Polyphenols show a spectrum of bioactive effects, including an influence on lipid metabolism. In this study, we performed activity-guided fractionations of black chokeberry (aronia), cranberry, and pomegranate extracts to identify the biologically active compounds. The extracts were prepared from fruit juice concentrates with the adsorbent resin Amberlite XAD-7 and were separated into a copigment and an anthocyanin fraction, followed by fractionation into a polymer and monomeric fraction by means of hexane precipitation. For further fractionation of the cranberry and pomegranate copigment fractions, high-performance countercurrent chromatography (HPCCC) was used. The compounds in each fraction were identified by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS), and the quantification was performed by ultra high-performance liquid chromatography-diode array detector (UHPLC-DAD) analyses. Each of the (sub-)fractions was tested in three in vitro assays: phosphodiesterase 3B (PDE) activity, lipid accumulation, and lipolysis in 3T3-L1 cells. The results showed that various fractions and subfractions can inhibit lipid accumulation and PDE activity as well as increase lipolysis, particularly copigments. Overall, our results indicate an influence of polyphenol-rich (sub-)fractions on the lipid metabolism. It is well known that fruits and vegetables are good sources of vitamins, fiber, and bioactive phytochemicals . CompounPomegranate contains another class of polyphenols, the so called hydrolyzable tannins, which are oligomers of ellagic and gallic acids ,6. The mSeveral studies have shown that polyphenols, especially anthocyanins and copigments, have antioxidant, anti-inflammatory, anti-bacterial, and anti-viral properties and are discussed in the context of influencing glucose and lipid metabolism in vitro as well as in vivo ,12,13,14Triglycerides are stored in cytosolic lipid droplets of fat cells (adipocytes) and are used as the main energy reserve. During lipolysis, triglycerides in fat cells are hydrolyzed; as a result, free fatty acids (FFAs) and glycerol are released. Lipolysis rates are regulated through biochemical or hormonal signals in response to changes in the nutritional state. Furthermore, lipolysis can be influenced by polyphenols, such as flavonoids, by inhibiting 3′,5′-cyclic adenosine monophosphate (cAMP)-phosphodiesterase (PDE), such as PDE 3B, which led to increased cAMP levels, as reported by Kuppusamy and Das  and DallThis paper describes the successful fractionation and identification of compounds from chokeberry, cranberry, and pomegranate extracts, influencing lipid metabolism in vitro. Therefore, we examined the effects of the (sub-)fractions on lipid accumulation, lipolysis, and PDE activity, which represent some parts of human lipid metabolism.2 • 8H2O), caffeine, Oil Red O, phosphodiesterase (PDE) 3B , resazurin sodium salt, saponine, and butanol (BuOH) were in HPLC grade from Sigma Aldrich (Steinheim Germany); -3′5′-cyclic adenosine monophosphate ammonium salt, 37 MBq/mL, was from Hartmann Analytic . Tris-(hydroxymethyl)-aminomethane (TRIS) was obtained from Carl Roth , and resveratrol was from Cayman Chemical . Formaldehyde solution of 37%; magnesium chloride hexahydrate (MgCl2 • 6H2O); zinc sulphate heptahydrate (ZnSO4 • 7H2O); and the standards of cyanidin-3-glucoside, quercetin-3-glucoside, chlorogenic acid, and punicalin were ordered from Merck . Isopropanol and tert-butyl methyl ether (tBME) in HPLC grade were purchased from Fisher Scientific GmbH ; dimethyl sulfoxide (DMSO) was from J&K Scientific . Acetonitrile (ACN) was purchased in HPLC and LC-MS grade from Honeywell Specialty Chemicals , and methanol (MeOH) was in HPLC grade from VWR Chemicals (Darmstadt Germany). The mouse embryo 3T3-L1 cell line was obtained from Zenbio . Cell culture consumable materials were obtained from Greiner Bio-One . The cinnamtannin A2 standard (epicatechin tetramer) was obtained from PhytoLab GmbH & Co. KG All chemicals and reagents used were of analytical grade. Adenosine monophosphate (AMP), and cyclic adenosine monophosphate (cAMP) were purchased from Alexis Biochemicals ; barium hydroxide octahydrate (Ba(OH)® XAD-7 adsorber resin from Sigma Aldrich (Steinheim Germany). The extracts were obtained from authentic juice concentrates from commercial sources: chokeberry with a °Brix of 65.4 and a juice dilution of 18 °Brix, cranberry with 49.8 °Brix and juice dilution of 8.8 °Brix, and pomegranate with 64.5 °Brix and a juice dilution of 15 °Brix.All extracts were generated using Amberlite® XAD-7 column. The column was washed with 4 L of water. The retained phenolic compounds were eluted with 2 L of a mixture of methanol/water (19/1 v/v). Solvents were evaporated under reduced pressure at 40 °C, and the XAD-7 extracts were freeze-dried with a Beta 2-8 LD plus from Christ .About 2 L of the juice concentrates were applied onto an Amberlitev/v). The membrane was washed with 2 L of NaOH (1 N) and equilibrated with 2 L of HCl (0.01 N) and 1.5 L of methanol/acetic acid (19/1 v/v). Then, the membrane adsorber was loaded with the dissolved extracts. The copigment fractions were eluted with a further liter of methanol/acetic acid (19/1 v/v). The retained anthocyanins were eluted with 1 L of a mixture of aqueous NaCl solution (1 N) and methanol (1/1 v/v). The membrane was washed with 2 L of NaOH (1 N) and equilibrated with a mixture of aqueous NaCl solution (0.1 N) and ethanol (8/2 v/v). The solvent of the copigment fractions were removed in vacuo, and the residue was freeze-dried. For removal of NaCl from the liquid anthocyanin fraction, a further extraction with Amberlite XAD-7 had to be carried out -cAMP), the mixture was incubated for 25 min at 37 °C to allow the reaction. The enzyme reaction was stopped by adding 250 µL of ZnSO4 (0.266°M) on ice. [3H]-5′-AMP was then precipitated by adding 250 µL of Ba(OH)2 (0.266°M). The tubes were then centrifuged at 13,000× g for 9 min at 25 °C, 450 µL of the supernatant was mixed with 3.5 mL of a scintillation cocktail, and the resulting radioactivity was measured using a liquid scintillation counter . Samples were dissolved in DMSO  or in assay buffer. Caffeine, the nonspecific PDE 3B inhibitor, served as a positive control. The experiments were performed in triplicate, and the half-maximal inhibitory concentration (IC50) values were determined after at least three independent experiments.The inhibitory effect of each fraction and subfraction on cAMP-specific PDE 3B was measured according to the method published by Pöch et al.  with slip < 0.05, p < 0.01, and p < 0.001) compared to the solvent-treated control by a Student’s t-test (one-sided).The results of the in vitro assays were presented as the mean ± standard deviation (SD) of at least three independent experiments. Statistical analyses were conducted using the Analysis Tool Excel of Microsoft 365 Apps for Enterprise  and Origin 2020 . The data of samples treated with fractions or subfractions were analyzed for significant differences , as well as pomegranate fractions (c = 5–100 µg/mL). All tested fractions  reduced cell viabilities in a concentration-dependent manner. A cell viability of around 80% was observed for all fractions of chokeberry and cranberry extracts at concentrations of 10–25 µg/mL and pomegranate extract at concentrations of 5–10 µg/mL (data not shown). In general, the pomegranate fractions showed stronger effects on cell viability than the fractions produced from chokeberry or cranberry extracts, and the cell viability decreased in the order of chokeberry ≈ cranberry > pomegranate. Based on this screening, the following concentration ranges for the lipid accumulation assay were selected: the fractions of chokeberry and cranberry extract were tested in concentrations of 10–50 µg/mL and the fractions of pomegranate extract in concentrations of 5–25 µg/mL.To evaluate non-cytotoxic concentration ranges for the lipid accumulation assay, the cells were incubated for 14 days (as in the lipid accumulation assay) with the chokeberry, cranberry (each c = 1–2 mg/mL), a cell viability of 80–100% was observed, while at higher concentrations , a decrease of up to 65% was shown.Since, for the lipolysis assay, the cells were incubated for 3 h, differentiated adipocytes (14 days post-differentiation) were treated with fractions at concentrations of 1–4 mg/mL. At lower concentrations  and 75% (c = 50 µg/mL), respectively, compared to the solvent control (100% lipid accumulation). Dose-dependent effects were detected with the fractions of the cranberry extract. The relative lipid content was decreased by up to 63% following treatment with the cranberry polymer fraction at a concentration of 50 μg/mL (c = 25 µg/mL) reduced lipid content to approximately 69% and 65% compared to the control, while the polymer fraction showed just a slight effect.Resveratrol can inhibit adipocyte differentiation of 3T3-L1 cells  and was 50 μg/mL b. The poAdipocytes were incubated with the fractions of chokeberry, cranberry, and pomegranate extracts to investigate whether they might reduce lipid content by increasing FFA release, which can be used as an indicator of adipocyte lipolysis. Isoproterenol was used as a positive control (PC) in this assay, since it stimulated lipolysis via beta adrenergic receptor activation and cAMP-dependent signaling . The resc = 235 µg/mL) was used as the positive control (PC) in this assay, since it is a well-known nonspecific PDE 3 inhibitor.The obtained fractions of chokeberry, cranberry, and pomegranate extracts were tested for their influence on cAMP-specific PDE 3B activity in vitro. Data are presented as the relative PDE activity in a percentage of the solvent control. Caffeine (c = 50–250 µg/mL) were in the same concentration range. Relative PDE 3B activities of 60–80% were shown by the chokeberry fractions at a concentration of 100 µg/mL. The highest tested concentration of these fractions (c = 250 µg/mL) decreased PDE activity to 16 ± 6%, 11 ± 1%, and 9 ± 1%, respectively, compared to the solvent control (All the tested fractions showed a dose-dependent inhibition of the enzyme PDE 3B in vitro . The PDE control .A similar reduced activity of the PDE 3B enzyme activity was observed for the three fractions of the cranberry extract compared to the solvent control . At the c = 6.25 µg/mL) was observed, and the impact on PDE activity was comparable to those seen with the PC caffeine  to 33 ± 1% (c = 100 µg/mL).The three fractions of the pomegranate extract showed the strongest impact on PDE 3B enzyme activity. For the anthocyanin fraction, a significantly diminished PDE activity at the lowest tested concentration (50) was calculated for each of the fractions investigated .Based on the results of the PDE 3B activity assays, a half-maximal inhibitory concentration  that could be injected in order to obtain a good fractionation. The total amount that had been separated was about 2.2 g. One of the HPCCC chromatograms is presented in Five subfractions (F1–F5), and the so-called coil fraction, could be collected. The coil fraction was the liquid solution that remained in the HPCCC after completion of the separation and could be collected after separation. Due to the low amount, the coil fraction of the cranberry copigment separation was not further examined. The amount and yield of each fraction are given in The separation of the pomegranate copigment fraction by HPCCC had to be repeated 28 times because, for a successful fractionation, only 200–300 mg could be injected. The total injected amount was about 6.1 g. One of the HPCCC chromatograms is presented as an example in The characterization of the HPCCC subfractions was performed by HPLC-DAD-MS/MS analyses. The identified compounds are listed in First, we determined the effects on cell viability of 3T3-L1 cells after treatment with the subfractions of the cranberry copigment fraction by the alamarBlue assay. After treating 3T3-L1 cells for 14 days with subfraction F1, a dose-dependent reduction of viability in comparison to the solvent control was observed; however, when the cells were treated with concentrations of 10–50 µg/mL of the subfractions F2–F5, no significant cytotoxicity was observed . Based on this screening, a concentration range of 10–50 µg/mL was used for the determination of lipid accumulation.The positive control resveratrol reduced lipid accumulation significantly at a concentration of 22.8 µg/mL, compared to the solvent control. Subfractions F4 and F5 were the most active subfractions in reducing lipid accumulation . In the The five subfractions (F1–F5) of the cranberry copigment fraction did not stimulate lipolysis of differentiated adipocytes (3T3-L1 cells) by increasing FFA release (data not shown).The results of the testing of the five subfractions of the cranberry copigment fraction on PDE 3B activity are presented in 50 values of the subfractions of the cranberry copigment fraction that we evaluated based on the results of the PDE 3B activity assay  and F4 (IC50 = 229 μg/mL). A weaker effect on PDE inhibition was shown by F3, and an IC50 value of 471 μg/mL was calculated.We calculated the ICty assay . The subThe seven subfractions (F1–F7) of the pomegranate copigment fraction were tested in the different in vitro assays, too. Since the copigment fraction of pomegranate extract did not induce FFA release in 3T3-L1 cells, we did not test the subfractions in the lipolysis assay.c = 5–10 µg/mL), with relative viabilities ≥78%. Treatment with concentrations of 25 µg/mL significantly decreased the viability of 3T3-L1 cells compared with the solvent control (data not shown). For the determination of lipid accumulation, the same concentration ranges of 5–25 µg/mL as for the fractions of the pomegranate extract, were used for the subfractions.There was minimal cytotoxicity of all seven subfractions treated with low concentrations  were able to reduce PDE activity . Subfrac50 values of the subfractions of the pomegranate copigment fraction are listed in 50 values less than 10 μg/mL were calculated. The less active ones were the subfractions F6 and F7, with IC50 values of 8.5 µg/mL and 9.0 µg/mL. The subfractions F1 (IC50 = 4.8 µg/mL) and F5 (IC50 = 3.8 µg/mL) showed a comparable inhibition level. All other fractions revealed nearly the same inhibitory potential against PDE, i.e., subfraction F2 , F3 (0.2 µg/mL), and F4 (0.5 µg/mL), respectively.The ICWithin the last years, it has been reported that polyphenols can influence the lipid metabolism in vitro and in vivo ,16,38,39First of all, XAD-7 extracts of commercially available concentrates from chokeberry, cranberry, and pomegranate were generated. These extracts were separated into an anthocyanin and a copigment fraction by membrane chromatography . It was We could demonstrate that all the tested fractions influenced lipid metabolism by decreasing lipid accumulation and inhibition of PDE 3B activity, whereas only the copigment fractions of the chokeberry and cranberry extracts tended to increase FFA release by lipolysis. Based on the effects of the in vitro assay, the fractions ranked as follows: pomegranate anthocyanin fraction ≈ pomegranate copigment fraction > pomegranate polymer fraction > cranberry polymer fraction > cranberry copigment fraction ≈ cranberry anthocyanin fraction ≈ chokeberry anthocyanin fraction ≈ chokeberry copigment fraction > chokeberry polymer fraction. However, when evaluating the relevance of these results, it could not be excluded that the effects observed in 3T3-L1 cells might have been amplified by reduced cell viability at the highest tested concentrations of the fractions.c = 25–100 µg/mL) significantly decreased lipid accumulation in 3T3-L1 cells without showing cytotoxicity [50 values between 120 ± 44 µM and 299 ± 56 µM [Some in vitro studies have also examined the effects of polyphenol-rich extracts on lipid metabolism, as well as one of our previous studies ,28,41. Ktoxicity . Comparatoxicity . We idenNot only the anthocyanin fractions, but also the copigment fractions, showed a strong influence on the lipid metabolism. The anthocyanin fractions of the chokeberry and cranberry extracts contained both cyanidin-3-galactoside and cyanidin-3-arabinoside, while in the cranberry anthocyanin fraction, additionally, peonidin-3-galactoside was identified, whereas cyanidin-3,5-diglucoside and delphinidin-3-glucoside were the major compounds in the pomegranate anthocyanin fraction. It was shown that the fruit-dependent inhibition of PDE 3B activity and lipid accumulation is mainly due to the copigments, e.g., hydrolyzable tannins, which were found in the pomegranate copigment fraction and were efficient inhibitors of PDE 3B activity and lipid accumulation, whereas for the stimulation of lipolysis via FFA release, copigments like phenolic acids and flavonols were responsible.Since the copigment fractions of cranberry and pomegranate extract showed strong effects on lipid metabolism in vitro, these two copigment fractions were further fractionated by HPCCC. Five subfractions (F1–F5) of the cranberry copigment fraction and seven subfractions (F1–F7) of the pomegranate copigment fraction were obtained. The first and second subfractions, F1 and F2, of the cranberry copigments contained mainly phenolic acids, such as caffeic acid, as well as hexosides of coumaric, ferulic, and sinapic acid. Subfraction F3 consisted of coumaric acid hexoside, myricetin hexoside, and coumaroyl iridoid hexoside, and subfraction F4 consisted of chlorogenic acid (hexoside), myricetin, and quercetin hexosides. The fifth subfraction (F5) included different quercetin derivates, coumaric acid, and myricetin-pentosides. It was more difficult to differentiate the subfractions of the pomegranate copigment extract. Punicalin occurred in the subfractions F1 and F2, punicalagin in the subfractions F1–F4, and pedunculagin in F1–F6. Ellagic acid hexoside was found in the subfractions F3–F5 and ellagic acid in F6 and F7. The last subfractions, F6 and F7, also included phenolic acids, such as caffeic acid, ferulic acid, and coumaric acid, and subfraction F7 included the flavonoids quercetin and luteolin hexoside.c = 1 µM) of quercetin significantly lowered stored lipid content in human Simpson–Golabi–Behmel syndrome (SGBS) adipocytes [All investigated subfractions prepared from the pomegranate copigment fraction by HPCCC reduced lipid accumulation in 3T3-L1 cells, whereas for the subfractions F2 and F3 of the cranberry copigment fraction, no influence on lipid accumulation was observed. The strongest inhibition of lipid accumulation could be detected for subfractions F3 and F4 of the pomegranate copigment fraction. Subfraction F3 consisted of ellagic acid hexoside, pedunculagin, and punicalagin, and subfraction F4 contained additionally granatin A and galloyl-O-punicalin. Similarly, Les et al. found that ellagic acid and punicalagin were able to reduce triglyceride accumulation in 3T3-L1 adipocytes . Respectipocytes . In compipocytes .50 = 37 ± 7 µg/mL–471 ± 67 µg/mL), the subfractions of the pomegranate copigment fraction were more active, and the IC50 values ranged from 0.2 ± 0.1 µg/mL to 9.0 ± 0.1 µg/mL. Within the cranberry copigment subfractions under study, F5 was the most potent PDE 3B inhibitor. We identified quercetin derivatives in subfraction F5 and determined an IC50 value of 37 ± 7 µg/mL. Quercetin, which was tested for the effect on PDE from isolated rat adipocytes by Kuppusamy and Das, showed an inhibitory potency, with an EC50 value of 32 µM, and additionally a dose-dependent increase in lipolysis was observed [The trends observed for the subfractions in the lipid accumulation assay were similar to those observed in the PDE activity assay. All investigated subfractions of the cranberry and pomegranate copigment fractions inhibited PDE 3B enzyme activity in vitro. Contrary to moderately active subfractions of the cranberry copigment fraction , we cannot fully explain any finding and mechanism being responsible for the effects influencing lipid metabolism at present and must address this matter in future studies. Moreover, it could be helpful to reach a better separation of the copigments so that it could be possible in the future to determine their biological activity with single compounds.Taken together, we were able to fractionate extracts (activity-guided) from fruit juice concentrates and could identify compounds in the fractions and subfractions, which are crucial for the effects on the lipid metabolism. However, the use of in vitro models provides only preliminary data, so further evaluation with additional studies, preferably in vivo, is essential, for example, by performing human intervention studies that investigate different parameters of lipid metabolism.


High correlations existed between embryogenicity and endogenous brassinosteroids (BRs) . Similarly, concentrations of endogenous BRs of the sample set correlated positively  to expression of somatic embryo (SE)-related genes, i.e. BBM, LEC2, ABI3, PLT2, and WOX2. Hierarchical cluster and weighted gene coexpression network analysis identified modules of coexpressed genes and network in 4820 differentially expressed genes (DEGs) from All-BR-Regulated Genes (ABRG). Moreover, exogenously-supplemented epiBR, together with 2,4-D and 6-BA, increased embryogenicity of GL-sourced callus, and expression of SE- and auxin-related genes, while brassinazole (BRZ), a BR biosynthesis inhibitor, reduced embryogenicity. Evidences obtained in this study revealed that BRs involved in phase change of leaf explants and may function in regulating gene expression and enhancing auxin effects. This study successfully established protocols for inducing somatic embryogenesis from leaf explants in hybrid sweetgum, which could facilitate the propagation process greatly, and provide theoretical basis for manipulating SE competence of explants in ornamental woody plants.Somatic embryogenesis is a preferred method for large-scale production of forest trees due to its high propagation efficiency. In this study, hybrid sweetgum leaves with phase changes from mature to embryogenic state were selected as experimental material to study somatic embryo initiation. Embryogenicity ranged from high to low,  BABY BOOM (BBM) [LEAFY COTYLEDON (LEC) [SOMATIC EMBRYOGENESIS RECEPTOR LIKE KINASE1 (SERK1) [ABA INSENSITIVE 3 (ABI3) [AGAMOUS-LIKE (AGL) [FUSCA3 (FUS3), PLETHORA (PLT), WUSCHEL (WUS) [LEC2, BBM, and WUS, enable somatic cells to maintain their embryonic characteristics in Arabidopsis [Arabidopsis, only a small number of SEis-related genes have been identified in forest trees and the regulatory mechanisms of these genes request further study.Somatic embryogenesis (SEis) is a process during which somatic cells form the structures similar to zygotic embryos without fertilization. As an ideal means, SEis has been used for efficient plant propagation and genetic transformation. It is also used, as a model system, for studies of reproductive biology and physiology in higher plants . The proOM (BBM) , LEAFY CON (LEC) , SOMATIC (SERK1) , ABA INS3 (ABI3) , AGAMOUSKE (AGL) , FUSCA3 EL (WUS) , 8, and bidopsis . CompareLiquidambar styraciflua × L. formosana) is a valuable ornamental species in horticultures due to its remarkable red leaves in the fall. It is also an economically important species for production of timber and bio fuel due to its fast growth [L. styraciflua) [Hybrid sweetgum (t growth . Currentaciflua)  and hybraciflua) . Accordiaciflua) . Actuallaciflua) . Inductiaciflua) . Howeveraciflua) , 13, 14., Coffea arabica [, cauliflower [, lettuce [Arabidopsis [PIN4 and PIN7, and an enzyme coding gene, CYP79B2, which could convert tryptophan to indole-3-acetaldoxime [IAA5, IAA19, IAA17, were induced by exogenously applied brassinolide (BL), while down-regulated in the BR biosynthetic mutant de-etilated2 (det2) [AUX/IAA, PINs, TIR1 and ARF [Brassinosteroids (BRs) are a class of plant steroid hormones playing pivotal roles in plant growth and development, such as hypocotyl and stem elongation, cell expansion, root development, vascular differentiation, and so on –18. Stud arabica , cauliflliflower , lettuce lettuce  and cott lettuce . Many mabidopsis . Moreovealdoxime , 26. Exp2 (det2) , 28. As  and ARF . Neverthin vitro cultures. Four types of explants were then selected and used for analyzing their embryogenicity, endogenous hormones, expression of selected genes, and etc. On the analytical results, exogenous BR and brassinazole (BRZ) were used to confirm its functions during SE induction. This research provides new insight into the mechanism underlying SEis from mature tissues. It could also benefit further studies and application practice in BR-induced SEis in woody plants.Aims of this research were to find key factors related to the phase change of leaf explants and SE initiation in hybrid sweetgum. We first found a special phenomenon of phase change of hybrid sweetgum leaves during BBM, ABI3, FUS3, PLT2 were not expressed in samples of GL, while LEC2, SERK1 and WOX2 expressed a little in GL. The genes BBM, LEC2, ABI3, PLT2 and WOX2 had the highest expression levels in EC and a relatively lower expression in WLI. These genes were also expressed in WLII. The expression of FUS3 in WLI was higher than that in EC.The normal leaves of somatic seedlings were green . When SE3), gibberellin A4 (GA4), zeatin riboside (ZR), dihydrozeatin riboside (dhZR), indolepropionic acid (iPA), and jasmonic acid methyl ester (JA-Me) were quantified (p < 0.05) between BRs and embryogenicity suggested an important role of BRs in SEis. The highest concentration of BRs was found in samples of EC at 14.70 ng g−1 FW, which was 1.93, 3.47 and 3.58 folds of those in the other three explants . Furthermore, concentrations of endogenous BRs correlated positively to the expression of SE-specific genes, i.e. BBM, LEC2, ABI3, PLT2, and WOX2, r = 0.93, 0.99, 0.87, 0.99, 0.96 respectively and P < 0.05 , indole-3-acetic acid (IAA), gibberellic acid  in n et al. , 7992 hoHierarchical cluster analysis of ABRGs was shown in AGL15 was mainly expressed in WLI and EC, but not expressed in GL. WOX8 was expressed in all four kinds of tissues, showing a similar declining pattern. WOX11 was specifically expressed in EC, WLI, and WL II, and the highest expression was found in WLI. AILs belongs to AP2-like ethylene-responsive transcription factor. AIL5 and AIL7 were specifically expressed in EC and WL tissues and had the highest level of expression in WLI. CUC3 was specifically expressed in EC and WLI, with the highest expression level in EC, while CUC2 was specifically expressed in EC. CLAVATA3/ESR (CLE)-related protein 16 (CLE16) had the same expression pattern, with the expression decreased from WLI, EC, to WLII, and had no expression in GL.Most of the selected auxin-related and SE-related genes were significantly down-regulated with lower embryogenicity , Tab. S5In order to capture key changes in the gene network of different embryogenic explants, weighted gene coexpression network analysis (WGCNA) was used to analyze the co-expression relationship among 4820 DEGs of ABRGs. After filtering, genes were divided into 6 modules, which correlate with a specific tissue cluster of genes . The turP < 0.05). Brassinazole (BRZ) of 1.0 μM decreased the SE induction , which included LEC2 at d5 and d15; FUS3, AUX22, SAUR50, CUC2 at d5, d10, and d15; ABI3, PLT2, YUC7, IAA4-L, LAX5, CUC3, CLE22 at d10 and d15; CUC3 at d5 and d15  produced SEs only after several sub-cultures and SE induction treatments. Manipulation of explants has been used extensively in different propagation methods, such as root cutting, grafting and micro propagation [Liquidambar. This experiment is on-going in our lab with more genotypes, especially the reluctant ones in SEis.This is the first report of inducing SEis from leaf-derived callus in pagation . The purpagation . For SEiArabidopsis, the ELS structure often appears in the abnormal expression of certain key SE-related genes, such as the over-expressed WUS and the ectopic postembryonic expression of LEC132. In our study, high expression of BBM, LEC2, ABI3, PLT2 and WOX2 could be the major cause for phase change and ELS formation. Results of the embryogenicity test could also reflect the phase change directly and obviously. It is the common knowledge that the best plant materials for initiating SEis should be of the least differentiated state, or embryogenic. The explants of EC of the highest SE induction rate, or embryogenicity was embryogenic. Explants of WLI were rejuvenated the most completely in the leaf explants, whereas the green leaf demonstrated no embryogenicity. During SE-induction treatment the GL explant produced no embryognic callus. After in vitro sub-cultures for several times, the GL-sourced callus could produce a few SE, which indicates further rejuvenation may have occurred during in vitro sub-cultures [Our transcriptomic evidences confirmed phase changes, or rejuvenation, indeed occurred in leaf explants of morphological changes in hybrid sweetgum. Several SE-related genes expressed from low to high in the explants changed from green to white in color. In cultures , 32.PLT2, AGL23, LEC2, FUS3, CUC2, and CUC3. Also, exogenously supplemented epiBR resulted in higher expression of those genes. This fact indicates that BRs play a role in the early stage of SEis by activating the expression of these genes. Both PLT2 and AIL5 promoted SEis, activating the expression of LEC1, LEC2 and FUS3 [AIL5 and AIL7 can also activate CUC1 and CUC2 in zygotic embryogenesis [WUSCHEL (WUS)-related homeobox (WOX) gene family mediated auxin-triggered SEis in Arabidopsis [BR6ox, WOX8, and GH3s, were identified by their connecting edges within the network. Future work showing the interaction of these genes may clarify the unique mode of action of BR in regulating somatic embryogenesis.Our results indicate that BR regulates SE induction by activating expression of genes related to embryogenesis and regulating the response to auxin. ABRGs may take part in regulation of gene expression in the process of BR-regulated SE induction. Several SE-related genes were regulated by BR, especially BBM, and FUS3 , 37. Expogenesis , 40. Thebidopsis . Our worARF, AUX/IAA, and SAUR family members [AUX22 and SAUR50 increased when the expression of auxin transport gene, LAX5, was up-regulated by BR treatment. Second, exogenous BR enhanced YUC7 expression  were germinated for two months. The germination medium contained improved Blaydes medium salts [−1) and gellan gum (3 g L−1) , adjusted to pH 5.7. Cultures were kept at 25 ± 2°C under 16 h light cycles in the culture room of Shen Zhou Lv Peng Agricultural Science & Technology Co. Ltd, Beijing, China. Four types of samples were collected: the normal green leaf (GL); the distal part of white leaf (WLI); the part between the white end and the green end of the same leaf (WLII); and embryogenic callus (EC). Tissue of EC was induced from immature zygotic embryos three years ago approximately, and maintained by regular subcultures once a month. Samples were divided into two parts; one part was preserved in liquid nitrogen for transcriptome analysis, plant hormones determination, and RNA extraction. Another part was inoculated in SE induction medium I in dark at 27 ± 2°C for 30 d and then used for embryogenic capacity evaluation. SE induction medium I contained improved Blaydes medium salts, inositol (0.1 g L−1), casein hydrolysate (1 g L−1), 2,4-dichlorophenoxyacetic acid  and 6-benzylamino-purine . Calli used for exogenous treatment were derived from green leaves, which were maintained by regular subcultures on fresh SE induction medium I once per month.Mature somatic embryos with two well-developed cotyledons of hybrid sweetgum  was added to SE induction medium I to achieve the final concentrations of 0, 0.1, 0.5 and 1.0 μM, respectively. Calli (1.0 cm × 1.0 cm) grew in SE induction medium I (20 each) for 30 days before the embryogenic capacity evaluation was made. Three biological replicates were carried out in this experiment.−1) and inositol (0.1 g L−1) without plant growth regulators. The culture was kept in dark at 27 ± 2°C for 90 days before SE induction. Embryogenic capacity was judged on the percentage of explants, such as calli, forming SEs to the total explants used. All experiments were repeated three times, and each replicate used at least 60 explants (20 explants/dish).Samples were transferred to SE induction medium II, which contained Blaydes medium salts with sucrose . Nikon ECLIPSE E100 microscopy and Nikon DS-U3 system was used for image acquisition.For SE induction viability analysis, callus from different treatments were stained with 0.01% fluorescein diacetate (FDA) solution diluted in water for 5 min and rinsed with water for 5 times. At least 0.2 g callus from 5 dishes for each treatment were evaluated. Photos were taken using Leica M205FA fluorescence stereo microscope. The experiment was based on three biological replicates, in which one 3.5 × 1.0 cm Petri dishes containing 1.5 mL water were considered as one replicate.EasyPure Plant RNA Kit . The cDNA was obtained using a TransScript One-Step DNA Removal and cDNA Synthesis SuperMix Kit . The qRT-PCR was performed using Trans Start Top Green qPCR SuperMix  on CFX Connect™ Real-Time PCR Detection System . The amplification condition for qRT-PCR was under the following program: denaturation at 94°C for 30 s, 40 cycles at 94°C for 5 s, 60°C for 15 s, 72°C for 15 s, with a melt cycle from 65 to 95°C. The relative gene expression in each sample was normalized to EF1 Ct value and calculated using the 2−ΔΔCt method. The gene-specific primers are listed in RNA was extracted using an L. formosana genome for the efficiency. StringTie was used to assemble the mapped date and analyze, based on uses of FPKM (Fragments Per Kilobase of transcript per Million fragments mapped) as an index to measure the expression levels of transcripts or genes. For analysis of differentially expressed genes, the DESeq2 was performed using Fold Change≧2 and FDR <0.01 as the criteria. Gene functions were annotated based on NR, Swiss-Prot, GO, COG, Pfam, and KEGG. Enrichment analyses were performed using Cluster Profiler. Coexpression networks were constructed using the WGCNA package in R [The total RNA was extracted using TRIzol reagent . The cDNA libraries were performed using the Illumina HiSeq4000 System . HISAT2 software was used to compare the clear reads with those of age in R . Eigengeage in R .P < 0.05 were considered statistically significant.Statistical analyses were performed using IBM SPSS Statistics version 20.0 . All statistical analysis was based on three independent replicates. Results were presented as means ± the standard error and analyzed using ANOVA, followed by Tuckey test. Differences with Web_Material_uhab047Click here for additional data file.


Mycobacterium tuberculosis warrant new strategies for TB control. The successful use of a bacteriophage cocktail to treat a Mycobacterium abscessus infection suggests that phages could play a role in tuberculosis therapy. To assemble a phage cocktail with optimal therapeutic potential for tuberculosis, we have explored mycobacteriophage diversity to identify phages that demonstrate tuberculocidal activity and determined the phage infection profiles for a diverse set of strains spanning the major lineages of human-adapted strains of the Mycobacterium tuberculosis complex. Using a combination of genome engineering and bacteriophage genetics, we have assembled a five-phage cocktail that minimizes the emergence of phage resistance and cross-resistance to multiple phages, and which efficiently kills the M. tuberculosis strains tested. Furthermore, these phages function without antagonizing antibiotic effectiveness, and infect both isoniazid-resistant and -sensitive strains.The global health burden of human tuberculosis (TB) and the widespread antibiotic resistance of its causative agent  Mycobacterium tuberculosis, the causative agent of human tuberculosis, has plagued humanity for nearly 9,000 years, with the earliest written records of the disease going back more than 3,000 years in India and China , mostly within the Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program  have been described  includes Mycobacterium africanum, Mycobacterium canettii, Mycobacterium bovis, Mycobacterium microti, Mycobacterium orygis, Mycobacterium caprae, Mycobacterium pinnipedii, Mycobacterium suricattae, and Mycobacterium mungi, in addition to M. tuberculosis  is closely related to L6, but is only found in eastern Africa. Epidemiological studies suggest that lineages 2 and 4 may be more virulent than lineages 1 and 3 , and propensity for cellular clumping present substantial challenges to detailed phage investigations using M. tuberculosis. Here, we screened an expanded panel for new phages that infect M. tuberculosis, enhanced potentially useful phages by genome engineering and host range manipulation, and defined variations in phage infection in a suite of M. tuberculosis clinical isolates. By defining patterns and mechanisms of phage resistance and interactions with antibiotics, we have assembled a five-phage cocktail that efficiently kills all of the tested M. tuberculosis strains and which can be used to evaluate phage therapy for human tuberculosis.The therapeutic potential of phages for treating tuberculosis has not been thoroughly explored, in part because relatively few phages are available. Thus, little is known about variation in susceptibility and killing of M. tuberculosis mc27000 (an avirulent derivative of H37Rv), but they belong to a few clusters/subclusters . Although phage BPs (cluster G1) does not efficiently infect mc27000, host range mutants containing single amino acid substitutions in the tail gene (gene 22) can be readily isolated   also infect H37Rv but are temperate and form extremely turbid plaques reflecting high lysogenization frequencies , D29 (A2), two host range mutants of BPs  also efficiently infect M. tuberculosis H37Rv   the intect H37Rv . Howeverquencies ; clear pis H37Rv . Adephagis H37Rv . The temain GD01 .M. smegmatis phages for those that infect M. abscessus GD01 identified phages Itos and Gabriela (both in subcluster L2) as potentially having a broad host range (M. tuberculosis H37Rv (−3). This is consistent with the report that the subcluster L2 phage Celfi infects M. tuberculosis mc26230, a derivative of M. tuberculosis H37Rv  were tested for sensitivity to phages that infect M. tuberculosis H37Rv , N1063 (L5), or lineage 6 strains, and Muddy, which does not efficiently infect any L1, L3, L5, or L6 strains, N0031 (L2), or lineage 4 strains N0136 and N1283  are strictly restricted to H37Rv infection, and do not efficiently infect any other strain  and N0052 , recovered on M. smegmatis, and further characterized. DNA sequence analysis (see below) showed the Muddy lysate derived from N0157 was a mixture of two phages carrying different mutations, which were separated and purified. Following purification, the three host range mutants (HRMs) were designated Muddy_HRMN0157-1, Muddy_HRMN0157-2, and Muddy_HRMN0052-1 , conferring amino acid substitutions G487W, T608A, and E680K, respectively, all within a predicted extended β-sheet at the C terminus of the gp24 protein  on strains N0004 (L3), N0145 (L2), and N0136 (L4)  , Table 3136 (L4) , Table 3M. tuberculosis L1 to L4 strains tested here. For strain N1063 (L5), all three mutations confer some improvement in infection, but for strain N1202, WT Muddy and the mutants infect at similarly reduced efficiencies , N0004 (L3), and N1283 (L4) showed that each had one of the same three substitutions in gp24. Plaques derived from strains N0072 and N0004 have the T608A and G487W substitutions, respectively, and plaques derived from N1283 had both the G487W and E680K mutations. Interestingly, although wild-type (WT) Muddy infects strain N1216 relatively well , and witciencies .M. smegmatismpr (multiple-phage-resistance) locus confers resistance of M. smegmatis to phages D29 and L5  of 1 to 10 in liquid culture, incubated for 1 week, and then plated on solid medium for bacterial growth. This analysis included H37Rv and a representative strain from lineages L1 to L4, with five phages from those identified above that infect these strains efficiently  efficiently kills all of the tested strains with nearly no survivors, a few very small colonies were observed, although these could not be further propagated and retested. We were similarly unable to recover genetically stable D29-resistant mutants (colonies either did not grow or retested as being D29 susceptible). In contrast, two resistant strains to AdephagiaΔ41Δ43 (from H37Rv and N1283), a Fionnbharth-resistant mutant of H37Rv, and three Fred313_cpm-resistant mutants (two in H37Rv and one in N1283) were isolated  coding for a cytidylate kinase   were propagated and tested for sensitivity to other s phages . In genes phages . Howevers phages ; the Ades phages  also rems phages . All of s phages  and C. Ister L2) . We noteM. tuberculosis strains were grown until visibly turbid , serially diluted, and incubated with individual phages in liquid medium for 96 h. These were then plated onto solid medium for growth of survivors . For strain N0004, growth was only observed for the least dilute sample of the control, and the killing efficiency is less clear. Muddy WT did not kill any strain well, and the Muddy host range mutants did not efficiently kill N0145  were incubated with the phage cocktail at a range of 107 to103 total PFU for 7 days and then plated on solid medium for bacterial growth (N0157-1 for Muddy HRMN0052-1) with strains N0052 (L4), N0054 (L4), and N1283 (L4) with similar results, and as few as 105 PFU input phage gave substantial killing within 24 h  isoniazid-resistant strain (mc24977) with H37Rv , we observed two healthy growing plaques (from ∼108 PFU input phage). These were purified, retested, and shown to be escape mutants (CG-REM-1 and CG-REM-2) that infect the resistant strain as efficiently as the parent H37Rv strain  could replace FionnbharthΔ45Δ47 as a means of further reducing resistance. A case can also be made for inclusion of DS6A, which broadly infects and kills the tested strains. Two potential caveats are that DS6A processes an integration cassette  but not the other (N1202) . HoweverOf the phages described here, only Muddy is a naturally lytic phage. All of the others are either naturally occurring or engineered lytic derivatives of temperate parent phages; all are siphoviral. Thus, the available phage “space” available for tuberculosis therapy is quite distinct from many other bacterial pathogens, for which lytic myoviruses and podoviruses have been widely used. This does appear to be an impediment, and engineering strategies can be used to convert the temperate phages into lytic phages through removal of the repressor gene. However, our finding that survivors of a Fred313_cpm challenge carry integrated phage genome segments suggests it is advisable to also remove the integrase genes. Fortunately, recombineering tools applied in the BRED and newer CRISPY-BRED methods provides simple and effective ways of doing so , 57.M. tuberculosis strains with seemingly low resistance frequencies, infrequent cross-resistance, and that work together with antibiotics and infect antibiotic-resistant strains, there are now few impediments to clinical evaluation of bacteriophages for relief of tuberculosis. Whether such therapy might be broadly applicable or restricted to a narrow spectrum of disease states is not clear, but with the excellent safety profile of phages in humans   and 0.05% Tween 80 until visibly dispersed (10 days to 3 weeks) at 37°C with shaking. Lineage 5 and 6 strains were further supplemented with 40 mM sodium pyruvate (Sigma). Strains were grown on solid Middlebrook 7H11 agar  supplemented with OADC and 1 mM CaCl2 for 2 to 6 weeks at 37°C .M. smegmatis mc2155 or an M. tuberculosis strain using Middlebrook 7H11 with 0.7% agar for M. tuberculosis and Middlebrook 7H10 with 0.35% agar for M. smegmatis. Plates were incubated at 37°C for 24 to 48 h for M. smegmatis or 2 to 8 weeks for M. tuberculosis, until visible lawns were obtained. Plates were photographed and analyzed for plaque formation.Phage lysates were 10-fold serially diluted and 3 μl were spotted onto top agar overlays containing 0.5 to 1 ml of M. tuberculosis strains. Lysates on M. smegmatis were amplified under BSL3 conditions and were filtered twice using 0.2-μm filters. Aliquots of lysates (1 ml) were serially diluted and plated onto agar lawns for isolated plaques. Isolated plaques (n = 8 to16) were picked using a 0.2 to 10 μl micropipette tip into 50 μl of phage buffer  as described previously . In a 96-well plate , 20 μl of each phage (one per row) was added to a total volume of 200 μl consisting of Middlebrook 7H9 supplemented with OADC and 1 mM CaCl2, and the bacterial strain, grown until visibly dispersed (OD600 of ≥0.1) and 10-fold serially diluted to 10−1 to 10−4. The bottom row of each 96-well plate contained bacteria and no phage. To assess killing of 104 PFU, the phage lysate was normalized to 105 PFU and then the same procedure was followed as detailed above. The plates were sealed and incubated without shaking at 37°C for 96 h. Each well was mixed by pipetting and then 3 μl was spotted onto Middlebrook 7H11 plates containing 1 mM CaCl2 and OADC and the plates incubated for 3 weeks at 37°C before imaging.To assess killing of individual phages at 108 PFU/ml and 20 μl of each phage were combined into a cocktail. Liquid bacterial cultures were grown and aliquoted into 96-well plates as described above; the cocktail was serially diluted such that each row contained from 107 to 103 PFU total phage. Approximately 20 μl of serially diluted M. tuberculosis (∼5 × 108 CFU/ml) from undiluted to a 10−4 dilution was added to each plate column. Plates were sealed and incubated standing at 37°C. At 24, 48, and 96 h and 1 week of time, the 96-well plates were centrifuged at 3,500 rpm for 2 min to remove condensation from the sealing film using a bio-liner swing bucket rotor (Thermo). Cultures were resuspended using a multichannel pipet and 3 μl aliquots were spotted onto Middlebrook 7H11 plates supplemented with OADC and 1 mM CaCl2 and incubated for 3 to 4 weeks at 37°C.Phage titers were normalized to 1 × 102 and 1 × 107 to 1 × 108 PFU of phage. After incubation with shaking (200 rpm) at 37°C for 1 week, cells were pelleted at 5,000 × g for 10 min, resuspended in 100 μl 7H9 OADC, and spread onto 7H11 plates containing OADC. Plates were incubated for 4 to 8 weeks and surviving colonies restreaked onto 7H11 OADC plates. Colonies that grew without evidence of lysis were inoculated into liquid culture and tested for phage sensitivity.Approximately 100 μl of bacterial cultures at OD of ∼0.1 to 0.2 was added to tubes containing 1 ml of 7H9 supplemented with OADC and 1 mM CaCl9 to 1011 PFU/ml) were spotted onto lawns of phage-resistant mutants and individual plaques picked and replated on the resistant mutant and M. smegmatis mc2155 to determine the EOP. Plaques were picked from the M. smegmatis mc2155 lawn and replated on the M. tuberculosis resistant mutant. True-breeding escape mutants were amplified and sequenced.Approximately 3 μl of phage lysates  or 0.2 μg/ml isoniazid . Phage lysate diluted to 10M. tuberculosis and phage DNAs was as described previously  reported previously for typing Copyright © 2021 Guerrero-Bustamante et al.2021Guerrero-Bustamante et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00973-21.2TABLE S2Table S2, PDF file, 0.05 MB.Oligonucleotides used in this study. Download Copyright © 2021 Guerrero-Bustamante et al.2021Guerrero-Bustamante et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00973-21.3TABLE S3Mycobacterium strains. Download Table S3, PDF file, 0.02 MB.Sequencing details of Copyright © 2021 Guerrero-Bustamante et al.2021Guerrero-Bustamante et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00973-21.4FIG S1M. tuberculosis lineages. (A) Dilutions of M. tuberculosis strains were prepared as in 41Δ43, Fred313_cpmΔ33, FionnbharthΔ45Δ47, Muddy_HRMN0157-1, and D29. The top rows contain a total of 107 PFU, and below are shown 10-fold serial dilutions of the phage input. Cocktail was spotted onto agar plates at 24 h, 48h, 96 h, and 1 week at 37°C as indicated. (B) The same experiment as in panel A, but using a cocktail containing AdephagiaΔ41Δ43, Fred313_cpmΔ33, FionnbharthΔ45Δ47, Muddy_HRMN0052-1, and D29. Download FIG S1, PDF file, 1.5 MB.Killing efficiencies of individual phages and the five-phage cocktail for Copyright © 2021 Guerrero-Bustamante et al.2021Guerrero-Bustamante et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


Elemental analysis indicated that the total amount of the organic phase of the prepared stationary phase (Sil−VPG15) was 38.0 wt%, and the quaternization degree of the pyridyl groups was determined to be 32.5%. Differential scanning calorimetric analysis of a methanol suspension of Sil−VPG15 indicated that the G moieties formed a highly ordered structure below the phase transition temperature even on the silica surface, and the ordered G moieties exhibited a gel-to-liquid crystalline phase transition. Compared with a commercially available octadecylated silica column, the Sil−VPG15 stationary phase showed high selectivity toward polycyclic aromatic hydrocarbons, and particularly excellent separations were obtained for geometrical and positional isomers. Sil−VPG15 also showed highly selective separation for phenol derivatives, and bio-related molecules containing phenolic groups such as steroids were successfully separated. These separation abilities are probably due to multiple interactions between the elutes and the highly ordered functional groups, such as the pyridinium and amide groups, on the highly ordered molecular gel having self-assembling G moieties.Chiral glutamide-derived lipids form self-assembled fibrous molecular gels that can be used as HPLC organic phases. In this study, HPLC separation efficiency was improved through the addition of branched amphiphilic glutamide lipids to the side chains of a terminally immobilized flexible polymer backbone. Poly(4-vinylpyridine) with a trimethoxysilyl group at one end was grafted onto the surface of porous silica particles (Sil−VP High performance liquid chromatography (HPLC) has been widely used for the analysis and separation of organic compounds containing polymers and bio-molecules, because it is convenient and quick analytical technique. The development of HPLC stationary phases is of importance to expand its applicability in various fields. So far, various surface-modified porous silica particles having organic phases such as ionic liquid analogues ,2,3,4,5,Molecular gels have been attracting significant attention because their highly oriented structures result in unique properties ,19,20,21The highly oriented structure of the self-assembled molecular gels can recognize the molecular shape of guest molecules through multiple interactions using regularly arranged functional groups. Directly immobilized lipophilic glutamide derivatives on the surface of silica microspheres recognize polycyclic aromatic hydrocarbons (PAHs), particularly the molecular planarity and the molecular length of PAHs ,30,31,322 g−1) for the stationary phase were purchased from YMC Co. Ltd. . All other chemicals used for the synthesis of Sil−VPG15 were purchased from Tokyo Chemical Industry Co., Ltd. , Fujifilm Wako Pure Chemical Co. , Nacalai Tesque, Inc. , and Sigma-Aldrich Japan , and were purified before use as needed.Porous silica microspheres . Chemical shifts (δ) are expressed in parts per million (ppm) using tetramethylsilane (TMS) as an internal standard (δ = 0.0 ppm). FT-IR spectra were obtained with a JASCO FT/IR-4100 spectrometer . A DR PRO410-M accessory (JASCO Co.) was used for diffuse reflectance FT-IR (DRIFT) measurements. Elemental analyses were carried out on a CHN Corder MT-6 Apparatus . Differential scanning calorimetry was performed using DSC 7000 . The aluminum open pan and the silver closed cell were used for solid state samples and suspension state samples respectively. The measurements were carried out the heating rate of 2 °C min−1 under the N2 gas atmosphere. The surface charge of the particles in suspension state were detected by Zetasizer Nano ZS .N1,N2-didodecyl-L-glutamide (G) was synthesized according to our previous report [G  and 11-bromoundecanoic acid  were dissolved in chloroform (300 mL), and the mixture was stirred in an ice bath. Triethylamine  and diethyl cyanophosphate  were added to the mixture, which was then stirred for 1 h in an ice bath and then for 24 h at 25 °C. The mixture was washed with 0.3 mol L−1 aqueous HCl solution (3 times), 5 wt% aqueous NaHCO3 solution (3 times), and deionized water (3 times). The solution was dried over anhydrous sodium sulfate. After removal of the sodium sulfate by filtration, the chloroform was removed by evaporation under reduced pressure. The residue was recrystallized from ethanol, and the precipitate was collected by filtration and dried in vacuo to obtain white crystals , 2923 cm−1 (νC–H), 2856 cm−1 (νC–H), 1637 cm−1 (νN–H (amide)), 1557 cm−1 (νN–H). 1H–NMR: δ 0.86–0.90 , δ 1.24 9CH3 and BrCH2CH2(CH2)6), δ 1.39–1.44 CH2CH2), δ 1.49–1.53 , δ 1.81–1.88 , δ 1.92–2.10 , δ 2.19–2.31 , δ 2.41–2.48 , δ 3.20–3.30 , δ 3.38–3.42 , δ 4.30–4.36 , δ 6.02–6.05 , δ 6.82–6.85 , δ 6.99–7.01 . Elemental analysis Calcd. (%) for C40H78BrN3O3: C 65.91, H 10.79, N 5.76; Found: C 64.91, H 11.76, N 5.62.The glutamide-derived lipid, s report . The gluals BrG, . Yield: 1H-NMR: δ 0.5–0.7 , δ 3.4–3.6 , δ 6.1–7.0 , δ 8.1–8.7 . The average polymerization degree of the poly(4-vinylpyridine) with a trimethoxysilyl reactive group at one terminus was determined to be 15 (VP15) from the integral ratio of the peaks of the methylene group at the γ-position of the terminal 3-mercaptopropyltrimethoxysilane and the pyridine ring.A mixture of 4-vinylpyridine  and 3-mercaptopropyltrimeth-oxysilane  was stirred with nitrogen gas bubbling through the solution for 20 min. Then, 2,2’-azobis(isobutyronitrile)  was added, and the mixture was stirred at 60 °C under a nitrogen atmosphere for 3 h. The obtained pale yellow viscous solution was dissolved in a small amount of chloroform, and the solution was added dropwise to diethyl ether. The precipitate that formed was washed with diethyl ether, dried in vacuo, and the product was obtained as a white powder. Yield: 7.10 g (67.6%). 15 (2.0 g) was dissolved in chloroform (35 mL) in a three-neck flask. Porous silica microspheres (4.0 g) were added to the solution and gently stirred at reflux for 3 days. The resulting surface-modified silica microspheres were filtered and washed with chloroform, then dried in vacuo  and BrG (7.01 g) were added to toluene (30 mL), and the mixture was heated at reflux for 3 days with stirring. The product was collected by filtration through a glass filter, washed with toluene, chloroform, and methanol, in that order, and dried under reduced pressure to obtain Sil−VPG15 , Fujifilm Wako Pure Chemical Co. , and Sigma-Aldrich Japan Inc. . HPLC-grade solvents were used for chromatographic separations. Octadecyl-functionalized porous silica particles (Inertsil ODS-3 packed column) were purchased from GL Science Inc.  and used as a reference stationary phase.G15 was packed into stainless steel columns (150 mm × 4.6 mm i.d.). Liquid chromatography was performed with acrylonitrile as the mobile phase at a flow rate of 1.0 mL min–1 under isocratic conditions. The chromatographic system included a JASCO 2089 pump and a JASCO MD-910 UV-Vis photodiode array detector. A 5 µL sample was injected through a Rheodyne Model 7725 injector  having a 20 µL loop. The column temperature was maintained using a column jacket. A personal computer connected to the detector with JASCO-Borwin (Ver 1.5) software was used for system control and data analysis. The separation factor (α) was determined by the ratio of retention factors (k). The retention time of D2O was used as the void volume (t0) marker. Sil−VPn) was prepared by telomerization of 4-vinylpyridine using 3-mercaptopropyltrimethoxysilane as a telogen.Poly(4-vinylpyridine) with a reactive group at one terminus  was determined to be 15. In our previous report, we confirmed that the separation behavior of the polymer-immobilized porous silica was almost the same in the polymer having a degree of polymerization of 10 or more . The obt−1 and from 1417 to 1467 cm−1, respectively [−1 that was due to the long alkyl chain of the G moiety appeared after quaternization.The quaternization reaction of the pyridyl side chain was confirmed by the FT-IR spectrum , in whicectively ,36. In aG was calculated as 32.5%. Because the pyridinium salt after quaternization is cationic, success of the quaternization reaction was also determined by measuring the zeta potential at the particle interface, which confirmed that the particle interface of Sil−VPG15. was more positively charged than Sil−VP15.Elemental analysis of the polymer-grafted porous silica  showed a15 and Sil−VPG15 (15) and quaternization (Sil−VPG15) showed no noticeable change in appearance of the spherical shape, which indicated that the polymer grafting and quaternization had occurred on the surface of the porous silica particles.Thermogravimetric analysis of Sil−VPil−VPG15 , in whicG15 could be dispersed in organic solvents such as methanol and acetonitrile. The surface charge of the porous silica particles was −11.4 mV in methanol, which changed to +7.7 after VP15 grafting (Sil−VP15) and +32.8 after quaternization with BrG (Sil−VPG15). These results suggest that the surface of Sil−VPG15 is amphiphilic and positively charged.The obtained Sil−VPG15 in the solid state  is higher than 68.8 °C, it is likely that the crystallization of the G moiety is perturbed by the polymer backbone. For Sil−VPG15 dispersed in acetonitrile, phase transition behavior was observed at 42.4 °C with a pre-transition at 23.1 °C  through a quaternization reaction. The amphiphilic side chains of the G moiety formed highly ordered structures on the silica surface even in polar organic solvents such as acetonitrile. Therefore, Sil−VPG15 was evaluated as a packing material in RP-HPLC retention studies using alkylbenzenes and PAHs as analytes. Octadecylated porous silica microspheres (ODS), which are commonly used for the stationary phase in RP-HPLC, were used as a reference. 0)/t0, where k is the retention factor, t is the retention time of the elute, and t0 is the void volume) and log P (P is the 1-octanol–water partition coefficient) for ODS and Sil−VPG15. The log k and log P plots of the alkylbenzenes and PAHs in ODS were almost identical. However, Sil−VPG15 showed a higher retention for PAHs than for alkylbenzenes. These results provided evidence that the ODS stationary phase can recognize only the hydrophobicity of the analytes, but Sil−VPG15 has specific interaction sites as well as molecular hydrophobicity for the retention of PAHs.The G15 also probably recognizes the π−electrons of the PAHs through multiple interactions with the ordered carbonyl groups on the oriented G moieties.We had previously reported that carbonyl group-containing stationary phases recognize the aromaticity of the elutes through carbonyl π−benzene π interactions ,38,39. PG moiety may have relatively less perturbation from the silica surface because the flexible polymer chain is branched. cis− and trans−stilbene; o−, m−, and p−sterphenyl; and triphenylene using Sil−VPG15 and a commercially available ODS. Sil−VPG15 showed baseline separations for these isomers whereas the ODS did not.We previously reported that self-assembling molecule-immobilized stationary phases, in which the glutamide derivative is directly grafted to the porous silica particles through a siloxane linkage, show high selectivity toward PAHs ,30,31,32G15 and ODS at 0 °C and 50 °C are summarized in G15 stationary phase than they were using the ODS stationary phase, which is probably due to the amphiphilic nature of organic phase. However, Sil−VPG15 showed extremely high selectivity toward these isomers at temperatures below (0 °C) and above (50 °C) its phase transition. These results indicate that the G moiety recognizes molecular shape even in a disordered state, and this is enhanced through orientation of the G moiety.The retention and separation factors of these molecules using Sil−VPtrans-stilbene/cis-stilbene, and triphenylene/o-terphenyl are shown in G moiety.The temperature dependency of the separation factors of naphthacene/fluorene, G15. As shown in G15. All monosubstituted benzenes with a polar group eluted later than toluene, indicating that retention was determined by a hydrophilic interaction (HILIC)/RP mixed-mode mechanism. It is noticeable that the organic phase of the Sil−VPG15 recognized not only hydrophilic groups but also hydrophobic groups due to its amphiphilic nature.Monosubstituted benzenes such as phenol, aniline, and benzoic acid, were examined for chromatographic separation using Sil−VPG15. As shown in G15 column recognized positional isomers of these substituted phenols.The positional isomers of monosubstituted phenols such as methyl phenols, dihydroxy benzenes, and aminophenols were also examined using Sil−VPG15 column compared with those obtained via an ODS column. Sil−VPG15 was able to separate the steroids based on their number of hydroxyl groups. The steroid  and at 42.4 °C (main phase transition) in an acetonitrile suspension even on the surface of a solid substrate. Compared with a conventional ODS column, Sil−VPG15 showed high selectivity toward PAHs, and particularly excellent separations were obtained for geometrical and positional isomers such as stilbenes and triphenylenes. This separation ability is probably due to multiple interactions between the elutes and functional groups on the highly oriented G moiety of the organic phase, such as pyridinium and amide bonding. Furthermore, Sil−VPG15 recognized substituted benzenes in the order of toluene (methyl group) < aniline (amine group) < phenol (hydroxyl group) < benzoic acid (carboxylic acid). Highly selective separations were obtained for positional isomers of phenolic compounds such as methyl phenols, dihydroxy benzenes, and aminophenols. We also demonstrated the separation of eight steroid compounds having different chemical structures and different numbers of hydroxyl groups. Isolation and purification of biological molecules such as peptides, proteins, saccharides and nucleosides are indispensable technology in the fields of proteomics, genomics, and glycomics [G15 is currently underway and will be reported in the future.Amphiphilic glutamide was successfully introduced as a self-assembling molecular gel to the side chain of a terminally grafted poly(4-vinylpyridine) on the surface of porous silica microspheres. The lycomics ,41,42. F


Ochratoxin A (OTA) is a mycotoxin frequently found in raw and processed foods. While it is considered a possible human carcinogen, the mechanism of action remains unclear. OTA has been shown to be hepatotoxic in both in vitro and in vivo models and oxidative stress may be one of the factors contributing to its toxicity. Hence, the effect of OTA on human hepatocellular carcinoma, HepG2 cells, was investigated on oxidative stress parameters. The cytotoxicity of OTA on HepG2 was time- and dose-dependent within a range between 0.1 and 10 µM; while 100 μM of OTA increased the intracellular reactive oxygen species (ROS) in a time-dependent manner. Additionally, the levels of glutathione (GSH) were increased by 9.7% and 11.3% at 10 and 100 nM of OTA, respectively; while OTA at 100 μM depleted GSH by 40.5% after 24 h exposure compared with the control. Finally, the mRNA level of catalase (CAT) was downregulated by 2.33-, 1.92-, and 1.82-fold after cells were treated with 1, 10, and 10 μM OTA for 24 h, respectively; which was linked to a decrease in CAT enzymatic activity. These results suggest that oxidative stress is involved in OTA-mediated toxicity in HepG2 cells. Ochratoxin A (OTA) is a fungal toxin produced by many species in the genera Aspergillus and Penicillium. Due to their diverse growth characteristics, OTA has been found in a wide variety of agricultural commodities and their processed products including major cereals, nuts, coffee, dried fruits, and wine ,2,3,4,5.2•− or H2O2) or serves as an electron donor for ROS-utilizing enzymes such as glutathione peroxidase (GPX). Antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT), and GPX. SOD converts O2•− to molecular oxygen (O2) and H2O2, whereas CAT converts H2O2 into oxygen and water, and GPX converts H2O2 into water [+, respectively [Until now, there is no consensus regarding its mechanisms of action, especially whether it acts through a genotoxic or non-genotoxic mechanism. Both in vitro and in vivo studies have shown that OTA toxicity may be related to the stimulation of oxidative stress as OTA increase levels of reactive oxygen species (ROS), oxidative DNA lesions, lipid and protein damage, and depletion of endogenous antioxidants such as glutathione and antioxidant enzymes ,15,16,17to water . In addiectively .The liver is the most important organ involved in drug metabolism. Drugs may accumulate in the liver and/or enzymes in the liver may form toxic drug metabolites, leading to hepatotoxicity . OTA hasp < 0.05) in cell viability was 1 μM at each exposure time . In general, OTA led to a dose- and time-dependent decrease in cell viability while dose-dependent decrease plateaued above 1 μM, as shown in ure time . Howeverp < 0.05) increase in ROS production by 27% and 35%, respectively. In contrast, TBHP at 250 μM increased ROS production by 74% and 169% at 4 h and 24 h, respectively. These results agree with previous studies, which have shown that OTA influences ROS production levels in a dose-dependent manner [To test the potential of OTA to induce oxidative stress in HepG2, cells were exposed to OTA concentrations ranging from 1 nM to 100 μM for 4 and 24 h, and ROS production was measured by the increase in 2’,7’-dichlorofluorescein (DCF) fluorescence . At botht manner ,27.p < 0.05) increased intracellular GSH levels by 9.7% and 11.3%, respectively, a higher dose of OTA (100 µM) significantly depleted GSH levels by 40.5%  downregulated upon exposure to OTA concentrations above 1 μM after 24 h, as mRNA levels ranged from 1.82- to 2.33-fold lower than the control  significantly decreased the CAT enzymatic activity after 24 h treatment in HepG2 from to 19% to 26%, respectively (p < 0.05).Since OTA decreased the expression of CAT mRNA, the effect of OTA on CAT enzymatic activity was also evaluated. HepG2 cells were exposed to increasing concentrations of OTA (10 nM to 100 μM) for 24 h, and the CAT activity was measured by monitoring Hectively . In conttRNA [The exact mechanism of OTA toxicity in the liver has not been elucidated yet. According to proposed mechanisms involved in OTA toxicity, acute and chronic toxicity of OTA are related directly or indirectly to: (a) its property of inhibiting mitochondrial respiration and ATP production through competitive inhibition of transporters located in the inner mitochondrial membrane ,30; (b) tRNA ,32; (c) tRNA ,33; (d) tRNA ,35; and tRNA ,36,37. BtRNA ,39. Howeoatp) has been shown to be involved in OTA uptake [It is known that the presence of serum in the culture medium influences OTA-induced cytotoxicity due to OTA ability to bind to serum proteins, and thus, lowering OTA cytotoxicity; however, in agreement with our results, previous studies have observed the cell viability plateau above 1 μM OTA, in both serum-free and serum-containing growth medium ,22,40. IA uptake . HoweverA uptake ,45.ROS are produced by the cell during normal aerobic metabolism and possess a dual function on the cell. While lower ROS levels are required for several physiological mechanisms such as host defense , excessic is released from the mitochondria, it may react with H2DCFDA, increasing DFC fluorescence [2•−) [2•− to H2O2, hydroxide radical (HO•) may lead to oxidative damage. This mechanism may corroborate depletion of GSH by OTA in cellular models [8-deoxyguanosine adducts [While an in vitro approach is often used to study ROS production/reduction to determine cytotoxicity, Zhu et al.  reportedrescence ,54. It irescence , which mce [2•−) . As a Fer models . In addi adducts .Mammalian cells handle ROS mainly through three crucial types of antioxidant enzymes: CAT, SOD, and GPX. We observed that OTA modulates the gene expression of these antioxidant enzymes differently. First, OTA had a binary effect on CAT, lower OTA doses (0.01 and 1 µM) upregulated CAT expression, while OTA concentrations above 1 µM downregulated it; second, OTA above 0.1 µM upregulated the expression of SOD1 in a dose-dependent manner; and third, OTA above 1 µM upregulated the expression of GPX1. Accordingly, our results agreed with previous in vitro studies with HepG2 and Vero (kidney) cells where OTA at 25 µM showed a reduction of CAT and an increase in SOD1 gene expression ,16,23. I2O2. Although the cell upregulated the expression of GPX1 after OTA exposure, it also decreased the expression of GSR, which led to an impairment of H2O2 detoxification by the glutathione peroxidase system. Additionally, it is noteworthy to mention that OTA mediated cytotoxicity in HepG2 cells did not follow a clear dose-response relationship, and for the first time, we have indicated that the maximum reported OTA solubility in water is 3 µM; which explains the plateau observed at higher OTA doses. In conclusion, the results presented in this study indicate that OTA may be involved in the development of oxidative stress through increasing intracellular H2O2 levels by impairing the primary antioxidant enzymes catalase and glutathione peroxidase.The most important finding about this study was that OTA is cytotoxic to HepG2 cells in a time-dependent manner, which could not be directly associated with an increase in ROS production, but a depletion of GSH and modulation on the mRNA expression of antioxidant enzymes and its activity. OTA significantly downregulated the gene expression of CAT, which was concomitant to a decrease in its enzymatic activity. Therefore, the cells rely on the GPX system for the removal of Htert-butyl hydroperoxide (TBHP), hydrogen peroxide (H2O2), protease inhibitor cocktail, catalase from bovine liver , sodium chloride, potassium chloride, magnesium chloride, calcium chloride, glucose, N-(2-hydroxyethyl)piperazine-2′-(2-ethane-sulfonic acid) (HEPES), and 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) were purchased from Sigma-Aldrich . Eagle’s minimum essential medium (EMEM), penicillin-streptomycin , Dulbecco’s phosphate-buffer , fetal bovine serum (FBS), 0.25% w/v trypsin-0.53 mM EDTA, 6-carboxy-2’,7’-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA), M-PER mammalian protein extraction reagent, Pierce Bicinchoninic Acid Protein Assay Kit (BCA), TRIzol reagent, Quant-iT RiboGreen RNA Assay Kit, and High-Capacity cDNA Reverse Transcription Kit were obtained from Invitrogen . RNeasy Plus Mini Kit was purchased from Qiagen .Ochratoxin A (OTA), 3--2,5-diphenyl-2H-tetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), N,N-dimethylformamide (DMF), reduced L-glutathione reduced (GSH), v/v) penicillin-streptomycin and 10% (v/v) FBS. Cells maintained at 37 °C in a humidified atmosphere with 5% CO2. HepG2 cells were seeded in each specific assay plate at a density of 1.0 × 105 cells/cm2. OTA was dissolved in pure methanol (24.76 mM) and further diluted in EMEM under serum-free conditions; methanol concentration did not exceed 0.1% (v/v) in the final assay.Human hepatocellular carcinoma derived cell line (HepG2) was obtained from the American Type Culture Collection (ATCC) and cultivated according to ATCC culture method recommendations. HepG2 cells were cultured in EMEM medium containing 1% (w/v SDS in DMF (pH 4.7), and the absorbance was read at 570 nm on a microplate reader . The cell viability was calculated with respect to non-treated cells as the control, and thus the results were expressed as percentage of control. The percentage of cell viability was calculated by the following formula: % cell viability = (sampleAbs − blankAbs)/(controlAbs − blankAbs) × 100, where Abs = absorbance value.Cell viability was determined using the MTT assay, which is a widely accepted method to enumerate viable cells . Briefly2DCFDA conversion to fluorescent 2′,7′-dichlorofluorescein (DCF) [2, 1.8 mM CaCl2, 25 mM glucose, and 10 mM HEPES, pH 7.4). Next, cells were incubated with 100 μM carboxy-H2DCFDA in KRH buffer for 30 min at 37 °C. Carboxy-H2DCFDA was removed by washing the cells once with KRH buffer, and then treated with increasing concentrations of OTA (1 μM to 100 μM). The fluorescent of the cells from each well was measured and recorded after 4 h and 24 h in a Sinergy 2 microplate reader  with a temperature maintained at 37 °C. The excitation filter was set at 485 ± 20 nm and the emission filter was set at 528 ± 20 nm. Tert-butyl hydroperoxide (TBHP) was used as the positive control for ROS. The results were expressed as percentage of control, which was calculated by the following formula: ROS (% of control) = (sampleF − blankF)/(ontrolFc − blankF) × 100, where F = fluorescence value.The redox state of the cells was assessed using carboxy-Hin (DCF) . Brieflyv/v) protease inhibitor cocktail, and after 5 min incubation, it was homogenized with a Teflon pestle for 30 s, followed by centrifugation at 12,000× g for 10 min at 4 °C. All assays were carried out in triplicate and the results were expressed as nmol GSH/mg protein. Glutathione concentrations were calculated based on a standard curve of reduced glutathione using linear regression, and protein content was determined by the Pierce BCA protein assay.Reduced glutathione was determined spectrophotometrically by quantifying the oxidation product (5′-thio-2-nitrobenzoic acid) upon the reaction between GSH and DTBN . BrieflyΔΔCΤ method [The relative expression of mRNA levels of selected genes encoding antioxidant enzymes was assessed by TaqMan gene expression assays. Cells were seeded in 100 mm culture dishes and allowed to grow for 24 h; afterward, cells were exposed to increasing concentrations of OTA (10 nM to 100 μM) in serum-free EMEM for 24 h. Cells were then washed with DPBS, and RNA was extracted using the TRIzol reagent following the manufacturer’s instructions. Total RNA was purified using RNeasy Plus Mini Kit according to the manufacturer’s procedure. Total RNA was quantified fluorometrically using the Quant-iT RiboGreen RNA Assay Kit according to the manufacturer’s procedure. First-strand cDNA was synthesized from 2 μg of RNA using the High-Capacity cDNA Reverse Transcription Kit according to the manufacturer’s procedure, and 10 ng of cDNA for gene expression analysis was used. TaqMan gene expression master mix with pre-validated TaqMan gene expression assays were SOD1 Hs00533490_m1), CAT (Hs00156308_m1), GPX1 (Hs00829989_gH), GSR (Hs00167317_m1), G6PD (Hs00166169_m1), and GAPDH (Hs99999905_m1). All assays were performed in triplicate and cycle threshold (CΤ) values were used for further analysis. The relative constitutive gene expression was calculated by the ΔCΤ method using a StepOne Plus real-time PCR system . Changes in gene expression in the groups treated with OTA were calculated by the 3490_m1, 2O2 per minute at pH 7.8 at 25 °C. Hydrogen peroxide decomposition was monitored at 240 nm using a Synergy 2 microplate reader .Cells were seeded in 6-well plates and treated as described in t-test for two group comparisons. Values represent means ± standard deviation (SD) of three independent experiments and subsequent cell passages. Differences were accepted as statistically significant at p < 0.05.Statistical calculations were performed using the Statistical Package for Social Sciences version 23.0 . The effect of different concentrations of OTA on individual parameters was analyzed by analysis of variance (ANOVA) and Dunnett’s test, and multiple comparisons of means was done with Tukey’s test or with the Student’s


However, α-tocopherol is diluted in ethanol, which can induce oocyte parthenogenetic activation (PA). This study aimed to evaluate the role of ethanol concentration on PA and the effect of α-tocopherol supplementation during IVM on the developmental competence and the expression of key genes in blastocysts derived from summer-collected oocytes. All in vitro embryo production was conducted at 5% O2, 5% CO2 at 38.5 °C. Experiment 1: oocytes were cultured with or without 0.05% ethanol. As positive PA control matured oocytes were subjected to 3% or 7% ethanol for 7 min. Oocytes from all groups were placed in fertilization medium (22 h) and culture medium (9 days). Ethanol at 0.05% during IVM did not induce oocyte PA, however, 3% and 7% ethanol were effective parthenogenetic inductors. Experiment 2: oocytes were cultured in maturation medium supplemented with 0, 50, 100 and 200 μM α-tocopherol, diluted in 0.05% ethanol. After in vitro fertilization and embryo culture, we assessed blastocyst apoptotic index and the transcription of a panel of genes. The results showed that supplementation with 100 μM α-tocopherol reduced apoptotic index and increased the expression of SOD2. In conclusion, 100 μM α-tocopherol, diluted in 0.05% ethanol, can be used during IVM to embryonic quality.The use of α-tocopherol during  Thus, st stress ,9,10,11  quality ,13.in vitro maturation (IVM). Vitamin E or α-tocopherol is a natural antioxidant that provides cell defense against oxidative stress [in vitro culture (IVC). Supplementation of IVM medium with α-tocopherol reduced apoptotic rate in rabbit [, pro- and anti-apoptotic activity (BAX and BCL2), and maternal recognition of pregnancy (IFNT2) has not been described in α-tocopherol-treated embryos, and its evaluation can provide insights into its mechanism of action [One of the mechanisms by which heat stress appears to induce damage to the oocyte and developing embryo is an increase in the production of reactive oxygen species (ROS) . Althouge stress . Previoue stress , pig [16e stress  with then rabbit  and porcn rabbit  cumulus f action .v/v) ethanol in culture media. Ethanol is a known parthenogenesis activator at concentrations above 3% [2 have been found to be inefficient in promoting parthenogenetic activation in bovine oocytes [v/v) ethanol can induce parthenogenetic activation in immature oocytes, in comparation with a high concentration of ethanol for a few minutes as a positive control, to promote parthenogenetic development in oocytes.Due to its hydrophobic nature, α-tocopherol is first diluted in absolute ethanol, to obtain a final 25–400 μM α-tocopherol concentration and 0.05%  does not promote parthenogenetic activation, but ethanol at 3 and 7% for 7 min is an effective inductor of parthenogenesis. Additionally, ethanol-diluted α-tocopherol improves the yield and quality of bovine embryos, which is reflected in the expression of target genes. The objectives of this study were to (1) measure the role of ethanol on parthenogenic activation during IVM of bovine oocytes collected during summer, and to (2) determine how α-tocopherol supplementation during in vitro maturation of oocytes, collected during summer, affects their nuclear maturation, fertilization rate, and the subsequent development and the expression of SOD2, CAT, HSPA1A, BAX, BCL2 and IFNT2 in the resulting embryos.Although the beneficial effect of α-tocopherol in mammalian oocytes has been investigated, to date there are no studies on the effect of α-tocopherol (and 0.05% ethanol) supplementation during IVM and their influence on the competence of bovine oocytes collected during summer. We hypothesized that during IVM the supplementation with ethanol at 0.05% , unless otherwise indicated.T is air temperature in degrees Celsius and RH is relative humidity [In this study, experiments were performed using bovine oocytes aspirated from ovaries obtained from a local slaughterhouse during summer in the southern hemisphere (between 21 December 2020 to 9 March 2021). Maximal and minimal temperatures and relative humidity were obtained from two meteorological stations of the National Research Institute for Agriculture (INIA-Uruguay), Agroclimatic Bank located in the north of the country: Salto  and Tacuarembó . The temperature–humidity index (THI) was calculated using the following equation:humidity .w/v) at 37 °C within 45 min of dissection. Cumulus-oocyte complexes (COCs) were aspirated from 2 to 8 mm-diameter follicles. For each repeat, around of 150 COCs with homogeneous cytoplasm and compact layers of cumulus cells were selected. All phases of in vitro embryo production were conducted at low oxygen tension (5% O2), a concentration closer to the oxygen content in the follicle and female reproductive tract [Pools of 35–45 ovaries were transported to the laboratory in a thermal container with 0.9% NaCl (ve tract .in vitro with ethanol at 0.05% (v/v) (n = 242) and without ethanol (n = 584) for 24 h. After in vitro maturation (IVM), 50–51 COCs per group were randomly denuded of cumulus cells, fixed, and processed for Hoechst DNA staining to determine meiotic progression. Matured COCs in the absence of alcohol were distributed into the control group (n = 180) and two groups that were activated with 3% (n = 176) and 7% (n = 178) ethanol (v/v) in holding media solution at 38 °C for 7 min. All groups were washed twice in holding media and cultured for 22 h in IVF-TL in absence of spermatozoa. After this period, a total of 48–53 oocytes per group were fixed and stained to determine pronuclei formation. The remaining oocytes (125–138 per group) were cultured in vitro for 9 days. Cleavage and blastocyst rates were determined on the third, seventh and ninth culture days, respectively. At the end of culture, embryos with a visible blastocoel were considered to be blastocysts. Parthenogenetic blastocysts of all groups (2–9 per treatments) were fixed and stained, and nuclei numbers were counted.For evaluation of parthenogenetic activation of bovine oocytes in the presence of ethanol, a total of 826 COCs were used in six independent repeats. Selected COCs were maturated v/v) for 24 h at 38.5 °C and 5% CO2, 5% O2 and 90% N2. After this period, a group of oocytes (n = 59–73 per treatment) were denuded of cumulus cells and stained to evaluate meiotic progression. Then, 1482 COCs were fertilized in vitro. From those, 353 presumptive zygotes (n = 66–73 per treatment) were denuded, fixed, and stained to determine fertilization rate. The remaining presumptive zygotes (n = 208–244 per treatment) were cultured in vitro and embryo development was assessed at day 3 (% cleavage) and day 9 . On day 9, 10 expanded blastocysts per treatment were used for measuring the relative expression of genes involved in oxidative stress (CAT and SOD2), heat shock (HSPA1A), maternal recognition of pregnancy (IFNT2) and apoptosis (BAX and BCL2). Finally, expanded (11–14 per treatment) blastocysts were used for total cell number and apoptotic index evaluation through Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL).To examine the effects of α-tocopherol during IVM on the developmental capacity of bovine COCs, a total of 1818 COCs were used in eight independents repeats, divided into five treatments, and placed in maturation medium supplemented with different concentrations  of α-tocopherol  supplemented with 1 μg/mL Folltropin-V®  (v/v), 5 UI/mL equine chorionic gonadotropin  (v/v), 10% fetal bovine serum (Gibco) (v/v), 5 μg/mL gentamicin (v/v) and 0.2 mM sodium pyruvate (w/v). For α-tocopherol treatment groups, IVM medium was supplemented with different α-tocopherol  concentrations . Antioxidant was first dissolved in absolute ethanol to obtain the stock solution of 0.1, 0.2 and 0.4 M concentration, stored in the dark at 4 °C, and diluted in maturation media to a final concentration of 50, 100 and 200 µM α-tocopherol, respectively, in 0.05% (v/v) ethanol, and prepared 4 h before culture.Groups of 45–50 COCs were matured v/v) in holding medium at 38 °C for 7 min. After activation, oocytes were washed three time with the same medium and transferred to 500 µL IVF-TL in absence of spermatozoa. Control group was cultured in IVF-TL in the same culture conditions, but in absence of ethanol.After IVM for 24 h, COCs were washed three times in holding medium  and then incubated in 3 and 7% ethanol (v/v) in modified phosphate-buffered saline (PBS) for 25 min and stained with 1 μg/mL Hoechst 33,342 for 5 min. These oocytes were placed on a slide and immediately observed under a fluorescence microscope Nikon Eclipse 50i equipped with a UV excitation filter at 330–385 nm and an emission filter at 420 nm. Oocytes were classified as mature (oocytes in metaphase II + polar body), immature , and degenerated (diffuse or degraded chromatin) [Briefly, IVM COCs were denuded of cumulus cells by pipetting in phosphate-buffered saline (PBS) supplemented with 200 μg/mL hyaluronidase for 5 min. Denuded oocytes were then fixed in 2.5% paraformaldehyde (romatin) .in vitro fertilization medium (TL-IVF) containing 10 µg/mL heparin (v/v), 10 µM hypotaurine (v/v) and placed in 4-well culture dishes. Sperm cells were selected with BoviPure density gradient  by centrifugation at 300× g during 10 min. IVF was performed by incubating COCs with 1 × 106 spermatozoa/mL in 500 μL TL-IVF medium for 22 h at 38.5 °C and 5% CO2, 5% O2, 90% N2 with maximum humidity.Matured COCs were washed twice in After 22 h in IVF-TL, COCs were denuded of cumulus cells, fixed, stained, and evaluated as in previous sections. In experiment 1, oocytes were classified according to the stage: one or two pronuclei (PN), telophase II, metaphase II or degenerated; while in experiment 2, presumptive zygotes with two PN were considered as having normal fertilization, those with three PN as polyspermic, those with one PN as asynchronous, and those without PN as unfertilized. Total fertilization rate was considered when the oocyte had one, two or more PN.w/v) BSA and 0.5% FCS (v/v). Presumptive zygotes were placed in groups between 20 to 25 in mSOF and overlaid with mineral oil at 38.5 °C and 5% CO2, 5% O2, 90% N2 with maximum humidity for 9 days. Culture media were replaced with fresh medium (replacement 50%) at 72, 120 and 168 h of culture. Cleavage, blastocyst rate, and total blastocyst rate was measured on day 3, 7 and 9 of culture, respectively.At 22 h of coculture, COCs and presumptive zygotes were totally denuded of cumulus cells by pipetting in holding medium and washed twice in modified culture synthetic oviduct fluid (mSOF)  containiBlastocysts on day 9 were fixed and stained with 1 µg/mL Hoechst 33342 at 38.5 °C for 10 min, washed twice in PBS and mounted on slides. They were observed under a fluorescent microscope, and the total number of nuclei for every blastocyst was counted. Stained blastocysts were photographed, and images were evaluated using ImageJ software.v/v) Triton X-100 in PBS for 40 min at 4 °C, and washed two times in PBS. Samples were incubated in TUNEL reaction mixture  at 37 °C for 1 h. TUNEL-stained blastocysts were washed with PBS and incubated in PBS containing 10 μg/mL Hoechst 33342 for 10 min. Blastocysts were washed and mounted onto a glass slide. Negative controls were prepared by omission of terminal deoxynucleotidyl transferase in the reaction mixture, and at the same time positive controls were prepared by pretreatment with 1 mg/mL DNase I  in 5 μL of Tris-HCl buffer, 1 μL of DNase, and 30 μL of H2O for 45 min at 37 °C. All samples were analyzed in a Nikon Eclipse 50i fluorescence microscope with filters for FITC (emission 520 and excitation 460–490) and Hoechst (emission 420 and excitation 330–385). TUNEL-positive blastomeres were fluorescently labeled green, indicating apoptosis. Blue (Hoechst) fluorescence indicated the presence of nuclei. Blastomeres were counted and analyzed using the open-source software Fiji/ImageJ. Apoptotic index was determined by dividing the total number of TUNEL positive blastomeres by the total number of blastomeres.TUNEL assay was performed as previously described . Hatched2+- and Mg2+-free PBS, and placed in 20 µL of RNA later™ Solution , snap-frozen in liquid nitrogen, and stored at −20 °C until gene expression analysis. Quick-RNA Microprep Kit  was used, for blastocyst RNA isolation followed by DNAse treatment using DNA-Free kit (Gibco), according to the manufacturer’s instructions. Total RNA concentration was determined by measuring the absorbance at 260 nm and its purity was evaluated at an absorption ratio of 260/280. For each sample, cDNA was synthesized by reverse transcription using a SuperScript III transcriptase  with random primers and 500 ng of total RNA as a template.Three replicates of expanded blastocysts treated with 0, 0.5% ethanol, 50, 100 and 200 µM α-Tocopherol were analyzed. All blastocysts were washed in CaIFNT2, HSPA1A, CAT, SOD2, BCL2 and BAX, and the endogenous control GAPDH are presented in GAPDH gene expression computed using the 2−∆∆CT method [The expression of target genes was quantified using real-time PCR (qPCR). Primer sequences and the expected product lengths for genes T method  in whichp < 0.05.Normality was checked with the Kolmogorov–Smirnov test, and homogeneity of variance was examined by Levene´s test. The Wilcoxon rank-sum test was performed for the data without normal distribution. Data were analyzed by one-way analysis of variance (ANOVA) with the Tukey test using SAS software . Results were expressed as the mean ± standard error of mean (s.e.m.). Differences between means were considered significant when p = 0.77), immature  and degenerate  oocytes IVM for 24 h in presence (0.05%) or absence of ethanol, respectively. After 22 h of culture in absence of spermatozoa, oocytes from control and ethanol at 0.05% groups showed a clear delay in the formation of pronuclei (p = 0.086) between control, 0.05%, 3 and 7% ethanol group .No significant differences were found in the proportion of mature . The highest blastocysts rate on day 9 was obtained in the α-100 group, but it only differed significantly from the α-200 group. Hatched blastocyst rates were similar in all groups , however, the addition of 100 μM of α-tocopherol during IVM reduced the proportion of TUNEL-positive blastomeres (SOD2 (p < 0.05) in blastocysts in the α-100 group, compared to the other treatment groups, while no differences were detected in the expression levels of IFNT2, HSPA1A, CAT, BCL2 and BAX  under 5% O2 during IVM does not promote parthenogenesis. The addition of α-tocopherol during IVM of summer-collected bovine oocytes affects blastocyst quality and yield in a dose-dependent manner. Supplementation of IVM medium with 100 μM α-tocopherol reduced the apoptotic index increased blastocysts quality and induced overexpression of SOD2 in expanded blastocysts.The results of the present study demonstrate for the first time that the use of ethanol at 0.05%  and blastocyst (1.5–2.8%) rates were similar to those reported by Méo et al. ,33. Sponv/v) for a few minutes was effective for parthenogenetic activation. Recent studies have reported similar cleavage and blastocyst rates after bovine oocyte parthenogenetic activation with 5 µM ionomycin and 2 mM 6-dimethylaminopurine [v/v), and 50 µM roscovitine improved pronucleus formation rates in parthenogenetic embryos, while the highest blastocyst yields were observed in the ionomycin and 6-dimethylaminopurine treatment [Our results suggest that a single ethanol treatment  into H2O2 [SOD2 [CAT, has been associated with higher embryo quality [SOD2 expression was downregulated in vitrified–thawed blastocysts [SOD2 expression, as shown in the present study, can be attributed to an effect of α-tocopherol on the expression of antioxidant genes [The addition of 100 µM α-tocopherol during IVM of bovine oocytes increased blastocyst rate, decreased apoptotic index and increased the expression of nto H2O2 . An overO2 [SOD2 , but not quality . In factstocysts  or blaststocysts . Recent nt genes .Dnmt1 and MLH1 gene expression and methylation [SOD2. However, the use of only two α-tocopherol concentrations in this study limited our understanding of the dose-dependent effect of α-tocopherol on oocyte maturation and competence.The study conducted by Borcąri et al.  showed thylation . We coulv/v) ethanol does not induce parthenogenetic activation of bovine oocytes, therefore, at this concentration, ethanol can be used as a safe vehicle for hydrophobic molecules in embryo production systems. In contrast, a single >3% (v/v) ethanol treatment for 7 min is an effective procedure for parthenogenetic activation. The addition of 100 µM α-tocopherol diluted in 0.05% ethanol during IVM improves oocyte competence leading to an increase in blastocyst production and higher embryo quality, without the risk of inducing parthenogenetic activation.In conclusion, 0.05% (