Background and Aims:Rabdosia japonica var. glaucocalyx is a traditional Chinese medicine (TCM) for various inflammatory diseases. This present work aimed to investigate the protective effects of R. japonica var. glaucocalyx glycoproteins on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and the potential mechanism.Methods: Glycoproteins (XPS) were isolated from R. japonica var. glaucocalyx, and homogeneous glycoprotein (XPS5-1) was purified from XPS. ANA-1 cells were used to observe the effect of glycoproteins on the secretion of inflammatory mediators by enzyme-linked immunosorbent assay (ELISA). Flow cytometry assay, immunofluorescence assay, and Western blot analysis were performed to detect macrophage polarization in vitro. The ALI model was induced by LPS via intratracheal instillation, and XPS  was administered intragastrically 2 h later. The mechanisms of XPS against ALI were investigated by Western blot, ELISA, and immunohistochemistry.Results:In vitro, XPS and XPS5-1 downregulated LPS-induced proinflammatory mediators production including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and nitric oxide (NO) and upregulated LPS-induced IL-10 secretion. The LPS-stimulated macrophage polarization was also modulated from M1 to M2. In vivo, XPS maintained pulmonary histology with significantly reducing protein concentration and numbers of mononuclear cells in bronchoalveolar lavage fluid (BALF). The level of IL-10 in BALF was upregulated by XPS treatment. The level of cytokines including TNF-α, IL-1β, and IL-6 was downregulated. XPS also decreased infiltration of macrophages and polymorphonuclear leukocytes (PMNs) in lung. XPS suppressed the expression of key proteins in the TLR4/NF-κB signal pathway.Conclusion: XPS was demonstrated to be a potential agent for treating ALI. Our findings might provide evidence supporting the traditional application of R. japonica var. glaucocalyx in inflammation-linked diseases. Rabdosia genus is an important genus of the Labiatae family, with about 150 species worldwide (Rabdosia japonica (Burm. f.) Hara var. glaucocalyx (Maxim.) is a synonym of Isodon japonicus var. glaucocalyx (Maxim.) H.W.Li. R. japonica var. glaucocalyx, distributed mainly in northern China, is a traditional Chinese medicine (TCM) for hepatitis, gastritis, mastitis, and cough. It is recorded in Chinese Materia Medica  were isolated from the aqueous extracts of this plant, and two homogeneous glycoproteins (XPS5-1 and XPS10-1) were purified from XPS  is a kind of respiratory disorder with high mortality and calls for researches on effective drugs. The lung injury is characterized by excessive accumulation of inflammatory cells, uncontrolled secretion of cytokines, and damages of the alveolar-capillary membrane including epithelium and endothelium . TentatiLPS activates TLR4, a member of the toll-like receptors (TLRs) family . After rThe lung injury induced by LPS is associated with the lung excessive inflammation, which causes the activation of alveolar macrophage polarization homeostasis . Alveola. Then, the beneficial effect of XPS on ALI mice was explored to seek the effective drug for ALI.Here, the anti-inflammatory activity of XPS and XPS5-1 was first elucidated in ANA-1 cells (a murine macrophage cell line)R. japonica var. glaucocalyx was obtained from Huqingyutang Pharmaceutical Co., Ltd.  by Prof. L.H.Wu from the Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, serving to authenticate. Monosaccharide standards  were obtained from Sigma-Aldrich . Diethylaminoethyl (DEAE) cellulose column and Superdex-200 were purchased from GE Healthcare Bio-Sciences AB .The commercial herb of Escherichia coli O127:B8), thiazolyl blue tetrazolium bromide (MTT), and dexamethasone (DEX) were purchased from Sigma, United States, for the in vitro experiments. RPMI 1640 and FBS were bought from Gibco, America. Dimethyl sulfoxide (DMSO) was purchased from Merck Millipore, Germany. Fluorescently labeled antibodies against mouse CD11b, F4/80, CD86, and CD206 were obtained from Biolegend . Arginase-1 (Cat. No. 93668), inducible nitric oxide synthase (iNOS) (Cat. No. 13120), and secondary antibodies (Cat. No. 4412 and 8889) were bought from CST Company . DAPI was obtained from Dalian Meilun Biotechnology Co., Ltd. (China).LPS (Escherichia coli O55:B5) was purchased from Sigma, United States, for the in vivo experiments. The ELISA kits were purchased from Boatman (China). Dexamethasone sodium phosphate injection (DEX) was purchased from Jiangxi Province Chuang Xin Pharmaceutical Group Co., Ltd. for the in vivo experiments. The ELISA kit for myeloperoxidase (MPO) was purchased from Lianke, Shanghai. Anti-mouse Ly-6G/Ly-6C (Cat. No. 108402) was purchased from Biolegend, United States. F4/80 (Cat. No. ab111101) was purchased from Abcam, Britain. Western blot kits were obtained from Beyotime . Antibody TLR4 (Cat. No. ab13556), TRAF6 (Cat. No. ab40675), and MyD88 (Cat. No. ab28763) were obtained from Abcam, Britain. GAPDH (Cat. No. AP0063) was purchased from Bioworld, China. Polyclonal antibodies NF-κB p65 (Cat. No. D14E12) and NF-κB p-p65 (Cat. No. S536) were bought from CST Company .LPS  was extracted with 50 L water at 100°C for 3 h and three times. The supernatant of water extract was combined and concentrated under reduced pressure. After centrifugation, four times 95% ethanol was added and the precipitates were collected and freeze-dried to obtain XPS  (The overground part of te 6.3%) . XPS (5 te 6.3%) . The totHigh-performance gel permeation chromatography (HPGPC) was performed with Agilent 1,260 instrument fitted with the GPC software and the determination of homogeneity and molecular weights of the glycoproteins sample. The system was linked to the series-connected serial column of Ultrahydrogel 1,000 and Ultrahydrogel 250, ID 7.8 mm, and length 300 mm , and the sample was eluted with 0.2 M NaCl at a flow rate of 0.80 ml/min at 40.0 ± 0.1°C. The gel filtration column was calibrated by pullulans with known molecular weight. The column temperature was kept at 40.0 ± 0.1°C. NaCl (0.2 M) was used as an eluant and the flow rate was kept at 0.8 ml/min. The sample concentration upon injection was 2 mg/ml and approximately 10 μL was injected .4 for 3 h at room temperature. After neutralization with acetic acid and evaporation to dryness, the residue was acetylated with acetic anhydride for 1 h at 100°C. The resulting alditol acetates were analyzed by GC-MS (The sample was hydrolyzed with 2.0 M trifluoroacetic acid (TFA) at 121°C for 2 h. TFA was completely removed by repeating evaporation with methanol. Water was added to dissolve the residue and reduced by NaBHby GC-MS .in vitro experiments. Cells were cultured in 10% FBS-RPMI 1640 medium .ANA-1 cells  are a murine macrophage cell line for 0 medium . The celThe cells were treated with XPS  or XPS5-1 (100 μg/ml) for 24 h and were stimulated by LPS (0.5 μg/ml) simultaneously. DEX (20 μM) was administrated as a positive control. All the supernatant was collected for nitric oxide (NO) and other inflammatory cytokines’ testing. The cell viability was tested by MTT and was presented as a percentage of viable cells to the control group .The production of NO was represented by its end product, nitrite in the cell culture supernatant. The supernatant was mixed with Griess reagent and shook for 15 min. The absorbance was recorded at 540 nm. The levels of TNF-α, IL-10, IL-1β, and IL-6 in collected supernatants were tested by ELISA kits.6 cells/well for 24 h. Cells were treated with XPS, XPS5-1 (both 100 μg/ml), and DEX (20 μM) for another 24 h after LPS (0.5 μg/ml) stimulation. Cells were collected to detect the expression of inflammation-related proteins (n = 4 for each group).The ANA-1 cells were plated in a 6-well plate as 1×10Total cell lysates were centrifuged at 4°C . Samples were loaded on 12% SDS-PAGE gel and were transferred to PVDF membranes. After blocking, the membranes were incubated with antibodies against iNOS, arginase-1, and GAPDH  for 10 h at 4°C and secondary antibody HRP-conjugated IgG (1:2000) for 2 h at room temperature. The protein bands were visualized by the enhanced chemiluminescence (ECL) method and quantified by the ImageJ software. The GAPDH protein was served as internal control .ANA-1 cells were cultured on a 24-well plate and then administrated with XPS, XPS5-1 (both 100 μg/ml), and DEX (20 μM) for another 24 h after LPS (0.5 μg/ml) was stimulated. Each representative experiment has three independent biological replicates.After being fixed in 4% paraformaldehyde and permeated with 0.3% Triton/PBS, the cells were incubated at 4°C with iNOS (1:500) and arginase-1 (1:200) antibodies for 12 h. Then they were incubated with fluorescent dyes conjugated secondary antibodies (green for arginase-1 and red for iNOS) for 1 h at room temperature. Nuclei were observed after staining with DAPI at 10 μg/ml for 15 min. The cells were immediately observed by a fluorescence microscope (Leica EL6000 Microsystems) .+, F4/80+, CD86+, and CD206+ was detected (n = 4 for each group) were analyzed using the FlowJo software .ANA-1 cells were cultured as previously described and expression of CD11bdetected . ANA-1 cdetected . Then thSix- to eight-week-old male BALB/C mice were obtained from the Shanghai SLAC Lab Animal Co. Ltd. All animal protocols in this experiment met the dictates of the National Animal Welfare Law of China and were in agreement with the Animal Ethical Committee of School of Pharmacy, Fudan University (approved identification: 2020-01-YL-LH-01).n = 6 for each group): sham, LPS-stimulated , LPS + XPS , and LPS + DEX (4 mg/kg) groups. The mice were intratracheally instilled with normal saline  or LPS. ALI mice were treated 2 h after instillation. The mice in the sham and model group were given NS, and the mice in the LPS + XPS groups were administered with XPS intragastrically. DEX was administered intravenously as a positive control. All mice were sacrificed by an overdose of urethane 24 h after treatment  were stained with hematoxylin and eosin (H&E) solution to perform histopathological assessments . The patAfter mice were sacrificed, trachea cannula was used to get the bronchoalveolar lavage fluid (BALF) from the left lungs. Collected lavage was centrifuged at 80 g for 15 min and retained for subsequent tests at −80°C.The protein concentration in BALF was tested by the BCA assay kit. The mononuclear cells were counted with a hemocytometer .MPO is a mark of neutrophils infiltration in the lungs . The MPOThe detection of immunohistochemical staining was performed according to our previous research . The sliLung tissues were homogenized and extracted with RIPA lysis reagent. The samples were loaded on gel for electrophoresis and transformed to PVDF membranes. After blocking, the membranes were incubated with primary antibodies against TLR4, TRAF6, MyD88, NF-κB p65, NF-κB p-p65, iNOS, arginase-1, and GAPDH  for 10 h at 4°C and anti-rabbit secondary antibody HRP-conjugated IgG  at room temperature for 2 h.post hoc test using the software of SPSS. p-value of less than 0.05 (p < 0.05) was considered to be significant.Values were expressed as means ± SD. Multiple group comparisons were performed using one-way analysis of variance (ANOVA) followed by Bonferroni R. japonica var. glaucocalyx by boiling water extraction and were characterized  were obtained from the overground part of cterized . XPS andas 49.9% . The proas 49.9% .p < 0.001, p < 0.05). The level of IL-1β was reduced by 100 μg/ml XPS treatment (p < 0.01). The production of IL-10 distinctly rose by LPS stimulation . DEX markedly decreased the level of NO, TNF-α, IL-6, and IL-1β (p < 0.001) but had less effect on IL-10 production. The effect of XPS5-1 (100 μg/ml) was similar to XPS.XPS, XPS5-1, and DEX had no effect on cell viability after LPS stimulation . Furtherp < 0.001, The location and expression level of proteins were tested by immunofluorescence assay and Western blot. Arginase-1 was normally expressed in the control group, whereas LPS obviously reduced its expression. As an inducible synthase, iNOS was less expressed in resting cells. The expression of it increased after LPS stimulation  in ANA-1 (p < 0.001). XPS and XPS5-1 obviously reversed the LPS-promoted M1 subtype (p < 0.001). The cells stimulated by LPS expressed less CD86–/CD206+ events (M2) (p < 0.001). XPS and XPS5-1 administration skewed the LPS simulated ANA-1 to an anti-inflammatory (M2) phenotype, characterized by the increased events of CD86–/CD206+  phenotype of macrophages, characterized by the increased events of CD86To estimate the effect of XPS treatment in LPS-stimulated mice at histological level, the lung tissues were tested by H&E staining. The sham group showed only mild changes caused by intratracheal instillation of NS. However, pulmonary congestion, alveolar wall thickness, and edema were observed in the model group. XPS or DEX administration obviously alleviated these damages . The LPSp < 0.001). XPS (40 and 80 mg/kg) and DEX (4 mg/kg) treatment significantly restrained these phenomena  and PMNs (expressing Ly-6G/Ly-6C (Gr-1)) in lung tissue was evaluated . The resp < 0.001, p < 0.01). XPS (40 mg/kg) only markedly inhibited the expression of TNF-α (p < 0.05). XPS 20 mg/kg mildly inhibited the release of cytokines with no significance. The production of IL-10 distinctly decreased in the model group . Treatment of XPS in 20 and 40 mg/kg only showed an upward tendency with no significance.Compared with the sham group, the level of TNF-α, IL-1β, and IL-6 was obviously elevated in the model group (p < 0.001), while XPS (40 and 80 mg/kg) and DEX markedly reduced such elevations , while only XPS 80 mg/kg significantly reduced MyD88 expression (p < 0.05).LPS obviously augmented the expression of TLR4, MyD88, and TRAF6 in the model group compared with the sham group (p < 0.001) and was downregulated by XPS . Arginase-1 expression reduced markedly in the model group . Moreover, iNOS (p < 0.05).The phosphorylation level of NF-κB p65 in the cytosol was expressed by the ratio of NF-κB p-p65 to NF-κB p65. The ratio  was marker, iNOS  expressiR. japonica var. glaucocalyx is a TCM prescribed to treat inflammatory diseases. The terpenoid and flavonoid in R. japonica var. glaucocalyx show the anti-inflammatory activity  (in vitro through modulating macrophage polarization with the reduction of inflammatory mediators.XPS was glycoproteins isolated from se (TDO) , an enzyse (TDO) . HoweverMacrophages have been widely studied as the center of inflammatory response in recent years . MacrophArginase-1 and iNOS are involved in two opposite macrophage phenotypes, M1 and M2 . Both en+ and less CD206+ .After LPS stimulation, TNF-α is produced first and upregulates the secretion of IL-1β . As an aXPS administration obviously decreased the production of TNF-α, IL-1β, and IL-6 with the increased production of IL-10 in the lung of ALI mice. XPS treatment alleviated the damages of the lung caused by excessive inflammation.LPS-TLR4/NF-κB signaling pathway is essential in inflammatory response . The myDThe expression of iNOS is motivated by the activation of the TLR4/NF-κB pathway . A largeOur testing results indicated that LPS stimulation upregulated the expression of TLR4, MyD88, TRAF6, and p-NF-κB. XPS administration decreased the expression of key proteins. The excessive iNOS and proinflammatory cytokines were reduced by XPS treatment. XPS might alleviate ALI by downregulating the LPS-TLR4/NF-κB signaling pathway.R. japonica var. glaucocalyx has been traditionally used in numerous inflammatory diseases. Our research suggested that the activity of R. japonica var. glaucocalyx might partly come from glycoproteins (XPS), an important ingredient in herbs. As homogeneous glycoproteins purified from XPS, XPS5-1 had similar activity in vitro. Consistent with the traditional application of R. japonica var. glaucocalyx, XPS might be a promising candidate for inflammatory diseases, especially for treating ALI.In vitro, XPS and its purified ingredients (XPS5-1) inhibited the secretion of proinflammatory mediators and polarized LPS-stimulated macrophages from M1 to M2. In vivo, XPS ameliorated LPS-induced ALI by reducing the inflammatory response and inhibiting macrophages and PMNs accumulation in lung tissues. These phenomena were related to the inhibition of the TLR4/NF-κB pathway. Glycoproteins from R. japonica var. glaucocalyx could be a potential agent for ALI.


Following this evidence, we evaluated CHIR99021, a GSK-3 inhibitor, as a potential drug for use in ES therapy. Our data highlight that, in ES cells, CHIR99021 induces cell cycle arrest, mitotic catastrophe (MC) and autophagic response, resulting in reduced cell proliferation. Our results support the potential efficacy of CHIR99021 in ES treatment and encourage further preclinical and clinical studies.Epithelioid sarcoma (ES) is a rare disease representing <1% of soft tissue sarcomas. Current therapies are based on anthracycline alone or in combination with ifosfamide or other cytotoxic drugs. ES is still characterized by a poor prognosis with high rates of recurrence. Indeed, for years, ES survival rates have remained stagnant, suggesting that conventional treatments should be revised and improved. New therapeutic approaches are focused to target the key regulators of signaling pathways, the causative markers of tumor pathophysiology. To this end, we selected, among the drugs to which an ES cell line is highly sensitive, those that target signaling pathways known to be dysregulated in ES. In particular, we found a key role for GSK-3β, which results in up-regulation in tumor  Epithelioid sarcoma (ES) is a rare soft tissue neoplasm described for the first time in the 1970s by Enzinger ,3. ES moFor improving outcomes and overall survival in ES patients, the signaling pathways and mechanisms involved in the progression of ES need to be clarified. From this view, several studies have aimed to better elucidate the molecular deregulations underlying ES pathogenesis. The disruption of pathway regulation by oncogenic mutations or the abnormal expression of key genes promotes tumor onset and progression. As for other kind of tumors, the identification of ES targetable molecular markers and their potential inhibitors will be useful for the development of tailored therapeutic regimens . In thisIn this study, we combined bioinformatics analyses and in-vitro validation to explore several multitarget chemical inhibitors as potential ES drugs using the cancerRXgene database , which cwww.cancerRxgene.org on 30 November 2020) collects datasets that annotate drug sensitivity response in cancer cell lines along with genomic characterization. Using as query VA–ES–BJ, the unique epithelioid sarcoma cell line included in GDSC, we received as output the drugs to which this cell line results sensitive. Among them, we selected five drugs characterized by the highest sensitivity (lowest Z score) and reported to inhibit well-known molecular targets involved in pathogenesis of cancer , NAE1 (HR: 2.1 p = 0.0008), CDK4 (HR: 1.8 p = 0.003), CDK6 (HR: 2.1 p = 0.0003) and HDAC1 (HR: 2.0 p = 0.01) genes  for GSK3B (HR: 1.7 1) genes .p = 0.02), GSK3B (HR: 1.8 p = 0.001) and NAE1 (HR: 1.5 p = 0.02) highlighting a negative impact of high gene expression levels on PFI . A significant relation arisen for ACVR1C  due to the contribution of higher GSK3B gene-expression levels. In addition to GSK3B, only age resulted in significant (p < 0.05) association with OS in both uni- and multivariate analyses. According to an Akaike information criterion (AIC)-based stepwise procedure, GSK3B expression alone (with age only with the OS) better models overall and progression-free survival. In particular, for both OS and PFI, the global p-value (log-rank) of the model was <0.001 with hazard ratios (HRs) 1.93-fold and 1.81-fold greater for GSK3B, in the OS and PFI models, respectively. Although significant, age HR was low (1.02-fold) in the OS model. Finally, GSK3B turned out to be an independent prognostic factor for both PFI and  OS.Moreover, we evaluated, by log rank test and multivariate cox analysis, the overall and the progression free survival associated with GSK3B and with the following clinical features: age, gender, histology, and radiotherapy. Interestingly, both full models (OS and PFI) were significant  .Remarkably, as shown in Overall, based on prognostic values in sarcoma patients, overexpression in soft tissue sarcomas as compared with normal tissues and the sensitivity of the epithelioid sarcoma cell line to CHIR99021, we considered GSK-3β a potential drug target worthy of further investigation.In order to validate GSK-3β as a cancer target, we characterized the effects of its inhibitor, CHIR99021 , on VA-Ep = 0.04).To elucidate the consequences of CHIR99021 exposure, we evaluated anti-proliferative effect performing a MTS assay at different concentrations and time points. We determined the IC50 value by treating cells with seven different concentrations, ranging from 2.5 µM to 150 µM for 24, 48 and 72 h. CHIR99021 demonstrated a dose-dependent effect on viability of VA-ES-BJ cell line, being significant at 100 µM. In contrast, no time effect was observed . Therefop = 0.0008) was observed.A similar trend was confirmed, also; after 48 h of treatment, a more significant decrease of live cells  to 52.8% ± 2.2 (CHIR99021) (p = 0.02). No differences were recorded for the S and G0/G1 phases at 24 h. Cell cycle analysis at 48 h revealed an increased percentage of G2/M cells varying from 14.4% ± 0.3 (DMSO) to 47.9 ± 4.1 (CHIR99021) (p = 0.01). Moreover, a concomitant decrease of cells at S phase was detected   C.To examine whether this cell-cycle-perturbing effect of CHIR99021 could also occur in non-cancer cells we used HDFa cells. Indeed, by sharing the mesenchymal origin with epithelioid sarcoma , they caThe results suggested that CHIR99021 impairs the cell cycle progression of ES cancer cells, resulting in it’s being well-tolerated by normal cells .Finally, we evaluated, by Western-blotting analysis, the expression levels of CHIR99021’s own targets, GSK-3α/β, as well as β-catenin, one of the main downstream signaling molecules.As reported in 2/M cell cycle phase [2/M phase [This was accompanied by a simultaneous increase of β-Catenin phosphorylated at Ser675, the transcriptionally active form . Interesle phase  , we evaluated the presence of giant micronucleate cells, a main feature of MC, by fluorescent staining of nuclei in VA-ES-BJ cell line exposed to CHIR99021.Under light filed microscopy evaluation, we observed a heavy alteration of cell morphology after CHIR99021 treatment. Since literature data reported that prolonged cell cycle arrest in Gp = 0.008) and Colcemid  as compared with vehicle (3.7% ± 1.9) or untreated controls (0.0% ± 0.0), respectively.We treated VA-ES-BJ cells with CHIR99021 100 µM or Colcemid 1 µg/mL, the latter by interfering with correct microtubule polymerization and mitosis completion represented the positive control. As shown in p = 0.01) vs. DMSO (6.2% ± 1.3) and Colcemid  vs. untreated (0.9% ± 0.9). No significant difference was observed between CHIR99021 and Colcemid demonstrating a comparable effectiveness of these two compounds.The same scenario, large cells with multiple micronuclei, resulted also after 48 h of treatment: CHIR99021 , also confirming the absence of time-dependent effects . Westernp = 0.01). After 48 h, live cells were 55.6% ± 6.9 vs. 94.4% ± 5.6 .As reported in 2/M phase after CHIR99021 at 50 µM compared with control (DMSO) (40.8% ± 3.6 vs. 23.8% ± 2.2 p = 0.02). No differences were recorded for S and G0/G1 phase at 24 h. The same scenario was observed at 48 h, being cells in G2/M phase 39.8% ± 1.4 and 19.8% ± 1.4 for CHIR99021 and vehicle, respectively (p = 0.0005). Significant difference was observed in G0/G1 phase at 48 h   C.p = 0.03) vs. 5.6% ± 2.8 (DMSO) and 37.9% ± 1.1  vs. 8.5% ± 2.6 (untreated). Similarly, after 48 h we found: 55.6% ± 3.8  vs. 4.9% ± 0.45 (DMSO) and 71.0% ± 8.5  vs. 12.8% ± 0.3 (untreated) . In particular, after 24 h of treatment, cells with MC-related features accounts for: 48.2% ± 10.2  D.The setting of an autophagy response was confirmed in NEPS cells by the evidence of LC3A/B-fluorescent puncta E. These To date, clinically helpful molecular targets for soft tissue sarcomas are still lacking, and even more so for epithelioid sarcoma. Moreover, due to the rarity of this tumor and poor long-term outcomes, little is known about the response to systemic treatments in ES patients and this knowledge scarcity highlights that current chemotherapy regimens are inadequate. Indeed, literature on ES treatment outcomes is limited to case reports or studies with a poor number of enrolled patients.Novel systemic treatments for ES remain a crucial objective to be pursued with high priority. In this regard, a major limit for research focused on this rare disease is the lack of patients’ samples, in-vitro and in-vivo models, high-throughput data, and large clinical trials. Given that, current ES treatments seems to be only an extended application of drugs effective in other kinds of tumors but not tailored for epithelioid sarcoma.In this scenario, we aimed to identify new possible molecular targets and their respective pharmacological inhibitors through an innovative approach considering the molecular features of ES.We first queried the GDSC database, which offers a large drug sensitivity dataset that contributes to discovery of new therapeutic biomarkers for cancer treatments. Among the outputted drugs, we selected those to which the VA-ES-BJ cell line resulted sensitive and those that target signaling pathways dysregulated in this type of cancer. Then, through bioinformatics analyses performed on data from TCGA Sarcoma project, we restricted the in-vitro evaluation to the drugs whose molecular targets’ gene expressions were associated with poor prognosis and were overexpressed in cancer versus normal tissues.GSK-3β resulted the only target gene satisfying the above-mentioned selection criteria, so we used CHIR99021, a selective inhibitor of GSK-3α/β, to investigate whether it could represent a suitable drug for the treatment of epithelioid sarcoma. GSK-3β is a fascinating enzyme, on which the literature is immense and quite often provides conflicting statements and observations. GSK-3β plays pivotal roles in the pathogenesis of several diseases, including cancer ,39. It i2/M phase after CHIR99021 treatment by flow cytometry. It is well known that G2/M transition is mediated by the activation of the CyclinB1/Cdk1 complex, which activity favors the entry in mitosis [2/M arrest was also supported by the increase of β-Catenin and phospho-Histone H3 (Ser10) protein levels, the latter playing a pivotal role in the regulation of mitotic catastrophe [Indeed, we confirmed a reduction of cell proliferation by MTS assay and an accumulation of cells in G mitosis ,46. Accoastrophe . Indeed,astrophe . Treatmeastrophe ,50.2 to M phase but did not complete mitotic division due to delaying the association between Cdk1 and CyclinB1 [Notably, we also found a strong reduction of p21 protein level supporting the idea that cells normally progress from GCyclinB1 ,52.Moreover, we observed a concomitant reduction of MAD2, as well as an increase of γ-H2A.X levels. These findings strongly reinforce, in line with a previous report , the hypIn order to define the signaling pathway responsible for triggering autophagy subsequent to GSK-3β inhibition, we firstly studied the possible cross-talk between GSK-3β and an autophagy upstream pathway regulator. Previous reports showed that GSK-3β negative regulation of AMPK activity has a fundamental role in autophagy control , so we dIn this study, we took a step back, starting from an in-vitro model to find new drugs selective for ES treatment. We also aimed to encourage the scientific community to move in this direction, in particular for those rare diseases that have not yet found optimal treatments capable of improving patients’ outcome.® CRL2138™), NEPS, and Primary Dermal Fibroblast; Normal, Human, Adult (HDFa). The VA-ES-BJ and HDFa cell lines were purchased from the American Type Culture Collection (ATCC), the NEPS cell line was provided by NU’s researcher; Hiroyuki Kawashima MD, PhD/Professor/Division of Orthopedic Surgery, Niigata University Graduate School of Medical and Dental Sciences [® PCS201012™) was cultured in Fibroblast Basal Media (ATCC PCS-201-030) supplemented with Fibroblast Growth Kit components (ATCC PCS-201-040). Cell lines were maintained in a humidified incubator at 37 °C and 5% CO2; however, they were passed no more than 4–10 times, after which, we again utilized cells from original stocks. NEPS cells were cultured in Roswell Park Memorial Institute 1640 medium  supplemented with 10% FBS and 1% penicillin–streptomycin and 1% l-glutamine. All cell lines were tested for mycoplasma infection upon receipt using LookOut® Mycoplasma PCR Detection Kit .The human cell lines used for this study were: VA-ES-BJ  .We performed a sensitivity assay using CHIR99021 (GSK-3α and GSK-3β inhibitor), which was purchased from Cayman Chemical . The stock solution was obtained resuspending the lyophilized form in dimethyl sulfoxide  and then diluted in fresh medium to the desired concentrations. The maximum DMSO volumes used were always <2%.3 cells/well into 96-well plates and grown overnight before treatments with the inhibitor, or vehicle for 24, 48 and 72 h. Cells’ viabilities were assessed using MTS assay. A solution of CellTiter 96® Aqueous MTS Reagent Powder  was added to each well. After incubation of 1.5 h at 37 °C the absorbance at 490 nm was measured and the percentage of viability in each well was calculated using the untreated cells as 100%. Three independent measurements were performed in triplicate for each assay. The CHIR99021 concentration effective to reduce cell proliferation by 50% (IC50) was estimated following the dose-response curves carried out using seven points, ranging from 2.5 to 150 μM.Cells were plated at a density of 3 × 10The trypan blue dye exclusion assay was also performed to determine the number of viable cells. Briefly, 10 μL of control and treated cell suspensions were added to 10 μL of trypan blue dye  and 10 μL of this solution was loaded in a Burker chamber and counted under 10× magnification using an inverted microscope. The results were expressed as the percentage of live and dead cells for each condition.2 flask and cultured to 70% confluence prior to be exposed with CHIR99021 100 μM or control media, containing an equivalent amount of DMSO, for 24 and 48 h. Briefly, cells were harvested and after washing twice in cold PBS were centrifuged at 1500 rpm at 4 °C and resuspended in 100 μL of binding buffer. Cells were double-stained with 5 μL FITC-Annexin V and 5 μL propidium iodide  and then incubated for 15 min at room temperature in the dark. Number of apoptotic cells was detected using a Navios flow cytometer . Data were analyzed by Kaluza analysis software 2.1.VA-ES-BJ cells were seeded in 25 cm5 cells in 25 cm2 culture flask. At 24 h after seeding, the cells were washed with PBS, and then CHIR99021 at 100 μM or 50 μM or DMSO was added. After 24 and 48 h, cells were harvested, washed and fixed in ice-cold 70% ethanol for 1 h. Fixed cells were then incubated with PI/RNaseA staining solution for 30 min at room temperature and dark. A total of 1 × 104 events were acquired by Navios flow cytometer performing the analyses using the Kaluza 2.1 software.VA–ES–BJ, NEPS and HDFa cells were seeded at 2.5 × 10® 20 for 1 h and then incubated separately with primary antibodies at 4 °C overnight. Membranes were then incubated with HRP-conjugated secondary antibody and stained with an enhanced chemiluminescence kit . The signals were acquired by ChemiDoc Imaging System XRS + (Bio-Rad). In order to detect the expression levels of GSK-3α/β, its signaling-related proteins were used: GSK-3α/β , phospho-GSK-3α/β  and rabbit monoclonal anti-phospho-β-Catenin (Ser675) . To detect the expression levels of cell cycle-related proteins, the following antibodies from Cell Signaling Technology , Santa Cruz Biotechnology  and Abcam  were used: Cdk1/Cdk2 (sc-53219), Cyclin B1  HistoneH3  and phospho-HistoneH3 (Ser10) . Furthermore, to evaluate the expression levels of the mitotic spindle check-point proteins MAD2 (sc-374131), the anti-gamma-H2A.X (ab-11174) and p21 Waf1/Cip1 (12D1)  antibodies were used. Finally, to detect autophagy marker expression and autophagy signaling related proteins the following antibodies were employed: LC3A/B , SQSTM1/p62 , AMPK , phospho-AMPK (Thr172) , mTOR (sc-517464), phospho-mTOR (ser2448) (ab-1093), phospho-Raptor (Ser792)  and phospho-ULK (ser757) . Mouse monoclonal anti-β-Actin  antibody was used as a loading control.Cells were harvested and the pellets were incubated in RIPA lysis buffer  containing protease and phosphatase inhibitors for 30 min on ice. Subsequently, at least 40 μg of extracted proteins were electrophoresed through 4–20% poly-acrylamide gels. After transferring the proteins to PVDF membranes using a TransBlot Turbo system , the membranes were blocked with 5% non-fat milk in PBS with 0.1% TWEENw/v) formaldehyde in PBS at room temperature for 15 min washed twice and permeabilized with 0.1% (v/v) Triton-X100  for 10 min at room temperature. After washing with PBS, non-specific binding sites were blocked by incubation in 5 % (w/v) bovine serum albumin (BSA) in PBS for 30 min at room temperature. For detection of autophagy related proteins cells were incubated with LC3A/B  overnight at 4 °C and subsequently with Goat anti-Rabbit IgG, Alexa Fluor 488  for 1 h at room temperature. Cell nuclei were counterstained with Hoechst 33,342 dye .About eighty-thousand cells were seeded on chamber slides and cultured for 24 and 48 h after CHIR99021 or DMSO treatment. A solution of Colcemid at 1 µg/mL was used as positive control to detected mitotic arrest. Cells were fixed with 4%  were obtained from UCSC Xena Browser as TPM (Transcripts Per Million) . The assp-value cut-offs at 1.5 (Log2FC = 0.58) and 0.05, respectively [Differential gene expression between tumor and adjacent normal tissue samples was assessed using GEPIA2 online tool, setting fold change and ectively . ExpressThe viability of cells was calculated by the normalization of absorbance values from MTS assay of treated cells against those from respective untreated controls. To calculate inhibitory concentration 50 (IC50), data were fitted with a log-logistic model using the drc R package . Time efp values < 0.05. Analyses were performed using R software.Counts from the trypan blue assay, the flow cytometer evaluations of the apoptosis and cell cycle phases, and number of cells with abnormal nuclei under fluorescent microscopy evaluation were reported as percent of total counts in each sample. Data were reported as mean ± SE. Differences between treatments groups were estimated by paired t test, considering significant 2/M arrest and consequent mitotic catastrophe that ends in autophagy-mediated cell death.Although CHIR99021 effectiveness on ES needs to be reinforced by in-vivo models to define therapeutic doses and possible drug-combination approaches to enhance antitumoral effects , this wo


Signatures of vortex unbinding Berezinskii–Kosterlitz–Thouless (BKT) transition are shown from both the conventional approach and the Fisher–Fisher–Huse dynamic scaling analysis, in which a characteristic Nelson–Kosterlitz jump is demonstrated. We also observe a non-Hall transverse signal exactly at the superconducting transition, which is explained in terms of guided motion of unbound vortices.In this study, we measure the in-plane transport properties of high-quality Ba(Fe Tc materials [2 planes in CuSCs. More similarities were manifested by the antiferromagnetism in parent compounds, superconducting phase diagrams with respect to chemical doping, and extreme type-II superconductivity with very high upper critical field, etc. However, FeSCs show unusually small anisotropy in the upper critical field [Tc superconductivity [Uncovering the underlying essential universality for high-temperature superconductivity is extremely important for understanding the superconducting mechanism as well as exploring the next high-aterials . The twoaterials ,2,3. Appal field ,5, whichal field . So far,al field , unlike al field  and the uctivity .2Sr2CaCu2O8 [2Cu3O7 [1.875Ba0.125CuO4 [Quasi-2D superconducting behaviors in CuSCs are demonstrated by a 2D Berezinskii–Kosterlitz–Thouless (BKT) topological phase transition  close tocrystals ,11,12,13crystals , and thecrystals . For CuStuations , or thertuations  in Josep2CaCu2O8 ,10, YBa24 [V0xy) ,19. The 4 [V0xy) , sheddin0.8F0.2 was reported to have a 3D character and extend far above Tc [0.55Se0.45 thin films, suggesting a quasi-2D characteristic in such systems [x1−Cox)2As2 [x = 0.086, via both the conventional approach and the Fisher–Fisher–Huse (FFH) [TBKT, pointing to the existence of thermally excited unbound vortices.As for FeSCs, either 2D or 3D of the nature of superconducting fluctuations is reported ,24,25,26 systems . This fiCox)2As2  with x =se (FFH)  dynamic 0.914Co0.086)2As2 crystals were grown by a self-flux method with procedures similar to Ref. [Tc = T(90%ρn) − T(10%ρn), where ρn is the normal-state resistivity at Tc value indicates that the crystal was in the optimally-doped regime, consistent with the cobalt content measured.The Ba, as shown in the left inset of Rxx = (RDC/AB + RBC/AD)/2, where RDC/AB (RBC/AD) equals to the potential VDC (VBC) divided by the current IAB (IAD) using ac transport option with a frequency of 13 Hz. The normal-state resistivity above Tc was ρn = πtRxx/ln2 ~ 0.09 mΩ cm, consistent with the previous report [I − Vxx characteristic were collected at a fixed temperature whose fluctuation was less than 1 mK, without any detectable heating effect during the measurement.The electro-transport measurements were performed on a Quantum Design Physical Property Measurement System (PPMS-9). We adopted a van der Pauw four-terminal configuration  for all s report . The datVxy, VBD/AC and VAC/BD were measured respectively by permutating the voltage and current electrodes [VBD/AC − VAC/BD)/2. Obviously, H+) and down (H−), respectively, For the measurement of transverse voltage ectrodes , so that by Ref. , Vxy0 = I → 0 A limit are often used to recognize a BKT transition. (1) Within a temperature region slightly above TBKT, the Ohmic longitudinal resistance has a unique temperature dependence [Rn is the normal-state resistance and b is a dimensionless parameter. The mean-field superconducting transition temperature TBKT obeys a power law α(T) has a “universal jump” from 1 to 3 upon approaching TBKT from above. Such a universal jump is regarded as the hallmark of BKT transition. In the BKT theory, α(T) is proportional to the superfluid density, therefore, the jump in α(T) means discontinuity in superfluid density [To study BKT dynamics in superconductors, there are two main approaches, the “conventional” approach and the dynamic scaling analysis . In the pendence ,(1)Rxx(erature) ,12,14, werature) . (2) Theepinning . (3) The density , which iR/Rn in logarithmic scale is plotted as a function of TBKT = 24.42 ± 0.01K and b = 2.10 ± 0.01. Note that the TBKT is very close to the zero-resistance temperature R(T) data in this region to either 2D or 3D forms of Aslamazov–Larkin theory [According to Equation (1),  systems ,10,11,12n theory . This suI − V characteristics at T = 24.40–24.72 K with I = 0.03–4 mA are displayed in a log–log plot shown in α, represented by the slope, changes with temperature. For T > 24.56 K, the α value is close to 1.0, namely, the I − V curves are basically Ohmic. When approaching TBKT, α increases abruptly, and it goes to 3.6 ± 0.3 at 24.40 K. The inset of α = 3.0 ± 0.2 at TBKT, which suggests the characteristic Nelson–Kosterlitz jump anticipated for a BKT transition.The isothermal I → 0 A. Thus it is necessary to perform a dynamic scaling analysis, which also holds for finite currents [z is the dynamic exponent, P+(−) is the scaling function for temperature above (below) TBKT, and TBKT.Strictly speaking, the above approach is valid only in the limit currents . Accordicurrents , the scacurrents ,38,(2)(Iξ/T vs. (I/T)(I/V)z1/, according to Equation (2). By setting the afore-determined TBKT = 24.42 K, and with the fitted parameters b = 2.1 ± 0.1 and z = 1.8 ± 0.2, all the I − V data points in T < TBKT are limited to one set of I − V data with T = 24.40 K). Since the critical I − V curve follows V ∝zI+1 at TBKT, the exponent is 2.8 ± 0.2 at the BKT transition, consistent with the result of above conventional approach. Besides, the value of parameter b is the same with that extracted by fitting R/Rn with Equation (1). Therefore, the FFH dynamic scaling analysis also suggests a BKT phase transition in Ba(Fe0.914Co0.086)2As2 crystals.TBKT, the thermally exited vortices are in pairs because of the attractive interaction. At TBKT, the vortex pairs start to unbind, and free vortices are generated due to the contribution of entropy to the free energy. It is the unbound free vortices that contribute to the nonzero longitudinal resistance expressed by Equation (1). Interestingly, such free vortices are able to induce an abnormal nonzero As we know, BKT transition is driven by the unbinding of vortex–antivortex pairs. Below in CuSCs ,19. So p0.914Co0.086)2As2 crystals. The Vxx for T < x1−Cox)2As2, the guided motion of vortices  are supported by the in-plane anisotropy [θ with respect to the current A → C as shown in the inset of fn refers to sheet density of free vortices, Φ0 is flux quantum, Fp is the weak pinning force along the guiding direction, η is the damping coefficient of vortex motion, and c is the speed of light. When permutating the voltage and current, the angle between the guided motion and current turns out to be (π/2 + θ), and the transverse field due to current B → D becomes,The non-Hall-type transverse voltage in the absence of external magnetic field at superconducting transition was explained by the guided motion of thermally excited vortices ,19,39. Iisotropy  and striisotropy . AssuminV = Ed , and VBD/AC − VAC/BD)/2, Since θ ≠ (k +1/4)π (k is an integer) in the presence of free vortices. The sinusoidal variation on θ qualitatively agrees with our experimental observation that θ changes into (π/2 − θ). Equation (6) gives fn. At fn decreases rapidly since the superconducting Cooper pairs dissociate. This explains the drop in Tc, such as the case in cuprate superconductors [Obviously, nductors .0.9Co0.1)2As2 crystal [We also measured the transverse voltage under external magnetic fields.  reports . At the  crystal . Here weα(T) = 3 at TBKT by itself. Besides, numerical simulations [Tc merely broadens the BKT transition without changing the universality class (z = 2), which agrees with our experimental observations.At the superconducting transition, however, the transverse voltage does not change sign upon reversal of magnetic field, especially under low magnetic fields. So, there exists a non-Hall-type signal Ta foils , the decogeneity . Howeverulations  indicatex1−Cox)2As2 bulk crystals suggests a quasi-2D nature for iron-based superconductivity. Indeed, 2D antiferromagnetic spin fluctuations, which are mostly believed to be the glue of Cooper pairing, were revealed in BaFe1.84Co0.16As2 by neutron scattering experiment [5H5N)0.2Fe2Se2 superconductor [xFeSe is also supported by both anisotropic transport and I − V curves [3 substrate [ξc, is shorter than 2.8 Å. Therefore, it is not so surprising that 2D superconducting behavior was manifested in Ba(Fex1−Cox)2As2, because the FeAs interlayer spacing is about 6.5 Å [ξc from the anisotropy ratio in Hc2 [ξc ~ 25 Å, may be misleading. This is because the measured Hc2 values are basically Pauli-limited  and FeSC are multi-band superconductors. For a specific superconducting pairing channel, ξc could be significantly smaller than the simple estimated from Hc2.The possible appearance of BKT-type phase transition in Ba(Feperiment . The 2D onductor . RecentlV curves . Furtherubstrate  directlyut 6.5 Å . Here weo in Hc2  using ξa0.914Co0.086)2As2. The observation of non-Hall transverse voltage, probably caused by the guided motion of thermally activated vortices, in turn, further indicates the BKT scenario with vortex–antivortex unbinding. Our results suggest that, similarly to CuSCs, two-dimensionality also plays an important role for high-temperature superconductivity in iron pnictides.To summarize, we have presented the possible evidence for BKT phase transition in a typical FeSC Ba(Fe


Glycine max) is an important food crop that is grown worldwide. Soybean yield is significantly affected by P deficiency in the soil. To investigate the molecular factors that determine the response and tolerance at low-P in soybean, we conducted a comparative proteomics study of a genotype with low-P tolerance  and a genotype with low-P sensitivity  in a paper culture experiment with three P treatments, i.e. P-free (0 mmol·L−1), low-P (0.05 mmol·L−1) and normal-P (0.5 mmol·L−1). A total of 4126 proteins were identified in roots of the two genotypes. Increased numbers of differentially expressed proteins (DEPs) were obtained from low-P to P-free conditions compared to the normal-P treatment. All DEPs obtained in L13 (660) were upregulated in response to P deficiency, while most DEPs detected in T3 (133) were downregulated under P deficiency. Important metabolic pathways such as oxidative phosphorylation, glutathione metabolism and carbon metabolism were suppressed in T3, which could have affected the survival of the plants in P-limited soil. In contrast, L13 increased the metabolic activity in the 2-oxocarboxylic acid metabolism, carbon metabolism, glycolysis, biosynthesis of amino acids, pentose phosphatase, oxidative phosphorylation, other types of O-glycan biosynthesis and riboflavin metabolic pathways in order to maintain normal plant growth under P deficiency. Three key proteins I1KW20 (prohibitins), I1K3U8  and C6SZ93  were suggested as potential biomarkers for screening soybean genotypes with low-P tolerance. Overall, this study provides new insights into the response and tolerance to P deficiency in soybean.Phosphorus (P) is an essential element for the growth and development of plants. Soybean ( Soybean is an important food crop but its yield is affected by soil phosphorus (P) deficiency. We conducted a comparative proteomics study of two genotypes with contrasting responses to low-P. We observed that important metabolic pathways such as oxidative phosphorylation, glutathione metabolism and carbon metabolism were suppressed in the sensitive genotype. In contrast, the tolerant genotype increased the metabolic activity in pathways such as 2-oxocarboxylic acid metabolism, carbon metabolism, glycolysis, biosynthesis of amino acids, in order to maintain a normal growth under P deficiency. This study provides new insights into the response and tolerance to P deficiency in soybean. Glycine max) is a worldwide important economic and nutritional crop. Its seeds are enriched with high levels of proteins (40–50 %), fats (20–30 %) and vital phytochemicals, i.e. anthocyanins, tocopherols, isoflavones and saponins . Five seeds were put in each bag in three groups for each genotype, i.e. P-free , low-P  and normal-P . Seeds with normal-P served as control for comparative proteomics. The seed bags were placed in a light culture room, and the cultivation conditions were 16-h light/8-h dark, 18–28 °C temperature and 72-h light. At this stage, distilled water was used as culture medium. Upon emergence of cotyledonary leaf, seedlings were transferred to nutrient solution with three P levels: , low-P  and normal-P . For other nutrients (compounds), concentrations were as follow: 4.5 mmol·L−1 KNO3, 1.2 mmol·L−1 NH4NO3, 3.6 mmol·L−1 CaSO4·2H2O, 0.25 mmol·L−1 MgSO4. Trace elements are: 9 μmol·L−1 H3BO3, 0.9 μmol·L−1 MnSO4, 0.9 μmol·L−1 ZnSO4, 1.5 μmol·L−1 CuSO4, 0.18 μmol·L−1 (NH4) 6Mo7O24 and 9 μmol·L−1 Fe-EDTA (pH adjusted to 5.8). Seedlings were supported on a 5-cm-thick styrofoam sheet at a spacing of 3 cm × 3 cm. The cotyledons were removed on third day of transfer to nutrient solution to minimize genotypic variation due to seed P content. Three replications with three seedlings each were maintained for all treatment combinations (Seeds of low-P-tolerant soybean genotype Liaodou 13 (L13) and low-P-sensitive genotype Tiefeng 3 (T3) previously studied by Nine days after the treatment, neat and consistent seedlings were selected for sampling in each treatment, and the average value of 3 plants per bag was taken as one repetition, and three repetitions were taken for each treatment. After taking out the whole plant, it was slowly washed with running water on the ground and root system of the plants was separated from the cotyledonary nodes. The roots were stored at −80 °C for proteomics study at: Chaya Biotech, Shanghai, China, following their standards procedures.The roots were taken from −80 °C and quickly ground to powder in a mortar with liquid nitrogen. Hundred milligram of lyophilized powder was taken into a 1.5-mL centrifuge tube and we added 800 μL sodium dodecyl sulfate + dithiothreitol + Tris-HCl protein lysate . It was kept in boiling water bath at 100 °C for 5 min, followed by ultrasonification in ice bath for 10 min, again followed by 100 °C boiling water bath for 5 min. The tubes were centrifuged at 14 000 g for 30 min, and finally the supernatant was collected and filtered with 0.22-μm ultrafiltration tube.BiCinchoninic acid assay was used to optimize the concentration of proteins in each sample. Twenty microgram protein samples were used for sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The remaining samples were divided into 300-μg portions and stored in refrigerator at −80 °C. Finally the SDS-PAGE gel was scanned to obtain the gel map.−1 DS buffer , centrifuged at 14 000 g for 30 min at room temperature. The same procedure was repeated two more times. Finally, the filtrate was discarded and 40 μL Trypsin buffer (6 μg Trypsin in 40 μL DS buffer) was added, and placed at constant temperature with gentle shaking . Then, centrifuged at 14 000 g for 30 min at room temperature to collect the filtrate, replaced with a new collection tube, added 40 μL DS, centrifuged at 14 000 g at room temperature for 30 min, took the filtrate and quantified at abs (280 nm).We took 300 μg of each sample for FASP enzymatic hydrolysis. Then, we added 200 μL of UA buffer  to the sample, mixed well, centrifuged at 14 000 g for 30 min at room temperature and discarded the filtrate. The same procedure was repeated two more times. We added 100 μL of IAA (50 mM IAA in UA), shook at 600 rpm for 1 min, incubated at 300 rpm for 30 min at room temperature, followed by centrifugation at 14 000 g for 30 min at room temperature. Next, 100 μL UA buffer was added, centrifuged at room temperature at 14 000 g for 30 min. The same procedure was repeated two more times. We added 100 μL of 25 mM·LWe took 100 μg of each group of sample, according to instructions of the manufacturer: TMT6plex™ Isobaric Label Reagent Set (Thermo Scientific) instructions for labelling. The labelling scheme was set as TMT6 -126 TMT6 -127 TMT6 -128 TMT6 -129 TMT6 -130 TMT6 -131. All the labelled peptides were mixed separately and we used the high-pH (HpH) reversed-phase to pre-fractionate the peptides. Column: Gemini-NX 4.6 × 150 mm column  . Buffer: Buffer A was 10 mM ammonium acetate, pH = 10.0; Buffer B was 10 mM ammonium acetate, 90 % ACN, pH = 10.0. Instrument: 1100 Series HPLC Value System (Agilent). After HpH fractionation, we collected 40 fractions of the flow-through and elution for each set of markers, and combined them into 15 fractions according to the HpH chromatogram. We stored them at −80 °C. We combined the fractions according to their abundance from the chromatograph. Low abundance peptides were combined into one fraction and high abundance peptides were kept as a single fraction.Liquid phase A was 0.1 % formic acid in water, and liquid B was 0.1 % formic acid in acetonitrile (acetonitrile is 100 %). The analytical column  was equilibrated with 95 % A solution. The sample was loaded on the Thermo Scientific EASY trap column  by the autosampler, and then separated by chromatographic column. The relevant liquid phase gradient was as follows: 0–45 min, the linear gradient of liquid B was from 5 to 28 %; 45–80 min, the linear gradient of liquid B is from 28 to 90 %; 80–90 min, the liquid B was maintained at 90 %. The enzymolysis products were desalted and separated by capillary high-performance liquid chromatography and then analysed by Q Exactive mass spectrometer (Thermo Scientific). Analysis time: 90 min, detection method: positive ion, precursor scan range: 350–1800 m/z, first-level mass spectrometry resolution: 70 000, AGC target: 3e6, first-level maximum IT: 30 ms, number of scan ranges: 1, dynamic exclusion: 60.0 s. The mass-to-charge ratio of peptides and peptide fragments was collected as follows: 20 fragment maps (MS2 scan) are collected after each full scan, MS2 activation type: HCD, isolation window: 1.6 m/z, secondary mass spectrometry resolution: 35 000, microscans: 1, level 2 maximum IT: 100 ms, AGC target: 2e5, normalized collision energy: 35 eV, underfill ratio: 0.1 %.G. max (soybean) protein database, carbamidomethylation of cysteine as fixed modification, oxidation of methionine, peptide mass tolerance of ±20 ppm, fragment mass tolerance of 0.1 Da and peptide charge +1, +2 and +3. For quadrupole time-of-flight (Q-TOF), peptide mass tolerance and fragment mass tolerance were 20 ppm and 0.05 Da, respectively. Proteome Discoverer 2.1 performed screening with false discovery rate (FDR) ≤ 0.01 based on peptide identification results and quantitative analysis based on peptide peak intensity values. The original protein quantification result was the median of the peptide quantification result and was corrected by the sum of the reported ion peak intensity values of all channels. The final quantitative results were then normalized by the ratio of each label to the average of all channel intensity values and the median of the ratio. Functional classification of the proteins was performed based on the Kyoto Encyclopaedia of Genes and Genomes (KEGG) database (https://www.genome.jp/kegg/). The KEGG enrichment pathway with Bonferroni-corrected P-value < 0.05 was considered significant by using the hypergeometric test.The raw data of mass spectrometry analysis were RAW file, and the built-in software SEQUEST Proteome Discoverer 2.1 (Thermo Scientific) was used for protein annotation and quantitative analysis. The parameters used in this search were as follows: no restrictions on protein molecular weight, two missed cleavage, trypsin digestion, monoisotopic mass values, UniprotKB t-test function in R software version 4.0.2. Differentially expressed proteins (DEPs) were identified with fold changes >1.5 or <0.67 and P < 0.05 , low-P  and normal-P . L13 is a low-P-tolerant genotype while T3 is a low-P-sensitive genotype as demonstrated by [see]. Sequence coverage of the majority of identified proteins was up to 60 %  to L13_P1 (low-P) and L13_P2  to L13_P0 (P-free). In L13_P2 vs. L13_P1, only seven DEPs were identified and all of them were upregulated in L13_P1 pathways . A crosspathways ; Table 1[see]. Out of these 23 DEPs, only four were annotated  protein, non-specific lipid-transfer protein and urease), while the remaining DEPs were uncharacterized proteins. Concerning T3_P2 vs. T3_P0, 127 DEPs were identified, all being downregulated in T3_P0 [see]. Majority of these DEPs are involved in oxidative phosphorylation, glutathione metabolism and carbon metabolism metabolic pathways  to T3_P1 (low-P) and T3_P2  to T3_P0 (P-free) were made in order to identify the DEPs involved in T3 response to P deficiencies in the soils. In T3_P2 vs. T3_P1, 23 DEPs were detected, including 22 downregulated and one upregulated proteins in T3_P1 pathways . The higpathways ; Table 2seeCollectively, 660 and 133 unique DEPs were detected in L13 and T3 genotypes in response to P deficiencies, respectively. We compared DEPs of the two genotypes in order to identify common and genotype-specific DEPs. The result showed that 71 DEPs were commonly regulated in both genotypes under P deficiencies , I1K3U8  and C6SZ93 ) constantly upregulated in L13 but not affected in T3 by low-P levels . Both I1Cajanus cajan), V7BRY5 (Phaseolus vulgaris), A0A4D6M2C2 (Vigna unguiculata)/C6SZ93: A0A445DA28 (Arachis hypogaea), A0A1S3TVI8 (Vigna radiata var. radiata), Q43681 (V. unguiculata)/I1K3U8: A0A1S3UMP3 (V. radiata var. radiata), A0A151QZQ6 (C. cajan), V7CR23 (P. vulgaris), A0A1J7HDZ3 (Lupinus angustifolius)); hence, we predict that this work could be translated to other botanical species.These three proteins could be tested in a large germplasm to confirm their capacity to discriminate low-P-tolerant and -sensitive soybean genotypes. Such biomarkers could facilitate and accelerate breeding efforts towards low-P tolerance in soybean. An interesting follow-up experiment may be to test T3 mutant plants over-expressing the three putative key adaptive proteins in P-limited conditions. Besides, we found homologues of these proteins in several other plant species (I1KW20: A0A151UI57 (The following additional information is available in the online version of this article—plab019_suppl_Supplementary_MaterialsClick here for additional data file.


We have characterised a loss-of-function zebrafish model for CDD, containing a nonsense mutation in cdkl5. cdkl5 mutant zebrafish display defects in neuronal patterning, seizures, microcephaly, and reduced muscle function caused by impaired muscle innervation. This study provides a powerful vertebrate model for investigating CDD disease pathophysiology and allowing high-throughput screening for effective therapies.Cyclin-dependent kinase-like-5 (CDKL5) deficiency disorder (CDD) is a severe X-linked neurodegenerative disease characterised by early-onset epileptic seizures, low muscle tone, progressive intellectual disability and severe motor function. CDD affects ∼1 in 60,000 live births, with many patients experiencing a reduced quality of life due to the severity of their neurological symptoms and functional impairment. There are no effective therapies for CDD, with current treatments focusing on improving symptoms rather than addressing the underlying causes of the disorder. Zebrafish offer many unique advantages for high-throughput preclinical evaluation of potential therapies for neurological diseases, including CDD. In particular, the large number of offspring produced, together with the possibilities for This article has an associated First Person interview with the first author of the paper. Summary: Characterisation of a novel loss-of-function zebrafish model for CDKL5 deficiency disorder, containing a nonsense mutation, demonstrates its utility for investigating disease aetiology and allowing high-throughput screening for potentially effective therapies. CDKL5). Patients with CDD often display a heterogeneous array of clinical phenotypes, including early infantile epilepsy, delayed motor function and intellectual disability  is a severe neurodegenerative disease caused by mutations in cyclin-dependent kinase-like-5  mice recapitulate many features of CDD, exhibiting severe neuronal impairment leading to deficits in learning, memory and social behaviour . These din vivo analysis of neurological development and CDD progression. Notably, the optical clarity and power of live imaging, combined with the accessibility of developing zebrafish larvae, provide novel ways for monitoring disease states. Development of both the brain and spinal cord is highly conserved between humans and zebrafish, as well as many signalling pathways involved in their function. In zebrafish, the skeletal muscle makes up ∼80% of the body mass, is functional as early as 20 h post fertilization (hpf) and, after 2 days post fertilization (dpf), the fish swim spontaneously. Muscle contractions controlling early spontaneous motor activity are likely to be directly modulated by embryonic central nervous system activity. At this time, the upper motor neurons in the primary cortex send their projections to the developing spinal cord, and it is only after the fast muscle is formed at ∼52 hpf that the lower motor neurons project to the myotome  are produced during zebrafish development, which differ in the inclusion of exon 16  and the notochord (or developing spine) at 3 dpf . We also used a sense cdkl5 mRNA probe and observed low and non-specific expression, unlike that observed for the antisense cdkl5 probe . The brain, spinal cord, eyes, muscle and kidney represent tissues that are affected in patients carrying CDKL5 mutations  creating a premature termination codon leading to a truncated and non-functional gene product  compared to both +/−cdkl5 (1.055±0.0647 mean±s.e.m.) and +/+cdkl5 fish (1.00±0.0469 mean±s.e.m.) (−/−cdkl5 fish (1.108±0.0892 mean±s.e.m.) compared to their siblings  . However, we did detect a significant decrease in the normalised duration spent swimming by −/−cdkl5 (0.774±0.0663 mean±s.e.m.) compared to +/+cdkl5 fish (1.00±0.0622 mean±s.e.m.), suggesting the reduced distance swum by −/−cdkl5 fish is due to reduced activity (−/−cdkl5 compared to +/+cdkl5 fish (Patients carrying ±s.e.m.) A. Intere±s.e.m.) B. We alsactivity C. We ana+/+ fish D,E at 2 +/−cdkl5 fish to a Tg(islet1:EGFP) zebrafish transgenic strain, labelling all of the motor neurons and ventral interneurons with enhanced green fluorescent protein (EGFP) (islet1:EGFP);−/−cdkl5 (244.6±2.65 mean±s.e.m.) compared to Tg(islet1:EGFP);+/+cdkl5 fish (246.2±5.96 mean±s.e.m.) (islet1:EGFP);−/−cdkl5 (7.004±0.626 mean±s.e.m.) compared to Tg(islet1:EGFP);+/+cdkl5 fish (9.600±0.499 mean±s.e.m.) .We then crossed n (EGFP) . Using t±s.e.m.) A,B, we o±s.e.m.) A,C. Thiscdkl5 mutants display seizures during early embryonic development, we used a Tg(HuC:H2B-GCaMP6s) transgenic strain, in which the genetically encoded calcium indicator GCaMP6s is expressed in mature neurons (HuC:H2B-GCaMP6s);−/−cdkl5 (0.875±0.214 mean±s.e.m.) compared to Tg(HuC:H2B-GCaMP6s);+/+cdkl5 fish (0.296±0.115 mean±s.e.m.) (−/−cdkl5 and +/+cdkl5 fish with a 20 mM pentylenetetrazole (PTZ) solution, a convulsant that affects GABA and glutamate receptors, which has previously been used as a seizure model in zebrafish (HuC:H2B-GCaMP6s);−/−cdkl5 (4.083±0.360 mean±s.e.m.) compared to Tg(HuC:H2B-GCaMP6s);+/+cdkl5 fish (2.974±0.342 mean±s.e.m.) (HuC:H2B-GCaMP6s);−/−cdkl5 fish and 1.031±0.004 for Tg(HuC:H2B-GCaMP6s);+/+cdkl5 fish; mean±s.e.m.] and after PTZ treatment   D, associ±s.e.m.) A-C. To eebrafish . Followi±s.e.m.) F over a ±s.e.m.] E,G.Fig.Fig. S4A). Based on these observations, we selected 2.0 ng MO to use in further experiments. To confirm that cdkl5 mRNA levels are reduced in Cdkl5 morphants, we performed RT-PCR analyses at 2 and 6 dpf and found that cdkl5 mRNA is decreased compared to standard control MO-injected fish . We performed locomotion assays at 6 dpf and found a significant decrease in swimming performance in Cdkl5 morphants (0.678±0.0336 mean±s.e.m.) compared to standard control MO-injected fish (1.00±0.0664 mean±s.e.m.) . We did not observe any defects in muscle patterning in standard control-MO injected fish or Cdkl5 morphants at 2 and 6 dpf , consistent with −/−cdkl5 fish.To confirm that a reduction in Cdkl5 causes defects in motor function and muscle innervation, we used a morpholino (MO) targeting the splice donor site of exon 5 (Cdkl5 ex5 MO) to knockdown Cdkl5 levels. To determine an effective dose for knockdown, we injected the MO at 0.5 ng, 1.0 ng or 2.0 ng into wild-type embryos and performed RT-PCR. We were able to detect a number of smaller sized amplicons by RT-PCR only in the 2.0 ng injected samples . However, in Tg(islet1:EGFP);Cdkl5 knockdown fish, there appeared to be a reduction in the density of axonal projections , recapitulating the pathologies observed in −/−cdkl5 fish  compared to +/+cdkl5 fish (3.793 mm±0.022 mean±s.e.m.)  compared to +/+cdkl5 fish  at 6 dpf ] (P<0.01) in total brain volume in Tg(HuC:EGFP)−/−;cdkl5  compared to Tg(HuC:EGFP)+/+;cdkl5 fish  (P<0.01) in the cerebellar volume of Tg(HuC:EGFP)−/−;cdkl5 (4.880±0.0153 mean±s.e.m.) compared to Tg(HuC:EGFP)+/+;cdkl5 fish (4.948±0.0162 mean±s.e.m.) .CDD patients have been reported to exhibit low muscle tone and a small head size, with some demonstrating cortical atrophy identified through brain imaging . To deteat 6 dpf A, and no±s.e.m.) A,B, therat 6 dpf A,C,D, reC:EGFP)] . We reco±s.e.m.) A,B, in l±s.e.m.) C,D. Addi±s.e.m.) C. We sta−/−cdkl5 and +/+cdkl5 fish. The cranial cartilage of the zebrafish is the first skeletal structure present and forms at ∼3 dpf. As zebrafish grow, the bones continue to ossify, with the first osteoblasts surrounding the cartilage and forming bone matrix by 7 dpf  compared to +/+cdkl5 fish  at 7 dpf  (HuC:EGFP) (HuC:H2B-GCaMP6s) (cdkl5 mutant line (sa21938) obtained from the Zebrafish International Resource Centre. For cdkl5 genotyping, Kompetitive allele-specific PCR technology (Geneworks) was used. For MO injections, the Cdkl5 ex5 (5′-AGATATAAACACTGTCATACCTCTG-3′) and standard control (5′-CCTCTTACCTCAGTTACAATTTATA-3′) MOs (GeneTools) were diluted in distilled water and co-injected with Cascade Blue-labelled dextran (Molecular Probes) into one- to two-cell wild-type  embryos. MO concentrations were calibrated according to Zebrafish were maintained according to standard protocols . Zebrafit1:EGFP) , Tg(HuC:uC:EGFP) , Tg(HuC:GCaMP6s)  and an Nin situ hybridization was carried out as described previously .Whole-mount eviously . Probes +/−cdkl5 incross, for three independent biological replicates. The tails were used for genotyping and the heads were used for RNA extraction, with 20 embryos per genotype per replicate. For MO experiments, whole embryos were analysed at either 2 or 6 dpf. Total RNA was extracted using TRIzol reagent (Invitrogen Life Technologies). cDNA was synthesised from 1 μg of each RNA sample in a 20 μl reaction using a Protoscript first strand cDNA synthesis kit (New England Biosciences), oligo(dT)20 and Random Primer Mix primers following the supplier's instructions. Primers used for RT-PCR were cdkl5RTF, 5′-CTCCGTACGCTCAAACAAGAC-3′, and cdkl5RTR, 5′-TGAGCTCTCCCAAAATACACC-3′; cdkl5shortF, 5′-CGCTTTTATGCTCAATGCAC-3′, and cdkl5shortR, 5′-AGCATCTTCAGCTCCCGTAA-3′; cdkl5longF, 5′-GCCAGAAGGTGGCAATAGTT-3′, and cdkl5longR, 5′-CCCGTCCCTAAGTTTCCATA-3′; cdkl5MOF, 5′-CTCCGTACGCTCAAACAAGAC-3′, and cdkl5MOR, 5′-TGAGCTCTCCCAAAATACACC-3′; βActF, 5′-GCATTGCTGACCGTATGCAG-3′, and βActR, 5′-GATCCACATCTGCTGGAAGGTGG-3′; and ef1αF, 5′-CTGGAGGCCAGCTCAAACAT-3′, and ef1αR, 5′-ATCAAGAAGAGTAGTACCGCTAGCATTAC-3′.For the qRT-PCR experiment, head and tails were separated from 3 dpf zebrafish, the progeny of a Locomotion assays were performed on 6 dpf zebrafish as per Immunofluorescence was performed on 2, 3 and 6 dpf zebrafish as per +/−cdkl5 fish were crossed to the Tg(HuC:H2B-GCaMP6s) line and raised to adulthood. Tg(HuC:H2B-GCaMP6s)+/−;cdkl5 fish were crossed to +/−cdkl5 fish, and their progeny were raised in embryo medium  containing 200 µM N-phenylthiourea  from 12 hpf to suppress melanocyte formation, changing medium every 24 h. Embryos were sorted for EGFP fluorescence at 2 dpf. At 4 dpf, fish were anaesthetised using 0.0016% tricaine methanesulfonate (Sigma-Aldrich) in embryo medium, and DNA was extracted from a small tail clipping and used to genotype embryos (as described above).0) was calculated from the first image taken for each fish in standard embryo medium. The final fluorescence values (F/F0) were normalised by dividing the mean fluorescence intensity (F) by the baseline value (F0).At 6 dpf, embryos were again anaesthetised and immobilised in 0.5% low-melting-point agarose in embryo medium. Embryos were first imaged for 5 min in standard embryo medium, after which point PTZ  dissolved in standard embryo medium  from 12 h to suppress melanocyte formation, changing the medium every 24 h. At 7 dpf, embryos were fixed in 4% paraformaldehyde and left overnight in methanol at 4°C. Embryos were rinsed in acid alcohol (70% EtOH:1% HCl) and stained overnight at room temperature in 0.1% Alcian Blue (80% EtOH:20% Acetic Acid). Embryos were washed for 6 h in acid alcohol and then rinsed in PBS plus Tween 20 before imaging.+/−cdkl5 fish were crossed to the Tg(HuC:EGFP) line and raised to adulthood. Tg(+/−HuC:EGFP);cdkl5 fish were crossed to +/−cdkl5 fish, and their progeny were raised in embryo medium containing 200 µM PTU (Sigma-Aldrich) from 12 hpf to suppress melanocyte formation, changing the medium every 24 h. Embryos were sorted for EGFP fluorescence at 2 dpf. At 4 dpf, fish were anaesthetised using 0.0016% tricaine methanesulfonate in embryo medium, and DNA was extracted from a small tail clipping and used to genotype embryos (as described above). At 6 dpf, embryos were again anaesthetised and set in 1% low-melting-point agarose in embryo medium containing tricaine in 0.8 mm fluorinated ethylene propylene tubing (Bola). Images were taken using a Thorlabs confocal microscope with an Olympus 20× water dipping 1.0 NA objective . Brain image registration and total brain and cerebellar volume analysis was performed as per +/−cdkl5 fish were crossed to the Tg(islet1:EGFP) line and raised to adulthood. Tg(islet1:EGFP)+/−;cdkl5 fish were crossed to +/−cdkl5 fish, and their progeny were sorted for EGFP fluorescence at 2 dpf. At 3 dpf, fish were anaesthetised using 0.0016% tricaine methanesulfonate in embryo medium, and DNA was extracted from a small tail clipping and used to genotype embryos (as described above). At 6 dpf, embryos were again anaesthetised and set in 1% low-melting-point agarose in embryo medium containing tricaine, and imaged using an LSM 710 confocal microscope (Zeiss) equipped with a 20× water dipping 1.0 NA objective and a 488 nm laser. Images were taken from comparable regions along the spinal cord for each fish. The total number of EGFP+ motor neuron cell bodies in Tg(islet1:EGFP) zebrafish were counted from a maximum projection image using the cell counter function in Fiji. The axonal density was determined by quantifying the surface of fluorescence above background. Each maximum projection Tg(islet1:EGFP) zebrafish image was cropped to remove any cell bodies. The background was normalised across images, and an upper threshold level of 64,000 and lower threshold level of 9000 was used for each image. The images were converted to a mask image and the percentage area of EGFP+ neuronal projections was determined using Fiji.+/+cdkl5 siblings. All statistical analyses were performed in GraphPad Prism 7 using a one-way ANOVA for qRT-PCR . Data in +/+cdkl5 embryo 4 sample from replicate 4), which was excluded from the analysis.For all experiments, the investigators were blinded to genotype during data capture and once the analyses were completed the genotypes of the fish were resolved. For swimming analyses, all values were normalised to the average value of  qRT-PCR D and cdkg assays A,B, a twanalyses C, seizuranalyses D-F, growurements B,C, braiurements B,C, Alciurements C and Cdk


TOTAL) of fresh pecan nuts maintaining oil characteristics and increasing phenolic compounds in the remaining by-product. However, there is no information regarding the PEF effect on dry pecan nuts. Dry kernels were pretreated at three specific energy inputs  and compared against untreated kernels and kernels soaked at 3, 20 and 35 min. OEYTOTAL, kernels microstructure, oil stability , oil stability index, phytosterols and lipoxygenase activity), along with by-products phenolic compounds , condensed tannins (CT)) and AC were evaluated. Untreated kernels yielded 88.7 ± 3.0%, whereas OEYTOTAL of soaked and PEF-treated kernels were 76.5–83.0 and 79.8–85.0%, respectively. Kernels microstructural analysis evidenced that the 0.8 kJ/kg pretreatment induced oleosomes fusion, while no differences were observed in the stability of extracted oils. PEF applied at 0.8 kJ/kg also increased by-products CT by 27.0–43.5% and AC by 21.8–24.3% compared to soaked and untreated kernels. These results showed that PEF does not improve OEYTOTAL when it is applied to dry pecan nuts, demonstrating that kernelsʹ moisture, oil content and microstructure play an important role in the effectiveness of PEF.Pulsed electric fields (PEF) have been reported to increase the total oil extraction yield (OEY Pulsed electric fields (PEF) involved the application of intermittent electric fields of varying intensity 0.1–50 kV/cm) and short duration (µs–ms) –50 kV/cm,3,4,5. TCarya illinoinensis (Wangenh. K. Koch)) are well-known for their high concentration of phenolic compounds, mono- and polyunsaturated fatty acids, phytosterols, and tocopherols [Pecan nut kernels , β-mercaptoethanol (β-ME), 2,2-diphenyl-1-picrylhydrazyl (DPPH), boric acid (H3BO3), hydrochloric acid (HCl), glutaraldehyde, osmium tetroxide, polyvinylpolypyrrolidone (PVPP), potassium hydroxide solution (KOH 0.1 M), sodium acetate (CH3COONa), sodium phosphate monobasic (NaH2PO4), sodium phosphate dibasic (Na2HPO4), Triton X-114 and uranyl acetate were acquired from Sigma-Aldrich  along with 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), linoleic acid, β-sitosterol, stigmasterol, campesterol, catechin, gallic acid and vanillin. Epoxy EMbed 812 resin was purchased from Electron Microscopy Sciences . Acetic acid (CH3COOH), ethanol (EtOH), sodium carbonate (Na2CO3) and KOH were purchased from DEQ .Acetonitrile, ethyl acetate, methanol (MeOH), hexane, water HPLC grade (HCarya illinoinensis) from Alesto brand were purchased in a local market in Lleida (Spain). In this study, dry kernels were separated in three experimental groups comparing PEF-treated kernels against reference and control kernels. No soaking nor PEF processing was performed on reference kernels, while control kernels were placed in tap water (1:3 w/w) for 3, 20 and 35 min to assess the effect of soaking at each PEF pretreatment. Kernels pretreated by PEF were immersed in tap water and processed. After a 10 min draining, control and PEF-treated kernels (1.0 g) were separated for moisture and microstructural analysis. Before freeze-drying, kernel samples (2.0 g) were taken for determination of lipoxygenase (LOX) activity. The remaining kernels were frozen , freeze-dried , and stored at −40 °C until oil extraction.Kernel halves of dry pecan nuts (w/w) to apply 10, 99 and 192 monopolar exponential-wave pulses at an electric field strength of 5.0 kV/cm. These electrical conditions equaled to specific energy inputs of 0.8, 7.8 and 15.0 kJ per kg of pecan nut kernels in wet basis , respectively.The application of PEF was performed in a batch-system using a 20 × 8 cm methacrylate container with stainless-steel parallel electrodes as the treatment chamber. The batch-system was equipped with a 0.1 µF capacitator , a pulse generator  and a TG-70 gas control unit. Based on a previous study , dry ker2 into the headspace and the generated cakes were placed in polyethylene bags and vacuum sealed. Oil and cake samples were stored at −40 °C until analyses.Oil extraction was performed on freeze-dried kernels (85.0 g) employing a screw-type press . Pecan nut oil was stored in 50 mL centrifuge tubes flushing NReference, control and PEF-treated kernels oil extraction yields  were calculated as follows:TOTAL, %) of control and PEF-treated kernels was estimated using the Total oil extraction yield  were homogenized  using an IKA® T25 ultraturrax , centrifuged  and supernatants collected. This procedure was repeated three times. Hexane was evaporated from pooled supernatants  and oil content was determined gravimetrically following the AOAC Official Method 960.39 [Oil content was analyzed as reported by Villarreal-Lozoya et al. . Samplesd 960.39 . Oil conControl and PEF-treated kernels were fixed employing the procedure reported by Kendall et al.  with modw/v) and Triton X-114 (0.05:100 w/v) in 50 mM phosphate buffer (pH 6.6). Kernels (2.0 g) and the extraction solution (10 mL) were homogenized , filtered using glass wool and centrifuged . Supernatants were collected for determination of lipoxygenase (LOX) activity according to the procedure reported by Li et al. [v/v/v) was employed as substrate stock solution to measure LOX activity. The stock solution (5 mL) was diluted completely in 20 mL of 0.2 M borate buffer and 5 mL of distilled water. The diluted solution (2 mL) and 0.2 M borate buffer (950 µL) were pipetted in a cell quartz and mixed by inversion. Next, enzymatic extracts (50 µL) were added and mixed by inversion. Absorbance was measured at 234 nm and registered every 10 s until 3 min of reaction in a Cecil CE 1010 UV-VIS spectrophotometer . LOX activity was calculated employing the molar extinction coefficient (ε) of hydroperoxides  to express it as µmol of hydroperoxide produced per L of LOX per s (µmol/L·s) [Kernels enzymatic extracts were obtained as described by Christopoulos and Tsantili . An extri et al. . Solutiomol/L·s) .Oil acidity was performed following the AOAC Official Method 940.28  and resuThe DPPH radical scavenging capacity method reported by Gao et al.  was usedThe stability index (OSI) of extracted oils was measured in a Rancimat 679 apparatus  . Oil samw/v) and incubated . The mixture was cooled in a water bath and 5 mL of 1.3% KOH was added and left to react . To phytosterols extraction, 2 mL of distilled water and 5 mL of hexane were included to the mixture, vortexed for 1 min, and centrifuged . The addition of distilled water and hexane was performed twice. Hexane was evaporated from pooled supernatants using a vacuum evaporator . Extracts were reconstituted in 0.5 mL of hexane for chromatographic analysis. A HPLC-ELSD system  equipped with a 5 µm, 4.6 mm × 500 mm Luna C8 column  was employed to identify and quantify phytosterols from pecan nut oil. Column and ELSD temperature were maintained at 40 °C and 50 °C, respectively. Aliquots (10 µL) were analyzed employing a mobile phase consisted of MeOH:H2O (95:5 v/v) at a flow rate of 1 mL/min and the detector was set at a gain of 16. Standard curves of β-sitosterol (0.2–1.2 mM), stigmasterol (0.2–1.2 mM), and campesterol (0.05–0.25 mM) were prepared to quantification. Concentrations were expressed as mg per kg of pecan nut oil (mg/kg).Extraction and quantification of phytosterols were done according to Domínguez-Avila et al. and Nair et al., respectively ,29. HCl After the determination of cakes oil content, the defatted portion was used for phenolic compounds analysis. Total phenolics (TP), condensed tannins (CT), and AC of reference, control and PEF-treated cakes were performed following the procedures reported by Rábago-Panduro et al. . Results® Version 18.1 software .PEF pretreatments, oil extraction and LOX activity were performed by triplicate while oil and cake analysis were done by duplicate to a total of six replicates per experimental group. One-way ANOVA and Dunnett test were performed with the Minitabp = 0.004 and p = 0.015, respectively). Regarding the oil content, reference kernels contained 69.4 ± 0.7 g/100 g db, decreasing to 62.7 ± 0.0 g/100 g db in control kernels and 62.7 ± 0.7 g/100 g db in kernels pretreated by PEF, containing 9.7% less oil than reference kernels. Likewise, the The moisture of dry pecan nuts used as reference was 2.4 ± 0.1 g/100 g db, while control kernels soaked at 3, 20 and 35 min had moisture contents of 13.5 ± 0.6, 21.1 ± 0.9, and 21.7 ± 0.7 g/100 g db, respectively. In PEF-treated kernels moisture contents also increased reaching values of 13.5 ± 1.0, 18.0 ± 0.1 and 19.3 ± 1.1 g/100 g db at 0.8, 7.8 and 15.0 kJ/kg pretreatments, respectively. However, the moisture of kernels processed at 7.8 and 15.0 kJ/kg was significantly lower than their respective control kernels . It seemed that the application of PEF at 0.8 kJ/kg did not promote lipid oxidation of pecan nut oil. These results agreed with acidity, AC, and OSI values of the oil extracted from kernels processed at 0.8 kJ/kg. However, it should not be discarded lipid oxidation via enzyme activation since the acidity of pecan nut oil increased at higher W.The LOX activity of reference, control and PEF-treated kernels was 4.52 ± 0.14, 4.83 ± 0.10 and 4.41 ± 0.20 µmol/L·s, respectively. Reference and PEF-treated kernels showed LOX activity values significantly lower than control kernels  was higher than those reported for pecan nut oil extracted by hydraulic pressing 56.4–58.9%) [% 9,35,3,335,36 aTOTAL of fresh kernels up to 74.8% being higher than OEY of kernels without soaking nor PEF pretreatment [The PEF pretreatments did not increase oil extraction from dry pecan nut kernels, contrasting with previous findings where the application of 0.8 kJ/kg increased OEYreatment . Sarkis reatment . ConsequThe analysis of oil stability showed that oils extracted from dry pecan nuts were within the acidity range accepted by the Food and Agriculture Organization for cold-pressed oils . FurtherTotal phenolics, condensed tannins, and antioxidant capacity of cakes generated from dry kernels were lower than those reported for fresh kernels . These dTOTAL of pretreated kernels  in comparison with kernels without soaking nor PEF processing (88.7%). These results could be due to changes in oleosomes characteristics and their localization within the cotyledon tissue of dry kernels submitted to water immersion. Kernels processed at 0.8 kJ/kg were used for the microstructural analysis showing a reduction of the cell size and oleosomes fusion in the intracellular space. These changes might help oil flow during mechanical extraction. Comparable values of acidity, AC, OSI and LOX activity between extracted oils showed that the application of PEF did not negatively affect pecan nut oil quality. Furthermore, the 0.8 kJ/kg pretreatment increased CT and AC of generated cakes compared to cakes from untreated kernels and kernels soaked for 3 min. These results showed that the effectiveness of PEF to increase OEYTOTAL of pecan nuts is dependent on not only kernelsʹ moisture and oil content but also on the drying process. It appears that the rehydration of dry kernels produced a negative effect on their microstructure and oleosomes characteristics. Thus, further research focused on these variables is necessary along with other microscopy techniques and analytical procedures to better understand the effect of PEF pretreatments on oil extraction from pecan nuts.The application of PEF to dry pecan nuts did not increase OEY nor OEY


Ehretia tinifolia Linnaeus (Boraginacea) and Sideroxylon lanuginosum Michaux (Sapotaceae) are wild fruits consumed in North America and are appreciated for their pleasant flavor and sweet taste. However, details regarding their composition and biological properties in the available literature are scarce. This study reports the phenolic composition, antioxidant, antiproliferative activities, and digestive enzymatic inhibition of amberlite-retained methanolic extracts from both fruits. Results revealed that these wild fruit extracts are rich in antioxidants. S. lanuginosum had lower phenolic but higher flavonoid contents (21.4 ± 1.5 mg GAE/100 g FW and 6.42 ± 0.9 mg CE/100 g FW) than E. tinifolia (64.7 ± 2.6 mg GAE/100 g FW and 5.1 ± 0.4 mg CE/100 g FW). HPLC-DAD-MS/MS analysis showed rosmarinic acid as a major polyphenol in E. tinifolia and quercetin glucoside in S. lanuginosum. Polyphenols content in E. tinifolia was related to a significant free radical scavenging ability: DPPH (EC50 = 0.32 ± 0.03 mg/mL), TEAC (4134 ± 9.7 μM TE/g dry extract), and hemolysis inhibition (IC50 = 58.55 ± 2.4 μg/mL). Both extracts were capable of inhibiting α-glucosidase, partially inhibiting α-amylase, and showed no inhibition against lipase, while showing antiproliferative activity against HeLa, HT-29 and MCF-7 cancer cell lines. Our study revealed that these wild fruit extracts are rich in health-beneficial phytochemicals and hold significant potential for elaborating functional foods. Ehretia tinifolia Linnaeus (Boraginacea), commonly called pinguica and Sideroxylon lanuginosum Michaux (Sapotaceae), known as Gum Bully, Black Haw, or Coma, are wild fruits appreciated for their pleasant flavor and sweet taste and have been consumed in North America by Native Americans since pre-Columbian times. Thus, wild plants have an important role in indigenous peoples’ lives −. These same fragments were found in compounds 27, 28, and 29, except they showed other signals that corresponded to fragments not determined as sugars. Compounds 20 to 25 were identified as lithospermic acid derivatives, which were isomers since they showed the same pseudomolecular ion at m/z 625, with the main fragment ion detected at m/z 537 (C27H22O12), which can be identified as lithospermic acid. Moreover, the fragment ion at m/z 493 [M-H]− can be formed by the loss of CO2 (44 Da) of the ion at m/z 537, whereas fragment ions at m/z 295 [M-H-CO2-C9H10O5]− and m/z 312 [M-H-C9H8O4]− were derived from the ion at m/z 493 due to the loss of C9H10O5 and C9H8O4 fragment ions. These fragments are those reported by Huang et al. − at m/z 301, was identified as quercetin (C15H10O7). The fragment ion at m/z 273 [M-H-CO]− was derived from the loss of a CO (28 Da) and that at m/z 179 ([M-H-C7H6O2]−) can be explained by the RDA fragmentation. Subsequently, fragment ions at m/z 151 [M-H-C7H6O2-CO]− originated from the ion at m/z 179 by the loss of a CO (28 Da) − after the loss of a glucoside molecule (162 Da). Similarly, compounds 23 and 24 also showed fragment ions at m/z 301 in their MS/MS analysis, formed after losing a glucuronic acid (176 Da). In contrast, compounds 25 and 26 were identified as quercetin-pentoside because of detecting the same ion fragment (m/z 301) after the loss of 132 Da (which can be attributed to a pentose moiety). Quercetin derivatives and, mainly quercetin glucoside, can be relevant for the biological activity of S. lanoginosum fruits, since this compound has been shown to protect DNA and erythrocytes from oxidative damage and exhibited anticancer activity [In the case of m fruits , althougm fruits . The strtituents . In addi (28 Da) . Compounactivity .E. tinifolia and S. lanuginosum extracts showed high inhibitory activities with IC50 values of 0.17 and 0.21 mg/mL, respectively  with IC50 values > 5 mg/mL. In other studies, polyphenol-rich extracts from different types of berries inhibited α-amylase in vitro, and the most effective were from raspberry and rowanberry . Thus, strawberry and raspberry extracts were more effective amylase inhibitors than blueberry and blackcurrant [α-Amylase enzyme is one of the key enzymes in the human digestive system, which degrades starch to monosaccharides and causes the rise of blood glucose . Naturalkcurrant . Other akcurrant . Our fru50 value of 0.04 μg/mL , MCF-7 (breast adenocarcinoma) and HT-29 (colon adenocarcinoma). Results showed that MCF-7 cell line was the most sensitive to the E. tinifolia extract, and the S. lanuginosum extract was against HT-29 cell line. The effect of E. tinifolia and S. lanuginosum extracts on cancer cell lines was dose-dependent and varied with the cell type and extract concentration. The concentration of 2 mg/mL of both extracts was the most effective. Nevertheless, E. tinifolia had the strongest effect on the three cell types. It revealed a marked decrease in the viability of the cancer cell lines after treatment. The 50% growth inhibition (IC50) of cells exposed to E. tinifolia was 0.99, 1.36 and 0.82 mg/mL, and for S. lanuginosum were 1.99, 3.22, and 1.97mg/mL for MCF-7, HeLa, and HT-29 cells, respectively . Though the extracts of Pyracantha coccinea (IC50 1.2 mg/mL) and Zosima absinthifolia (IC50 1.5 mg/mL) had less antiproliferative activity than E. tinifolia for HeLa cells [E. tinifolia extract was rosmarinic acid, and it could be responsible for the antiproliferative activity, as described by Niu et al. [Rosmarinus officinalis with rosmarinic acid and concluded that rosmarinic acid was more active than the plant extract against HeLa cells.Antiproliferative activity of ectively . It can La cells ,67. The u et al. . They coE. tinifolia and S. lanuginosum extracts showed better antiproliferative activity against MCF-7 cells than an apple extract (IC50 = 70.7 mg/mL) [50 value of QG in inhibiting MCF-7 cell growth was 46.4 μM [S. lanuginosum extract and could be responsible for inhibiting MCF-7 cells.Breast cancer is the most frequently diagnosed cancer in women. An alternative strategy to reduce the risk of cancer is through dietary modification. 7 mg/mL) . Moreove 46.4 μM . This flE. tinifolia and S. lanuginosum inhibited proliferation of colon cancer cells HT-29. Similar effects have been shown of Borago extracts with CI50 values within the 250–300 μg/mL range after 72 h of cell exposure. Like in E. tinifolia, rosmarinic acid was the main phenolic compound detected in all Borago taxa [50 of ~1000 μg/ mL [Phenolic compounds of ago taxa . In anot0 μg/ mL .Sideroxylon lanuginosum (coma) presented the higher content of total flavonoid (21.4 ± 1.5 mg GAE/100g FW); the most abundant flavonoid detected was the quercetin glucoside. In comparison, Ehretia tinifolia (pinguica) had higher total phenol content (64.7 ± 2.6 mg GAE/100g FW), and the most abundant phenolic detected was rosmarinic acid. These major compounds could be related to the interesting biological activity of both extracts. E. tinifolia showed the best effect in antioxidant/antiradical activity both in vitro , ex vivo assay (IC50 = 58.55 ± 2.4 μg/mL), and the inhibition of α-glucosidase (IC50 = 0.17 ± 0.1 mg/mL). Additionally, the extract of E. tinifolia showed a better effect on the growth inhibition of cancer cells, particularly against those of the human colon (IC50 = 0.82 ± 0.09 mg/mL) and breast cancer (IC50 = 0.99 ± 0.01 mg/mL). These results contribute to the scant information available on these wild fruits and contribute to revalue their use as a natural source of antioxidants with potential therapeutic application in diseases caused by free radicals. Further investigations using animal models are needed to confirm the beneficial effects of the studied extracts as supplementary treatments for oxidative diseases.