), 69 (35), 41.1 (50) : 3081, 2940, 2860, 1786, 1732, 1458, 1382, 1129, 992, 987. 1H, 13C NMR, and 2D NMR data (see Table Compound ) Figure . HRMS : 211.1 (50), 165.1 (100), 137.1 (55), 109.1 (10), 89.0 (20), 41.1 (30)  Figure . HRMS (n5): white powder. HRMS (positive ESI) m/z calcd for [M + Na]+, 305.1365, found 305.1366. GC-EI-MS, m/z (%): 222.1 (70), 179.1 (30), 150.1 (55), 93.1 (40), 43.1 (100) : 3243, 2988, 2860, 1734, 1459, 1391, 1198, 1137, 985. 1H and 13C NMR data are consisted with those reported  was added to all wells then incubated for 48 h at 37 °C in a gas chamber with 1% oxygen, 3% carbon dioxide and 96% nitrogen. A single-step lysis and bioluminescence assay to determine growth relative to control was carried out as previously described  of log10-transformed concentration versus normalized growth (%) using GraphPad Prism v5.0 .Parasite strain Dd21). Aspergillus niger was found to be most efficient to produce different products and selected for preparative scale biotransformation with artemisinin, while others did not yield detectable products. TLC analysis  high-resolution (HR) mass spectrometry (MS) analysis of the extract of A. niger after incubation with artemisinin (1) confirmed the formation of a number of transformation products (2–5)  and/or analytical high-performance liquid chromatography (HPLC) of the ethyl acetate extract of microbial cells after incubation with artemisinin . These compounds were identified using HR-ESI–MS, GC–MS, FT-IR, 1D and 2D NMR. Among them, compound 2 is a novel metabolite of artemisinin  as evidenced by GC–MS  indicating the presence of four unsaturation units. This is consistent with the mass for compound 2 found in LC-HRMS . 1H NMR  and two quaternary carbons. There is an ester carbon , three oxygenated carbons  and a methoxy group . 1H NMR, HSQC and 1H-1H COSY ; 0.94 ) and a OCH3 group ). HR ESI MS/MS showed a strong fragmentation ion at m/z 245.1152 [MCH3OH]+ after collision of ion at m/z 277.1512 [M + Na]+, which indicates a loss of CH3OH group (a mass difference of 32.0360).Compound S Figure . High-reS Figure  determinR Figure , 13C NMRT Figure  and HSQCT Figure  analysisY Figure  support 1H and 13C NMR of 2 with those of compound 6, 4-hydroxy-9,10-dimethyloctahydrofuro--isochromen-11(4H)-one  on the NOESY spectrum  and at 105.1 (C-4) on the HMBC spectrum  -isochromen-11(4H)-one, a new compound  from its molecular ion m/z at 240.1. After comparison of 1H and 13C NMR data of 3 with those of compound 2 -isochromen-11(4H)-one (compound 6)  by A niger, the transformation products were monitored using 1H NMR spectroscopy  in the medium at 30 ºC after 14 days without addition of A. niger  were observed as well as the formation of compounds 2 and 3. This indicated that certain enzymes from the fungi could catalyze these transformation processes. The levels of compounds 2, 3, 4 and 5 hardly changed from day 2 to day 14. Compound 7 decrease from day 2 to day 8, and artemisinin (1) was almost fully consumed on day 8. Interestingly and surprisingly, on day 11 and 14, artemisinin was found to be reformed and increased, while compound 7 was significantly decreased  and Penicillium chrysogenum (ATCC 9480) to deoxyartemisinin and 3- α-hydroxydeoxyartemisinin, respectively, was first reported in 1989  by Aspergillus showed to be an efficient machinery to metabolize artemisinin. Artemisinin was converted to deoxyartemisinin, 3-α-hydroxydeoxyartemisinin and 1-α-hydroxydeoxyartemisinin by Aspergillus niger AS 3.1858  by A. niger resulted in 5-β-hydroxyartemisinin and 7-β-hydroxyartemisinin  and 3-α-hydroxydeoxyartemisinin by A. niger VKM F-1119   and 3-α-hydroxydeoxyartemisinin  of artemisinin was identified, which is structurally similar to artemisinin G (7), previously found from a biotransformation product of artemisinin by A. niger VKM F-1119  to compound 4 in the culture medium was reported before  was initially formed via ring rearrangement of artemisinin (1) as also found previously by A. niger , suggesting that on day 8 the fungus A niger might produce an unique enzyme allowing the convertion of the degradation product (7) back to the original artemisinin via unususal ring closure and rearrangement reactions. This is somehow consistent with our large scale of biotransformation of artemisinin, where a portion of artemisinin was recovered over the purification process while compound 7 was not isolated at all. The possible enzymes involved and associated mechanisms are under further investigation.In this study, a novel metabolite  was 28.1 ng/mL with the antiplasmodial activity almost completely ablated following biotransformation for compounds 2, 3 and 5. Whilst one fraction has antiplasmodial activity at a reduced potency  compared to the artemsininin, this fraction contains both compound 1 (artemisinin) and compound 4 as a 1:1 mix. This suggests that the antiplasmodial activity of this fraction likely results from the artemsisinin content, and that compound 4 has no or almost none antiplasmodial activity. The antiplasmodial activity data is in agreement with repored data for compound 4 and 5 , this peroxide-containing moiety was removed and thus antiplasmodial activity lost. Nevertheless, the biotransformaton produced new molecular entities which are not easily accessible by chemical synthesis. Further derivation of these derivatives as precursors using chemical or biological means and additional biological activities including cytotoxicty against cancer are worth investigating. Our studies along with others indicated that A. niger could produce mainly the deoxyartemisinin derivatives, which can cause the significant loss of antiplasmodial activity. However, fortunately other microorganisms such as Mucor polymorphosporus  along with three known compounds has been generated using mild microbial transformation of atemisinin with a strain of A. niger CICC 2487, whose structure was elucidated using extensive spectroscopic analysis. The antiplasmodial activity of all the transfomed products signficantly decreased due to the loss of the peroxide moiety in artemisinin. Hydroxylation artemisinin derivatives retaining a peroxide bridge through microbial transformation should be the future focus, which will allow facile chemical conjugation of artemisinin with other antimalarial compounds to tackle artemisinin-sensitive or artemisinin-resistant malaria through dual or multiple mechanisms.In conclusion, a novel atemisinin derivative Supplementary file1 TLC analysis, GC–MS, NMR data and ICBelow is the link to the electronic supplementary material.


Streptomyces sp. SH-1312 for secondary metabolite production, with strong pharmacological activity, along with pharmacokinetics profile, was firstly investigated. The optimum metal stress conditions consisted of actinobacteria strain Streptomyces sp. SH-1312 with addition of mix metals (Co2+ + Zn2+) ions at 0.5 mM in Gause’s medium. Under these conditions, the stress metabolite anhydromevalonolactone (MVL) was produced, which was absent in the normal culture of strain and other metals combinations. Furthermore, the stress metabolite was also evaluated for its anti-oxidant and cytotoxic activities. The compound exhibited remarkable anti-oxidant activities, recording the IC50 value of 19.65 ± 5.7 µg/mL in DPPH, IC50 of 15.49 ± 4.8 against NO free radicals, the IC50 value of 19.65 ± 5.22 µg/mL against scavenging ability, and IC50 value of 19.38 ± 7.11 µg/mL for iron chelation capacity and the cytotoxic activities against PC3 cell lines were recorded with IC50 values of 35.81 ± 4.2 µg/mL after 24 h, 23.29 ± 3.8 µg/mL at 48 h, and 16.25 ± 6.5 µg/mL after 72 h. Further mechanistic studies have revealed that the compound MVL has shown its pharmacological efficacy by upregulation of P53 and BAX while downregulation of BCL-2 expression, indicating that MVL is following apoptosis in varying degrees. To better understand the pharmacological properties of MVL, in this work, the absorption, distribution, metabolism, excretion, and toxicity (ADMET) were also evaluated. During ADMET predictions, MVL has displayed a safer profile in case of hepatotoxicity, cytochrome inhibition and also displayed as non-cardiotoxic. The compound MVL showed good binding energy in the molecular docking studies, and the results revealed that MVL bind in the active region of the target protein of P53 and BAX. This work triumphantly announced a prodigious effect of heavy metals on actinobacteria with fringe benefits as a key tool of MVL production with a strong pharmacological and pharmacokinetic profile.Elicitation through abiotic stress, including chemical elicitors like heavy metals, is a new technique for drug discovery. In this research, the effect of heavy metals on actinobacteria  Microorganisms quickly adapt and retort to the variations in the availability and concentrations of metals within their harsh and dynamic habitat ,2. MicroLatterly, the usage of drugs derived from natural origin has increased dramatically because of their high safety profile and potency compared to synthetic pharmaceuticals ,14. EvenCurrently, a lot of researchers have been driven towards abiotic stress-discovery of natural products by using metals triggering plants ,19, and Streptomyces sp. SH-1312 strain to elicit secondary metabolites in the presence of metal ions in the culture medium. Furthermore, the metal-elicited produced compound was evaluated for its anti-oxidant and cytotoxic activities, including Absorption, distribution, metabolism, excretion, and toxicity (ADMET).In this study, we used a metal-stress approach to explore the ability of the terrestrial actinobacteria 2+, Zn2+, and mix metals (Co2+ + Zn2+) were chosen as initial elicitors premised on their previous ability to produce secondary metabolites in microbes. The cultivation experiment of strain SH1312 was carried out in Gause’s medium having Co2+, Zn2+, and (Co2+ + Zn2+) ions with four different initial concentrations in a rotatory shaker at 180 rpm for 10 days at 28 °C. The two metals Zn2+ and Co2+ did not elicit any compound at any concertation, but a new peak was elicited in (Co2+ + Zn2+)  was detected in metal treated culture but was nearly undetectable in non-metal culture (2+ + Zn2+) stressed broth, triumphantly announcing that a stress metabolite was triggered by mix metals stress.The impacts of elution mode, mobile phase, detection wavelength, and column temperature were conducted to determine the ideal HPLC setting for purification of the stressed metabolite of actinobacteria. Two different kinds of metals Co + Zn2+) . The alt culture . The 0.5 culture . There w culture . A new pStreptomyces sp. SH-1312 with molecular formula C6 H8 O2. 1H NMR  the proton NMR showed a total number of 4 signals δ2.04 , δ2.46 , δ4.40 , δ5.80   the 13C NMR spectrum showed the presence of 6 signals, including 1 carboxyl group at δ166.1 (C-1), 1 quaternary carbon at δ160.6 (C-3) (3) group at δ21.5 (C-6), one methane (CH) group at δ115.1 (C-2), and two methylene (CH2) groups at δ28.5 (C-4) and δ66.1 (C-5)  . 13C NMR.6 (C-3) . DEPT135.1 (C-5) . From th.1 (C-5)  21]. Th. ThStrep50 for MVL in DPPH was recorded as 19.65 ± 5.7***µg/mL while for ascorbic acid 6.52 ± 4.92 µg/mL was recorded. MVL showed 80.05 ± 3.88% inhibition of NO free radicals in comparison to ascorbic acid (81.69 ± 2.69% inhibition) with IC50 of 15.49 ± 4.8**** for MVL and 8.44 ± 4.17 µg/mL for ascorbic acid. Similarly, MVL has shown significant scavenging ability against OH● radicals and inhibited 72.69 ± 4.93% radicals at 100 µg/mL concentration compared to gallic acid (83.15 ± 3.67%) used as standard. IC50 for MVL and gallic acid was recorded as 19.65 ± 5.22*** and 6.26 ± 6.39 µg/mL, respectively. To gauge the chelation power of compounds to scavenge radicals, the iron chelation capacity of the compounds was evaluated. MVL was found to chelate 70.96 ± 5.79% (IC50 19.38 ± 7.11***µg/mL) iron radicals in comparison to EDTA with 81.52 ± 4.67% chelation capacity and IC50 of 10.20 ± 6.54 µg/mL. All the observations are exhibited in  used as standard. ICbited in  , the MTT method was followed. Prostate cancer cells (PC3) were treated with multiple concentrations of MVL for 24 h, 48 h, and 72 h duration. Cabazitaxel was used as a standard drug. A significant decrease in cell viability has been examined on 24 h, 48 h, and 72 h exposure with MVL. MVL showed significant time-dependent inhibition in PC3 cell line with ICreatment . While Creatment , Table 2Stress compound MVL was tested for cell migration of PC3 using an established in vitro scratch test for 24 h. MVL (10 µg/mL) appeared to induce a significant reduction in cell migration at 12 and 24 h treatment of PC3. This was estimated by comparing the scratch area at every observation time as a percentage of control at 0 h. The area of scratch at 24 h treatment with MVL was recorded as 61.10 ± 5.2% compared to control (16.62 ± 4.4%) .To unveil the possible mechanistic reason behind the anti-proliferation of cancer cell colonies in MTT assays, expression analysis of apoptotic proteins P53, BCL-2 and BAX were assessed via western blot technique . GAPDH sTo evaluate toxicity, blood lymphocytes were treated with 10 and 20 µg/mL MVL samples, 20 µg/mL EMS (positive), 1% DMSO in PBS (negative control), and comet assay was performed. The slides were envisioned under a fluorescent microscope, and the extent of DNA damage was evaluated by analyzing the photomicrographs via CASP 1.2.3.b image analysis. Analysis of 50–100 cells from each sample was performed to assess comet length, head length, tail length, tail moment, DNA content in the head, and tail of lymphocytes. MVL (10 and 20 µg/mL) showed no significant damage to nuclear material compared to EMS used as standard genotoxic control. The tail length (18.5 ± 1.4 µm) of EMS-treated DNA clearly indicates the production of nicks in nuclear content, representing a greater extent of genotoxicity. While the stressed compound MVL showed no significant toxicity to DNA having the least tail content compared to control. All the fluorescent photomicrographs of the treated lymphocytes and their comet parameters are presented in Absorption, distribution, metabolism, excretion, and toxicity (ADMET) prediction studies were conducted for the stress compound MVL.Po/w) XLOGP3 < +5.0, size (SIZE) 150 g/mol < MV < 500 g/mol, polarity (POLAR) 20Å2 < TPSA < 130 Å2, Insolubility (INSOLU) 0 < Log S (ESOL) < 6, Insaturation (INSATU) 0.25 < Fraction Csp3 < 1 and flexibility (FLEX) 0 < No. of rotatable bonds < 9. The Topological polar surface area (TPSA) scores of MVL were in the range of 20Å2 to 130Å2, suggesting MVL provides good transport properties in vivo. The physicochemical properties of the compounds are mentioned in . AccordiHIA and CNS absorption are important parameters checked for every biomolecule before its entry for drug formulation in the pharmaceutical or clinical trials field . The BloKp  isoforms. These enzymes play a crucial role in drug excretion, and these isoforms are metabolizing almost 75% of market available drugs. Inhibition of any of these isoforms results in causing some significant pharmacokinetics-based drug-drug interactions ,26. As mBefore any drug enters the clinical trials phase or pharmaceutical industry manufacturing phase, it is imperative to consider the drug’s toxicology profile . The strhttp://www.way2drug.com/gusar/acutoxpredict.htmL, accessed on 1 October 2021). Furthermore, for the prediction of lethal dose (LD50), the compound MVL had a score of more than 300 mg/kg and was classified as class 4; therefore, it is considered “harmful if swallowed” (300 < LD50 ≤ 2000)  values for rats with oral administration was conducted by the General Unregulated Structure-Activity Relationships (GUSAR) programme ( ≤ 2000) . In addi+ channels has been linked to fatal cardiac arrhythmias and before any biomolecule is elected as a drug candidate, it is mandatory by the FDA to check its hERG safety. The probability map of MVL derived after predicting cardiac toxicity by pred-hERG is given in The blockage of the hERG KThe docked complexes of MVL against BAX and P53 are formed and evaluated based on minimum energy values and ligand interactions patterns. Results showed that MVL showed good binding energy, value bind in the active region of the target protein .The generated docking complexes showed that MVL was confined in the active binding pocket of BAX, as mentioned in In P53 docking, MVL is confined in the active binding pocket of the target protein, as mentioned in This research focused on isolating a natural product MVL as a stressed compound from actinobacteria by abiotic stress technique. Furthermore, evaluating the in vitro pharmacological profile of MVL as an anti-oxidant and anti-cancer agent, including In-silico studies of ADMET and molecular docking, strengthened its profile.The metal-stress technique opens the road towards the most promising directions and increases the chances for novel secondary metabolite production. As proved from the previous findings, stress-elicited compounds are generally absent in normal culture method but after inducing abiotic stress the new compound with its unique carbon skeletons appeared in stressed culture ,5,17. Th50 value of 19.65 ± 5.7 µg/mL in DPPH, IC50 of 15.49 ± 4.8 against NO free radicals, the IC50 value of 19.65 ± 5.22 against scavenging ability, and IC50 value of 19.38 ± 7.11µg/mL for iron chelation capacity. These results clearly indicate that MVL has a robust anti-oxidant profile. Furthermore for the cytotoxic activities as mentioned in 50 values of 35.81 ± 4.2 µg/mL after 24 h, 23.29 ± 3.8 µg/mL at 48 h, and 16.25 ± 6.5 µg/mL after 72 h. To achieve the mechanistic studies for the compounds’ pharmacologic profile it was observed that MVL has shown its cytotoxic response by upregulation of P53 and BAX while downregulation of BCL-2 expression, indicating that MVL is following apoptosis in varying degrees.Nowadays, many natural products researchers have driven their research towards untapping the secondary metabolites with promising anti-oxidant and anti-cancer properties ,13,29,30ADMET is a crucial stage for every kind of biomolecule before its biotransformation into a drug . AccordiAs evident from our study, MVL has shown encouraging results in terms of p53 and BAX inhibition in the sense of molecular docking. Therefore, in these ways, special attention should be placed on investigating this process’s therapeutic importance.Thus, the current study revealed the potential advantages of Stress driven compound MVL and may be useful against different diseases. The research relied on in vitro and computational tools that documented pharmacological properties and bioactivities predictions. Moreover, clinical studies are necessary to confirm the findings of the present work. Nonetheless, the results of this work will provide future guidance for the design and development of new lead compounds as anti-oxidant and anti-cancer agents.High-performance liquid chromatography (HPLC) system was used with a set of Waters 996 Photodiode Array Detector and a Waters 717 plus Autosampler . On a Bruker AVANCE DMX 500 NMR spectrometer with TMS as an internal standard, 1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were measured at 25 °C.Soil sediment samples were obtained from the lower Orakzai Agency, Pakistan, at 300 feet in the bushy mountains. The sediments were collected in unpolluted areas near the lower Orakzai Agency in Pakistan. The samples were then taken to the lab and kept at 4 °C in the refrigerator.−1 dilution, the fresh soil sample of about 1–2 g was immediately subjected to the pre-sterilized glass vials and diluted with simulated seawater. The mixture was sonicated for 1 min to liberate microorganisms tied to the soil particles, then shaken for 15 min at room temperature. The serial dilutions of up to10−2, 10−3, and 10−4 were then prepared. To prevent fungal contamination, nystatin (0.05 g/L) was added to the pre-prepared isolation media (Gause’s synthetic agar). Following the preparation of the dilutions, 100 µL aliquots of each dilution were inoculated on each media and disseminated with the pre-sterilized spreader. For 15–20 days, the plates were incubated at 28 °C.To obtain a 10Streptomyces sp.Purified colonies of actinobacteria were collected and kept on agar media and stored at 4 °C. The actinobacteria were identified by their morphology, most commonly their colors. Based on the ITS 16S segment, the SH-1312 strain was recognized as a 2, ZnSO4, and (CoCl2 + ZnSO4) were added to the stressed culture media for SH-1312. The mycelium was withdrawn, and the culture broth was extracted twice with EtOAc in an equal volume.The metal stress and normal cultures of strain SH-1312 were conducted simultaneously in 500 mL flasks containing 200 mL liquid Gause’s medium for ten days at 28 °C in a rotatory shaker at 180 rpm. As a control, a standard broth culture of strain SH-1312 was conducted in two flasks. Additional 0.5 mM, 1 mM, 2 mM, and 4 mM/L CoCl2O/MeOH gradient from 20–100% for 0–30 min, 100% MeOH from 30–50 min with a constant flow rate of 0.8 mL/min using 210 wavelengths. The stress-induced metabolite was purified on preparative HPLC by setting a constant mobile phase of 70% MeOH for 28 min with a 9 mL/min flow rate.The analysis of isolated compounds was done through a reversed-phase HPLC-UV equipped with a C18 column. The isolation procedure was set on an HPLC with an H2O/MeOH gradient from 20%–100% for 0–30 min. As mentioned in 2O (70:30)”. The purified compound was then subjected to its 1D NMR . The structure of the stressed compound Anhydromevalonolactone (MVL)  was identified by 1D NMR.EtOAc (2 × 200 mL) was used to extract the 13 L of fermented broth. After the solvent had evaporated, the crude residue was dissolved in methanol and centrifuged at 120,000 rpm for ten minutes before being exposed to analytical HPLC. After subjecting to the HPLC the initial screening was conducted by setting an HTo evaluate the antioxidant potential of the stress compound, a multifaceted antioxidant evaluation of a stress compound was performed via DPPH scavenging assay, OH● radical scavenging assay, NO scavenging assay, and Iron chelating assay by following pre-described protocols .v/v heat-inactivated fetal bovine serum (HIFBS), 0.25 g/mL amphotericin B, and 100 IU/mL penicillin G sodium. 20 µL of a compound in 1% DMSO in PBS and 180 µL of PC3 cancer cells were combined in a 96-well plate to reach a final concentration of 20 µg/mL for evaluating the cytotoxicity on PC3 cancer cells. The cells were loaded at an assay density of 1 × 104 cells/mL. The plate was incubated in humidified 5% CO2 at 37 °C for 24 h, 48 h, and 72 h. Cabazitaxel was utilized as a positive control, and 1 percent DMSO in PBS was employed as a negative control. Cabazitaxel was procured from Enzo life sciences, Fisher Scientific  CAS-183133-96-2 (ENZCHM2170010). A stock solution (4 mg/mL) of Cabazitaxel was prepared in DMSO and diluted in 1× PBS to make it 1% DMSO. Then serial dilutions of the standard compound were prepared in such a way that wells received 50, 25, 12.5, 6.25, and 3.12 µg/mL of Cabazitaxel with ≤1% DMSO. After that, 20 µL of pre-filter sterilized MTT solution was added to plates and incubated at 37 °C for 4 h in a CO2 incubator. Colored formazan crystals were detached by deliberately removing the supernatant and dissolved in 100 µL of DMSO with absorbance measured at 540 nm using Elx 800 microplate reader. Cytotoxicity was calculated using the following formula:As and Ac show the absorbance of sample and negative control, respectively. Cytotoxicity was calculated with GraphPad Prism 5 software.MTT assay was used to assess cytotoxicity against the human prostate PC3 cell line . Non-adhIn vitro wounds were induced by a modified protocol described , and thePC3 cells were cultured in a T75 flask (1 × 106/flask). After 48 h, cells were treated with NA (20 µM and 40 µM) for 12 and 24 h. Following treatment, media was aspirated and cells were washed with cold PBS whose pH was maintained at 7.4 trypsinized and pelleted in 15 mL falcon tubes, and cold lysis buffer was added to the pellet. Protein extraction and western blot analysis were done by following the protocol described previously .DNA mutilation was evaluated by following the protocol as described by Peter et al. . To remohttp://www.swissadme.ch/index.php, accessed on 1 October 2021) was used to obtain ADMET properties of the stress compound [http://biosig.unimelb.edu.au/pkcsm/prediction, accessed on 1 October 2021) was used to predict the pharmacokinetic scores.ADMET  are the essential measurement tools for any compound before being elected as a drug candidate. The online web tool swiss ADME  was used for its early detection of potential hERG blockers and non-blockers [Blockage of the hERG Kblockers .Repossession of BAX and P53 from PDB www.rcsb.org, accessed on 1 October 2021), respectively. Energy minimization of target structures were carried out by using conjugate gradient algorithm and amber force field in UCSF Chimera 1.10.1 [The crystal structure of BAX and P53 was retrieved from the Protein Data Bank (PDB) having PDBIDs 2LR1 and 4MZI, . The grid box center values of  and size values were adjusted as  for BAX and P53, respectively for better conformational position in the active region of target proteins. The ligand was docked separately against BAX and P53 with a default exhaustiveness value = 8. The predicted docked complexes were evaluated based on the lowest binding energy  values and structure-activity relationship (SAR) analyses. The three-dimensional (3D) graphical depictions of all the docked complexes were accomplished by Discovery Studio (2.1.0) and UCSF Chimera 1.10.1 .The secondary metabolite patterns of an actinobacterial strain can alter under the presence of heavy metals supplemented to the fermentation medium, as proven by two chemical and pharmacological screening approaches. This work created a simple, quick, and easy approach for screening such kinds of secondary metabolites that were totally absent at the normal culture under optimal conditions. Strains isolated by the metal stress technique with enhanced pharmacological spectrum open a new road for the researchers to re-screen existing microbial strain libraries for unique secondary metabolites that emerged under the effects of heavy metals. The findings of this study triumphantly revealed that stress-driven discovery of potent biomolecule from the actinobacteria is an efficient technique for uncovering the microorganisms’ undiscovered pool of small molecules.