Non-toxic concentrations of CGT oil and β-bisabolol (1.6–50.0 µg/mL) significantly inhibited the production of the inflammatory mediators in a dose-dependent manner. Maximal inhibition by β-bisabolol was 55.5% for NO, 62.3% for PGE2, and 45.3% for TNF-α production in RAW cells. β-Bisabolol induced a level of inhibition similar to an equal concentration of α-bisabolol (50.0 µg/mL), a known anti-inflammatory agent. These results suggest β-bisabolol exerts similar in vitro effects to known topical anti-inflammatory agents and could therefore be exploited for cosmetic and therapeutic uses. This is the first study to report the in vitro anti-inflammatory activity of β-bisabolol in CGT essential oil.Natural α-bisabolol has been widely used in cosmetics and is sourced mainly from the stems of Candeia trees that have become endangered due to over exploitation. The in vitro anti-inflammatory activity of cotton gin trash (CGT) essential oil and the major terpenoid (β-bisabolol) purified from the oil were investigated against lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages as well as the 3t3 and HS27 fibroblast cell lines. Nitric oxide (NO), prostaglandin E Durined cells . Inflammed cells ,5. Normaed cells ,6. Howeved cells ,8. Regul2, interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin 8 (IL-8) are some examples of cell signaling molecules that promote prolonged inflammation [2, one of four known prostaglandins, is a lipid inflammatory mediator produced as a result of activity of the cyclooxygenases (COX1 and COX2) and prostaglandin synthases [2 is the most abundant prostaglandin (PG) produced in the body and its biosynthesis increases in inflamed tissues, thereby initiating the production of other pro-inflammatory mediators such as interleukin 17 cytokine (IL-17), which activates neutrophils and monocytes to sites of infection [Inflammation mediators such as NO, PGEammation ,10,11, aammation . Initiatammation ,13 as weammation . PGE2, oynthases ,16. Theyynthases . PGE2 isnfection . The pronfection ,20 initinfection ,22, and nfection ,24.Production of these mediators are specific to certain cells including macrophages  and fibrFibroblasts are also active in the process of inflammation. In addition to their mechanical and structural roles in tissues, they also promote inflammation by producing cytokines  and chemokines  when stimulated ,35,36, aPlant-derived natural compounds are constantly being investigated for anti-inflammatory properties ,41, part2, TNF-α, IL-6, and IL-8 in in vitro anti-inflammatory studies [Plant derived essential oils are exploited for their anti-inflammatory properties and have been shown to be effective using different cell lines ,46. The  studies . Inhibit studies . Another studies  to inhibIn our previous publication , essentiThe extraction yield of the essential oil was about 0.1% of CGT (m/m), similar to our previously reported yield.  The domiPercentage composition of terpenoids calculated from the total area under peaks of all volatiles identified in the CGT oil extract .50) of CGT oil was calculated to be 129.4 µg/mL for RAW 264.7, 90.2 µg/mL for 3t3, and 107.2 µg/mL for HS27 cells.Fibroblasts (HS27 and 3t3) and leukemic macrophages (RAW 264.7) were exposed to concentrations of CGT oil ranging from 2.0 µg/mL to 1000.0 µg/mL to determine a non-toxic concentration range of the oil for further anti-inflammatory assays. The highest concentration of CGT oil (1000.0 µg/mL) induced a similar toxic effect as the positive control chlorambucil (1 mg/mL) . The halBased on these data, the non-toxic range was deemed to be from 1.6 µg/mL to 50.0 µg/mL. The non-toxic concentration of β-bisabolol and α-bisabolol was selected following the anti-inflammatory assay of Maurya et al. , who repp < 0.05). The effectiveness of CGT oil to regulate the production of NO was such that there was an observed reduction of 12.0% at the lowest concentration of the oil (1.6 µg/mL). At the maximum concentration of CGT oil (50.0 µg/mL), NO was inhibited by 48.8% compared to 71.9% NO inhibition induced by 2.5 µM of the anti-inflammatory drug, dexamethasone (DEX). CGT oil and β-bisabolol significantly regulated the production of nitric oxide by the macrophages in a dose-dependent manner  after 24Nitric oxide production was reduced by 29.8% by the lowest concentration of β-bisabolol (1.6 µg/mL) and inhibition of NO remained unchanged when RAW 264.7 macrophages were exposed to 3.1 µg/mL and 12.5 µg/mL β-bisabolol . The hig2 when stimulated by 100 ng/mL LPS. Oil extracted from CGT reduced the production of PGE2 in a dose-dependent manner (p < 0.05) (2 production by 24.0% and 50.0 µg/mL by 62.3%.Leukemic macrophages (RAW 264.7) used in this study were able to release PGE < 0.05) . The sam < 0.05)  as 1.6 µUncontrolled secretion of Interleukin 6 (IL-6) can result in chronic inflammation and disease. In this study, mouse 3t3 cells produced IL-6 when stimulated with 100 ng/mL LPS after 24 h. Exposure of the cells (3t3) to increasing concentrations of CGT oil revealed an increase in IL-6 inhibition . Exposurp < 0.05), with an EC50 of 27.2 µg/mL and 4.3 µg/mL, respectively. Comparing the response of LPS-stimulated cells to 50.0 µg/mL of either β-bisabolol or α-bisabolol indicated there was a significant difference between the two responses with the presence of α-bisabolol resulting in greater inhibition of IL-6 (87.2%), 9.5% more than its isomer β-bisabolol, which inhibited IL-6 production by 77.7%. Nevertheless, inhibition of IL-6 production by β-bisabolol is indicative of its anti-inflammatory activity. The effect of CGT oil and β-bisabolol on 3t3 IL-6 production was significant -stimulated human fibroblasts (HS27) also produced IL-6 and inhibition of the cytokine was observed after 24 h exposure to CGT oil and β-bisabolol. Hydro-distilled CGT oil concentrations inhibited IL-6 production by up to 49.5% at the highest concentration of 50.0 µg/mL . β-BisabCGT oil and β-bisabolol downregulated IL-8 production in stimulated human HS27 cells in a dose-dependent manner . Inhibitp > 0.05) in inhibitory activity of β-bisabolol (45.3%) and α-bisabolol (47.4%) at 50 µg/mL  produced by LPS-stimulated RAW 264.7 macrophages was inhibited by different concentrations of CGT oil and β-bisabolol . CGT oil50 µg/mL . The resα-Bisabolol and β-bisabolol differed in their effects on NO and TNF-α production in LPS-stimulated macrophages. β-Bisabolol inhibited NO production to a greater extent than α-bisabolol at 12.5 µg/mL, however, the difference between α- and β-bisabolol diminished at 50.0 µg/mL A. In con2, IL-6, and TNF-α has continued to be the focus of research for alleviating symptoms of chronic inflammation and subsequent chronic diseases. In vitro anti-inflammatory assays involve controlling the overproduction of these mediators in stimulated cells. In this study, CGT oil extracted by hydro-distillation and the principal component of the oil, β-bisabolol, were investigated for their anti-inflammatory properties using the 3t3, HS27, and RAW 264.7 cell lines. It was found that a non-toxic concentration range (1.6 µg/mL to 50.0 µg/mL) of CGT oil extracted by hydro-distillation inhibited overproduction of inflammatory mediators by LPS stimulated cells. The anti-inflammatory effect of CGT oil can be attributed to the different bioactive compounds in the oil, which can either work individually or synergistically to cause the inhibition. The process of inhibition of inflammatory mediators involves the regulation of different effectors to the process of inflammation in the body [The use of essential oils and their chemical components for preventing the overproduction of pro-inflammatory mediators such as NO, PGEthe body . These eKB), which also stimulates cells during inflammation to produce NO and other pro-inflammatory cytokines, is also regulated in the presence of essential oils [Synthesized from L-arginine and molecular oxygen, the signaling molecule nitric oxide is actively involved in innate immunity and inflammation ,57 and iial oils . It can 2 observed in the study also followed a similar pattern to what was observed for NO production in RAW macrophages. Production of PGE2 by immune cells contributes to inflammation by promoting the further production of cytokines that are involved in the immune response. Expression of either pro-inflammatory or anti-inflammatory mediators by macrophages depends on the type of stimuli inducing inflammation [2 secretion by LPS-stimulated macrophages in the presence of CGT oil were compared with other PGE2 assays that report the efficacy of essential oils and terpenoids in reducing inflammation [2 production during inflammation is triggered by the activity of the enzymes COX-1 and COX-2. The dose dependent control of PGE2 production in this study may therefore be linked to the inhibition of COX enzyme activity in the cell. This assumption is supported by other studies that report PGE2 and COX inhibition in blood cells and RAW 264.7 macrophages by essential oils from plant materials such as Chamaecyparis obtusa [Trachydium roylei [2 was such that at the very low concentration of 1.6 µg/mL, there was an observed inhibition of 35.2% for the oil. Likewise, the high inhibition of PGE2 production by β-bisabolol  at 50 µg/mL further supports the efficacy of β-bisabolol against prolonged inflammation.Inhibition of PGEammation . Inhibitammation ,48,54. Ss obtusa  and Trac2 [The cytokines TNF-α and IL-6, which are well known mediators in the inflammation process, have also been investigated in anti-inflammatory assays when screening oils and compounds for therapeutic properties. Interleukin 6 (IL-6) is one of the cytokines produced by fibroblasts and macrophages that can simultaneously act as a stimulant for the production of other cytokines and acute-phase proteins during inflammation ,36. TNF-2 . The obsReduction in cytokine production by CGT oil can also be attributed to the presence of bioactive terpenoids in the oil. This assumption is due to reports from previous anti-inflammatory studies on essential oils from plant materials such as eucalyptus and rosemary inhibiting both IL-6 and TNF-α production in inflamed cells . AccordiInhibition of IL-8, a member of the CXC family of chemokine proteins and a chemoattractant ,67, was The current study suggests β-bisabolol in CGT oil is one of the main contributors to the anti-inflammatory property of the oil. The sesquiterpene alcohol, which made up approximately one-fourth of the total volatile extractives, was effective against all inflammatory mediators investigated. β-Bisabolol has not been investigated for any anti-inflammatory activities prior to this study, even though its isomer α-bisabolol has been extensively studied for anti-inflammatory and other biological activities. Activities associated with α-bisabolol include anti-inflammatory ,54, antiComparing the level of β-bisabolol activity with α-bisabolol activity as presented in this study, it appears β-bisabolol was more active against NO production in RAW 264.7 cells than α-bisabolol. One of the properties of α-bisabolol that enhances its anti-inflammatory function is its strong binding affinity to the active sites of pro-inflammatory proteins . The hig50 values of 1.5 µg/mL (6.8 µM) and 4.3 µg/mL (19.6 µM) for PGE2 and IL-6 inhibition, respectively, is indicative that β-bisabolol at low concentrations can be as effective as other anti-inflammatory agents such as resveratrol, dexamethasone, and quercetin [2, but limited information on its activity on IL-8 production. The inhibitory effect of α- and β-bisabolol on IL-8 production presented in this study shows that both isomers of bisabolol regulate IL-8 production during prolonged inflammation. Downregulation of inflammatory mediators by β-bisabolol at a level comparable to the similar concentration of α-bisabolol in this study gives a clear indication that β-bisabolol could be used for similar purposes as α-bisabolol. The reported low ECuercetin . There aAnti-inflammatory activity of β-bisabolol in this study represented by inhibition of cytokines clearly indicates that this sesquiterpene has the property to induce downregulation of the enzymes INOS and COX-2, which are upregulated during inflammation in the body system. Although not investigated in this study, there is a published report on the mechanism of action of α-bisabolol, an isomer of beta-bisabolol . The stuAlthough α-bisabolol can be extracted from cultivated chamomile flower, most of it is still sourced from the bark and stems of wild harvested candeia trees due to the high concentration of α-bisabolol  and loweThere may be a synergistic anti-inflammatory effect for the major terpenoid compounds in the CGT distilled oil. However, the main purpose of this current study was to find a sustainable source of bioactive bisabolol compounds. The distilled CGT oil will unlikely be used directly as an anti-inflammatory agent as it may have harmful contaminants from the CGT, a by-product of cotton processing. For a large industrial-scale production of bisabolol, the extract from CGT will have to go through a process of cleaning and purification to remove any contaminants, and such a process should ideally also remove compounds other than β-bisabolol. Therefore, the synergistic anti-inflammatory effects of major terpenoid compounds extracted from CGT was not investigated in this study.Essential oil was extracted from pesticide-free CGT, which was comprised mainly of the calyx (bracts). Pesticide-free CGT was kindly supplied by the Australian Cotton Research Institute, Narrabri, NSW, Australia. Milled samples were extracted by means of hydro-distillation, as outlined in Egbuta et al. , and extEscherichia coli lipopolysaccharide (LPS), dexamethasone (DEX), chlorambucil, and α-bisabolol standard were purchased from Sigma Aldrich Co. Ltd., Burlington, MA, USA.Human foreskin fibroblast cells , mouse embryo fibroblast  cells, and murine leukemic macrophages RAW 264.7 (TIB-71) cells were purchased from American Type Culture Collection (ATCC), Manassas, VA, USA. Dulbecco’s modified Eagle medium (DMEM), L-glutamine, sodium pyruvate, phosphate buffered saline (PBS), thiazolyl blue tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), calf serum, Greiss reagent, penicillin-streptomycin solution, 0.25% trypsin- 0.2 g EDTA, 2 Corning® T surface culture flasks  containing DMEM, 10% calf serum, 2 mM glutamine, 1 mM sodium pyruvate, and penicillin-streptomycin solution. Cells were incubated at 37 °C in a 5% CO2 incubator for proper attachment and proliferation. Culture medium was replaced regularly every two days and the cells were checked under a microscope until proliferated up to 90% confluency. When cells achieved approximately 90% confluence, culture medium was removed, and cells washed with 1 mL PBS. Fibroblasts were harvested by trypsinization for the HS27 and 3t3 lines using 3 mL trypsin-EDTA and by scraping for RAW 264.7 cells. Cells were sub-cultured to obtain the required number of cells for subsequent assays. Cell passages used in this study were between passage 2 and passage 8.Under sterile conditions, Hs27, 3t3, and RAW 264.7 cells were added to 75 cm5 cells per mL of culture medium (phenol red free) and incubated for 24 h to allow the cells to attach to the bottom of the wells. The seeding density was determined after harvesting the cells and performing cell counting. After 24 h incubation, the cells reached between 85–95% confluency and were then exposed to concentrations of CGT oil between 2.0 µg/mL and 1000.0 µg/mL for a duration of 24 h. Alternatively, cells were exposed to α-bisabolol and β-bisabolol in a range of concentrations between 1.6 µg/mL to 100.0 µg/mL. Positive and negative controls used in the assay were 0.1% DMSO and 1 mg/mL chlorambucil, respectively. Test samples including CGT oil, α-bisabolol, β-bisabolol, and chlorambucil were first dissolved in DMSO and added to culture medium prior to exposure to the cells. An equal concentration of 0.1% DMSO was maintained in CGT oil, α-bisabolol, β-bisabolol, and chlorambucil dilutions. After 24 h exposure to the test compounds, culture media containing test compounds and controls were replaced with 0.3 mg/mL tetrazolium dye (MTT) and incubated for 3 h in the dark at 37 °C. Insoluble formazan crystals formed by viable cells after exposure to MTT for 3 h were solubilized with 200 µL DMSO added to each well and colorimetric absorbance determined at 570 nm with a KC4 multi-detection microplate reader  after 15 min of incubation. Cell viability was calculated using the following equation,A cell viability assay was performed to determine the non-cytotoxic concentration range ideal for anti-inflammatory assays. Harvested cells were transferred into 96-well plates in triplicate at a seeding density of 1.2 × 102 production by RAW 264.7 and HS27 cells was conducted using a Cayman PGE2 Express ELISA Kit . Cells were seeded at 1.2 × 105 cells per mL of phenol-red free culture medium in 96-well plates and left to incubate overnight for 24 h. After 24 h incubation, the cells were exposed to non-toxic concentrations of CGT oil, β-bisabolol, α-bisabolol, and dexamethasone. Positive (2.5 µM (1.0 µg/mL) DEX) and negative (0.1% DMSO) controls were used and 100 ng/mL LPS was used to stimulate the cells 1 h after adding the test compounds [2 production was analyzed following the manufacturer’s instructions. Measurement of PGE2 production was by absorbance at 405 nm with a PerkinElmer VICTOR X4 2030 Multilabel plate reader . A standard curve was constructed from dilutions of the PGE2 standard and used to determine the concentration of PGE2 produced by cells in the assay.Measurement of LPS-induced PGEompounds . After a5 cells per mL of phenol-red free culture medium and incubated for 24 h at 37 °C in a CO2 incubator. Cells were incubated with test compounds and controls 1 h before the addition of 100 ng/mL LPS [Nitric oxide production was measured by quantifying nitrite in the cell supernatant according to previously published procedures ,77,78 wig/mL LPS . Dexamet5 cells per mL of phenol-red free culture medium and left to incubate for 24 h. The cells were then exposed to non-toxic concentrations of CGT oil, α-bisabolol, and β-bisabolol in triplicate. Controls for the assay were 2.5 µM (1.0 µg/mL) DEX (positive) and 0.1% DMSO (negative). One hour after the addition of test compounds and controls, 100 ng/mL LPS was added to the wells and left to incubate for another 24 h [A cytokine inhibition assay was conducted to understand the effect of CGT oil and β-bisabolol on LPS-induced production of pro-inflammatory cytokines in HS27, 3t3, and RAW 264.7 cells. Cells were seeded at a density of 1.2 × 10her 24 h . Cell su2 assays were analyzed using GraphPad Prism version 4.0 to calculate the concentration of inflammation mediators, IC50 and EC50 of CGT oil and β-bisabolol. Microsoft Excel 2013 was used to calculate the mean concentration and standard deviation of the different inflammatory mediators measured. Analysis of variance (ANOVA) was performed using GenStat 64-bit Release 18.1 (18th edition) with the Duncan multiple range test. p values ≤ 0.05 were considered to indicate statistical significance.Data obtained from cell viability, NO, and PGE2, TNF-α, IL-6, and IL-8 in RAW 264.7, 3t3, and HS27 cells in a dose-dependent manner. β-Bisabolol was the most abundant terpenoid in CGT oil and the primary contributor to the anti-inflammatory activity of the oil. The sesquiterpenoid, β-bisabolol had similar anti-inflammatory effects as its isomer α-bisabolol and in some cases was more effective than α-bisabolol as observed for NO and IL-6 inhibition. Compared to the composition reported for α-bisabolol (7.7–16.4%) in chamomile flower extracts, the higher concentration of β-bisabolol in CGT oil (23.5%) and its high anti-inflammatory activity first reported in this study suggests that this terpenoid could be used as an alternative to its isomer α-bisabolol for the treatment of inflammatory conditions. The observed activity of β-bisabolol provides an indication of its potential anti-inflammatory activity when investigated in an in vivo setting against the well-established bioactive α-bisabolol. Although not investigated in the present study, an in vivo anti-inflammatory assay of β-bisabolol alongside α-bisabolol will further validate the efficacy of β-bisabolol as an anti-inflammatory agent. This assay can be performed in the future following ethical considerations and strict adherence to EU limitations on in vivo assays using cosmetic products.CGT oil and β-bisabolol isolated from CGT oil inhibited the production of NO, PGE


The Chromatin Assembly Factor 1 is a heterotrimeric complex responsible for the nucleosome assembly during DNA replication and DNA repair. In humans, the largest subunit P150 is the major actor of this process. It has been recently considered as a tumor-associated protein due to its overexpression in many malignancies. Structural and functional studies targeting P150 are still limited and only scarce information about this subunit is currently available. Literature data and bioinformatics analysis assisted the identification of a stable DNA binding domain, encompassing residues from 721 to 860 of P150 within the full-length protein. This domain was recombinantly produced and in vitro investigated. An acidic region modulating its DNA binding ability was also identified and characterized. Results showed similarities and differences between the P150 and its yeast homologue, namely Cac-1, suggesting that, although sharing a common biological function, the two proteins may also possess different features. From yeast to human, genomic DNA is well packaged into a compact and ordered entity named nucleosome, the single unit of chromatin ,2. This H. sapiens, D. melanogaster, M. musculus, C. elegans, S. cerevisiae, and A. Thaliana and consists of three subunits in a 1:1:1 ratio in all these species [CAF-1 has been found in  species ,23,33,34 species , whereas species . Among the three subunits, yeast Cac-1 has been the most investigated, and the reported results highlight its importance from a functional point of view. This protein, which consists of 606 residues, contains Cac-2 ,37,38 anHere, we focused our attention on P150 , the largest subunit of the human CAF-1 complex. This protein was first cloned by Kaufman and coworkers in 1995  and lateRecently, Zhang and coworkers identified a DNA-binding domain in human and mouse P150, corresponding to the Cac-1 WHD, and proved its ability to bind 58 dsDNA . Here, f721–860) was designed, cloned, expressed, and subsequently purified pLysS strain at 16 °C with 1 mM isopropyl-thio-D-glactosidase (IPTG). Upon three steps of purification, a purity level greater than 95% with a final yield of 2.5 mg/L of growth medium was achieved. Mass spectrometry analyses were carried out to confirm protein identity and purity. Results . This excision, due to the methionylamino peptidase [E. coli [Zhang and coworkers identified the region 727–854 of human P150 as the one corresponding to Cac-1 WHD . Interespurified . Briefly Results A showed eptidase , is a wi[E. coli ,67.721–860 was assessed by circular dichroism (CD). Results showed negative molar ellipticity values at 208 nm and 222 nm in addition to a positive ellipticity below 198 nm  or length (16 and 58 dsDNA). Obtained results highlighted that P150721–860 is able to bind dsDNA no matter the composition (Electrophoretic mobility shift assay (EMSA) was carried out to explore the propensity of P150position  and lengposition , consistD value of 10.4 ± 0.5 µM was estimated , a complex in a 1:1 molar ratio was clearly identified  and lengths (dsDNA 16 and 58 bp) with a binding affinity in the micromolar range, in accordance with values already reported for WH proteins such as isolated Cac-1 (KD = 2 µM) [D = 12 µM) [D = 7 µM) [D > 20 µM) [721–860 and 58 bp dsDNA was evaluated in vitro giving a 1:1 molar ratio, in contrast to that observed for Cac-1, which binds an 18 bp dsDNA in a molar ratio of 2:1 (Cac-1:DNA) [721–860, carried out with the online program PHD , reveals that, although this protein retains the typical elements of the WHD present in Cac-1, significant differences are also present. In particular, P150721–860 contains a big insertion between predicted helices α2 and α3 with respect to Cac-1 WHD; moreover, it is longer and contains, at the C-terminus, an additional coil region followed by a β-strand  , Ash2L (= 12 µM) , FoxM1 ( = 7 µM) , and Rtt> 20 µM) . The abs> 20 µM) . Bindingc-1:DNA) . Taken tβ-strand . The obsβ-strand . 721–860 to DNA is modulated by an acidic region which precedes the WHD. Accordingly, an enlarged construct comprehensive of the ED-rich region, namely P150575–860, is not able to bind DNA. It is assumed that the negatively charged residues of the ED domain sequestrate the WHD, making it inaccessible to DNA binding [The binding ability of P150 binding . Similar binding . 721–860 DNA binding region. Results indicated the typical features of a WHD containing both alpha and beta secondary structure elements. P150721–860 directly interacts with dsDNA [In conclusion, we investigated, for the first time, the biochemical and structural features of the P150th dsDNA  in vitro721–860 and P150575–860 cDNAs , optimized for expression in E. coli, were amplified by PCR using forward and reverse primers as listed in NcoI and XhoI enzymes . This vector was chosen for its feature to encode a six-histidine tag (His-tag) at the C-terminus. The integrity of the sequence was assessed by DNA sequencing upon appropriate digestion with restriction enzymes.P150721–860 or P150575–860) was expressed by transforming E. coli BL21(DE3)pLysS strain (kind gift from EMBL Heidelberg) with the corresponding plasmid. Cells were grown in LB and induced with 1 mM IPTG at 16 °C or 22 °C for the expression of P150721–860 and P150575–860, respectively. After an overnight culture, cells were pelleted by centrifugation (7500 rpm for 20 min at 4 °C). Identically for both proteins, pellets were resuspended in 20 mM Tris-HCl, 500 mM NaCl, pH 7.5 supplemented with PMSF (1 mM), DNaseI (5 µg/mL), lysozyme (0.1 mg/mL), and 1 μg/mL of the following protease inhibitors . Cells were then sonicated on ice and centrifuged . Supernatant was purified at room temperature on ÄKTA FPLC . P150721–860 was purified by two affinity chromatography steps (TALON and heparin) and a Superdex 75 size exclusion chromatography (SEC), while P150575–860 purification consisted of a TALON affinity chromatography and SEC. Protein quality was assessed by 20% SDS-PAGE and ESI-TOF MS analysis . Deconvolution was carried out by means of the Agilent MassHunter Qualitative software.Each recombinant protein  LC/MS system . The LC module Agilent 1290 was coupled to a photodiode array (PDA) detector and a 6230 time-of-flight MS detector, along with a binary solvent pump degasser, column heater, and autosampler. Chromatographic separation was performed using, as solvent A, 0.01% TFA in H2O (v/v), and as solvent B, 0.01% TFA in CH3CN (v/v). A fully reduced sample with 10 mM DTT was also analyzed. Deconvolution was carried out by means of the Agilent MassHunter Qualitative software.A C4 Biobasic 50 × 2.1 mm ID column  was used to load P150721–860 in 1 mM Tris-HCl, pH 7.5. Raw spectra were corrected for buffer contribution, converted to mean molar ellipticity per residue (θ) (deg cm2 dmol−1), and visualized using GraphPad software. For thermal stability experiments, P150721–860 was heated from 20 to 100 °C with a temperature increase of 1 °C/min and spectrum recorded at 222 nm, as above described. The molar ellipticity values at 222 nm were plotted as function of the temperature using GraphPad software. Additionally, the three spectra  were recorded as previously described once the fixed temperature was reached within ± 0.1 °C set by a Peltier device. Circular dichroism (CD) experiments were carried out as previously described ,80. Spec721–860 or 58 dsDNA, 475 µg and 330 µg, respectively. The third run consisted of mixing together the previously mentioned quantities achieving a 3.2:1 molar ratio (protein: DNA) incubating the mixture on ice for 45 min before loading the column. Runs were performed in 20 mM Tris-HCl, 150 mM NaCl, and pH 7.5 at room temperature. When specified, LS was carried out in 20 mM Tris-HCl, 500 mM NaCl, pH 7.5. Data analysis was carried out using ASTRA 5.3.4.14 software (Wyatt Technology Corporation). SEC–MALS–QELS (size exclusion chromatography–multi-angle light-scattering–quasi-elastic light scattering) analyses were carried out as previously described . Briefly721–860 in 0–15 molar ratio. Similar quantities were used in the AT- and GC-rich binding experiments. In the case of 58 dsDNA, 2 µM of DNA were mixed with P150721–860 or P150575–860 in 0–25 molar ratio. When staining with Sybr Gold, 1 µM of dsDNA was mixed with P150721–860 in 0–80 molar ratio. Binding reaction was incubated 45 min on ice. Gel loading dye, purple  and glycerol , were added to the mixture, prior to the loading into a 6% PAGE pre-run in 0.5X TBE buffer. The pre-run and the run were performed at 75 V on ice, for 30 and 45 min, respectively. Gels were stained either with ethidium bromide (EtBr) or with Sybr Gold (Thermofisher) prior to visualization with UV light. Each fraction bound was calculated by quantifying the density of each lane by ImageJ [The 16 and 58 bp oligonucleotides  used fory ImageJ . The obt


A steep increase in locomotor activity was seen after a modest increase in the methiopropamine dose from 10 to 12.5 mg/kg, suggesting that a small increase in dosage may engender unexpectedly strong effects and heighten the risk of unintended overdose in NPS users. In vitro studies revealed that methiopropamine mediates its effects through inhibition of norepinephrine and dopamine uptake into presynaptic nerve terminals , while the plasmalemmal serotonin uptake and vesicular uptake are affected only at high concentrations (IC50 > 25 µM). In summary, methiopropamine closely resembles methamphetamine with regard to its pharmacokinetics, pharmacodynamic effects and mechanism of action, with a potency that is approximately five times lower than that of methamphetamine.Methiopropamine is a novel psychoactive substance (NPS) that is associated with several cases of clinical toxicity, yet little information is available regarding its neuropharmacological properties. Here, we employed in vitro and in vivo methods to compare the pharmacokinetics and neurobiological effects of methiopropamine and its structural analog methamphetamine. Methiopropamine was rapidly distributed to the blood and brain after injection in C57BL/6 mice, with a pharmacokinetic profile similar to that of methamphetamine. Methiopropamine induced psychomotor activity, but higher doses were needed (E The novel psychoactive substance (NPS) methiopropamine (1-thiophen-2-yl)-2-methylaminopropane) emerged on the European drug market in 2011. Methiopropamine is a structural analog of methamphetamine where the benzene ring of methamphetamine is replaced with a thiophene ring . Methiop-yl-2-metN-demethylated metabolite nor-methiopropamine and the parent compound were detected 18 h after self-reported intake of 200 mg methiopropamine -(DA) ([3H]DA), NE HCl DL-[7-3H(N)] ([3H]NE), 5-HT [3H(G)] ([3H]5-HT) and UltimaGoldTM liquid scintillation cocktail were purchased form from PerkinElmer Inc. . HEPES was purchased from Thermo Fischer Scientific  and sodium heparin was purchased from Leo Pharma .Drugs: Methiopropamine hydrochloride (HCl) (mol. wt. 191.7) was purchased from Cayman Chemicals  and (+)methamphetamine HCl (mol. wt. 185.7) was purchased from Chiron AS . Chemicals: 5-HT HCl, L-NE HCl, 5-HIAA HCl, 3-MT HCl, ephedrine-DMale C57BL/6J mice  and Sprague-Dawley rats  were housed with 4–8 mice/cage and 2 rats/cage in the animal facility at the Norwegian Institute of Public Health . Animals were given free access to water and commercial food pellets for rodents. Temperature, humidity and lighting were regulated . The animals were acclimatized for at least five days prior to the experiments. The animal experiments were approved by the Norwegian Animal Research Authority  and performed in accordance with the laws and regulations controlling experiments on animals in Norway.max) or as the total run distance during the 240 min session.Locomotor activity was assessed using the Versamax optical animal activity monitoring system , as described in detail by Andersen et al. . BrieflyMice were given a single i.p. injection of methiopropamine (12.5 mg/kg) or methamphetamine (3.75 mg/kg) and anaesthetized with isoflurane prior to blood and brain tissue sampling. To assess the level of anesthesia, the pedal reflex was tested by a firm toe pinch. Blood sampling by heart puncture was performed at different time points after drug injection (5–240 min) using a syringe prefilled with heparin . Blood samples (50 or 100 µL) were transferred to tubes containing 100 µL ice-cold 5 mM ammonium formate buffer, pH 3.1, with sodium fluoride , and were immediately frozen in liquid nitrogen. After cervical dislocation, the cerebrum was quickly removed, homogenized (4 mL per g tissue) in ice-cold 5 mM ammonium formate buffer, pH 3.1, containing sodium fluoride (4 mg/mL), aliquoted (50 or 100 µL) and frozen in liquid nitrogen. Blood and brain samples taken 240 min after drug injection were from animals included in the locomotor activity study. The samples were stored at −80 °C until sample preparation and determination of drug concentrations.3 and methamphetamine-13C6 in 5 mM ammonium formate buffer, pH 3.1) was added to all samples followed by immediate agitation on a multitube vortexer. A borate buffer  and ethyl acetate/heptane mixture  were added, the samples shaken for 10 min and then centrifuged for 10 min at 3900× g at 4 °C. The organic layer was transferred to glass tubes and 0.1% HNO3 (30 µL in methanol) was added before drying under a stream of nitrogen at 40 °C. The samples were reconstituted ) and thoroughly shaken prior to transfer to autosampler vials.Stock solutions of analytes and internal standards were prepared in methanol and stored at −20 °C. Working solutions for seven calibrators and four quality control (QC) samples were prepared in 5 mM ammonium formate buffer. Calibrators (0.10–20 µM) and QC samples (0.10–15 µM) were prepared independently by fortifying human whole blood or rat brain tissue homogenate with working solutions. Samples and solutions were kept on ice throughout the experiment. An internal standard  system , applying an Acquity™ UPLC HSS T3 column  with an Acquity™ UPLC HSS T3 VanGuard pre-column . The mobile phase consisted of 10 mM ammonium formate buffer, pH 3.1 (A) and methanol (B). The separation was carried out using a 7-min gradient with the following profile: 0–4.0 minutes; 2.5–22.5% B, 4.0–4.1 min; 22.5–100% B, 4.1–6.0 min; 100% B, 6.0–6.01 min; 100–2.5% B, and 6.01–7.0 min; 2.5% B. The flow rate was 0.5 mL/min, the column temperature 65 °C and the injection volume 0.5 µL.13C6 (156.2 > 125.1) and ephedrine-d3 (169.1 > 117.0). The retention times for methiopropamine, nor-methiopropamine and ephedrine-d3 were 3.05, 2.82 and 2.97 min, respectively, while methamphetamine and methamphetamine-13C6 both eluted at 3.86 min. Quantification was performed using MassLynx 4.2 software (Waters). Calibration curves were constructed by plotting the calibrator concentration against the analyte/internal standard peak height ratio. The internal standard used for methiopropamine and nor-methiopropamine was ephedrine-d3 and the internal standard used for methamphetamine was methamphetamine-13C6. The calibration curves were linear, and the QC sample results were satisfactory .A Waters Quattro Premier XE tandem mass spectrometer (MS/MS), or a Xevo TQ triple quadrupole mass spectrometer, equipped with a Z-spray electrospray interface, was used for the analyses. Positive ionization was performed in the multiple reaction monitoring mode, with two transitions for all analytes , and one transition for methamphetamine-Mice were given a single i.p. injection of methiopropamine (12.5 mg/kg), methamphetamine (3.75 or 12.5 mg/kg) or saline, anesthetized  were added to plastic tubes and vortexed for 20 s. The samples were centrifuged for 10 min at 14,500× g and 4 °C, and the supernatant transferred to amber colored autosampler vials. For calibrators and QC samples, 50 µL was added to 25 µL internal standard in amber colored vials. The concentrations of neurotransmitters and neurotransmitter metabolites in NAc and DS were determined by an UPLC-MS/MS method described by Bergh et al. DA (0.5 µCi), 0.014 µM [3H]NE (0.1 µCi) and 0.06 µM NE, or 0.012 µM [3H]5-HT (0.5 µCi) and 0.08 µM 5-HT  in a total volume of 500 µL. After 9 min incubation, the reactions were terminated by dilution with ice-cold wash solution (0.15 M NaCl and 0.05% (w/v) bovine serum albumin) and rapid filtration through GF/B glass microfiber filters . The filters were washed 4×, dissolved in 4 mL UltimaGoldTM liquid scintillation cocktail and counted for the retained radioactivity in a liquid scintillation analyzer . Non-specific uptake tubes were treated similarly, but incubation was performed either in the presence of the DA transporter inhibitor benztropine mesylate (0.01 µM), the NE transporter inhibitor reboxetine mesylate (0.05 µM) or the 5-HT transporter inhibitor fluoxetine (0.05 µM). The specific uptake was defined as the total uptake minus the non-specific uptake.Synaptosomal uptake of neurotransmitters was determined as described by Bogen et al. . Synapto4, 0.19 M sucrose, 15 mM HEPES/KOH, pH 7.4) with different concentrations of methiopropamine (0.1–50 µM) or methamphetamine (0.1–50 µM). The uptake was initiated by adding 1.8 mM ATP and either 0.025 µM [3H]DA (0.5 µCi), 0.068 µM [3H]NE (0.5 µCi) or 0.012 µM [3H]5-HT (0.5 µCi) in a total volume of 330 µL. After incubation for 3 min, the reactions were terminated by dilution with ice-cold wash solution (0.15 M KCl) and rapid filtration through MFTM membrane filters . The filters were washed 4×, dissolved in 4 mL UltimaGold™ and counted for retained radioactivity in a liquid scintillation analyzer. Non-specific uptake tubes were treated similarly, but incubation was performed in the presence of 25 µM reserpine. The specific uptake was defined as the total uptake minus the non-specific uptake.Synaptic vesicles (12.5 µL) were pre-incubated for 15 min at 30 °C in incubation buffer  [50 values were calculated by nonlinear regression  using Graph Pad Prism version 7.02 for Windows . Statistical analysis of drug-induced locomotor activity over time was performed using the General Linear Model for repeated measures, followed by Dunnett’s post hoc test. One-way ANOVA was performed for comparison of total locomotor activity, Emax, effects on neurotransmitter concentrations and concentrations ratios, with a significant F value followed by Dunnett’s post hoc test. Independent samples t-tests were performed for comparison of the IC50 values and comparison of the brain:blood ratios for methiopropamine and methamphetamine. P values less than 0.05 were considered statistically significant. All statistical tests were performed using SPSS, version 26 .Data are expressed as mean ± SEM, or mean + SEM, unless stated otherwise. Animals were randomly assigned to the different experimental groups. The pharmacokinetic analyses and neurotransmitter uptake experiments were performed in duplicate samples. Elimination half-lives were calculated from the equation tt2 − t1) . For cal


APOE4) variant is the strongest genetic risk factor for Alzheimer disease (AD), while the APOE2 allele is protective. A major question is how different APOE genotypes affect the physiology of astrocytes, the main APOE-producing brain cells. Here, we differentiated human APOE-isogenic induced pluripotent stem cells (iPSCs)  to functional “iAstrocytes”. Mass-spectrometry-based proteomic analysis showed genotype-dependent reductions of cholesterol and lipid metabolic and biosynthetic pathways (reduction: APOE4 > E3 > E2). Cholesterol efflux and biosynthesis were reduced in APOE4 iAstrocytes, while subcellular localization of cholesterol in lysosomes was elevated. An increase in immunoregulatory proteomic pathways (APOE4 > E3 > E2) was accompanied by elevated cytokine release in APOE4 cells (APOE4 > E3 > E2 > KO). Activation of iAstrocytes exacerbated proteomic changes and cytokine secretion mostly in APOE4 iAstrocytes, while APOE2 and APOE-KO iAstrocytes were least affected. Taken together, APOE4 iAstrocytes reveal a disease-relevant phenotype, causing dysregulated cholesterol/lipid homeostasis, increased inflammatory signaling, and reduced β-amyloid uptake, while APOE2 iAstrocytes show opposing effects.The apolipoprotein E4 ( •Human astrocytes show strong proteomic differences depending on their APOE genotype•APOE-KO and lowest in APOE4 astrocytes (KO > E2 > E3 > E4)Aβ uptake is highest in •APOE4 astrocytes show exacerbated pro-inflammatory reactions (APOE4 > E3 > E2 > KO)•APOE4 astrocytesCholesterol synthesis and efflux are reduced in  APOE genotype-dependent differences in the proteomic profile of APOE-isogenic iPSC-derived human astrocytes. They provide evidence for a pro-inflammatory state of APOE4 iAstrocytes, while APOE2 and APOE-KO iAstrocytes show reduced inflammatory signaling. They further show the APOE genotype-dependent regulation of β-amyloid uptake capacity and cholesterol/lipid homeostasis and a differential reaction to inflammatory stimuli.In this article, Tackenberg and colleagues show  Apolipoprotein E (APOE) is part of a family of lipoprotein chaperones, binding lipoprotein complexes and facilitating their uptake through low-density lipoprotein receptors (LDLRs), or lipoprotein receptor 1 (LRP1), providing lipids and cholesterol to the cell . In turnAPOE mainly exists in three allelic variants, APOE2, E3, and E4, which differ in a single amino acid change at position 112 or 158: APOE2 , APOE3 , and APOE4  . The CysAPOE4 allele is the major genetic risk factor for developing sporadic Alzheimer disease (AD), where one allele confers a 3-fold increase and two alleles a 12-fold increase in risk. The APOE2 allele is protective for AD; however, its biological role is heavily understudied and not yet understood  iAstrocytes display distinct phenotypes in homeostatic functions, cholesterol and lipid metabolism, lysosomal function, and inflammatory regulation.We aimed to understand the fundamental role of the three different APOE isoforms , in human induced pluripotent stem cell (iPSC)-derived astrocytes. After successfully deriving non-proliferative astrocytes at a resting state, we show that There is currently an abundance of differentiation protocols for iPSC-astrocytes. However, the majority are laborious, require tedious cocktails of small molecules, require sorting methods presuming previous expertise, or use fetal calf serum (FCS), resulting in activated astrocytes at baseline . By simpAPOE iPSCs were obtained from the European Bank for Induced Pluripotent Stem Cells (EBiSC), and show mono-allelic expression of APOE, or APOE deletion  between APOE2 and APOE4 (173), less between APOE2 and APOE3 (130), and least between APOE4 and APOE3 (72) (APOE-KO iAstrocytes (To investigate OE3 (72) A. We anatrocytes B and 2C.trocytes .Figure 2APOE4 iAstrocytes taking up the lowest amount of glutamate while APOE2 and APOE-KO took up the highest amounts  , which primarily blocked LRP1 and very-low-density lipoprotein receptor (VLDLR), in order to induce a mild reduction in Aβ uptake, resulting in a measurable difference between genotypes. Blocking LDLR family members caused a significant decrease in Aβ uptake in APOE-KO, E2, and E3, but not in E4 iAstrocytes (p = 0.23). Quantification of LRP1 and VLDLR showed no significant difference, albeit a minor decrease of average LRP1 levels in APOE4 cells   F. IntereE4 cells G and 2H.id (CSF) . We furtAPOE4 compared with APOE3 or APOE2 iAstrocytes, while these were upregulated in APOE2 compared with APOE3  (APOE4 compared with E3. Cellular PE showed higher levels in E2 and E4 and triacylglycerol (TAG) displayed an isoform-dependent increase (E4 > E3 > E2 = KO). Note that TAG was not present in the secreted fraction, so cellular TAG represents the total amount. Interestingly, secreted cholesterol and CE were significantly decreased only in APOE4 iAstrocytes  treatment and subsequent Filipin III staining, as well as additional Dextran-Alexa 555 treatment, to determine lysosomal localization of cholesterol. We found that non-membrane-bound cholesterol was increased in APOE4 iAstrocytes (E4 > E3 = E2 = KO)   of proteomic data showed significant downregulation of pathways involved in lipid biosynthesis/metabolism and sterol biosynthesis/metabolism in th APOE3 A. Leadin pathway . A secon pathway B and 3C.trocytes A and S2Btrocytes E. The reE2 = KO) F. FurtheE4 > E2) G.Figure APOE4 iAstrocytes, and lowest in E2  (APOE3 and APOE4 was significantly lower than in APOE2 and APOE-KO cells (KO = E2 > E3 = E4)  D and 4E.E3 = E4) F. Thus, APOE genotype on the inflammatory state of astrocytes, we treated iAstrocytes with IL-1β and performed proteomic analysis. At baseline, pathways involved in inflammatory regulation were highest in APOE4 compared with E3 and E2 and lower in E2 compared with E3 (E4 > E3 > E2) . In APOE4 and APOE3 iAstrocytes, inflammatory pathways were predominantly upregulated after IL-1β treatment, while the opposite was found in APOE2 cells. Interestingly, the cell aging pathway was significantly upregulated in E4; enriched, but not significantly, in E3; and even less in E2. Lastly, cell-cell junction was significantly downregulated in E4 but only slightly in E2 and E3, indicating that an inflammatory microenvironment affects astrocytes, especially E4, on a fundamental level, from cell aging to cellular communication. Interestingly, APOE4 iAstrocytes showed the most differentially expressed pathways (both up- and downregulated), more than APOE3, and APOE2 iAstrocytes showed almost no significantly regulated pathways, indicating that APOE4 iAstrocytes are most susceptible to pro-inflammatory stimuli -1β or tumor necrosis factor alpha (TNF-α), to induce a cellular inflammatory cascade, and activate astrocytes. To assess the role of E3 > E2) A. When sE3 > E2) B. On the stimuli C. Using  stimuli D.Figure APOE4 iAstrocytes. We then assessed whether IL-1β treatment also affected homeostatic functions by measuring Aβ42 uptake after IL-1β treatment of the iAstrocytes, but, besides the previously shown allele-dependent degree of uptake, no significant effect of the treatment was observed , except for IL-8 . This cyobserved . These dAPOE4 versus APOE2 and APOE3 at baseline , which is likely facilitated by APOE-bound Aβ42 clearance; a complex formation that is genotype dependent (E2 > E3 > E4) . Interes of Aβ42 . APOE-deAPOE4 allele induces a gain of (toxic) effect. APOE2 and APOE-KO iAstrocytes show a similar phenotype, indicating differential homeostatic mechanisms by different APOE isoforms, or lack thereof, in human iAstrocytes. The APOE allele-dependent effect on glutamate uptake and LRP1-dependent Aβ42 uptake can provide a mechanism explaining the biological role of APOE in homeostatic functions such as regulation of glutamate levels, but also pathological events such as extracellular Aβ42 buildup.Altogether we provide evidence that the presence of the APOE4 compared with APOE3 and APOE2 iAstrocytes, whereas lipid catabolic processes show opposite effects. To functionally validate these findings, we performed in-depth HPTLC-based lipid profiling. Triacylglycerols are subject to lipid catabolism, where they are broken down into glycerol and fatty acids, followed by fatty acid β-oxidation , indicating potential LD accumulation, which is in agreement with a study showing increased LDs in APOE4 compared with APOE3 astrocytes , showed very strong allele-dependent downregulation (E4 > E3 > E2 = KO), indicating that indeed these cells have a decreased capacity to produce cholesterol  and efflux (E2 = E3 = KO > E4), in line with our proteomic findings. The first committed enzyme in lesterol . At the lesterol . Furthermembrane . We furtAPOE4 iAstrocytes (E4 > E3 = E2 = KO), which is in agreement with previous observations of increased cholesterol in APOE4 compared with APOE3 astrocytes , and even IL-6 and IL-8 release from carriers .APOE4 iAstrocytes, while the opposite effect was seen in APOE2 iAstrocytes. APOE3 cells showed a decrease in sterol metabolic pathways upon activation, while lipid and fatty acid metabolism were not regulated uniformly. These results oppose the findings at baseline. The significant difference between intracellular and secreted cholesterol in APOE4 iAstrocytes was abolished after IL-1β treatment but had no effect on CE or PE, indicating that IL-1β treatment specifically affects cholesterol homeostasis. It has been shown that cytokine stimulation can regulate cholesterol synthesis by activating SREBP1  complex activation; when intracellular cholesterol is increased, LXR/RXR increases ABCA1 expression to induce lipid efflux, APOE expression, and inhibition of SREBP activation , which can also induce ABCA1 and APOE expression. In line with our results, PPARγ signaling was decreased in APOE4 transgenic mouse brain, and increased in APOE2  upon IL-1β treatment, which was not present in APOE4, we conclude that the effect of IL-1β seen is due to an increase of total cholesterol in the APOE2 and E3 iAstrocytes, but not in APOE4.In addition, IL-1β treatment abolished the allele-dependent non-membrane-bound increase in cholesterol load that was observed at baseline by Filipin III staining. Considering the significant increase in cellular APOE2, E3, E4, and KO iAstrocytes show distinct phenotypes in homeostatic functions, cholesterol and lipid metabolism, lysosomal function, and inflammatory regulation. While several cellular functions, such as glutamate uptake, seem to display a clear allele-dependent decrease in capacity, the interplay between cholesterol metabolism and immune regulation indicates that it is not a mere decrease of function in APOE4 cells: APOE2, APOE3, and APOE4 iAstrocytes may utilize different mechanisms altogether. Further mechanistic insights into the difference in APOE2 and E4 iAstrocytes are imperative to continue the understanding of fundamental APOE biology, which simultaneously proves its inherent relevance in AD. Our human isogenic mature iAstrocyte cell model represents a powerful tool to further address these open questions.Together, we show that APOE2, APOE3, or APOE4 allele generated using CRISPR, or an APOE-KO generated by an insertion-deletion mutation in exon 2 , harboring a single n exon 2 , 2020. IiPSCs were maintained on Vitronectin (StemCell Technologies)-coated plates in TesRE8 medium (StemCell Technologies) and passaged using ReLeSR (StemCell Technologies). iPSCs and subsequent cell types were frozen in BAMBANKER serum-free cryopreservation medium . NPCs were derived as described  with minBetween passages 1 and 4, NPCs were differentiated to astrocytes by the application of primary AM  for 30 days according to Astrocytes were grown on coverslips for 1–2 days and fixed with 4% paraformaldehyde (Electron Microscopy Science) in PBS plus 4% sucrose (Sigma-Aldrich) for 20 min at room temperature (RT), and washed three times with PBS. Cells were blocked in 10% donkey serum with 0.1% Triton X-100 in PBS for 1 h at RT, and subsequently incubated with primary antibody in 3% donkey serum with 0.1% Triton X-100 in PBS overnight at 4°C. Primary antibodies were used accordingly: S100β , GJA1 , GFAP , RAB5 , LAMP2 , Aβ 1-42 . Cells were washed three times with PBS and incubated with the respective secondary antibody at 1:500 in 3% donkey serum with 0.1% Triton X-100 in PBS for 2 h at RT . Cells were washed once and incubated with 0.2 μg/mL DAPI  for 10 min at RT. Cells were washed twice with PBS before mounting with Mowiol 4-88 (Sigma-Aldrich).g for 5 min at 4°C, and diluted at 1:4 in Diluent 2 from the MSD MULTI-Spot Assay system, pro-inflammatory panel 1 (human) kit. With this kit, 10 cytokines were measured according to protocol: IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-α.Astrocytes or NPCs were plated on 24-wells plates 24 h prior to treatment. Both cell types were incubated with medium only, additionally astrocytes were treated with 10 ng/mL IL-1β or TNF-α (StemCell Technologies), for 24 h. Supernatant was transferred to fresh Eppendorf tubes, and cells were harvested for protein samples and used in label-free MS. Supernatant was centrifuged at 21,000 × APOE3.Astrocytes were seeded onto μ-Slide Angiogenesis slides, and were additionally treated with Dextran-Alexa 555 (Thermo Fisher) if needed, otherwise cells were immediately used for Filipin III staining. Incubation with 0.2 mg/mL Dextran-Alexa 555 was performed overnight, and followed by a 14-h chase period . Astrocytes were then treated with 10 mM methyl-β-cyclodextrin for 30 min, followed by fixation and treatment with the Cholesterol Assay Kit (Abcam), according to protocol with minor adjustments. After fixation, astrocytes were incubated with Filipin III for 2 h, followed by washing steps accordingly. Filipin images alone were acquired with fluorescence microscopy, Dextra-Alexa 555, and Filipin double labeling imaged with confocal microscopy. For the colocalization analysis, the area of Dextran staining was calculated of the total area of Fillipin III signal, normalized to Confocal images were acquired with a Leica SP8 microscope, equipped with an HC PL APO CS2 20× objective (NA 0.75), using the Leica Application Suite X software. Fluorescent images were acquired with a Zeiss Axio Observer 7 equipped with an ORCA-flash4.9 camera and Plan-Apochromat 20× objective (NA 0.8), using the Zen 3.1 pro software. Image analysis and post processing was done in ImageJ or Imaris (Oxford Instruments).post hoc Dunn's multiple comparisons test, or a one-way followed by post hoc Tukey's multiple comparisons testing, or two-way ANOVA with post hoc Holm-Šídák's multiple comparisons test. The statistical tests used in the different experiments are annotated in the respective figure legends.Data are presented as mean plus or minus standard deviation (SD). All datapoints (n-numbers) are plotted individually in each bar graph. Statistical analysis was performed with GraphPad Prism (GraphPad Software) and RStudio. Proteomic datasets were analyzed and visualized using edgeR . ExperimConceptualization, S.M.d.L. and C.T.; methodology, S.M.d.L. and C.T.; software, R.R. and W.W.; validation, S.M.d.L., A.W.T.K. and K.L.; formal analysis, S.M.d.L., A.W.T.K., K.L., R.R., W.W., and C.T.; investigation, S.M.d.L., A.W.T.K., K.L., and V.B.; resources, A.C.G. and R.M.N.; writing, S.M.d.L. and C.T.; visualization, S.M.d.L., R.R., W.W., and C.T.; supervision, A.-C.G. and C.T.; project administration, C.T.; funding acquisition, C.T.The authors declare no competing interests.


Vaccinium uliginosum × (V. corymbosum × V. angustifolium). The percentage of seed germination in in vitro culture was 88% for V. uliginosum, form No. 8 × (V. corymbosum × V. angustifolium), SC5-8, while it was 42% for V. uliginosum, form No. 8 × (V. corymbosum × V. angustifolium), ‘Northcountry’. The analysis of mean value showed that the multiplication rate increased and the shoot height decreased as the 2-isopentenyl adenine (2iP) concentration was increased in the nutrient medium of the studied hybrids. The maximum rate was achieved using 15 μM 2iP. A detailed analysis of the hybrids indicated that the hybrid variant reliably affected growth and development indicators. Inter simple sequence repeat analysis demonstrated that all analyzed hybrids inherited DNA fragments of the parent plants in various combinations, confirming their hybrid nature. Thus, the use of in vitro methods for the propagation and further selection of genotypes is demonstrated as being an effective approach for developing interspecific hybrids of V. uliginosum × (V. corymbosum × V. angustifolium).The most serious problem of intergeneric and interspecific hybridization is related to overcoming the reproductive isolation of different species. We assessed the efficiency of reproduction under in vitro conditions and the ex vitro growth capacity of interspecific hybrids of  Vaccinium corymbosum × V. angustifolium, 2n = 48) and a local species of bog blueberry . However, the most serious problem of intergeneric and interspecific hybridization is related to overcoming the reproductive barrier that separates species. The use of in vitro techniques makes it possible to obtain viable hybrids from seeds that have an underdeveloped embryo and to overcome the negative effect of various factors on seed germination and seedling viability at the initial stages of development. In vitro embryo culture can be used to overcome the barriers of nonbreeding in interspecific hybridization of Vaccinium species with different levels of ploidy [V. corymbosum × V. ashei [A promising area of blueberry breeding in Siberia is interspecific hybridization involving the most productive resistant varieties and selected forms of half-highbush blueberry (f ploidy . FurtherV. uliginosum (section Vaccinium) and V. corymbosum L. (section Cyanococcus). Similar interspecific hybrids were obtained in Poland, Russia, and other countries [V. uliginosum (2n = 6x = 72), and V. corymbosum ‘Bluecrop’ [V. uliginosum as a seed parent in comparison with parental pollen, and unilateral cross incompatibility occurred in reciprocal crosses. The resulting hybrid seeds germinated after pretreatment with gibberellin in in vitro culture or in nurseries after stratification [V. uliginosum, which determined the low pollen quality. Therefore, this species is best used as a maternal plant. Earlier, it was shown that tetraploid bog blueberry  easily crosses with tetraploid high blueberry and half-high blueberry  [Rousi  providedountries ,5,6, andfication  revealedctively) . In intectively) .V. uliginosum × (V. corymbosum × V. angustifolium) since previous studies have not examined the in vitro culture of seeds of these hybrids.This research was carried out to assess the possibility of using in vitro techniques to propagate interspecific hybrids of V. uliginosum, form No. 8 × (V. corymbosum × V. angustifolium), ‘Northcountry’ (V.ul.8 × ‘NC’) and 88% for V. uliginosum, form No. 8 × (V. corymbosum × V. angustifolium), ‘SC 5-8’ (V.ul.8 × ‘SC 5-8’) (V.ul.8 × ‘NC’ and 22 seedlings (variants) of V.ul.8 × ‘SC 5-8’. The germination rate of seeds in the soil substrate was 80%, and the survival rate was no more than 2%. A comparison of plants obtained in vitro and sown in soil was not carried out due to the unrepresentative sample of the latter. The experimental scheme is shown in V.ul.8 × ‘NC’ and 7 variants (32%) of V.ul.8 × ‘SC 5-8’ were capable of morphogenesis (shoot formation) in in vitro culture. Significant differences in the multiplication rate among the studied hybrid variants were noted at the second passage. As for the hybrid of V.ul.8 × ‘NC’, its maximum and minimum multiplication rates were 42.5 shoots/explant for variants No. 4-5 and 1.3 shoots/explant for No. 4-1  a. In tot No. 4-1 a. The maNo. 7-11 b. Furthep ≤ 0.05) for hybrids of V.ul.8 × ‘NC’ and V.ul.8 × ‘SC 5-8’. A detailed analysis of this parameter for variant No. 6-3 showed no notable differences in the multiplication rate in all tested media modifications. However, other variants showed reliable differences when using 15 µM 2iP. Among the variants of V.ul.8 × ‘SC 5-8’, the differences among the experimental variants were demonstrated to be insignificant (p ≤ 0.05) for variant No. 7-9. However, significant differences from the control were shown only on media with 15 µM cytokinin for variant No. 7-3; hybrid No. 6-2 was characterized by a considerable increase in the multiplication rate as the 2iP concentration increased. The highest multiplication rate was obtained with the nutrient medium to which 15 µM 2iP was added, which had a multiplication rate of 8.9 shoots/explant for variant No. 1-2 of the V.ul.8 × ‘NC’ and 3.4 shoots/explant for variant No. 6-2 of V.ul.8 × ‘SC 5-8’. Variant No. 4-6 formed only a conglomerate of shoots in this medium. The factor analysis confirmed that both the 2iP concentration and the variant of hybrid influenced the multiplication rate of the studied hybrids . Variant Nos. 4-2, 4-8, and 5-3 of the V.ul.8 × ‘NC’ hybrids had no notable differences between the experimental and the control groups. In the nutrient medium lacking growth regulators, variant Nos. 1-1, 1-2, 1-5, and 2-2 had the highest shoot heights of 72.4, 72.2, 73.4, and 71.4 mm, respectively. A detailed analysis of the shoot height parameter in hybrid variants showed that all variants of the hybrid of V.ul.8 × ‘SC 5-8’ had significant differences in only the media with 15 µM 2iP. The highest height was observed for variant No. 6-2 when using nutrition without growth regulators, reaching 43.7 mm. The factor analysis confirmed that the shoot height of the studied hybrids was influenced by both 2iP concentration and variants of hybrids (p ≤ 0.05). It should be noted that the combination of factors (concentration × variant) did not significantly affect the height of shoots of V.ul.8 × ‘SC 5-8’ at p ≤ 0.05  . FragmenV.ul.8 × ‘NC’, while it was 31.8% for V.ul.8 × ‘SC 5-8’ hybrid. In vitro technologies made it possible to overcome the negative effect of various factors on seed germination and seedling viability at the initial stages of development, thus allowing the preservation of unique genetic material. Pathirana et al. [Vaccinium species with different ploidy levels. In addition, treatment with colchicine allowed viable amphiploids of V. corymbosum ×  V. ashei hybrids to be obtained in in vitro culture [According to our data, the germination rate was 80% when sowing seeds of similar hybrid combinations into a soil substrate, but only a few specimens were viable (no more than 2%). In in vitro culture, seed germination was comparable, but the survival rate was 78.6% for the hybrid of V. uliginosum. Although there are a number of reports of in vitro propagation of Vaccinium representatives [V. uliginosum micropropagation. Cüce et al. [V. uliginosum plants  were successfully propagated in vitro on Woody Plant medium (WPM) supplemented with 1.0 mg/L zeatin and 0.1 mg/L β-indolebutyric acid (IBA). The multiplication rate was 3.46 ± 0.59 shoots/explant, and the shoot height was 34.98 ± 1.51 mm. Zong et al. [V. uliginosum  on Murashige and Skoog medium (MS) with the addition of 0.5 mg/L zeatin and 0.2 mg/L IBA (multiplication rate of 5.3 shoots/explant). Gu et al. [V. uliginosum cv. Zishuijing. The optimal medium was WPM supplemented with 6-benzyladenine (6-BA) 1 mg/L and zeatin 1 mg/L. We previously showed that for the propagation of four varieties of V. uliginosum, two-stage cultivation is optimal: 2 weeks on Anderson’s nutrient medium supplemented with 20 µM 2iP, and then on the same medium with 5 µM 2iP. The multiplication rate was from six to seventeen shoots/explant, depending on the variety, and the shoot height was 30–35 mm [The choice of nutrient media was based on the fact that the mother plant of the obtained hybrids was ntatives ,14,15,16e et al.  showed tg et al.  successfu et al.  noted thu et al.  develope30–35 mm ,22,23.V.ul.8 × ‘NC’, and 3.4 shoots/explant for variant No. 6-2 of V.ul.8 × ‘SC 5-8’. Generally, the hybrid of V.ul.8 × ‘NC’ had greater shoot height for all tested variants of nutrient media compared with V.ul.8 × ‘SC 5-8’. The greatest height of 43.7 mm occurred for the hybrid shoots on a nutrient medium without growth regulators for variant No. 6-2 of V.ul.8 × ‘SC 5-8’, while the heights of variant Nos. 1-1, 1-2, 1-5, and 2-2 of V.ul.8 × ‘NC’ were 72.4, 72.2, 73.4, and 71.4 mm, respectively. Our data indicate that the hybrid variant of V. uliginosum × (V. corymbosum × V. angustifolium) considerably affected the growth and development indices, which should be considered to optimize nutrient media and cultivation conditions further. At the same time, there were data showing the reverse trend. It was demonstrated that the growth medium—and not the genotype and physical conditions of cultivation—primarily affected the shoot height and multiplication coefficient of five genotypes of the genus Vaccinium spp. [Our study showed that the maximum reproduction factor of the studied hybrids was obtained on Anderson nutrient medium with 15 µM 2iP, producing 8.9 shoots/explant for variant No. 1-2 of the ium spp. .V. uliginosum varieties are characterized by different rooting abilities in in vitro culture [We previously observed that  culture . Some vaV. uliginosum × (V. corymbosum × V. angustifolium) in controlled crosses. In addition, ISSR markers allow for the easy, fast, inexpensive, accurate, reliable, and simultaneous detection of polymorphisms at multiple loci in the genome using low quantities of DNA. These properties make the markers useful for the genetic analysis of various plants. ISSRs are found to be effective in diversity analyses of lowbush blueberry [V. uliginosum × (V. corymbosum × V. angustifolium).Our results confirmed that ISSR markers are efficient tools for the discrimination of F1 hybrids of lueberry  and linglueberry . To the The experimental scheme is shown in V.ul.8 × ‘SC 5-8’, and 5 samples of V.ul.8 × ‘NC’, obtained in 2017 at an experimental site of the Laboratory for Food Plant Introduction of the Central Siberian Botanical Garden of the Siberian Branch of the Russian Academy of Sciences (CSBG SB RAS) . Each hybrid variant (seedling) was given an ordinal number (V. uliginosum were collected in 2003 in the Kolyvan’ district of Novosibirsk region (the upper reaches of the Shegarka river), and studied at the CSBG SB RAS’ experimental site. The origins of the paternal forms are as follows: ‘Northcountry’ is an interspecific hybrid of V. corymbosum (B6) × (V. corymbosum × V. angustifolium) (R2P4) obtained in Minnesota, USA, in 1973; SC 5-8 is a spontaneous hybrid of V. corymbosum × V. angustifolium, from which seeds were collected by T. Paal on Prince Edward Island, Canada, the seedlings were grown in the vicinity of Tartu city (Estonia), and were transferred to CSBG SB RAS in 2006.Seven samples of seeds from three-species hybrids were placed in in vitro culture: 2 samples of l number . The ori−2s−1. After 30 days of cultivation, the seedlings were placed on Anderson nutrient medium [Seeds were surface sterilized with 20% Domestos solution for 20 min, followed by three times rinsing in sterile distilled water. Seeds were germinated on 0.6% water agar at 24 ± 1 °C under a 16/8 h photoperiod with lighting of 54 μmol mt medium  supplemeSphagnum moss at 24 ± 1 °C under a 16/8 h photoperiod with lighting of 54 μmol m−2s−1 for 1.5 months. A high relative humidity (above 80%) was maintained for the first 14 days. The acclimatized plants were planted in a greenhouse in plastic seedling trays with a substrate containing neutral and acidic peat in a 2:1 ratio for further 4 weeks and, finally, they were transferred to the nursery.Healthy plants with well-developed shoots and roots were removed individually from the culture vessels, washed gently under running tap water and transferred to plastic pots containing Taq buffer, a mixture of 0.2 mM each dNTP, 2.5 mM free Mg2+, 1 µM ISSR primer, 1.5 ng/µL total DNA, a unit of HS Taq DNA polymerase . The amplification was carried out as follows: predenaturation at 94 °C—4 min; 38 cycles denaturation at 94 °C—25 s, annealing at 60 °C—30 s, elongation at 72 °C—2 min; final elongation—10 min at 72 °C, and storage at 4 °C until further use. PCR aliquot containing the amplified fragment was stained with SYBR Green I, and analyzed by electrophoresis in 1.7% agarose gel using 1 × TAE buffer at 4 V cm−1 voltage. ISSR markers  were used: UBC825 (Sequence (5′-3′): (AC)8T, Tm: 60 °C), UBC811 (Sequence (5′-3′): (GA)8C, Tm: 60 °C). The sizes of the PCR products were compared with the molecular size standard 100+ bp DNA ladder .DNA was isolated of fresh leaves using a modified CTAB method . Polymerp ≤  0.05) . Data are presented as mean and standard errors (M ± SE). To study the different quality plants and leaves, the following indicators were taken into account: plant height (mm), leaf length (mm), leaf area (mm2), leaf elongation coefficient. The processing of images of seeds was carried out using the SIAMS Photolab system for obtaining and processing images .Measurements were replicated twice using 100 microshoots per experiment. Statistical analysis was carried out in Statistica 6.0  hybrids in in vitro culture greatly exceeded their viability in a soil substrate. Genotypic differences in the multiplication rate and shoot height among the studied hybrid variants were identified. We showed that the maximum multiplication rate was achieved when using Anderson nutrient medium containing 15 µM 2iP. The factor analysis confirmed that both the 2iP concentration and the variant of hybrid influenced the multiplication rate and shoot height of the studied hybrids. The efficiency of using 1/2 A hormone-free nutrient medium was evident at the stage of rooting. We did not find any regularities between plant heights in in vitro and ex vitro culture. ISSR demonstrated that all analyzed hybrids inherited DNA fragments of the parent plants in various combinations, which confirmed their hybrid nature. Thus, this study demonstrates that applying in vitro methods for the propagation and further selection of genotypes is an effective approach in programs to obtain interspecific hybrids of V. uliginosum × (V. corymbosum × V. angustifolium). These hybrids may be useful as breeding material for creating new high-quality varieties in the future.Thus, the study revealed that the survival rate of seedlings of


H-tetrazolium bromide (MTT) microtiter assay. Euphorbia schimperiana, Acokanthera schimperi, Kniphofia foliosa, and Kalanchoe petitiana exhibited potent antiproliferative activity against A427, RT-4, MCF-7, and SiSo cell lines, with IC50 values ranging from 1.85 ± 0.44 to 17.8 ± 2.31 µg/mL. Furthermore, these four extracts also showed potent antiproliferative activities against LCLC-103H, DAN-G, A2780, KYSE-70, HL-60, and U-937 cell lines, with IC50 values ranging from 0.086 to 27.06 ± 10.8 µg/mL. Hence, further studies focusing on bio-assay-guided isolation and structural elucidation of active cytotoxic compounds from these plants are warranted.Medicinal plants have been traditionally used to treat cancer in Ethiopia. However, very few studies have reported the in vitro anticancer activities of medicinal plants that are collected from different agro-ecological zones of Ethiopia. Hence, the main aim of this study was to screen the cytotoxic activities of 80% methanol extracts of 22 plants against human peripheral blood mononuclear cells (PBMCs), as well as human breast (MCF-7), lung (A427), bladder (RT-4), and cervical (SiSo) cancer cell lines. Active extracts were further screened against human large cell lung carcinoma (LCLC-103H), pancreatic cancer (DAN-G), ovarian cancer (A2780), and squamous cell carcinoma of the esophagus (KYSE-70) by using the crystal violet cell proliferation assay, while the vitality of the acute myeloid leukemia (HL-60) and histiocytic lymphoma (U-937) cell lines was monitored in the 3--2,5-diphenyl-2 Cancer is one of the leading causes of death worldwide. Mainly due to the rapid growth in the human population and increment in the prevalence of risk factors associated with economic transition, cancer incidence and death are rising in Africa . MoreoveMedicinal plants have been traditionally used in Ethiopia for the treatment of various diseases, including cancer ,4,5. HowA. schimperi, E. schimperiana, K. foliosa, and K. petitiana—showed negative T/Ccorr. values, indicating relevant cytotoxic activity at 50 µg/mL , and two suspensions (HL-60 and U-937) cell lines. PBMCs were included to compare the results obtained by the HL-60 and U-937 to primary noncancer cells. This should allow an initial assessment of the selectivity of the extracts. Accordingly, concentration-response curves of the extracts against 10 cell lines were generated and IClculated , Table 250 ˃ 50 µg/mL).Selectivity is a desired property of active lead anticancer agents . DiffereA. schimperi: potent activity was observed when the extract of leaves of A. schimperi was tested on all ten cell lines with IC50 values ranging from 1.87 ± 0.4 to 10.31 ± 3.45 µg/mL. This plant is reported to contain acolongifloroside K as its major active principle, as well as cardiotonic glycosides ouabain and acovenoside A as minor constituents; in fact, it is used by Maasai people in east African as an arrow poison [A. schimperi have been reported for their antiviral [w poison . Young lw poison , hepatitw poison , and tonw poison  in Ethiontiviral , antibacntiviral , antiprontiviral , and in ntiviral  activitintiviral .E. schimperiana: the methanolic extract of E. schimperiana showed moderate cytotoxicity activity against the MCF-7 cell line, with an IC50 value of 25.2 µg/mL [Euphorbia (E. turcomanica) also reduced the viability of HT-29 cells, with an IC50 value of 43 µg/mL [E. umbellata latex sap showed potent cytotoxic activity on hepatocellular carcinoma tumor cells (Hepa1c1c7), at IC50 values ranging from 2-12 µg/mL [E. tirucalli, showed cytotoxic activity against a wide range of human cancer cell lines, including esophageal squamous cells (11.08 µM) and pancreatic carcinoma cells (6.84 µM) [O-β-D glucoronside, and 3-methyl-qurecetin-7-O-β-D-glucoronside), isolated from the 80% methanol extract of E. schimperiana, showed antioxidant activities [E. schimperiana showed potent cytotoxic activity against A427, SiSo, and RT-4 cell lines at concentrations ranging from 1.85 ± 0.44 to 3.28 ± 1.2 µg/mL, and moderate cytotoxic activities against A2780 (26.54 ± 18.5 µg/mL)..2 µg/mL . The met43 µg/mL . Similar12 µg/mL . Euphol,tivities . In the K. petitiana: K. petitiana, known for its traditional use as a treatment of breast and skin cancer in Ethiopia [50 values ranging from 2.09 ± 0.43 to 10.41 ± 5.59 µg/mL. Another plant from the same genus, K. crenata, has been reported to possess potent cytotoxic activity, with IC50 values of 2.33 and 28.96 µg/mL against mesothelioma (SPC212) and hepatocarcinoma (HepG2) cell lines, respectively [K. gracilis, on the other hand, showed only weak antiproliferative activity (IC50 = 136.85 ± 2.32 µg/mL) against human hepatocellular carcinoma (HepG2) [K. pinnata, was reported for its potent cytotoxic activity against the human nasopharyngeal (KB) cell line, with the ED50 value of 80 ng/mL [K. tubiflora were reported to displayed strong cytotoxic activity against A549, Cal-27 , A2058 (melanoma), and HL-60 (promyelocytic leukemia) cell lines [Ethiopia , also shectively . The chl80 ng/mL . Furtherll lines .K. foliosa: K. foliosa inhibited the proliferation of all cell lines, with IC50 values ranging from 14.54 ± 4.14 to 27.06 ± 10.8 µg/mL. Knipholone anthrone, a 4-phenylanthraquinone isolated from this plant, has been reported to possess cytotoxic activity against human acute monocytic (THP-1) and promonocytic (U-937) leukemic cell lines, with IC50 value of 0.9 ± 0.09 and 0.5 ± 0.05 µM, respectively [ectively .http://www.theplantlist.org, accessed on 15 April 2021). Each plant materials were shade dried and ground into powder.Different parts of 22 plant species  were col®®, R-200and R-210, Duisburg, Germany) with heating  at 37–40 °C, followed by freeze-drying  the aqueous concentrate. The dried powder (200 g each) was macerated in 1 L (80% methanol) and shaken for 48 h. The macerated plant material was then filtered through Whatman No.1 filter paper by using a Buchner funnel. The crude methanol extracts were concentrated with a rotary evaporator , in a humid atmosphere of 5% CO2 at 37 °C [v/v) heat-inactivated fetal bovine serum  and supplemented with 30 mg/L penicillin and 40 mg/L streptomycin. Cells were incubated in a 5% CO2 humidified incubator , at 37 °C, and passaged weekly.The cancer cell lines in this study, MCF-7, A427, RT-4, SiSo, LCLC-103H, DAN-G, KYSE-70, A2780, HL-60, and U-937 were obtained from the German Collection of Microorganisms and Cell Culture  . The A27at 37 °C . Cells w®®) with 3.2% sodium citrate as an anticoagulant. Whole blood was 1:2 diluted with phosphate-buffered saline (PBS) . Afterward, 10 mL Histopaque®®-1077  were overlaid with 10 mL of the diluted cell suspension and centrifuged without brake (400× g) for 30 min at room temperature. Subsequently, the PBMC layer was transferred to a new 50 mL centrifugal tube, washed with 10 mL PBS , and centrifuged for 10 min at 250× g. The obtained cell pellet was then washed three times with PBS (5 mL) and centrifuged at 250× g for 10 min. After isolation, the PBMCs were cultured in RPMI 1640 , supplemented with 2 mM L-glutamine  1% v/v antibiotics  and 10% v/v heat-inactivated fetal calf serum. The viability and number of cells were assessed by the trypan blue exclusion assay. Trypan blue was added in a 1:1 ratio to the cell suspension and counted with a Neubauer counting chamber. Cell viability was always >90%. Cells were adjusted to a density of 1 × 106 cell/mL for the following MTT assay. The assay was performed as described, with extract concentrations ranging from 50 to 3.13 µg/mL.Peripheral blood from healthy humans was provided by the blood bank of the University Medicine Greifswald. All donors signed informed consent forms that their blood could be used for research purposes, and all samples were randomized so as to conceal the identity of the donors. Blood samples were collected by venipuncture in tubes (BD Vacutainerv/v) was used as a solvent. The plates were incubated for an additional 72 h at 37 °C. After 96 h, the culture medium was discarded and replaced with a 1% glutaraldehyde buffer saline for 20 min and then stored under Dulbecco’s buffer solution (pH 7.4) at 4 °C. On the day of staining, the buffer solution was removed, and the cells were stained with 0.02% crystal violet in deionized water (100 µL/well) for 30 min. Excess dye was discarded by washing the plates for 15 min in fresh water. The cell-bound dye was redissolved in 70% (v/v) ethanol/water and the optical density was measured at λ = 570 nm with a Spectramax 384 Plus plate reader  or a Sunrise plate reader . The IC50 values were calculated by linear least-squares regression of the T/C corr values versus the logarithm of the added extract concentration and extrapolating to the T/C corr. values of 50% [T is the mean optical density (OD) of the treated cells, ODC the mean OD of the controls, and ODC,0 the mean OD of seeded cells at the time the drug was added.Crystal violet assay was used for both primary and secondary screening of extracts, as described previously . This asH-tetrazolium bromide (MTT) assay was used in this experiment to determine the inhibition of the viability of suspension cells by the extracts [The 3--2,5-diphenyl-2extracts . Briefly50 values were calculated with the software GraphPad Prism 7.0a by determining the inflection point of the simulated sigmoidal curves. The results are presented as means ± standard error of mean where appropriate.All tests were independently performed in triplicate. ICA. schimperi, E. schimperiana, K. petitiana and K. foliosa, showed cytotoxic activity against all ten cell lines. Moreover, these extracts possessed selective cytotoxicity toward suspension cell lines (HL-60 and U-937) when compared to their effect on PBMC, consistent with their traditional use in anticancer therapies. In addition to these four plants, C. abyssinica and G. involucrata also showed selective cytotoxic/antiproliferative activities against some of the human cancer cell lines used in this study. These encouraging results have motivated us to begin isolating and identifying the active components of these four plant extracts, which may contain novel lead compounds for the treatment of cancer.The results of this study indicate that crude extracts of 4 out of the 22 plant species have good cytotoxic activity against human cancer cell lines, as depicted in


Cellulase plays a key role in converting cellulosic biomass into fermentable sugar to produce chemicals and fuels, which is generally produced by filamentous fungi. However, most of the filamentous fungi obtained by natural breeding have low secretory capacity in cellulase production, which are far from meeting the requirements of industrial production. Random mutagenesis combined with adaptive laboratory evolution (ALE) strategy is an effective method to increase the production of fungal enzymes.Trichoderma afroharzianum by exposures to N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), Ethyl Methanesulfonate (EMS), Atmospheric and Room Temperature Plasma (ARTP) and ALE with high sugar stress. The T. afroharzianum mutant MEA-12 produced 0.60, 5.47, 0.31 and 2.17 IU/mL FPase, CMCase, pNPCase and pNPGase, respectively. These levels were 4.33, 6.37, 4.92 and 4.15 times higher than those of the parental strain, respectively. Also, it was found that T. afroharzianum had the same carbon catabolite repression (CCR) effect as other Trichoderma in liquid submerged fermentation. In contrast, the mutant MEA-12 can tolerate the inhibition of glucose (up to 20 mM) without affecting enzyme production under inducing conditions. Interestingly, crude enzyme from MEA-12 showed high enzymatic hydrolysis efficiency against three different biomasses , when combined with cellulase from T. reesei Rut-C30. In addition, the factors that improved cellulase production by MEA-12 were clarified.This study obtained a mutant of  T. reesei, which provides a new choice for processing of bioresources in the future.Overall, compound mutagenesis combined with ALE effectively increased the production of fungal cellulase. A super-producing mutant MEA-12 was obtained, and its cellulase could hydrolyze common biomasses efficiently, in combination with enzymes derived from model strainThe online version contains supplementary material available at 10.1186/s13068-021-02072-z. In biorefinery process, cellulase plays a key role in converting cellulosic biomass into fermentable sugar to produce chemicals and biofuels . Many miClassic physical or chemical mutagenesis is a kind of strategy that can effectively improve the performance of strains, especially for increasing enzyme yields , 7. The Trichoderma spp. have catabolite repression [Adaptive laboratory evolution (ALE) also can achieve the phenotypic optimization. ALE refers to the realization of microbial evolution by imposing human interference and controlling the growth environment of microbes . Comparepression . High copression . TherefoT. afroharzianum. Subsequently, it was found that the mutant can tolerate the inhibition of glucose (up to 20 mM) without affecting enzyme production under inducing conditions. Interestingly, crude enzyme from the mutant showed high enzymatic hydrolysis efficiency against three common biomasses , which were pretreated by cooking with active oxygen and solid alkali (CAOSA), when combined with cellulase from T. reesei Rut-C30. In addition, real-time reverse transcription quantitative PCR (RT-qPCR) was employed to analyze the transcription levels of major cellulase genes and transcription factors genes in isolated mutant.In this study, MNNG/EMS/ARTP mutagenesis combined with ALE method was used to improve cellulase production in To optimize the screening efficiency of high-yield cellulase mutants, a strategy of compound mutagenesis combined with microbial adaptive laboratory evolution (ALE) was established Fig. a. By artThe mutagenesis was performed in three rounds: (1) one round of MNNG, 2) one round of EMS, and (3) one round of ARTP. Before each round of mutagenesis, a preliminary experiment on the relationship between lethality and positive mutation rate was done. In each round of mutagenesis, with the increased of time, the lethality rate of the strain increased, while the positive mutation rate reached a peak. In the first round of MNNG mutagenesis, when the mutagenesis time was 50 min, the lethality rate of the strain exceeded 99% . This appears to be attributable to the compound mutagenesis combined with microbial ALE strategy, which avoided the defect that the repeated processing of a single mutagenesis technique leading to reducing the efficiency of mutagenesis . The selTo evaluate the genetic stability of the mutant MEA-12, the cellulase activity was measured in the fermentation medium after seven consecutive subcultures. No significant change in cellulase activities was observed after the subcultures  reached the maximum on the day 6, while that of the mutant MEA-12 was on the day 4  endoglucanases (EGs); (2) cellobiohydrolases (CBHs); 3) β-glucosidases (BGs) . In this β-glucosMoreover, the specific activities (IU/mg) of strain MEA-12 which were calculated based on protein mass increased 1.44, 1.51, 1.29 and 1.05 times in FPase, CMCase, pNPCase and pNPGase, respectively, compared with that of the parental strain Fig. . The incTrichoderma [T. afroharzianum, as far as we know. In this study, activities of all four enzymes from parental strain TA were profoundly inhibited with the increase of glucose concentration  is widely present in microorganism growth process . When thchoderma . Howeverion Fig. , which m mM Fig. . It was T. reesei Rut-C30 (TR) and Novozymes were compared by SDS-PAGE analysis. The composition of two protein mixtures in parent strain TA and mutant MEA-12 was similar, but the relative concentrations of different components were significantly different. Specifically, the band corresponding to cellulose-degrading enzyme cellobiohydrolase II (CBH II)  [T. reesei Rut-C30 was slightly thicker than that of MEA-12. This matched the superior power of the cellulase production model strain T. reesei Rut-C30. T. reesei Rut-C30 has been developed for decades and is currently an industrial strain with a relatively complete cellulase system [T. reesei Rut-C30 and the higher production of β-glucosidase by MEA-12   in MEA-1e system . The comcbh1, cbh2, eg1, eg2 and bgl1) and transcriptional regulatory genes  were measured by RT-qPCR analysis. Compared to the parental strain TA, the transcriptional levels of the major cellulase genes  in MEA-12 were all significantly up-regulated in both 24 h and 48 h induction and the relative expression reached 2.48, 4.54, 1.61, 7.16 and 3.23 at 48 h, respectively .In addition, transcriptional levels of major cellulase genes , in the future, comparative genomics can be used to analyze the mechanism of mutants’ high cellulase production. Further improve cellulase production of the target strain through the inverse metabolic engineering.Since the whole genome analysis of T. reesei Rut-C30 (TR) is recognized as an excellent cellulase-producing strain. However, its lower β-glucosidase activity causes cellobiose to accumulate, which affects the efficiency of enzymatic hydrolysis and saccharification, thereby reducing the yield of ethanol [Cornstalk, bamboo and reed as non-wood and non-food biomass materials, depending on their different fiber processing technology, can turn them into glucose and other wide range of products, which has been widely used in China, the United States and some of European countries . Current ethanol . TherefoAs shown in Fig. Trichoderma and Aspergillus [Trichoderma, is possible to co-culture with T. reesei and provides a better choice for the compounding of commercial cellulase.The enzymatic hydrolysis efficiency of pretreated biomass hinges on the composition of the enzyme mixture . After mergillus . HoweverT. afroharzianum mutant strain, MEA-12, was isolated and resulted in 4.33, 6.37, 4.92 and 4.15 times increase in FPase, CMCase, pNPCase and pNPGase, respectively, with higher insensitivity for catabolite repression. Crude enzyme from T. afroharzianum MEA-12 showed high enzymatic hydrolysis efficiency against common biomasses , when combined with cellulase from T. reesei Rut-C30. Furthermore, the factors that improved cellulase production by MEA-12 were clarified. This study confirmed that compound mutagenesis combined with ALE method was an effective tool to increase the cellulase production of filamentous fungi and provided a new choice for processing of bioresources in the future.In this study, a T. afroharzianum (CICC 40189) and T. reesei Rut-C30 (CICC 13052) were obtained from the China Center of Industrial Culture Collection (CICC). Fresh spores were collected from T. afroharzianum and T. reesei Rut-C30 cultured on potato dextrose agar (PDA) plates for 4 days and 6 days, respectively.The T. afroharzianum (1×107–1×108 spores) were diluted and spread on the screening medium (SM), cultured at 30 ℃ for 4 days. The SM contains 2.0 g/L KH2PO4, 1.4 g/L (NH4)2SO4, 0.75 g/L peptone, 0.3 g/L urea, 0.3 g/L CaCl2, 0.3 g/L MgSO4·7H2O, 0.25 g/L yeast extract, 2.0 g/L Triton X-100, 20.0 g/L agar and 1.0 mL/L trace element nutrient solution [4·7H2O, 2.0 g/L CoCl2·6H2O, 1.6 g/L MnSO4·4H2O, 1.4 g/L ZnSO4·7H2O [For mutagenesis, solution . The traSO4·7H2O . The SM T. afroharzianum (1×108 spores) and T. reesei Rut-C30 (1×108 spores) were cultured in preculture medium 2SO4, 1.0 g/L CaCl2, 0.8 g/L MgSO4·7H2O, 2.0 g/L tween 80, 1.0 mL/L trace element nutrient solution, pH 4.8), at 180 rpm and 30 °C, for 2 days, respectively. Then, it was inoculated into fermentation medium 2SO4, 1.4 g/L CaCl2, 1.2 g/L MgSO4·7H2O, 2.0 g/L tween 80, 1.0 mL/L trace element solution, pH 4.8) in 10% (v/v) inoculum size at 180 rpm and 30 °C, for 4 days. Centrifuge at 10,000×g for 15 min and collect the supernatant for measuring enzyme activity and enzymatic hydrolysis of bamboo pulp.For fermentation of enzyme production, T. afroharzianum (1×108 spores) were cultured in preculture medium for 2 days, then transferred into fermentation medium, at 180 rpm and 30 °C, for 24 and 48 h. Centrifuge at 12,000×g for 10 min to collect the mycelia and grind with liquid nitrogen to extract total RNA.For RT-qPCR analyses, All the above media were sterilized at 121 °C for 20 min before use.g for 3 min, and the new spore suspension was prepared by washing and the precipitation with 1% normal saline for 2 times. After the dilution treatment, post-mutagenesis spore suspension was spread on the SM1 and cultured at 30 °C for 4 days. Strain with apparent positive mutation, which the ratio of transparent circle diameter to colony diameter was higher than that of the parental strain.MNNG (0.1 g) was dissolved in 1 mL acetone to prepare 10% solution for treatment. The spore suspension (970 μL) and 10% MNNG solution (30 μL) placed in a constant temperature shaker, shaking at 30 °C and 180 rpm, for 0–90 min, to determine the optimum treatment time. After the treatment of mutagenesis, the supernatant was discarded after the centrifugation at 12,000×g for 3 min, and the new spore suspension was prepared by washing and precipitation with 1% normal saline for 2 times. After the dilution treatment, post-mutagenesis spore suspension was spread on the SM2 and cultured at 30 °C for 4 days. Colony that became darker and larger in diameter was selected as the apparent positive mutation strain.For EMS treatment, 50% EMS solution was prepared by mixing EMS and acetone at the volume ratio of 1:1. The spore suspension (970 μL) and 50% EMS solution (30 μL) were mixed in a constant temperature shaker, shaking at 30 °C and 180 rpm, for 0–90 min, to determine the optimum treatment time. After the treatment of mutagenesis, the supernatant was discarded by centrifugation at 12,000×For ARTP treatment, the spore suspension (10 μL) was smeared on the slides and placed in an ARTP-IIS Bio-breeding Machine , for 0~270 s, to determine the optimum treatment time. After the mutagenesis, the slides were taken out and placed in an EP tube with 1 mL 1% normal saline. After the dilution treatment, post-mutagenesis spore suspension was spread on the SM3 and cultured at 30 °C for 4 days. Strain with apparent positive mutation was chosen according to the ratio of transparent circle diameter to colony diameter, which was higher than that of the parental strain.After determining the optimal treatment time , perform the combined treatment as described above.For catabolite repression studies, add different concentrations of glucose (0–100 mM) to the fermentation medium. Fermentation lasted for 1–7 days and the optimum fermentation time with the highest enzyme activity was recorded for comparison.g for 15 min. Total soluble protein was quantified using the Modified Bradford Protein Assay Kit  according to the manufacturer’s instructions. FPase and CMCase activities were assayed using the standard method recommended by the International Union of Pure and Applied Chemistry (IUPAC) [The supernatant was collected from fermented broth by centrifugation at 10,000× (IUPAC) . pNPCase (IUPAC) . One uni (IUPAC) .T. afroharzianum and MEA-12 cultured in fermentation medium were collected at the 4th day and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Proteins secreted by TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix  according to the manufacturer's instructions. RT-qPCR primers of the tested genes were designed as described in a previous study  method. The actin gene was chosen as the endogenous control to normalize the expression levels. All experiments were performed in triplicates.Culture conditions of the strains used for RT-qPCR were same to those described above. Total RNA was extracted using the RNA pure total RNA rapid extraction kit  and then cDNA synthesis using the L) × 0.9)/(Substrate (g)×Cellulose content (%)).Cornstalk pulp , bamboo pulp  and reed pulp   and ** (p<0.01)).All the experiments values presented in figures are mean±SD calculated with Excel 2019. All experiments were conducted in triplicate and all the multiple comparison tests were performed with Student’s Additional file 1: Table S1. Primers used for RT-qPCR. Figure S1. Enzyme activity of MNNG mutagenesis re-screening. a diameter ratio (hydrolysis circle diameter/strain diameter) of TA and its mutants, b FPase activities of TA and its mutants, c CMCase activities of TA and its mutants. Figure S2. Enzyme activity of EMS mutagenesis re-screening. a FPase activities of M-84 and its mutants, b CMCase activities of M-84 and its mutants, c pNPCase activities of M-84 and its mutants, d pNPGase activities of M-84 and its mutants. Figure S3. Enzyme activity of ARTP mutagenesis re-screening. a FPase activities of ME-10 and its mutants, b CMCase activities of ME-10 and its mutants, c pNPCase activities of ME-10 and its mutants, d pNPGase activities of ME-10 and its mutants. Figure S4. Common biomasses after pretreatment for hydrolysis.