Phaeodactylum tricornutum is now realized as a promising producer for commercial Fx production due to its higher content of Fx than brown seaweeds with easily artificial cultivation and Fx extraction. In the present study, to improve Fx production in P. tricornutum, the mixotrophic cultures were applied to optimize initial cell density, light intensity, light regime and nitrogen supplementation. The results showed that the higher initial cell density (1 × 107 cells mL–1) and lower light intensity (20 μmol m–2 s–1) were favorable for biomass production and Fx accumulation. The maximal Fx content [16.28 mg g–1 dry weight (DW)] could be achieved under blue light (BL), but the highest biomass concentration (5.53 g L–1) could be attained under red: blue light  in the batch culture. A novel two-phase culture approach was developed to increase the biomass concentration to the highest value (6.52 g L–1) with the maximal productivity of Fx (8.22 mg L–1 d–1) through light shift from R:B ratio (6:1) in phase 1 to R:B ratio (5:1) by enhancing BL and tryptone addition in phase 2. The content and intracellular amount of Fx were also increased 8% and 12% in phase 2 compared to phase 1. The expression levels analysis revealed that genes encoding phytoene synthase (PSY), zeaxanthin epoxidase (ZEP), and fucoxanthin-chlorophyll-protein b (FCPb) were upregulated significantly, with downregulation of the gene encoding violaxanthin de-epoxidase (VDE), leading to the improvement of Fx in phase 2. The present study demonstrated the two-phase culture strategy could promote Fx productivity through enhancing biomass production and increasing Fx content, indicating that strengthening BL coupled with adding tryptone were effective to facilitate Fx production by mixotrophic cultivation of marine diatom P. tricornutum.Fucoxanthin (Fx), a kind of primary carotenoids in brown seaweeds and diatoms, has attractive efficacy in human’s healthcare including loss weight, the prevention of diabetes and Alzheimer’s disease. Marine diatom  Phaeodactylum tricornutum is realized as a promising producer for commercial production of Fx since it grows fast and contains higher amount of Fx [1% to 6% of dry weight (DW)], which is over one hundred-fold of brown seaweeds  could enhance Fx accumulation , aiming to reveal the metabolic regulation in two-phase culture process.In the present work, the mixotrophic P. tricornutum CCMP 1327 was kindly provided by Dr. Hanhua Hu in Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China. The seed culture was applied in 250-mL Erlenmeyer flasks containing 100-mL modified f/2 medium (–2 s–1 under white LED light and rotating speed of 150 r/min in shaking incubator. The modified f/2 medium contained (per liter): 20 g sea salt, 9.20 g glycerol (0.10 mol L–1), 10 mg NaH2PO4.H2O, 30 mg Na2SiO3.9H2O, 3.15 mg FeCl3.6H2O, 4.36 mg Na2EDTA.2H2O, 9.80 μg CuSO4.5H2O, 6.30 μg Na2MoO4.2H2O, 22 μg ZnSO4.7H2O, 10 μg CoCl2.6H2O, 180 μg MnCl2.4H2O. In addition, 1.17 g L–1 of tryptone and 0.30 g L–1 of urea  were added into the medium to final concentration of total nitrogen (TN) at 0.02 mol L–1. The medium was autoclaved at 121°C for 20 min followed by urea addition using stock solution filtered through 0.45 μm membrane. The seed culture in late logarithmic phase was harvested by centrifugation and re-suspended in the medium above as the inoculum for subsequent experiments.Marine diatom 2 medium  under mi6, 4 × 106, 7 × 106, 1 × 107 cells mL–1) were investigated by adding the inoculum and culturing for 14 days. Then, various light intensities  were evaluated at initial cell density of 1 × 107 cells mL–1 by cultivation for 12 days. The other culture conditions were same as the seed culture. Cell density, glycerol and nitrogen concentration were measured every 2 days during the cultivation. Biomass, content and volumetric concentration of Fx were measured at the end of cultivation.A series of initial cell densities  were evaluated at initial cell density of 1 × 10Two-phase culture approach was investigated to promote biomass and Fx production by light shift and nitrogen supplementation through two batch cultures.–2 s–1 was used in the mixotrophic cultivation for 6 days in phase 1, and then shifted to BL at 20 μmol m–2 s–1 with tryptone (T), urea (U) or the mixture  addition in the medium to final concentration of TN (0.02 mol L–1) in phase 2. The culture without nitrogen addition in phase 2 was set as the control.In batch 1, R: B light (6:1) at 20 μmol m–2 s–1 was used to culture for 6 days in phase 1, then BL was strengthened alone to form various R: B lights  at 25 μmol m–2 s–1, or shifted to pure green light (GL) at 20 μmol m–2 s–1 with tryptone addition in phase 2. The cell growth and nutrient consumption were monitored every 2 days during the cultivation. Fx production were analyzed at the end of culture. The cells in the culture of R: B light (5:1) at 25 μmol m–2 s–1 on the 6th, 8th, 10th, 12th day were collected for Fx detection and total RNA isolation.In batch 2, the two-phase culture with light shift to BL and tryptone addition (BL + T) was set as the control. In the experimental groups, R: B light (6:1) at 20 μmol m–1) of cells was calculated by the following formula:Cell density was determined by CytoFLEX flow cytometry . Specific growth rate  at time t(d) and time t0 (d) (where Ne t0 (d) .Biomass, mg L–1 d–1) was calculated by following formula:2-mL cell suspension was collected in a pre-weighed tube and centrifuged at 3300 × g for 3 min. The pellet was washed twice and dried in a 60°C oven to a constant weight for biomass measurement. The biomass productivity (P0 and DWt are the biomass concentration (g L–1) at time t0(d) and time t(d) (where DWime t(d) .–1) was determined by M-100 Biosensors . The TN concentration  was determined by DR2700 spectrophotometer  with reagent No. 2714100 (−1 d−1) and biomass yield per TN consumed  were calculated by following formula:Glycerol concentration (g L 2714100 . The nutt and GC0 are the glycerol concentration (g L–1) at time t (d) and time t0 (d).where GCt and NC0 are the TN concentration (mg L–1) at time t(d) and time t0(d), NC1 and NC2 are the TN concentration (mg L–1) before and after the nitrogen addition on the 6th day in two-phase cultivation.where NC0 and DWt are the biomass concentration (g L–1) at time t0 (d) and time t (d), NCt and NC0 are the TN concentration (mg L–1) at time t (d) and time t0 (d), NC1 and NC2 are the TN concentration (mg L–1) before and after the nitrogen addition on the 6th day in two-phase cultivation.where DWNatural pigments were extracted by organic solvents, and the qualitative and quantitative analysis were carried out by high performance liquid chromatography (HPLC) method modified from references . BrieflyTM Carotenoids column  was employed for pigments analysis. The column temperature was maintained at 30°C, the flow rate was 0.8 mL min–1, and detection wavelength was 440 nm. Methanol and methyl tert-butyl ether were employed as mobile phases A and B, respectively. The gradient program was as followed: 0∼6 min, A: 95%→80%, B: 5%→20%; 6∼12 min, A: 80%→60%, B: 20%→40%; 12∼19 min, A: 60%→55%, B: 40%→45%; 19∼20 min, A: 55%→95%, B: 45%→5%; 20∼23 min, A: 95%, B: 5%. The peaks of pigments were characterized according to the retention time of Fx and Chl a standards , and external standard curve was used for quantification. The volumetric concentration of Fx , intracellular amount of Fx  and Fx productivity  were calculated by the following formula:High performance liquid chromatography system  equipped with PDA detector and YMCFx0 and VCFxt are the volumetric concentration of Fx (mg L–1) at time t0(d) and time t(d).where VCPro Taq HS qPCR Kit  were employed. qRT-PCR analysis was performed on CFX96 TouchTM Deep Well Real-Time PCR Detection System . The gene coding β-actin was selected as an internal control  group on the 6th, 8th, 10th, 12th day was carried out using Plant RNA Kit  for qRT-PCR analysis. Evo M-MLV RT Kit for gDNA clean and SYBR Green Premix  control . The seqt method .t-test with SPSS V22.0 software. Significant differentiation level were set at ∗p < 0.05 and ∗∗p < 0.01 by compared with control group.All data were performed in biological triplicates and presented as mean ± standard deviation (SD). Origin V9.0 software was used to plot figures. The statistical analysis was performed by one-way analysis of variance (ANOVA) and LDS p < 0.01) (7 cells mL–1) and the maximal biomass concentration (3.73 g L–1) were achieved at the highest initial cell density (1 × 107 cells mL–1) (–1) and intercellular amount of Fx (1.23 pg cell–1) were attained at 4 × 106 cells mL–1   . The higls mL–1) . Even thlls mL–1 , the higls mL–1) . Therefo–2 s–1 (–1) and Fx content (16.03 mg g–1) could be reached at light intensity of 20 μmol m–2 s–1  was significantly increased from 20 μmol m–2 s–1 in this study (p < 0.05)  . Since Cee Chl a , the inccornutum . The traa and Fx . When cea and Fx . In diata and Fx . Under ha and Fx , which da and Fx . Therefo–1) with productivity (351.39 mg L–1 d–1) could be achieved at R: B light (6:1), which was 1.22- and 1.38-fold higher than WL (p < 0.01)  and R:B (30:70), which was consist with this study  reached to 28.49 mg mg–1 at R: B light (6:1) (–1) and Chl a content (32.68 mg g–1) were attained under BL (p < 0.01), respectively. These results were similar with that the Fx and Chl a contents were significantly higher under BL than WL in autotrophic P. tricornutum and Coscinodiscus granii (–1) under BL was the lowest one  were shown in  < 0.01) . Even th < 0.01) , which mis study . The incis study  was due is study . The maxht (6:1) . Moreoveunder BL , which ws granii . Howeverwest one  in this P. tricornutum produced more biomass under higher proportion of red light in mixture light under autotrophic condition , respectively. The highest biomass productivity achieved 373.61 mg L–1 d–1 under tryptone addition, which was significant higher than BL + U and the control group  and volumetric concentration (76.58 mg L–1) of Fx were attained by tryptone addition in phase 2, with the consumption rate of glycerol (233.33 mg L–1 d–1) and TN (13.69 mg L–1 d–1). Besides, the intercellular amount of Fx was significantly increased up to 0.61 pg cell–1 in BL + T group in phase 2, which was 1.42-fold higher than the control group (BL)  for 6 days in phase 1, then light shifted to pure BL for induction with or without supplementation of nitrogen  in different groups in phase 2. As shown in addition , which wol group . In contglycerol . This phoup (BL) . Compareoup (BL) , the biooup (BL) , respect–1), which was 17% higher than the control group (BL + T) (p < 0.01), was achieved with the maximum productivity (402.78 mg g–1 L–1) in R: B (5:1) + T group in phase 2  + T group in phase 2 (p < 0.05), the intercellular amount of Fx was similar in the two groups (–1) achieved the highest level (p < 0.05)  for 6 days in phase 1, various light shifts were evaluated in phase 2 compared with the control group (BL + T). Among them, BL was enhanced solely to form R: B lights , or light shifted to pure green light (GL). All of the groups were combined with tryptone addition in phase 2. The light spectrums were shown in  phase 2 . Glycero phase 2 , respecto groups  but the  < 0.05) . Therefoa was more inhibited compared to carotenoids under green light induction  + T group in the batch 2, the biomass and Fx production, nutrient consumption were analyzed in the two-phase culture. The cell samples were taken from the culture in phase 2 at four time points  to evaluate the Fx content and key genes expression in Fx biosynthesis pathway.–1 d–1) in phase 1, and then the cells utilized glycerol more quickly for rapid growth from 4th day, which might be due to the exhaustion of available amino acids in tryptone as carbon source of prior utilization. With the rapid consumption of glycerol in phase 2, the biomass productivity reached to 597.22 mg L–1 d–1, which was 2.87-fold higher than phase 1 (p < 0.01)  on the 8th and 12th day, respectively (p < 0.05)  . The glyntration . Howeverntration , which mectively . At the  < 0.05) . Throughd so far .DXS and PSY could raise the content of Fx by 2.40- and 1.80-fold in P. tricornutum, respectively  from the 10th day. Similarly, the previous report indicated that BL induction was proved to upregulate the expression of PSY under autotrophic condition in P. tricornutum . In the previous report, the accumulation of Fx was not synchronized with the abundance of PSY transcripts, while the Fx content at the stationary phase was correlated with the amounts of PSY transcripts at the exponential phase  on the 12th day (It is known that biosynthesis of Fx involved in methylerythritol phosphate (MEP) pathway, IPP pathway and Fx formation . Howeverectively . Under tectively , which i 6th day , suggest 6th day . In contcornutum . One posht shift , the othcornutum , and theal phase . Therefo12th day , which pZEPs were significantly upregulated  when the culture shifted to phase 2. Among them, ZEP1 was the most sensitive gene in response to the BL induction (ZEP1 transcript level was over 50-fold higher than ZEP2 and ZEP3 under BL induction after dark treatment (P. tricornutum did not have the specific enzymes of Dtx epoxidase/diadinoxanthin de-epoxidase (DEP/DDE) in Ddx cycle, and the enzymes of ZEP/VDE in violaxanthin cycle played the same role instead  were significantly regulated by BL strengthening and tryptone addition, which had positive effects on Fx accumulation, leading to the a great improvement of Fx production.In a word, the expression levels of key genes involving Fx biosynthesis (P. tricornutum, which achieved the highest level of ever reported so far. The analysis of gene expression levels involving Fx biosynthesis revealed that PSY, ZEPs, and FCPb were upregulated while VDE was downregulated under BL strengthening and tryptone addition, indicating the possible regulatory mechanism on the enhanced Fx production in phase 2. This study developed a novel approach of two-phase culture to produce Fx efficiently by the mixotrophic P. tricornutum, which facilitate the scale-up production of Fx by photo fermentation in the future.In this study, the combination of red: blue light at a favorable ratio in phase 1 and light shift with tryptone addition in phase 2 was employed to significantly improve Fx production by the mixotrophic All datasets presented in this study are included in the article.RY and DW conceived and designed the experiments and drafted the manuscript. RY performed the experiments and analyzed the data. DW contributed to the funding. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Soluble sugar levels increased before germination, but subsequently decreased, whereas starch contents were relatively unchanged. Changes to organic acids were observed at 120 h after sowing, including a decrease and increase in citrate and malate levels, respectively. Similarly, soluble protein contents increased at 120 h, but the abundance of most free amino acids decreased at 48 h. Moreover, the total amino acid levels increased only in response to VGB at 0 h. Accordingly, GABA and VGB altered the balance of carbon and nitrogen metabolism, thereby inhibiting chestnut seed germination. These results suggested that changes to GABA levels in chestnut seeds might prevent seed germination. The study data may also help clarify the dormancy and storage of chestnut seeds, as well as other recalcitrant seeds.Recalcitrant chestnut seeds are rich in γ-aminobutyric acid (GABA), which negatively regulates adventitious root development by altering carbon/nitrogen metabolism. However, little is known regarding the role of this metabolite in chestnut seeds. In this study, we investigated the effects of GABA changes on the germination of chestnut seeds treated with exogenous GABA and vigabatrin . Both treatments significantly inhibited seed germination and primary root growth and resulted in the considerable accumulation of H Castanea; family Fagaceae), which is a major nut crop in East Asia and Southern Europe, is unique among temperate nut crops because its seed is starchy rather than oily, making it ecologically and economically valuable [Cryphonectria parasitica [Chestnut  cultivar “Yanshanzaofeng” seeds with GABA and vigabatrin  to investigate seed germination changes, as well as the central carbon/nitrogen metabolic activities, which demonstrated that both treatments inhibited chestnut seed germination and might by altering the balance of carbon and nitrogen metabolism, which would provide a better understanding for elucidating the role of GABA during the storage and germination of recalcitrant chestnut seeds. The effects of GABA on chestnut seed germination remain unclear. Thus, on the basis of our previous findings that GABA regulates stress responses ,23 and aCastanea mollissima) cultivar “Yanshanzaofeng” seeds were collected from healthy trees growing in the core chestnut-producing region of Qianxi county, Tangshan city, Hebei province, China, after which they were stored at 0–1 °C until further use. Relatively uniform chestnut seeds were washed five times with sterile water and air-dried. The seeds were then soaked in sterile water (control/CK), 10 mM GABA , or 100 μM vigabatrin  for 15 h at 25 °C before they were placed evenly on a tray (33.5 cm × 26 cm × 11 cm) containing sterilized sand. A germination test was conducted in a climate chamber with a 16 h light (25 °C): 8 h dark (20 °C) cycle and 60% relative humidity. The seed germination rate was calculated, and the root length was measured after 2, 5, 8, 15, and 30 days. Five seeds per replicate were collected at 15 h before the treatment  and at 0, 48, and 120 h after sowing. After discarding the seed coats, the kernels were ground into a powder and stored at −80 °C for the subsequent physiological measurements. Each treatment was completed with three replicates, each comprising 50 seeds.Chinese chestnut , the supernatant was mixed with a titanium sulfate solution and concentrated ammonia. After another centrifugation (4000× g for 10 min at 25 °C), the sediment was dissolved in concentrated sulfuric acid and incubated at room temperature for 5 min. The absorbance of the reaction solution was measured at 415 nm, and the H2O2 content was recorded as μmol/g fresh weight (FW).The Hescribed . Brieflyg for 10 min at 25 °C), the diluted supernatant was added to a reaction mixture comprising anthranone in ethyl acetate and a concentrated sulfuric acid solution and then heated for 10 min at 95 °C. The absorbance of the reaction solution was measured at 620 nm, and the soluble sugar content was recorded as % FW.The total soluble sugar content was measured with a commercial assay kit  based on the anthrone-sulfuric acid method as previously described . Brieflyg for 5 min at 25 °C), and the sediment was dissolved in 0.5 mL sterile water and heated at 95 °C for 15 min. After cooling, 0.35 mL perchloric acid and 0.85 mL sterile water were added, and the resulting solution was thoroughly mixed and then centrifuged (3000× g for 10 min at 25 °C). A 50 μL aliquot of the solution was mixed with 250 μL reaction mixture (3.75 mL anthranone solution and 21.25 mL concentrated sulfuric acid) and then heated at 80 °C for 10 min. The absorbance of the reaction solution was measured at 620 nm, and the starch content was recorded as % FW.The starch content was quantified with a commercial assay kit (Comin Biotechnology) based on a previously described method . Brieflyg for 10 min at 4 °C). The diluted supernatant was mixed with Coomassie brilliant blue R-250, after which the absorbance was measured at 595 nm. The total soluble protein content was recorded as mg/g FW.The total soluble protein content was measured with a Bradford Protein Assay Kit  based on the Coomassie brilliant blue method. Briefly, 0.1 g fresh powder were suspended in 5 mL phosphate buffer (pH 7.2) and centrifuged (8000× g for 5 min at 4 °C), the supernatant was added to a reaction solution consisting of 100 μL sodium acetate-glacial acetic acid, 100 μL ninhydrin, and 10 μL ascorbic acid. After boiling for 15 min, the absorbance of the reaction solution was measured at 570 nm, and the total amino acid content was recorded as mg/g FW.The total amino acid content was measured with a commercial assay kit (Comin Biotechnology) based on a previously described method . BrieflyTo analyze the organic acid and amino acid contents, 0.4 g fresh powder were suspended in 2 mL sterile water and boiled at 100 °C for 90 min. After a centrifugation, the supernatant was filtered (0.45 µm pore size). The organic acid and amino acid contents of the filtrate were analyzed by high-performance liquid chromatography with the 2695 Separations Module  as previously described ,42. All 3OH + 97% H2O as the mobile phase . The organic acid contents were recorded as mg/g FW.Organic acids: The citrate, succinate, and malate contents of the prepared extract were measured at 214 nm with the Waters 2487 UV detector, a Thermo ODS HYPERSIL (4.6 mm × 200 mm) chromatographic column, and 3% CHAmino acids: The prepared extract was analyzed with a pre-column derivatized with the AccQ Fluor reagent. The mobile phase consisted of 140 mM NaAc solution  and a 60% aqueous acetonitrile solution. A Waters 2475 fluorescence detector and an AccQ Tag amino acid analysis column (3.9 mm × 150 mm) were used. The separate amino acids were detected at excitation and emission wavelengths of 250 and 395 nm, respectively. The amino acid contents were recorded as μg/g FW.3 (pH 9) buffer was added, and the solution was incubated for 1 h at 60 °C. The solution contents were separated on a C18 SunFire column (4.6 mm × 250 mm), and the eluted products were detected with the Waters 2487 UV detector. The column temperature was maintained at 35 °C, and the mobile phase comprised phosphate buffer (pH 7), water, and acetonitrile, with a flow rate of 0.1 mL/min. The separated GABA was detected at 360 nm. The GABA content was recorded as μg/g FW.GABA: The prepared extract was mixed with acetonitrile containing 1% 2,4-dinitrofluorobenzene, after which an equal volume of NaHCOp < 0.05 or p < 0.01 levels. A principal component analysis (PCA) was performed with the command prcomp in R (http://www.r-project.org/), as previously described [Data were compared and analyzed with ANOVA , and multiple comparisons were made with SPSS 16.0 . Differences were scored as significant at the escribed ,44.p < 0.05). These results indicated that both treatments had significant inhibitory effects on chestnut seed germination and early root growth.The CK, 10 mM GABA, and 100 μM VGB solutions did not induce seed germination on Day 2. The germination rate of the CK seeds increased after five days, whereas the 10 mM GABA and 100 µM VGB treatments inhibited chestnut seed germination and primary root growth at five days after sowing A. The ge2O2 contents, the CK level slightly increased. Surprisingly, the H2O2 contents increased considerably after the GABA and VGB treatments. After the 15 h imbibition, the H2O2 contents increased sharply by over 94.0% and 163.0% in response to GABA and VGB treatments, respectively, at 0 h, and remained high until 120 h  A and aftChestnut seeds are widely considered to be healthy for humans . EarlierRecent genetic and physiological studies have implied that GABA is involved in responses to abiotic stresses ,47, as wH. ammodendron [Previous studies revealed that endogenous GABA concentrations increase during germination ,17,52. Iodendron  seeds. Modendron . Thus, tCastanea sativa seeds [Generally, exogenous GABA promotes germination ,21 and eva seeds , and theva seeds . Accordiva seeds , which cva seeds . In this2O2  can enhance the germination of Kabuli chickpea seeds, which coincides with an increase in amino acid levels [2O2 also promotes the germination of eggplant seeds, accompanied by enhanced amino acid biosynthesis and protein expression [Brassica napus seedlings [In many plant species, most amino acids accumulate during seed germination . For exad levels . Exogenopression . Therefopression ,58,59,60eedlings , but theThe importance of GABA for human health and plant development has been confirmed. In this study, high GABA levels were detected in seeds before germination, implying GABA not only could influence chestnut seed germination, but also could act as a potential compound of functional chestnut food. This should be examined in greater detail in future studies. The application of exogenous GABA and VGB inhibited chestnut seed germination and early primary root growth, possibly by altering the balance between carbon and nitrogen metabolism, especially the free amino acid contents before germination . The dat


This study assessed whether adaptation to acidic pHe enhances the metastatic phenotype of tumor cells. The low metastatic variant of Lewis lung carcinoma (LLCm1) cells were subjected to stepwise acidification, establishing acidic pHe-adapted (LLCm1A) cells growing exponentially at pH 6.2. These LLCm1A cells showed increased production of matrix metalloproteinases (MMPs), including MMP-2, -3, -9, and -13, and pulmonary metastasis following injection into mouse tail veins. Although LLCm1A cells exhibited a fibroblastic shape, keratin-5 expression was increased and α-smooth muscle actin expression was reduced. Despite serial passage of these cells at pH 7.4, high invasive activity through Matrigel® was sustained for at least 28 generations. Thus, adaptation to acidic pHe resulted in a more invasive phenotype, which was sustained during passage at pH 7.4, suggesting that an acidic microenvironment at the primary tumor site is important in the acquisition of a metastatic phenotype.Acidic extracellular pH (pHThe online version of this article (10.1007/s10585-019-09990-1) contains supplementary material, which is available to authorized users. In the presence of oxygen, lactic acid is the main cause of extracellular acidification, a process called the “Warburg effect” or “aerobic glycolysis” [+ symporters, also known as monocarboxylate transporters (MCTs), the hypoxia-inducible subtype MCT4 is primarily responsible for the secretion of lactic acid. MCT4 exports lactate, thereby affecting the proliferation of tumor cells [2 by tumor carbonic anhydrase IX [e also induces the production of vascular endothelial cell growth factor (VEGF)-A [e microenvironment, whether independent of, in addition to, or synergistically with hypoxia, may support the malignant phenotype of cancer cells and play a role in metastasis.Extracellular pH -A , interle(VEGF)-A , and VEG(VEGF)-A  through e can act as a feed-back stimulator of a metastatic phenotype. Our investigations of the association of acidic pHe with the metastasis-related activities of mouse B16 melanoma variants, including the induction of matrix metalloproteinase-9 (MMP-9) expression, found that MMP-9 induction correlated with the metastatic activity of B16 variants and the acceleration of tumor invasion through type IV collagen sheets [e resulted in a switch from an epithelial to a mesenchymal phenotype, called an epithelial-mesenchymal transition (EMT) [e 5.9–6.8 was found to potentiate the invasive and metastatic activities of these cells [e in mouse B16-F10 melanoma xenografts with CEST-MRI [e of most early stage tumors ranged between pH 6.0–6.2, whereas the pHe of most late stages tumors ranged between pH 5.7–6.7, with 10% of the area of late stage tumors having a pHe < 5.5. These findings suggested that primary tumors were continuously influenced by pHe 6.0–6.2 over a long period and that adaptation of tumor cells to this pHe range is an important step in tumor metastasis.Tumor-derived acidic pHn sheets . Transieon (EMT) –13. Transe cells , 14–19. CEST-MRI  showed te. Tumor cell lines have been subjected to chronic extracellular acidification and/or adaptation to pHe 6.7 for 2 weeks to 3 months [e 6.2 could be attained [e increased tumor metastatic activity and whether the metastatic phenotype could be sustained at neutral pHe.Because an acidic microenvironment can chronically affect tumor cells in vivo, studies are needed to evaluate the chronic effects of pH3 months –23. We fattained . In thisTaq II was from Takara Bio , fetal bovine serum (FBS) was from Hyclone , and sodium pentobarbital was from Kyoritsu .Dulbecco’s modified Eagle’s medium (DMEM), Ham’s F12 medium, and High Capacity RNA-to-cDNA kits were purchased from Thermo Fisher Scientific . SYBR Premix Ex 3PO4 1.0 g/L NaHCO3, 100 units/mL penicillin G, and 0.1 mg/mL streptomycin sulfate, and its pH was adjusted with NaOH or HCl [2 incubator.A low metastatic variant of Lewis lung carcinoma (LLCm1) was established in our laboratory using an experimental lung metastasis method through tail vein injection . Basal mH or HCl . Cells we by serial passage through media of stepwise decreasing pH  until pH 6.2 was reached. The cells were maintained for 2–4 weeks at each pH and passaged 2–3 times per week, depending on growth rate. Adaptation to each pHe was confirmed by showing exponential growth after seeding cells at 2.5 × 105 cells/60 mm dish. Finally, acidic pHe-adapted cells (LLCm1A cells) were established by more than 40 passages (more than 3 months) through medium at pH 6.2 in the presence of 10% FBS. Where indicated, LLCm1A cells were passaged 3–10 times in medium at pH 7.4 in the presence of 10% FBS.Cells were adapted to acidic pH1 − T0)/log2 (N1/N0), with N0 and N1 defined as the number of cells at the initial time (T0) and after cultivation for time T (T1), respectively.Cells were suspended in medium at pH 7.4 containing 10% FBS and seeded onto 24-well plates. After 3 h, the medium was changed to medium of various pH containing 10% FBS. At this time, cells in some wells were counted and determined as the cell number at day 0. Cells were harvested using trypsin/EDTA and the number of cells in each well counted using the trypan blue dye exclusion method. Doubling time was calculated as ) and resuspended in ice cold PBS(-). In experimental metastasis assays [5 cells in 200 µl PBS(-) were injected into the tail vein of each 7-week-old male C57BL/6 mouse . Each experimental group consisting of 6 mice was housed in a cage. Animals were maintained in the barrier facility for laboratory animals with a 12 h light–dark cycle and allowed food and water ad libitum. Three weeks later, the mice were sacrificed by intraperitoneal injection of sodium pentobarbital (120 mg/kg). Their lungs were removed and the numbers of metastatic foci at lung surfaces were counted [All animal experiments were performed in accordance with the guidelines of the Ministry of Education, Culture, Sports, Science and Technology, the Ministry of Health, Labor and Welfare of Japan and ARRIVE . The exps assays , 25, 26,Taq II in a Thermal Cycler Dice Real Time System  using the specific primers listed in Table S1. The level of expression of each target gene was normalized relative to the level of Actb mRNA in the same samples. The data were analyzed by the Total RNA was purified using the acid-guanidinium-thiocyanate-phenol-chloroform (AGPC) method and reverse-transcribed to cDNA using a High-Capacity cDNA Reverse Transcription Kit. Target sequences were amplified by SYBR Premix Ex t method , with no2 for 24 h at 37 °C, and stained with Coomassie Brilliant Blue R-250.MMP-2 and -9 activities were determined by gelatin-zymography, as described , 12, 26.Wound healing assays were performed as described . Briefly®-coated polycarbonate porous filters (8 μm pores) mounted onto transwell chambers  as described [5 cells/100 μl/chamber on an insert consisting of a Matrigel® (37.9 μg/cm2)-coated filter. This insert had been mounted onto a well of a 24-well plate, which had been filled with 600 μl of 20% FBS-containing medium adjusted to the same pH as the chemoattractant. After incubation for 18 h, non-invasive cells were removed with a cotton swab and the invasive cells were fixed in 100% methanol, stained with Giemsa solution, and counted under a light microscope (× 200).In vitro invasive activity was determined using Matrigelescribed . Brieflyt-tests, and more than two samples compared by ANOVA and the Holm method [Results were expressed as mean ± SE. Two independent samples were compared by Student’s m method . Data ofe-adapted, or LLCm1A, cells, LLCm1 cells were conditioned by stepwise reductions in pHe, with the recovery of proliferative capacity confirmed at each pHe. Although LLCm1 cells continuously grew at pHe 6.5, they were unable to grow at pHe 6.2. A critical point was observed between pHe 6.5 and pHe 6.2. These cells were maintained at pHe 6.2 by medium renewal alone until significant growth was observed. Overall, more than 3 months were required to obtain proliferating LLCm1A cells at pHe 6.2. Acclimation involved the seeding of LLCm1 cells onto 24-well culture plates at pHe 7.4, followed 3 h later by replacement with medium at different pH; thereafter culture media were renewed every day. An obvious reduction in growth rate was not seen until pHe 6.5. However, cells showed almost no growth in medium at pHe 6.2. , showing that these cells had high seeding efficiency. LLCm1A cells had a fibroblastic shape and cell-to-cell contact was dispersed. In contrast, parental LLCm1 cells showed a cobblestone like morphology -MMP, was lower in LLCm1A than in LLCm1 cells -like changes, rather than EMT, occurred partly by adaptation to acidic pHe.Because LLCm1A cells had a spindle shape with little cell-to-cell contact, their expression of mesenchymal and epithelial cell markers was investigated. Unexpectedly, the expression of lls Fig. . Althouge dependent secretion of MMP-2 and -9 showed that the production of both enzymes was highly enhanced at pHe 6.8  conditions . In contrast, these cells spread well and could be maintained in serum-free and serum-reduced (2% FBS) conditions at pHe 6.5. MMP-2 and -9 levels and invasive activity were high under acidic conditions (pHe 6.5–6.8) without switching to neutral pH . Although MMP activities were reduced as pHe increased, these activities were significantly higher than in medium at pHe 7.4. These results seemed complementary to the transient increases in MMP expression  at neutral pHe, suggesting that the sustained invasive phenotype of these cells was likely not due to a memory effect but rather to an acquired phenotype. Thus, the acidic pHe-mediated acquisition of metastatic phenotype can likely be sustained in the circulation in vivo.Metastatic activity has been associated with the tumor microenvironment, which consists of growth factors, the extracellular matrix, hypoxia, and acidic pHnt cells . This mals [+ [+ . Initialhenotype , 31, 32. effects , 33, cal effects . This ste. Although Krt5 mRNA expression was higher in acidic pHe-adapted LLCm1A than in LLCm1 cells, it was lower in the latter cells exposed to transient acidification. In contrast, Trpm5 mRNA, which encodes a molecule involved in sensing acidic pHe and whose overexpression in patients with melanoma and gastric cancer has been associated with shorter survival [e-induced EMT [e-adapted LLCm1A cells unexpectedly showed reduced expression of Act2 mRNA, which encodes a mesenchymal marker, and increased expression of Krt5 mRNA. Our working hypothesis was that cells of primary tumors affected for a long time by acidic microenvironments metastasize through the circulation. EMT is an important step, especially for dissemination of cells from primary tumors, whereas MET is involved in the establishment of secondary tumor formation [e 7.4. The acquired metastatic potential of acidic pHe-adapted tumor cells was sustained at physiological pH, with these cells playing an important role in secondary tumor formation through MET-like conversion.We also observed differences between cells exposed to transient acidification and those adapted to acidic pHsurvival , was notuced EMT , 12, 35,ormation . This ste may also be regulated by these epigenetic alterations. Because highly proliferative cells consume glucose to generate ATP, and deoxyribose from the pentose-phosphate pathway, adaptation to extracellular acidification resulted in an escape from glucose dependence [e may have a partial CSC phenotype and may be a therapeutic target as much as CSCs [Transient and chronic extracellular acidification have been reported to affect metabolic pathways through epigenetic alterations, including histone acetylation and DNA methylation , 37–39. pendence . CSCs arpendence , suggest as CSCs .® [KRT5 mRNA expression was higher in early than in late passage cells of the human mammalian epithelial MCF10A cell line, with late passage cells having a more mesenchymal phenotype than early passage cells [The number of passages of cultured cells has been reported to affect tumor phenotype. Serial long-term or late passage was found to increase the metastatic activity of rat mammary adenocarcinomas , whereas® , and lat® . Moreovege cells , indicatge cells . In the e. Despite having growth potential at acidic pHe with high MMP production, LLCm1 cell clone 4 did not have invasive activity, suggesting that the acquisition of invasive and metastatic ability is likely due not only to a simple effect of serial passage, but to adaptation to acidic pHe. Because our experiments could not completely distinguish between simple clonal selection and adaptation to acidic pHe, both remain possible. Our results showed, however, that acidic pHe altered the tumor microenvironment, shifting tumor heterogeneity to the accumulation of a metastatic population. Because acidic pHe was reported to induce the expression of sterol regulatory element-binding protein 2 (SREBP2) in pancreatic cancer cells [These parental cells were serially passaged in our laboratory and showed a stable phenotype, as assessed by morphology, MMP production, in vitro invasiveness and experimental metastasis. These activities were not increased by serial passage, in contrast to previous findings . Moreoveer cells , lipid he.In conclusion, these findings suggest that prolonged tumor cell acidification induced a sustained invasive phenotype through a mechanism differing from that resulting from transient exposure to acidic pHSupplementary material 1 (DOCX 15 kb). Table 1S: Primer sequences.5 cells/cm2 per well in 24-well plates. After 1 day, the cells were cultured in medium at pH 7.4 (open circle), pH 6.8 (open square), or pH 6.2 (open diamond), each containing 10% FBS (arrow), and the medium was renewed on day 3. LLCm1A cells were cultured at a density of 1.5 × 105 cells/cm2 in medium at pH 6.2 (filled diamond), with medium renewed on day 2. Representative results of two independent experiments are reported as mean ± SE. In some cases, error bars are hidden by the data symbol due to small values (n = 3)Supplementary material 2 TIFF 215 kb). Figure 5 kb. FigA. Cell morphology after culture for 9.5 h. Bar, 100 μm. B. Zymographic analysis (Upper and Middle panels). The intensity of the gelatin-lysed clear zone was determined using Image J software . Data expressed as % of maximum ). In vitro cell invasiveness (Lower panel). Because cells incubated in serum-free medium at pH 6.2 became round in shape and detached rapidly from the culture dish, the cells were pre-incubated in medium containing 2% FBS for 18 h at each pH . Cells (5 × 105) were harvested, resuspended in medium containing 20% FBS at each pH value and placed onto Matrigel®-coated filters in transwell chambers. Cells that passed through to the lower surface of the filter were counted after Giemsa staining. In some cases, error bars are hidden by the data symbol due to small values. Representative results of two independent experiments are reported as mean ± SE (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001Supplementary material 3 (TIFF 193 kb). Figure 2S: LLCm1A cells showed high potentials for MMP production and in vitro invasion regardless of neutralization. Cells were pre-incubated in the presence of 2% FBS at the desired pH, with no switch to physiological pH for 18 h. Below is the link to the electronic supplementary material.


Germacrone, a monocyclic sesquiterpene, exerts marked antitumor effects in a variety of cancers, including hepatocellular carcinoma, gastric cancer, and breast cancer. However, the mechanism underlying the effects of germacrone on gastric cancer remains unclear. In this study, we show that germacrone inhibited gastric cancer cell proliferation in a dose-dependent manner, and induced G0/G1-phase cell cycle arrest and apoptosis in these cells. Moreover, germacrone increased the expression of LC3II/LC3I. And LC3II/LC3I was significant increased after germacrone treatment compared with germacrone and bafilomycin A1 (Baf A1) treatment, which suggested germacrone promoted the formation of autophagosomes. Proteomic analysis was then used to identify molecular targets of germacrone in gastric cancer. A total of 596 proteins were screened, and the top hit was identified as late endosomal/lysosomal adaptor and MAPK and MTOR activator 5 . Overexpression of HBXIP delayed the germacrone-induced cell cycle arrest, induction of apoptosis, and inhibition of autophagy. Combined, our results indicate that germacrone suppresses gastric cancer cell proliferation by inhibiting HBXIP, and this process is related to G0/G1-phase arrest and apoptosis. Gastric cancer is a leading cause of cancer-related deaths worldwide, and the prognosis for these patients remained dismal –3. The cCurcuma zedoaria is an important traditional herb that is widely used as a herbal medicine in China, India, and other Asian countries. It is indicated to exert antitumor effects in breast cancer, hepatocellular carcinoma, and gastric cancer through suppression of cell proliferation, metastasis, and angiogenesis in vitro , a protein that is highly expressed in several types of human cancer –23. We f2 at 37°C. Cells in the exponential growth phase were used in the experiments. The experiments were divided into four groups: controls, DMSO , germacrone , and Baf A1 (0.01 µM) or rapamycin . For Baf A1 or Rap and germacrone cotreatment, SGC7901 and MGC803 cells were first incubated with Baf A1 or Rap for 4 h, and then germacrone was added.Human gastric adenocarcinoma SGC7901 and MGC803 cells  were grown in DMEM supplemented with 10% fetal bovine serum  in a humidified atmosphere containing 5% COPlated SGC7901 and MGC803 cells were cultured in the presence or absence of germacrone  for 24, 48, and 72h. Proliferation was measured by MTT assay (Beyotime Biotechnology) to determine the effects of germacrone on cell proliferation. After germacrone treatment, MTT solution (5 mg/ml in PBS) was added (20 μl/well) and the plates incubated for another 4 h at 37°C. The formed purple formazan crystals were dissolved in 150 μl/well of DMSO. After 10 min, the absorbance at 490 nm was read using a microplate reader .SGC7901 and MGC803 cells were treated with germacrone for 24 h. Cytotoxic activity was determined by a standard lactate dehydrogenase (LDH) release assay. The percentage of cytotoxicity was calculated as described by the manufacturer (Beyotime Biotechnology).SGC7901 or MGC803 cells  were cultured for 24 h in the presence or absence of germacrone. The medium was changed and the culture continued until clear cell colonies were formed. Cells were then stained using the Wright-Giemsa Stain Kit .The proteins were separated by SDS–PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes  by electroblotting. The membranes were incubated overnight at 4°C with antibodies against GAPDH , KI67 , cyclin-D1 , CDK4 (Cat. No. ab108357), CDK2 , cyclin-E1 , cleaved caspase-3 , p62 , LC3 I/II , ANXA 1 , HSP70 , and HBXIP . The proteins were detected with an ECL chemiluminescence detection kit.Paraffin-embedded sections were dewaxed at high temperature using xylene and ethanol. Antigen retrieval was performed by boiling in sodium citrate solution, following which the samples were incubated with an anti-HBXIP antibody at 4°C overnight. After incubating the biotin-labeled goat anti-mouse/rabbit IgG and horseradish enzyme-labeled streptavidin working solution in this order, Staining was revealed with DAB . Stained sections were observed under a microscope and imaged. Staining values were calculated according to the semi-quantitative integration method. For to the proportion of observed positive cells, the values 0,1,2,3, and 4 represented ≤5%, 5%–25%, 26%–50%, 51%–75%, and ≥75% positive cells, respectively. The assigned values were then multiplied by the staining intensity . Value between 0 and 3 were considered as low expression, while a score >3 was considered as high expression.Flow cytometry (FCM) analysis was carried out on SGC7901 and MGC803 cells. For cell cycle analysis, cells were stained with propidium iodide (PI). DNA content was analyzed by FAC Scan. For the cell apoptosis analysis, cells were first stained with Annexin V–FITC and PI and detected by FCM. .Cells were seeded on sterile slides, cultured and treated, and the medium discarded. After washing 3 times with PBS, cells were fixed in 4% paraformaldehyde for 10 min. The appropriate amount of Hoechst 33258 (Beyotime Biotechnology) staining solution was added and the samples were incubated for 5 min in the dark. After washing 3 times with PBS, the plate was protected from light, and observed and imaged under a fluorescence microscope.Cells were centrifuged and collected, washed 3 times with PBS, and the supernatant discarded. To fix the cell morphology, 4% glutaraldehyde was slowly added and the cells were incubated overnight at 4°C. The next day, the cells were incubated in ethanol , air dried, and coated with gold. Cell morphology was then assessed by SEM.After treating MGC803 cells with 0.15% DMSO and 150 µM germacrone for 24 h, total protein was extracted. Protein lysates were precipitated overnight with 4 times volume of cooled acetone. The next day, the acetone was discarded and the lysates incubated with DTT (10 mM) and IAM (30 mM). Proteins were cleaved into peptides by adding 1 µg/µl trypsin . The collected enzymatic hydrolysates were desalted with a ZipTip pipette  and concentrated in a freeze-dryer . The precipitates were reconstituted with 0.1% formic acid.m/z scan range was 350 to 2,000 Da. The RAW data generated by Q-Exactive was outputted via the Xcalibur workstation (Thermo Fisher Scientific).LC–MS/MS: A nano-ESI HPLC system  equipped with a C18 column  was used for separation, and the injection volume was 4 μg/8 μl. Mobile phase A was 5% acetonitrile (ACN)  and mobile phase B was 95% ACN . The gradient elution program was as follows: 0–5 min, 5% buffer B; 5–45 min, 5%–35% buffer B; 45–50 min, 35%–90% buffer B; 50–55 min, 90% buffer B; 55–56 min, 90%–5% buffer B; 56–65 min, 5% buffer B. Q-Exactive (ThermoFisher Scientific): Intact peptides were detected in the Orbitrap at a resolution of 70,000 FWHM. Peptides were selected for MS/MS using high-energy collision dissociation (HCD) operating mode with a normalized collision energy setting of 27%; ion fragments were detected in the Orbitrap at a resolution of 17,500 FWHM. A data-dependent procedure that alternated between one MS scan followed by 15 MS/MS scans was applied for the 15 most abundant precursor ions above a threshold ion count of 20,000 in the MS survey scan with an ensuing Dynamic Exclusion duration of 15 s. The electrospray voltage applied was 1.6 kV. Automatic gain control (AGC) was used to optimize the spectra generated by the Orbitrap. The AGC target for full MS was 3e6 and 1e5 for MS2. For MS scans, the ftp://ftp.uniprot.org/), the relevant information was obtained for the detected protein. After excluding redundant proteins and filtering to an FDR <1%, we matched 4014 proteins. Filter by: unique peptides >2, abundance ratio>1.2, P<0.05, we screened suitable proteins. Filter by: unique peptides >2, abundance ratio >1.2, P<0.05, we screened suitable proteins.Data was imported into Proteome Discoverer . After searching the Human Proteome Database (2015.3.4) downloaded from the Uniprot website  and purified by sequential washing with chloroform, isopropanol, and 75% ethanol. The cDNA was synthesized by using Superscript First-Strand Synthesis System kits . HBXIP forward 5’-CGAGGTTTGCGGTGAAGG-3’and reverse 5’-CCACGGCAGCCCAGATTA-3’; GAPDH forward 5’-GAAGGTGAAGGTCGGAGTC-3’, and reverse 5’-GAAGATGGTGATGGGATTTC-3’. The sequences were amplified according to the instruction .si-RNA directed against HBXIP (si-HBXIP-1 and si-HBXIP-2) were supplied by Life Technologies . MGC803 and SGC7901 cells were transfected with 20 nM oligonucleotide using Lipofectamine™ RNAiMAX (Life Technologies). HBXIP siRNA sense-1: CGGAAGCGCAGUGAUGUUUdTdT; HBXIP siRNA sense-2: GCAGCUAAGGCAGCUAAGCUAACCUCUGTT.Cells were seeded on sterile slides, cultured and treated. Cells were fixed with 2% paraformaldehyde for 10 min and blocked by 5% BSA for 30 min, then incubated with anti-LC3 antibody at 4°C overnight. The appropriate anti-FITC (Beyotime Biotechnology) staining solution was added and covered the cells 1 h in the dark. Observed and imaged under the fluorescence microscope.t-test. Results were considered significant if P<0.05.All experiments in this study were repeated more than 3 times and the data were presented as means ± standard deviation. Statistical analysis was performed using SPSS 17.0. Comparisons between multiple groups were performed by one-way analysis of variance. Comparisons between two groups were performed using Dunnett’s Following germacrone treatment, SGC7901 and MGC803 cell viability was significantly inhibited in a dose-dependent manner  identified 111 proteins related to the cell cycle, apoptosis, and autophagy. These proteins were analyzed for molecular function, cellular component, and biological function (https://string-db.org/cgi/input.pl) and Cytoscape to analyze protein interactions and construct an interaction network .Five hundred ninety-six suitable proteins among the 4,014 proteins have been identified. A total of 350 proteins were down-regulated and 246 up-regulated in the germacrone-treated group compared with the DMSO group. Analysis using the Database for Annotation, Visualization, and Integrated Discovery , cyclin-dependent kinase inhibitor 2A (CDKN2A), mitogen-activated protein kinase 3 (MAPK3), and late endosomal/lysosomal adaptor and MAPK and MTOR activator 5  Table 1.We collected cancerous and matched paracancerous tissues from 21 gastric cancer patients following surgical resection, and found that HBXIP was expressed in gastric cancer tissue from all the patients. What’s more, we found that HBXIP expression was significantly up-regulated in gastric cancer tissues -3,7-dimehyl-10-propan-2-ylidenecyclodeca-3,7-dien-1-one] is a well-characterized anticancer terpenoid isolated from g et al. , reporteoduction . Germacroduction . Additioin vivo. In clinical studies, HBXIP was elevated in normal gastric mucosa, as well as in gastric cancer tissue and adjacent, noncancerous tissue , p14 (LAMTOR2), MP1 (LAMTOR3), C7orf59 (LAMTOR4), and HBXIP (LAMTOR5). Among these, HBXIP-C7orf59 is responsible for the initial nucleation of the complex and stabilization of the p18 conformation, allowing the subsequent binding of MP1-p14  via the iProX partner repository: PXD021980.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: ProteomeXchange Consortium , the National Natural Science Foundation of China (No 81273245) and the Natural Science Foundation of Anhui Province, China (1908085QH378).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Nitrogen supplementation increased the plant water status, photosynthetic pigment synthesis, and gas exchange attributes. Addition of nitrogen to the saline media significantly decreased the generation of lethal oxidative stress biomarkers such as hydrogen peroxide, lipid peroxidation, and electrolyte leakage ratio. The activity of the antioxidant defense system was upregulated in both saline and non-saline growth media as a result of nitrogen application. Furthermore, nitrogen supplementation enhanced the accumulation of osmolytes, such as soluble sugars, soluble proteins, and free amino acids. This established the beneficial role of nitrogen by retaining additional osmolality to uphold the relative water content and protect the photosynthetic apparatus, particularly in the salt-sensitive genotype. In summary, nitrogen application may represent a potential strategy to overcome the salinity-mediated impairment of cotton to some extent.Increasing soil salinity suppresses both productivity and fiber quality of cotton, thus, an appropriate management approach needs to be developed to lessen the detrimental effect of salinity stress. This study assessed two cotton genotypes with different salt sensitivities to investigate the possible role of nitrogen supplementation at the seedling stage. Salt stress induced by sodium chloride (NaCl, 200 mmol·L Salt stress is the foremost environmental factor that drastically decreases the crop yield in different geographic areas, especially in arid and semi-arid regions ,2. This Gossypium hirsutum L.) is considered a moderately salt-tolerant and non-halophytic plant that may also be used as a pioneer crop for soil reclamation of saline-alkaline land. However, the growth and productivity of cotton are adversely affected by high salinity levels and their tolerance level depends on the genetic background of plants and their developmental stage [+ and Cl−), oxidative damage, and/or nutritional disorders in plant tissues, which subsequently lead to weakened plant growth and endurance [Cotton , leaf number (LN) per plant, and total biomass production compared with the non-saline environment . Moderata (Chl a), chlorophyll b (Chl b), and total chlorophyll (TChl) contents of cotton seedlings  compared with the untreated control environment of both genotypes. The seedlings subjected to 2.5 mmol·L−1 nitrogen treatment alone had significantly increased gas exchange parameters compared with the control. Furthermore, nitrogen supplementation in the salt-stressed seedlings amends this detrimental effect of NaCl. A, E, gsw, and Ci increased by 22.6%, 71.3%, 54.1%, and 30.2% in ST genotype and 25.0%, 73.3%, 69.5%, and 39.5% in SS genotype, respectively, compared with salt-treated seedlings alone  as shown in Nitrogen significantly alleviated the damaging effect of hydrogen peroxide and, therefore, averting lipid peroxidation (MDA content) and electrolyte leakage (EL) ratio . Salt stNitrogen availability in the growth media markedly increased the antioxidant activities in cotton seedlings by upregulating SOD, CAT, and POD activities . CompareHeat map analysis clearly identified the overall variations among all four treatments . NitrogeHordeum vulgare [Carrizo citrange [Spartina alterniflora [Sorghum bicolor [Brassica [−1 nitrogen increased the SL, NL per plant, and total biomass in both ST and SS genotypes under salt stress  under salinity, the availability of effective nitrogen fertilizer alleviated the damage caused by salinity, particularly in the SS genotype, by enhancing both antioxidant enzyme activity and osmotic regulation . It willThis study showed that nitrogen supplementation was beneficial for cotton plants and helped to alleviate the toxic effects of salinity stress on the growth of two genotypes. Salt stress noticeably decreases the photosynthetic efficiency of plants by influencing ROS generation. In contrast, nutritional supplementation with nitrogen mitigated the detrimental effects of salt stress and boosted cotton-seedling growth by upregulating the activity of antioxidant enzymes and accumulated organic solutes. Additionally, this comprehensive study provided evidence that the SS genotype more effectively alleviated the salt-induced damage than the SS genotype upon nitrogen co-application in the saline media. Therefore, nitrogen application may be suggested as a vital strategy for strengthening the salt tolerance of cotton plants at the seedling stage.v/v) sodium hypochlorite solution for 15 min, followed by washing with double-distilled water four times. After disinfection, seeds were sown into a germination pot containing sterilized wet sand (10 × 10 × 10 cm). After seven days of germination, identical seedlings were transplanted to 7-L plastic boxes, covered with a black foam board, containing half-strength modified Hoagland’s nutrient solutions (12 plants per box). During the second week of growth, full-strength modified Hoagland’s nutrient solutions (2.0 mmol·L−1 Ca(NO3)2, 2.0 mmol·L−1 KCl, 0.5 mmol·L−1 KH2PO4, 2.0 mmol·L−1 MgSO4, 0.1 mmol·L−1 EDTA·Fe·Na, 46.2 µmol·L−1 H3BO3, 9.1 µmol·L−1 MnCl2·4H2O, 0.8 µmol·L−1 ZnSO4·7H2O, 0.3 µmol·L−1 CuSO4·5H2O, and 1.0 µmol·L−1 (NH4)6Mo7O24·4H2O) was supplied. The pH of the nutrient solution was attuned to 5.8–6.2, using KOH or HCl solutions. The temperature, relative humidity, and photoperiod of the growth chamber were 28 ± 2 °C, 60% ± 5%, and 16 h light/8 h dark, respectively. After two weeks of transplanting or three weeks after germination, cotton seedlings (with three true leaves) were subjected to treatments with NaCl and nitrogen. Ca(NO3)2·4H2O was used as nitrogen source, according to Dai et al. [−1 N without NaCl), NaCl (0.25 mmol·L−1 N with 200 mmol·L−1 NaCl), Nitrogen (2.5 mmol·L−1 N), and NaCl + N (200 m mol·L−1 NaCl and 2.5 mmol·L−1 N). Each treatment was conducted in three replications. To evade osmotic shock of cotton seedlings, NaCl was dissolved in the nutrient solution via a three-step process over a 3-day period. The nutrient solution and dH2O were replaced once per week throughout the experimental duration and the solution was constantly aerated. Moreover, the plastic boxes were frequently moved to a new position to decrease the impact of micro-environmental effects. The evaporated water was replenished every two days to re-attain the target volume. After 14 days of treatment, cotton seedlings were harvested, and the relevant parameters were analyzed.The experiments were conducted in the greenhouse of the Cotton Research Institute, Chinese Academy of Agricultural Sciences, Anyang, Henan, China. The two cotton genotypes used in this study were Z9807  and Z0102 , which were selected during an earlier screening study (data unpublished). The processes of seedling culture and experimental treatment of this study are depicted in After 14 days of treatment, cotton seedlings were harvested and their shoot length (SL) was measured from cotyledon to shoot apex. The number of leaves (LN) of every single plant was counted. Six cotton seedlings were used to measure all morphological traits for each replication.The treated cotton seedlings were thoroughly washed with deionized water and the water on leaf surfaces was wiped off with absorbent paper. The seedlings were divided into shoot and root parts. After that, plant materials were oven-dried at 105 °C for 30 min, followed by 80 °C for the next 24 h until a constant weight was reached. The total biomass was measured.Approximately 0.05 g of fresh leaf tissue was extracted with 95% ethanol to determine the content of photosynthetic pigments (chlorophyll a and chlorophyll b). All samples were analyzed using a spectrophotometer  at wavelengths of 663 and 645 nm . The who2 concentration (Ci), were measured using portable photosynthesis instruments  at the top third fully expanded leaf. The measurement was conducted on 6 cm2 of total leaf area. The CO2 concentration of the chamber was 400 µmol·mol−1, the airflow was constant at 700 µmol·s−1, and the relative humidity of the chamber was 55%.Gas exchange attributes, e.g., net photosynthetic rate (A), stomatal conductance (gsw), transpiration rate (E), and intercellular CO2O2 concentration was measured by extracting 0.1 g fresh leaf samples in 0.1% TCA according to the instructions of Velikova et al. [The Ha et al. . The optLipid peroxidation as malondialdehyde (MDA) was measured from the third fully expanded frozen leaf, following the procedure reported in Shi et al. . The absElectrolyte leakage (EL) indicates damage of the cell membrane and was conducted following the protocol described by Larka et al. . The EL The soluble sugar content was determined according to the anthrone sulphuric acid method as described by Irigoyen et al. . The sugAccording to Bradford , the solTo estimate the total free amino acid content, the ninhydrin reagent method was adopted according to Lee and Takahashi  and the The leaf relative water content (LRWC) was determined according to Bars and Weatherly  based on−1 sodium phosphate buffer (pH 7.8) containing 1% polyvinyl pyrrolidine, 0.2 mmol·L−1 ethylenediaminetetraacetic acid, and 10 mmol·L−1 magnesium chloride. The homogenate was then used as an enzyme source after centrifugation at 12,000× g for 25 min at 4 °C.The antioxidant enzyme assay was prepared by homogenizing 0.5 g fresh leaves in a pre-chilled mortar and pestle, using 50 mmol·LThe activity of SOD (EC 1.15.1.1) was determined according to Giannopolitis and Ries . FinallyTo assay CAT (EC 1.11.1.6) activity, the Havir and McHale  method wPOD (EC 1.11.1.7) activity was assayed with guaiacol as substrate and the increased absorbance of guaiacol oxidation was measured at 470 nm .p < 0.05) were analyzed by ANOVA followed by Duncan’s multiple range test.The data was presented as the mean value of three replicates per treatment. The statistical analysis was performed by SPSS statistics 25.0 software  and treatment effects (


Notably, the pretreatment with CoQ10 loaded in ethosomes exerted a consistent protective effect against oxidative stress, in both models, fibroblasts and in reconstituted human epidermis respectively.Ethosome represents a smart transdermal vehicle suitable for solubilization and cutaneous application of drugs. Coenzyme Q10 is an endogenous antioxidant whose supplementation can counteract many cutaneous disorders and pathologies. In this respect, the present study describes the production, characterization, and cutaneous protection of phosphatidylcholine based ethosomes as percutaneous delivery systems for coenzyme Q10. CoQ10 entrapment capacity in ethosomes was almost 100%, vesicles showed the typical ‘fingerprint’ structure, while mean diameters were around 270 nm, undergoing an 8% increase after 3 months from production. An ex-vivo study, conducted by transmission electron microscopy, could detect the uptake of ethosomes in human skin fibroblasts and the passage of the vesicles through 3D reconstituted human epidermis. Immunofluorescence analyses were carried on both on fibroblasts and 3D reconstituted human epidermis treated with ethosomes in the presence of H Finally, the inhibition of the oxidative damage induced by H2O2 has been evaluated also on reconstituted human epidermis (RHE) treated with CoQ10 loaded ETHO.Coenzyme Q10 (CoQ10) is a lipophilic endogenous antioxidant involved in the production of cellular energy, having a key role in the mitochondria electron transport chain . ParticuCoenzyme Q10  was purchased from Sigma-Aldrich . The soybean lecithin (PC) (90% phosphatidylcholine) used for ETHO preparation was Epikuron 200 from Lucas Meyer, Hamburg, Germany. Solvents were HPLC grade and all other chemicals were analytical grade.® - Alltima, Alltech, Bareggio, Milano, Italy). A mobile phase constituted of acetonitrile:tetrahydrofuran:water  has been eluted at a 1 mL/min flow rate, retention time was 5.7 min. The method was validated for linearity (R2 = 0.995), repeatability , and limit of quantification (0.04 µg/mL). Analyses were conducted in triplicate, mean and standard deviations values were calculated.Solubility of CoQ10 in water, ethanol, methanol, dimethylsufoxide, and acetonitrile was determined by saturating each solvent or solvent mixture with an excess of drug. The obtained saturated solutions were mixed in an horizontal shaker (100 rpm) in the dark for 3 h. Afterwards 1 mL of samples were withdrawn and filtered through a Millex-LCR Filter, 0.45 μm, hydrophilic PTFE, 25 mm . CoQ10 concentration was determined by reverse phase high performance liquid chromatography (RP-HPLC) analysis. HPLC analyses have been conducted using a quaternary pump , a UV-detector operating at 275 nm, and a 7125 Rheodyne injection valve with a 50 μL loop. Samples have been loaded on a stainless steel C-18 reverse-phase column (15 × 0.46 cm) packed with 5 μm particles (Gracew/v) was dissolved in ethanol and heated up to 30 ± 1 °C in a water bath in a closed vessel. Afterwards, twice-distilled water was slowly added to the ethanolic solution up to a final 70:30 (v/v) ratio, under continuous stirring at 700 rpm by an IKA Eurostar digital . Mechanical stirring was performed for 30 min at room temperature in the dark. In the case of CoQ10 containing ETHO (ETHO-CoQ10), CoQ10 (1 mg/mL) was added to PC ethanol solution before the addition of water.For the preparation of ETHO, PC  and analyzed using a GMS 1.9 software . Samples were vitrified as previously described . Particuv:v ratio. Data were analyzed using the “CONTIN” method [Vesicle size analysis has been conducted using a Zetasizer Nano S90  with a 5 mW helium neon laser and a wavelength output of 633 nm. Measurements were taken at 25 °C at a 90 ° angle and a run time of at least 180 s. Samples have been diluted with twice distilled water in a 1:10 −1. 2D data were corrected for background, detector efficiency, and sample transmission were then radially averaged to derive I(Q) vs. Q curves [Small angle X-ray scattering (SAXS) experiments were performed at the bioSAXS beamline B21 . CoQ10 loaded and unloaded ETHO were transferred into PCR tubes in an automated sample changer. The samples were then delivered into a temperature-controlled quartz capillary and exposed for 1s, acquiring 30 frames at 20 °C in order to check equilibrium conditions and to eventually monitor radiation damage. Data were collected using a Pilatus Dectris 2 M detector with a 3.9 m sample–detector distance and X-ray wavelength λ = 1.0 Å. The explored Q-range  extended from 0.0004 to 0.035 nmQ curves .CoQ10 is the total amount of CoQ10 employed for ETHO production.The entrapment capacity (EC) of CoQ10 in ETHO has been determined after 1, 30, and 90 days from production of ETHO-CoQ10. Namely a 500 µL sample was loaded in a centrifugal filter  and subjected to ultracentrifugation  at 8,000 rpm for 20 min. Hundred microliters of ETHO-CoQ10 in the supernatant have been diluted with ethanol  and maintained under magnetic stirring for 30 min . After f2 until 80% confluence. Stock solutions containing CoQ10 (500 μM) were further diluted to reach the final concentrations, ranging from 1 to 41.3 μM. For LDH assay seeded cells were treated for 24 h with the different vehicles at various concentrations. Cytotoxicity was evaluated by spectrophotometric quantification of the LDH released in culture medium, using a commercial kit , as previously described [Fibroblast cells  were grown in Dulbecco’s modified Eagle’s medium (DMEM) Low Glucose, supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin and 2 mM L-glutamine. Cells were incubated at 37 °C for 24 h in 95% air/5% COescribed ,37. For escribed . In tryp2 culture flask for 10 h and then in EpiLife™  basal medium containing low calcium level (60 µM CaCl2) which was renewed after 2 days. When keratinocytes were subconfluent , cells were harvested by trypsinization and centrifuged for 5 min. The pellet was resuspended in EpiLife™ submerged medium containing 1.5 mM CaCl2. Polycarbonate culture inserts (0.47 cm2 of diffusion area and 0.4 µm pore size)  were placed in a 24-well plate containing 1.5 mL of pre-warmed submerged medium, using a specific carrier system . Each insert received about 500 µL of keratinocyte suspension to reach a cell density of 375.000 cells/cm2. After three-day incubation at 37 °C in a humidified atmosphere containing 5% CO2, cells were exposed to the air–liquid interface by removal of the upper compartment medium. The 1.5 mL of submerged medium in the lower compartment were replaced with 1.5 mL EpiLife™ air-liquid interface medium containing 1.5 mM CaCl2, vitamin C, and keratinocyte growth factor, and renewed every 2–3 days for 14 days.Human epidermal equivalents (HEEs) were reconstituted according to the CITYCARE HEE protocol from Dijkhoff et al . BrieflyFibroblasts treated with ETHO for 24 h and non-treated cells, used as control, were grown to confluent monolayer on 10 cm Petri dish. RHE tissues were topically treated with ETHO for 6 h and non-treated RHE were used as control. Fibroblasts and RHE tissues were fixed in 2.5% glutaraldehyde in cacodylate buffer for 3 h at 4 °C. Then fibroblasts and tissues were post-fixed in 1% osmium tetroxide for 2 h at 4 °C, dehydrated in a graded series of alcohol, embedded in Araldite resins, and polymerized in oven for 48 h at 60 °C. Ultrathin sections of 60 nm were cut with an ultramicrotome , stained with uranyl acetate and lead citrate and examined in a Philips CM100 transmission electron microscope .2O2 for 1 h. After H2O2 exposure, the medium was changed with fresh medium and the cells were analyzed at three different time points . Afterwards, cells were fixed in 4% paraformaldehyde for 10 min at room temperature (RT). Cells were then permeabilized for 5 min at RT with PBS containing 0.2% Triton X-100, then the coverslips were blocked in PBS containing 1% BSA at RT for 1 h. Cells were then incubated with primary antibody for 4HNE  (1.200) in PBS containing 0.5% BSA at 4 °C overnight. After washing, coverslips were incubated with appropriate secondary antibody (1:100) for 1 h at RT. Nuclei were stained with 1 μg/mL DAPI  for 1 min. Coverslips were mounted onto glass slides using anti-fade mounting medium 1,4 diazabicyclooctane (DABCO) in glycerin and examined by the Leica light microscope equipped with epifluorescence at 40× magnification. Negative controls for the immunostaining experiments were performed by omitting primary antibody. Images were acquired and analyzed with Leica software [Fibroblasts were grown on coverslips at a density of 1 × 105 cells/mL, after 24 h of treatment with plain ETHO and ETHO-CoQ10, cells were exposed to 50 μM Hsoftware .2O2 for 90 min and analyzed. Formalin-fixed paraffin-embedded (FFPE) sections were used to quantify 4-HNE expression in the RHE models with a thickness of 4 µm. FFPE sections were deparaffinized, rehydrated, and rinsed with water. An antigen retrieval step was performed by incubating the FFPE sections in 10 mM citrate buffer pH 6 at 90 °C for 20 min. All FFPE sections were firstly washed twice in phosphate buffer saline (PBS), then blocked for 1h in PBS containing 0.2% bovine serum albumin and 0.02% Triton X-100, further incubated overnight at 4 °C with 4-HNE antibody  (1:100). Tissue sections were washed in PBS containing 0.2% bovine serum albumin and 0.02% Triton X-100 before incubation with Donkey Anti-Goat IgG H&L  (1:200) for 2h. After several washes, sections were finally stained with a diamidino-2-phenylindole dye  300 mM solution for 5 min, then washed thrice in TBS-T, a mixture of tris-buffered saline (TBS) and polysorbate 20 (aka Tween 20). Coverslips were subsequently mounted in an aqueous mounting medium  and tissue sections observed under a fluorescence microscope  equipped with a fluorescein isothiocyanate (FITC) filter. DAPI and FITC pictures were finally overlapped [RHE was topically pretreated with 50 µL of ETHO-CoQ10 for 24 h, then exposed to 200 μM Herlapped .Statistical differences of in vivo data have been calculated by repeated-measures analysis of variance (ANOVA) and the Bonferroni–Dunn post hoc pairwise comparison procedure. The software Prism 5.0, Graph Pad Software Inc.  has been employed. Probability values less than 0.05 have been considered significant in this study.w/v) to ethanol enabled to increase CoQ10 solubility up to 1 mg/mL, the inclusion of the drug in ETHO vesicles has appeared the most appropriate strategy.In order to design a nanotechnological formulation suitable for the delivery of the lipophilic CoQ10 , its sow/w on ETHO size, as reported in PC is a natural amphiphilic molecule widely employed for its emulsifying properties. In addition, PC can self-organize in bilayers in excess water, giving rise to supra-molecular structures, such as direct and inverted micelles; hexagonal, cubic, and lamellar phases; as well as stable vesicles. Thus, the addition of water to PC ethanol solution under stirring spontaneously led to ETHO, appearing as milky homogeneous dispersions . Differew/w. Thus the lowest the PC concentration, the highest the size stability, as found by other authors [It was found that both ETHO opalescence and mean diameter increased with the increasing of PC concentration, particularly ETHO mean diameters, measured by PCS, were comprised between 227 and 274 nm. Notably, ETHO size distribution was homogeneous, being characterized always by one peak, as indicated by the intensity distribution and by the low dispersity indexes . ETHO si authors .w/w has been selected in order to adequately entrap CoQ10, while maintaining the vesicle size stability.Despite PC 0.6% enabled to better control ETHO size stability, since previous studies have demonstrated that phospholipid concentration in ETHO significantly affects drug entrapment efficiency ,45,46, aw/w ethanol solution, before water addition at 22 °C. This strategy, devoid of high energy input and heating, appeared particularly suitable for encapsulation of labile drugs such as CoQ10. The size analysis of ETHO-CoQ10 confirmed a unimodal size distribution also in the presence of the drug, with a 16 nm (6%) increase in the vesicle mean diameter, passing from 254 to 271 nm (0.9 (panel a) and ETHO-CoQ10 (panel b), clearly showing spherical vesicles. In both panels a mixed population of unilamellar, multilamellar, as well as multivesicular vesicles can be noted, particularly, a ‘fingerprint-like’ structure due to the presence of phospholipid bilayers, can be detected in both panels, as previously described by Touitou and Godin [Therefore, for ETHO-CoQ10 production the drug has been solubilized in PC 0.9% o 271 nm . It is nnd Godin . Notably0.6, ETHO0.9 and ETHO1.5, showing a broad band (Q ≈ 1.5 nm−1) and two shoulders (Q ≈ 0.075 and 0.2 nm−1). The broad band, whose position roughly corresponds to a bilayer thickness of 4.2 nm, is indicative of the presence of PC bilayers [0.9  and the bilayers—symmetrically constituted by three shells with constant electron densities corresponding to the polar head (thickness R1 and electron density ρ1), the aliphatic chain (R2 and ρ2), and the terminal methyl group (R3 and ρ3) domains—are separated by water layers of thickness c and electron density ρ0. Moreover, the bilayer distribution is described by the paracrystal theory [c. The best fitting curve is reported in SAXS analyses have been conducted to shed light on the inner ETHO organization. bilayers ,49, whilfunction ) surrounl theory , with di−1 (4.2 nm) to 1.38 nm−1 (4.6 nm) and for the disappearance of the first shoulder at Q = 0.075 nm−1. The change in the broad-band position suggests an increase of the lipid bilayer due to the CoQ10 presence, while the absence of distinct Bragg peaks confirms the disordered structure of ETHO vesicles. Model fitting results, reported in SAXS profile of ETHO-CoQ10 b was simThe EC of CoQ10 in ETHO was almost total, namely 98 ± 0.2% of the employed drug has been found within ETHO-CoQ10. This high EC value was expectable, indeed the preparation modalities prevented drug loss on mechanical devices, as well as thermal or light degradation. In addition many authors have demonstrated that the presence of ethanol and PC organized in multilamellar vesicles enables to increase drug loading with respect to other nanosystems ,22,47. ACoQ10 stability has been studied evaluating EC values for 3 months in ETHO-CoQ10 stored in the light at 22 °C. It is noteworthy that, despite CoQ10 photo instability, EC was 90% and 60% after 30 and 90 days from production, thus higher with respect to similar studies conducted on nanostructured lipid carriers containing CoQ10 . It can 0.9 and ETHO-CoQ10 (referring to loaded CoQ10) on primary dermal fibroblasts from healthy controls, using three different techniques: trypan blue exclusion, MTT, and LDH assays. The concentration range was selected in order to confirm the safeness and biocompatibility of ETHO constituents and of the loaded drug. 0.9 with ETHO-CoQ10. Both formulations display similar behavior, with no cytotoxicity up to the highest concentration tested. In detail, cell viability detected by trypan blue exclusion assay  has been selected for further ex-vivo experiments.The in vitro cytotoxicity of ETHO-CoQ10 has been assessed with different concentrations of ETHOon assay a was higver, MTT b and LDHver, MTT c assays 0.9 in treated fibroblasts, TEM analysis was performed, comparing the ultrastructural morphology with untreated cells , or 2 (T2h) and 6 (T6 h) h post-exposure. 4HNE protein adducts were analyzed by immunofluorescence (2O2 induced a significant increase in 4HNE protein adducts formation (green fluorescence), in CTRL and cells treated with ETHO0.9 cells , is the main hurdle for the substance passage to the deeper skin layers . In thisd by TEM . Figure d by TEM a. The im2O2. Particularly, the effect of H2O2 oxidative stress was detected in RHE pretreated with ETHO-CoQ10 (ETHO-CoQ10- H2O2-RHE) and compared to RHE treated with ETHO-CoQ10 and not exposed to H2O2 (ETHO-CoQ10-RHE), or to RHE exposed to H2O2 (CTRL- H2O2-RHE). 4HNE protein adducts levels have been analyzed by immunofluorescence (In order to investigate the ability of CoQ10 loaded ETHO in protecting skin tissue against oxidative stress, an immunohistochemical analysis for 4HNE protein adducts has been performed on RHE treated with ETHO-CoQ10 and exposed to Hrescence a and quarescence b.2O2 induced a significant increase in 4HNE protein adducts, as evidenced by the green fluorescence in CTRL-H2O2-RHE (2O2-RHE, suggesting that ETHO-CoQ10 are able to protect skin tissue after exposure to oxidative stress (stratum basale. It can be supposed that after application, ETHO-CoQ10 first pass through the upper SC, then they reach the deeper layers, confirming the mechanism of skin penetration previously described for ETHO [The exposure to HH2O2-RHE a. Notable stress b. It is for ETHO ,54. Indefor ETHO ,54.The present investigation has demonstrated the suitability of ETHO as a transcutaneous delivery system for CoQ10. The preformulatory study has underlined the importance of size distribution analysis for selecting the composition of ETHO vesicles. SAXS and cryo-TEM evidenced the multilamellar structure of vesicles based on phosphatidylcholine and ethanol.Notably, due to their peculiar supramolecular organization, ETHO were able to increase the EC of CoQ10 and to better control its stability with respect to other nanosystems previously investigated in other studies, confirming the tremendous potential of ETHO vesicles for solubilization and delivery of lipophilic drugs. The composition and preparation modalities of ETHO described in this study could be applied to entrap molecules not only with antioxidant properties in order to improve their absorption and to delay their degradation.stratum basale corroborates the efficiency of ETHO as transdermal delivery systems, resulting as a promising vehicle for CoQ10 encapsulation. Remarkably, RHE can be considered reliable models to investigate the effect of topical formulation on skin; nevertheless, further in vivo studies will be required to evaluate not only the comparable antioxidant delivery against other carrier systems, but also the prolonged antioxidant effect of ETHO-CoQ10.Ex-vivo studies have corroborated the hypothesis of using ETHO as nanosystems for CoQ10 delivery through the skin. Indeed, TEM analyses confirmed the ETHO fibroblasts uptake, as well as their passage through the more complex model RHE. Furthermore, the decrease of fluorescence signal intensity down to the


We are just beginning to understand the unintended effects of antibiotics on our microbiomes and health. In this study, we aimed to define an approach by which one could obtain a comprehensive picture of (i) how antibiotics spatiotemporally impact commensal microbes throughout the gut and (ii) how these changes influence host chemistry throughout the body. We found that just a single dose of antibiotic altered host chemistry in a variety of organs and that microbiome alterations were not uniform throughout the gut. As technological advances increase the feasibility of whole-organism studies, we argue that using these approaches can provide further insight on both the wide-ranging effects of antibiotics on health and how to restore microbial communities to mitigate these effects. Antibiotics are a mainstay of modern medicine, but as they kill their target pathogen(s), they often affect the commensal microbiota. Antibiotic-induced microbiome dysbiosis is a growing research focus and health concern, often assessed via analysis of fecal samples. However, such analysis does not inform how antibiotics influence the microbiome across the whole host or how such changes subsequently alter host chemistry. In this study, we investigated the acute (1 day postadministration) and delayed (6 days postadministration) effects of a single parenteral dose of two common antibiotics, ampicillin or vancomycin, on the global metabolome and microbiome of mice across 77 different body sites from 25 different organs. The broader-spectrum agent ampicillin had the greatest impact on the microbiota in the lower gastrointestinal tract (cecum and colon), where microbial diversity is highest. In the metabolome, the greatest effects were seen 1 day posttreatment, and changes in metabolite abundances were not confined to the gut. The local abundance of ampicillin and its metabolites correlated with increased metabolome effect size and a loss of alpha diversity versus control mice. Additionally, small peptides were elevated in the lower gastrointestinal tract of mice 1 day after antibiotic treatment. While a single parenteral dose of antibiotic did not drastically alter the microbiome, nevertheless, changes in the metabolome were observed both within and outside the gut. This study provides a framework for how whole-organism -omics approaches can be employed to understand the impact of antibiotics on the entire host.IMPORTANCE We are just beginning to understand the unintended effects of antibiotics on our microbiomes and health. In this study, we aimed to define an approach by which one could obtain a comprehensive picture of (i) how antibiotics spatiotemporally impact commensal microbes throughout the gut and (ii) how these changes influence host chemistry throughout the body. We found that just a single dose of antibiotic altered host chemistry in a variety of organs and that microbiome alterations were not uniform throughout the gut. As technological advances increase the feasibility of whole-organism studies, we argue that using these approaches can provide further insight on both the wide-ranging effects of antibiotics on health and how to restore microbial communities to mitigate these effects. Clostridioides difficile colitis  by 16S rRNA sequencing . Compared to control mice, the centered log ratio (clr)-transformed proportion of Gram-positive bacteria was significantly reduced in the fecal pellet, certain sections of the stomach and colon of AMP.d1 and VAN.d1 mice, and certain sections of the cecum in AMP.d1 mice .10.1128/mSystems.00340-20.2FIG S2P < 0.05 after Benjamini-Hochberg correction for multiple comparisons). Error bars represent the 95% confidence interval (CI). Download FIG S2, TIF file, 1.1 MB.Antibiotics reduce levels of Gram-positive bacteria. Asterisks indicate a significant difference in the centered log ratio (clr)-transformed proportion of Gram-positive bacteria compared to the control  and VAN.d1 mice (duodenum and stomach), differentially abundant sub-operational taxonomic units (sOTUs), also known as amplicon sequence variants or exact sequence variants, were seen in both AMP.d1 and VAN.d1 mice, and the level of one .d6 mice  and b. A.d6 mice . Specifi.d6 mice  for each.d6 mice . AMP.d1 .d6 mice . AMP hadTo evaluate the global impact of antibiotics on metabolome composition, we calculated effect size using Bray-Curtis distance comparing each treatment group to controls and mapped the effect-size data onto a three-dimensional (3D) mouse model to provide a spatial visualization . For eacm/z 100 to 1,500.Molecular networking analysis  of the r10.1128/mSystems.00340-20.3FIG S3FIG S3, TIF file, 2.6 MB.Molecular network of ampicillin and ampicillin network features. (a) GNPS molecular network displaying the molecular family in which ampicillin was annotated based on spectral library matching (blue) with unannotated analogs (green). Circles outlined in orange indicate a putative ampicillin analog. Neutral-charge chemical structures proposed are indicated. (b) MS/MS spectra for AMP. Putative fragments ions resulting from fragmentation of the proposed neutral-charge chemical structures are indicated using color. Download Copyright © 2020 Vrbanac et al.2020Vrbanac et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00340-20.4FIG S4FIG S4, TIF file, 1.7 MB.Ampicillin network features correlate with changes in the metabolome and microbiome. (a) The sum of the peak area of putative ampicillin network features moving down the GI tract; error bars represent the 95% CI. (b) Pearson correlation of ampicillin network features with metabolome pairwise effect size to control (Bray-Curtis) for gut samples  only from the ampicillin 1-day group. (c) Pearson correlation of ampicillin network features with the change in Shannon diversity  relative to control samples for gut samples  only from the AMP.d1 group. The shaded area represents 95% CI. Download Copyright © 2020 Vrbanac et al.2020Vrbanac et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the In the lower GI tract, differential abundance testing revealed an increase in small di- and tripeptides in AMP.d1 and VAN.d1 mice. To investigate this further, the metabolomics data were processed through PEAKs  to yield10.1128/mSystems.00340-20.5FIG S5P value of <0.05 by Mann-Whitney U test. (b) Abundances of peptides from histones, both H2A and H2B, were elevated in the lower GI tract of acute antibiotic-treated mice (AMP.d1 and VAN.d1) compared to other groups; significance testing was by dsFDR. Error bars represent the 95% CI. Download FIG S5, TIF file, 1.5 MB.Small peptides are elevated in the lower GI tract day 1 postantibiotic exposure. (a) Sum of peptide spectral abundances along the lower GI tract comparing AMP.d1- and VAN.d1 (acute antibiotic)-treated mice to control mice and mice at day 6 posttreatment (nonacute). Significance indicates a Copyright © 2020 Vrbanac et al.2020Vrbanac et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the R2 = 0.59, P = 0.0033) and H2B , and the most important features in the regression model for H2A and H2B were mainly from the Clostridiales order and positively associated with histone abundance, except for one Anaeroplasma sOTU, the 6th most important feature for H2B, which was negatively correlated with both H2A and H2B abundance. For the metabolomics data, the regression performance was improved for H2A  and H2B , although annotated features were not among the top 20 most important features. The regression was repeated with the metabolome data filtered to only contain putatively annotated features for H2A  and H2B . As expected, di- and tripeptides were among the top 10 annotated features for both H2A and H2B. Another top feature for H2A was phytomonic acid, a saturated fatty acid present in bacterial plasma membranes, which was positively associated with histone abundance; accumulation of phytomonic acid is associated with ethanol stress in Oenococcus oeni , an enzyme that acetylates maltose and other sugars, and a variety of other sugar transport systems for the Coprococcus sOTU , suggesting the potential for the consumption of sugars. Differential abundance testing recapitulates some of these findings , although, in general, we did not observe alterations in the abundance of bile acids or sugar-related molecules. Studies with longer antibiotic courses report altered differential abundance of bile acids and sugars/carbohydrates  and VAN.d1 mice (both family S24-7) compared to the control. Annotated metabolites that had the highest and lowest cooccurrence probabilities with these sOTUs are displayed. Download FIG S6, TIF file, 2.5 MB.Cooccurrence probabilities for microbes and metabolites cluster by metabolite class. (a) Log conditional cooccurrence probabilities for all microbes and putatively annotated metabolites. The microbial phylum is indicated on the left by row, and general metabolite class is indicated on the top by column . (b) A multinomial regression model was used to find sOTUs that were most associated with the AMP.d1 mice  Experimental design. Thirty-seven-week-old female C57BL/6 mice (Jackson Laboratory) were randomized to 3 or 2 mice per cage upon receipt and allowed to acclimatize for 6 days before antibiotic administration. AMP and VAN were administered by i.p. injection in doses of 100 mg/kg of body weight, dissolved in 100 μl PBS. Control mice were given 100 μl PBS via i.p. injection. Cohoused mice were administered the same antibiotic or PBS alone to avoid coprophagy across treatment groups. All mice, including controls, were dissected at both day 1 and day 6 time points. See (ii) Sample collection and processing. Mice were dissected at both the day 1 and day 6 postantibiotic administration time points, alternating between treatment groups. The dissection protocol was as follows. Mice were sacrificed by CO2 asphyxiation, followed by cardiac puncture. Dissection was carried out under an open flame with forceps and scissors cleaned with 70% ethanol after the removal of each organ. Fresh fecal samples were collected prior to CO2 asphyxiation and organs removed in the following order: spleen, gallbladder, liver, pancreas, stomach, duodenum, jejunum, ileum, cecum, colon, kidneys, adrenal glands, urinary bladder, ovaries, uterine horns, uterine body, cervix, vagina, thymus, heart, lungs, esophagus, trachea, and brain. Organ subsections  were collected in separate tubes for a total of 77 samples per mouse.Samples were flash frozen in 2-ml Eppendorf tubes in an isopropanol dry ice bath (−77°C) and stored at −80°C. All samples were weighed, and 10 μl of sterile water per milligram of tissue was added to each tube . For samples under 15 mg, 150 μl of water was added. Samples were homogenized in a Qiagen TissueLyser II using stainless steel beads.(iii) Sample plating scheme. As the large number of samples required an extensive amount of LC-MS/MS instrument time, which was not available in a continuous block, we separated the samples into three organ groups to avoid issues with comparing treatment groups across metabolomics runs. These groups included “gut” samples , “reproductive” samples , and “circulatory” samples . To avoid plate effects biasing the results, samples were plated by cycling through treatment groups, resulting in every 96-well plate containing samples from multiple treatment groups. Both the metabolome and sequencing plates were prepped at the same time for the gut samples to avoid extra freeze-thaw cycles.(iv) Metabolomics preparation and data acquisition. Five milligrams of tissue homogenate (50 μl) was added to 96-well plates containing 150 μl of 70% high-performance liquid chromatography (HPLC)-grade methanol (chilled) spiked with 5 μM sulfamethoxine . Plates were sealed, vortexed, and stored at 4°C overnight (12 h) for extraction. To remove insoluble material, plates were centrifuged at 2,000 rpm at 4°C for 10 min, and 130 μl from each well was aliquoted to a new 96-well plate. Plates were stored at −80°C, and, immediately prior to running on the LC-MS/MS instrument, were centrifuged at 2,000 rpm at 4°C for 10 min; 100 μl was aliquoted to a new 96-well plate. LC-MS/MS data acquisition was performed on a Vanquish ultrahigh-performance liquid chromatography (UPLC) system using a core-shell silica C18 column  coupled to a Q Exactive Orbitrap mass spectrometer . Five microliters of sample was injected and run at 0.5 ml/min on a gradient of solvent A (HPLC-grade water with 0.1% formic acid) and solvent B (HPLC-grade acetonitrile with 0.1% formic acid). The column was maintained at 40°C. The UPLC elution gradient ran for 12.5 min per sample: 5% B from 0 min to 1 min, a linear gradient of 5 to 100% B over 8 min, a hold at 100% B for 2 min, a return to 5% B over 0.5 min, and a hold at 5% B for 2 min to equilibrate the column for the next sample. The flow was directed into a heated electrospray ionization source operated in positive ionization mode with the following parameters: an auxiliary gas flow rate of 14 arbitrary units (a.u.), sweep gas flow rate of 3 a.u., sheath gas flow rate of 52 a.u., spray voltage of +3.5 kV, capillary temperature of 270°C, auxiliary gas heater temperature of 435°C, and S-Lens RF level of 50. The data-dependent acquisition mode was used to acquire the data in which MS1 scans from m/z 100 to 1,500  were followed by an MS2 scan, specifically a product ion scan produced using stepped normalized collision energy higher-energy collisional dissociation, of the five most abundant ions from the prior MS1 scan.(v) LC-MS/MS raw data processing. Raw data were uploaded to MassIVE (https://massive.ucsd.edu/), converted to .mzML files, imported into MZmine2 (m/z 1500 and a 9.5-min retention time (RT). Due to some retention time shift while running the larger gut sample set, parameters are slightly modified versus non-gut sample sets, as indicated in parentheses. The parameters for the gut samples used in MZmine2 are as follows. Mass detection was performed with a noise threshold of 2.0e5 for MS1 and 2.0e3 for MS2 (1.0e3 for non-gut samples) in centroid mode, and chromatograms were built with a 0.05-min time span, 1.0e6 minimum height, and 10-ppm m/z tolerance. Chromatograms were deconvoluted with the baseline cutoff algorithm and a minimum peak height of 1.0e6, peak duration of 0.05 to 1.0 min, and baseline of 1.0e4. Isotope peak removal was performed with 15-ppm m/z tolerance, 0.3 retention time tolerance (0.05 RT for non-gut samples), and maximum charge of 4, and peaks were aligned with 10-ppm m/z tolerance, 75 weight m/z tolerance, and 0.4 RT tolerance (0.05 for non-gut samples). Gap filling was also performed with 10% intensity tolerance, 15-ppm m/z tolerance, and 0.3 RT tolerance (0.1 for non-gut samples). Peaks were also filtered to remove singletons found in only one sample. Both MS1 and MS2 feature tables were exported, and the “export for GNPS” feature was used to generate a .mgf file for GNPS  Metabolite annotation. GNPS (on. GNPS  was used(vii) Peptidomics data processing. LC-MS/MS .mzXML formatted files were loaded into PEAKS Studio 8.51 . Data were imported and refined according to PEAKS settings for Orbitrap instruments under the collision-induced dissociation fragmentation mode. Error tolerance parameters were set to 15-ppm parent mass error tolerance and 0.02-Da fragment mass error tolerance. The search settings included no added restriction enzymes as well as variable modifications for dehydration, acetylation , oxidation (M), and formylation . The maximum number of variable posttranslational modifications per peptide was set to 3. The label-free quantification mode was used, and the final false discovery rate (FDR) for peptide spectral matches was reported to be 2.9%. Quantification was normalized to the total ion chromatograph.dio 8.51  for the (viii) Microbiome sample processing. DNA was extracted from 100 μl of each gut sample using a MoBio PowerMag soil DNA isolation kit  by following the Earth Microbiome Project protocol (protocol . Samples(ix) Microbiome and metabolome data processing and analysis. Raw sequencing data were transferred to Qiita (https://doi.org/10.1101/133462). Bugbase requires an OTU table picked against the GreenGenes database, and closed reference OTU picking was performed with the GreenGenes database (v13_8)   at a 97% (v13_8) , the clo (v13_8)  analysis(x) Statistics and data visualization. SciPy . dsFDR  (n. SciPy  was usedn. SciPy  was used). dsFDR  was used). dsFDR  beta-grof adonis . Figuresf adonis  and seabf adonis . The 3D f adonis  from a mmbl.de/) . Random mbl.de/)  using 10ERP121045.Raw data are available on MassIVE under the data sets MSV000082049, MSV000082157, and MSV000082048. Sequencing data are available under EBI accession number