Methylovulum psychrotolerans, isolated from geographically remote low-temperature environments in Northern Russia, were grown at three different growth temperatures, 20, 10 and 4°C and were found to be capable of oxidizing methane at all temperatures. The three M. psychrotolerans strains adapted their membranes to decreasing growth temperature by increasing the percent of unsaturated fatty acid (FAs), both for the bulk and intact polar lipid (IPL)-bound FAs. Furthermore, the ratio of βOH-C16:0 to n-C16:0 increased as growth temperature decreased. The IPL head group composition did not change as an adaption to temperature. The most notable hopanoid temperature adaptation of M. psychrotolerans was an increase in unsaturated hopanols with decreasing temperature. As the growth temperature decreased from 20 to 4°C, the percent of unsaturated M. psychrotolerans bulk-FAs increased from 79 to 89 % while the total percent of unsaturated hopanoids increased from 27 to 49 %. While increased FA unsaturation in response to decreased temperature is a commonly observed response in order to maintain the liquid-crystalline character of bacterial membranes, hopanoid unsaturation upon cold exposition has not previously been described. In order to investigate the mechanisms of both FA and hopanoid cold-adaption in M. psychrotolerans we identified genes in the genome of M. psychrotolerans that potentially code for FA and hopanoid desaturases. The unsaturation of hopanoids represents a novel membrane adaption to maintain homeostasis upon cold adaptation.Three strains of aerobic psychrotolerant methanotrophic bacteria  Methylovulum psychrotolerans is a recently described species of aerobic methanotrophic bacteria, isolated from several low-temperature habitats in Northern Russia, such as cold methane seeps and subarctic freshwater lake sediments  (n-C16 fatty acids (C16 FAs), and in particular, unsaturated n-C16 FAs  . The lip-C16 FAs . Howevern-C14 FA . β-hydron-C14 FA  and thein-C14 FA , which an-C14 FA . Methanon-C14 FA . BHPs haositions . BHPs  were grown at three different growth temperatures, 20, 10 and 4°C. In order to examine growth temperature-driven changes in their lipid distribution, the FA distribution (bulk and IPL-bound) was examined at each growth temperature. The changes in hopanoids were examined by analysis of the hopanols formed by Rohmer degradation of intact BHPs.In this study, three strains of Methylovulum psychrotolerans isolated from different geographical locations in Northern Russia were used in this study. The type strain of this species, Sph1T, was obtained from a cold methane seep located in the valley of the river Mukhrinskaya, Irtysh basin, West Siberia (60°53.358′ N 68°42.486′ E). Strain OZ2 was isolated from sediments of a subarctic, shallow, unnamed freshwater lake in Archangelsk region (67° 36.567′ N 53°35.317′ E). These two strains were characterized in detail by T and OZ2. In addition, the 16S rRNA gene sequence determined for strain Sph56 (GenBank Accession number MH701868) displayed 99.5 % identity to the corresponding gene sequences from strains Sph1T and OZ2  of a trace elements stock solution containing (in grams per liter) EDTA, 5; FeSO4.7H2O, 2; ZnSO4.7H2O, 0.1; MnCl2.4H2O, 0.03; CoCl2.6H2O, 0.2; CuCl2.5H2O, 0.1; NiCl2.6H2O, 0.02; and Na2MoO4, 0.03. The medium pH was 6.8. The flasks of a total volume 500 ml were filled to 20 % capacity with NMS medium, sealed with rubber septa, and CH4  was added to the headspace using syringes equipped with disposable filters (0.22 μm). The cultures were grown in a Multitron Pro shaker incubator  at 4, 10 and 20°C, respectively, and were harvested in late exponential growth phase.For lipid analysis, the three strains were cultivated in NMS medium containing (in gram per liter) MgSOT, OZ2 and Sph56 were maintained in NMS medium at 4, 10 and 20°C, respectively, for one month, with regular transfers after 5–7 days. Methane oxidation rates were determined as described by 8 cells ml−1 with 0.5 mM phosphate buffer. Chloramphenicol (1.25 mg l−1) was added to the experimental flasks to inhibit further cell growth. Aliquots (10 ml) of cell suspensions were added to 500-ml flasks, which were sealed gas-tight with butyl rubber septa. Methane was added to final mixing ratios of 0.5% (v/v). The flasks were fixed in a horizontal position on a rotary shaker with the temperature settings of 4, 10 and 20°C, respectively, and shaken at 110 rpm. The decrease of methane concentration in the headspace was followed over time using a gas chromatograph  equipped with a flame ionization detector. Methane oxidation rates were estimated by linear regressions of CH4 concentrations versus time.Prior to kinetic experiments, strains Sph12. Before analysis an aliquot was treated with BSTFA in pyridine to derivatize alcohol groups and then brought to a final volume with ethyl acetate. FA methyl ester (FAME) identification was carried out using gas chromatography-mass spectrometry (Thermo Finnigan TRACE GC-MS). FAMEs were separated using a CP-SIL 5CB capillary column  with the following oven conditions: initial temperature 70°C, increasing to 130°C by 20°C min−1, then increasing to 320°C by 4°C min−1 and held for 10 min. MS operating parameters were: electron multiplier 1663 V; source temperature 250°C; full scan m/z 50–800; scan time 0.33 s. MS data were acquired and processed using the Thermo Finnigan Xcalibur software. FAMEs were identified based on literature data and library mass spectra. Double bond positions were determined, where possible, using dimethyldisulfide (DMDS) derivatization of the FAMEs. To this end, extracts were derivatized in hexane (100 μl) with DMDS  and I2/ether  and heated overnight at 40°C. Hexane (400 μl) was then added with Na2S2O3  to deactivate the iodine. The hexane layer was removed, and the aqueous phase washed with hexane (× 2). The hexane layers were combined and analyzed by GC-MS as described above.For (bulk) fatty acid (FA) analysis, aliquots of the lyophilized cells from duplicate cultures were hydrolyzed by refluxing with 1.5N HCl:MeOH solution for 3 h. After adjustment of the pH to 4–5 with a 2 N KOH/MeOH  solution, extraction with dichloromethane (DCM) was carried out. The resulting extract was methylated with diazomethane in diethyl ether which was removed under a stream of N2SO4, and evaporated to dryness. The residue was treated with 20 mg of NaBH4 in 1 ml methanol by stirring at room temperature for 1 h. After addition of a solution of KH2PO4 , the hopanols were extracted with DCM. The obtained reaction mixture was methylated with diazomethane and separated over a small column with activated Al2O3 into an apolar and a polar fraction using DCM and DCM-MeOH  as eluent, respectively. The polar fractions were silylated with N,O-bis(trimethylsilyl)-fluoroacetamide in pyridine at 60°C for 20 min and analyzed by GC and GC-MS. The distribution of hopanols was obtained by integration of the appropriate peaks. GC and GC-MS analysis was performed as described above for fatty acid analysis.For the detection of the presence of biohopanoids, lyophilized cells were directly treated with periodic acid/sodium borohydride to convert bacteriohopanepolyols (BHPs) into GC-amenable hopanoid alcohols (hopanols) following procedure 2 described by 2 gas. Before analysis the extract was redissolved in a mixture of MeOH:DCM  and aliquots were filtered through 0.45 μm regenerated cellulose syringe filters . Analysis of extracts was carried out using an Ultra High Pressure Liquid Chromatography-High Resolution Mass Spectrometry (UHPLC-HRMS) according to the reversed phase method of 2O:formic acid:14.8 M NH3aq (85:15:0.12:0.04 [v:v:v:v]) and (B) IPA:MeOH:formic acid:14.8 M NH3aq (50:50:0.12:0.04 [v:v:v:v]). The elution program was: 95% A for 3 min, followed by a linear gradient to 40% A at 12 min and then to 0% A at 50 min, this was maintained until 80 min. The flow rate was 0.2 mL min−1. Positive ion ESI settings were: capillary temperature, 300°C; sheath gas (N2) pressure, 40 arbitrary units (AU); auxiliary gas (N2) pressure, 10 AU; spray voltage, 4.5 kV; probe heater temperature, 50°C; S-lens 70 V. Target lipids were analyzed with a mass range of m/z 350–2000 , followed by data-dependent tandem MS2 , in which the ten most abundant masses in the mass spectrum were fragmented successively . The Q Exactive was calibrated within a mass accuracy range of 1 ppm using the Thermo Scientific Pierce LTQ Velos ESI Positive Ion Calibration Solution. During analysis dynamic exclusion was used to temporarily exclude masses (for 6 s) in order to allow selection of less abundant ions for MS2. The relative abundance of peak areas does not necessarily reflect the actual relative abundance of the different compounds, however, this method allows for comparison between the samples analyzed in this study. The IPL groups were identified through comparison with fragmentation patterns of authentic standards, as described in 2 spectra  was extracted using a modified Bligh-Dyer procedure . Briefly spectra . The peaM. psychrotolerans Sph1T  from Saccharomyces cerevisiae as queries. The sequences were aligned with MAFFT (http://www.ebi.ac.uk/Tools server (The sequences of putative fatty acid desaturase (POZ50891.1) and sterol desaturase (POZ53527.1) of th MAFFT  in the hs server  using ths server  and the 4 concentration in the experimental flasks incubated at different temperatures is shown in −15mol CH4 h−1 cell−1) were observed at 20°C, which is consistent with the original description of Methylovulum psychrotolerans as showing growth optimum at 20–25°C : C14:1ω7, C14:0, C15:0, C16:1ω8c, C16:1ω7c, C16:1ω6c, C16:1ω5tr, C16:0, βOH-C16:0. C16:1ω8c was previously considered unique to type I methanotrophs , grown at 20, 10 and 4°C, were examined for their intact polar lipid (IPL) distribution. The IPLs detected had either phosphatidylglycerol (PG) or phosphatidylethanolamine (PE) head groups, with a range of FA combinations . The hea2 . Where t2  an infor30:0 (homohopanol), C30:1 (homohop-11-enol), C31:0 (homohopan-31-ol), C31:1 (homohop-11-en-31-ol), C32:0 (bis-homohopan-32-ol) and C32:1 (bis-homohop-11-en-32-ol). The double bond position in the three unsaturated hopanols was confirmed as Δ11 by both the absence of m/z 119 ion peaks and the presence of M - 192 Da ion peaks in their EI mass spectra . The total percent of unsaturated bulk-FA . The total percent of unsaturated IPL-bound FAs behaved similarly, to the bulk-FAs: it increased with decreasing growth temperature, from 69% at the 20°C to 80% at 10°C and 81 at 4°C.The bulk-FA distributions of the three strains were so similar to each other at each temperature, that we discuss them further only as an average of the 6 cultures (three strains in duplicate). For the data of the individual strains see Clostridium psychromophilum . The major change between the 20 and 10°C was that the relative percentage of C32:1 increased significantly, from 11 ± 1.1 % to 45 ± 4.6%. It was slightly lower at 4°C (34 ± 7.8%) but still significantly higher than at 20°C. This relative increase was countered by concomitant decreases in the other hopanols (30 (saturated and unsaturated), C31 and C32. On average, the C30 sum decreased from 32 ± 10 at 20°C to 17 ± 6.0 at 10°C and 18 ± 4.2 at 4°C. The total C31 also decreased from 22 ± 5.7% at 20°C to 7.4 ± 1.4% at 10°C and 14 ± 2.8% at 4°C. Consequently, there was an increase in the total C32, from 46 ± 5.0% at 20°C to 76 ± 6.2% at 10°C and 69 ± 5.2% at 4°C.The majority of the hopanols at the temperature optimum of 20°C were Chopanols . As was 30 and C31 and concomitant increase in C32. In a mesocosm study of symbiotic methanotrophs in Sphagnum moss (32 hopanol) increased as temperature decreased (from 25 to 5°C), while both aminotetrol BHP (corresponding to C31 hopanol) and aminopentol BHP (corresponding to C30 hopanol) decreased. Similarly, 32 hopanol), while aminopentol BHP decreased and aminotetrol BHP remained constant (Methylomonas methanica) the relative percentage of C32 hopanol remained the same as temperature decreased, while the C30 hopanol increased and the C31 hopanol decreased. Overall, based on our results and the limited reports in the literature, it would seem that methanotrophic bacteria adapt their BHP distribution to adapt to colder temperatures, by increasing the relative amount of tetrafunctionalised BHPs (either in the saturated or unsaturated form).Overall, there was an increase in the percent of unsaturated hopanols as temperature decreased, along with a decrease in both the total Cnum moss , no unsaconstant . In contM. psychrotolerans strains diplopterol was not detected in the cultures grown at 20°C but the ratio of diplopterol to the sum of the six hopanols was on average at 0.1 ± 0.0 at both 10 and 4°C. During hopanoid biosynthesis, the cyclisation of squalene results in either the production of diploptene or diplopterol, but it is diploptene that is reported to be the pre-cursor in the biosynthesis of BHPs. The relative increase in diplopterol with decreasing temperature suggests an additional membrane adaption separate from that of the BHPs.In all three M. psychrotolerans bulk-FAs increased from 79 to 89% while the total percent of unsaturated IPL-bound-FAs increased from 69 to 81% . In order to investigate the enzymatic mechanisms responsible for hopanoid unsaturation, we searched for genes that potentially code for proteins involved in this reaction. We identified a gene AADEFJLK_00554 coding for a potential sterol desaturase POZ53527 protein, belonging to the FA hydroxylase (FAH) superfamily of proteins. Within this group, a wide set of enzymatic activities related to hydroxylation, desaturation and oxidation have been described  of B. subtilis has been characterized and it is known to be transcriptionally regulated in response to decreased temperature  . Their sperature . Proteinperature   and hopanoids. The bulk FA ratio of βOH-C16:0 to C16:0 increased as growth temperature decreased. The hopanol composition of M. psychrotolerans contains C30:0, C31:0 and C32:0 along with their unsaturated forms. The most notable hopanoid adaption M. psychrotolerans exhibited to lower growth temperatures was an increase in C32:1 hopanols . The total sum of C32 hopanols increased as temperature decreased. Similar results have been reported in other studies. Diplopterol was not detected in the cultures grown at 20°C but was present at 10 and 4°C.Three strains of M. psychrotolerans. Further work would be needed to produce the biochemical evidence of the enzymatic activity of the proposed membrane-adaption genes in M. psychrotolerans.Our results demonstrate that not only FAs, but also hopanoids can be remodeled to maintain bacterial membrane homeostasis upon cold adaptation. We identified genes that potentially code for FA lipid desaturases and for hopanoid unsaturation in the genome of NB carried out data analysis and wrote the manuscript. WR carried out extractions and lipid analysis. DS-C carried out genomic analyses. IO and SB cultured bacteria and measured methane oxidation rates. SD and JSD designed and supervised the study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bombus terrestris) cytochrome P450s of the CYP9Q subfamily, CYP9Q4 and CYP9Q5, are important biochemical determinants of sensitivity to neonicotinoid insecticides. Here, we report the characterisation of a third P450 gene CYP9Q6, previously mis-annotated in the genome of B. terrestris, encoding an enzyme that metabolises the N-cyanoamidine neonicotinoids thiacloprid and acetamiprid with high efficiency. The genomic location and complete ORF of CYP9Q6 was corroborated by PCR and its metabolic activity characterised in vitro by expression in an insect cell line. CYP9Q6 metabolises both thiacloprid and acetamiprid more rapidly than the previously reported CYP9Q4 and CYP9Q5. We further demonstrate a direct, in vivo correlation between the expression of the CYP9Q6 enzyme in transgenic Drosophila melanogaster and an increased tolerance to thiacloprid and acetamiprid. We conclude that CYP9Q6 is an efficient metaboliser of N-cyanoamidine neonicotinoids and likely plays a key role in the high tolerance of B. terrestris to these insecticides.Recent work has shown that two bumblebee (  •Bombus terrestris.A cytochrome P450 gene CYP9Q6 is identified as a potential N-cyanoamidine neonicotinoid metaboliser in •in vitro in insect cell lines.CYP9Q6 efficiently metabolized N-cyanoamidine neonicotinoid insecticides when expressed •D. melanogaster expressing CYP9Q6 in vivo had increased tolerance to N-cyanoamidine neonicotinoids.Transgenic •Amino acid substitutions in the P450 active site may be important determinants of binding and metabolism of these compounds. Domesticated colonies are used for managed pollination of crops in orchards and greenhouses and are one of the main commercial alternatives to the western honey bee, Apis mellifera  supplied by Biobest (Belgium). Colonies were kept in the dark at 27±1 °C, 50% relative humidity. All chemicals were supplied by Sigma-Aldrich unless stated otherwise.Worker bees used for the experiments came from 2.2® III reverse transcriptase  with OligodT(15) primer .DNA and RNA extractions were done on single adult worker bees. DNA was extracted using the E.Z.N.A. Insect DNA kit  following the manufacture's protocol. Total RNA was extracted using Triazol reagent  following the manufacturer's protocol. 4  μg of RNA was used for cDNA synthesis using SuperScript2.3pfu polymerase  in 25 μl reactions containing; 1  μl of cDNA or genomic DNA template, 10 pmol of each forward and reverse primer , and 0.25 μM of each primer  buffer, and total RNA was prepared from tissues of a single female worker as described above. Two μg of RNA was used for cDNA synthesis as described above. PCR reactions (15 μl) contained 4 μl of cDNA (diluted 1:10 v/v), 7.5 μl of SYBRh primer . Cyclingt method  in MS ext method  (Hornakot method . Data wa2.5B. terrestris CYP9Q6 and house fly NADPH-dependent cytochrome P450 reductase (CPR) (GenBank accession no. Q07994) were codon optimised for expression in lepidopteran cell lines, synthesised by GeneArt® , and inserted into the pDonor221 expression vector (Invitrogen). The recombinant baculovirus DNA was synthesised in vitro and transfected into Trichoplusia ni High five™ cells using the BaculoDirect C-term expression kit , according to the manufacturer's protocol. The titre of the recombinant virus was determined following protocols detailed by the supplier. High Five™ cells , grown to a density of 2 × 106 cells ml−1, were co-infected with recombinant baculoviruses containing CYP9Q6 and CPR with a range of MOI (multiplicity of infection) ratios to identify the optimal conditions. Control cells were co-infected with the baculovirus containing vector with no insert (ctrl-virus) and the recombinant baculovirus expressing CPR using the same MOI ratios. Ferric citrate and δ-aminolevulinic acid hydrochloride were added to a final concentration of 0.1  mM at the time of infection and 24 h after infection, to compensate for the low levels of endogenous heme in the insect cells. Cells were harvested, washed with PBS, and microsomes prepared according to standard procedures (ocedures  48 h posocedures . The pro2.6N-desmethyl acetamiprid) were monitored. The peak integrals were calibrated externally against a standard calibration curve. The linear range for the quantification of neonicotinoids and their hydroxylated (thiacloprid) and N-desmethylated (acetamiprid) metabolites was 0.1–1000  ng ml−1. Recovery rates of parent compounds using microsomal fractions without NADPH were normally close to 100%. All statistical calculations and substrate turnover from two independent reactions were plotted vs. Controls and Michaelis-Menten kinetics determined using GraphPad Prism version 7 .Metabolism of thiacloprid and acetamiprid were assessed by incubating recombinant P450/CPR (50μg/well) or control virus/CPR (50μg/well) with a range of substrate concentrations (200-3.1  μM) in the presence of an NADPH regeneration system  at 30 ± 1 °C, shaking, for 1 h. Three replicates were performed for each data point and the total assay volume was 200 μl. The breakdown rate was assayed exactly as described above with a single concentration of substrate (200 μM). Samples incubated without NADPH served as an additional control. The reactions were terminated by the addition of 800 μl of ice-cold acetonitrile, centrifuged for 10 min at 3000 g and the supernatant subsequently analysed by tandem mass spectrometry as described previously . For chr2.7Cyp9Q6 was amplified from B. terrestris genomic DNA and cloned into the attB-carrying plasmid pUAST (pUASTattB_EF362409) using the EcoRI and XbaI restriction sites. Injection mixes containing 0.5x phosphate buffer pH 6.8 , 300 ng/μl of the pUAST-Cyp9Q6 construct and 200  mg l−1 fluorescein sodium salt were delivered into pre-blastoderm embryos of an integration strain ZH-2A; P{CaryP}. attp40) sourced from the Fly Facility, University of Cambridge, using an inverted microscope  equipped with a 10x/0.25 lens, 10x/22 eyepiece and fluorescence illumination and a FemtoJet express micro-injector  controlled by a motorised TransferMan NK2 micromanipulator . Injection needles were prepared according to white gene, which generates flies with orange eyes. Homozygous stocks for the integration of pUAST-Cyp9Q6 at attP40 were generated by inter-crossing orange eyed flies and selecting for red eyed individuals. Control flies carrying an empty pUAST plasmid were generated following the same procedures described above.All Drosophila strains were maintained on standard fly food (Bloomington formulation) at 25 °C, on a 12/12 h light/dark cycle and 65% relative humidity. 2.8Cyp9Q6. Male flies, homozygous for pUAST-Cyp9Q6 (UAS::CYP9Q6) or an empty pUAST plasmid inserted at the same genomic location (UAS:empty), were crossed to virgin females of a heat-shock inducible GAL4 driver strain  to generate UAS::CYP9Q6/ + ;Hsp::GAL4/ + and UAS::empty/ + ;Hsp::GAL4/ + strains, respectively. Overexpression of Cyp9Q6 in UAS::CYP9Q6/ + ;Hsp::GAL4/ + flies was confirmed via qPCR with the UAS::CYP9Q6 parental strain used as a control for the relative comparisons. Reactions were setup as presented in section UAS::CYP9Q6/ + ;Hsp::GAL4/ + and UAS::empty/ + ;Hsp::GAL4/ + ) were selected and used in contact/feeding bioassays. 24 h prior to bioassays, flies were heat-shock treated three times for 30 min at 37 °C, with 1-h intervals, to induce the expression of Cyp9Q6. Groups of 20 flies were transferred to vials, previously coated with 100 μl of an insecticide dilution, containing 3 ml of a settled agar-sucrose solution. Each bioassay used five different insecticide concentrations, five replications per concentration and the number of dead flies was recorded after 48 h. Control mortality was assessed using vials containing agar-sucrose without insecticide. Lethal concentrations  and 95% confidence limits were calculated by probit analysis using Genstat version 16 . Statistical significance was calculated using two tailed t-test in GraphPadprism version 7 .The UAS/GAL4 system was used to drive the expression of 3A. mellifera had three complete CYP9Q genes while B. terrestris had only two (the previously characterised CYP9Q4 and CYP9Q5). To investigate if an additional CYP9Q gene was present in B. terrestris we performed an in silico re-analysis of P450 sequences in the B. terrestris genome.Phylogeny  of the C3.1XP_020718536.1 and XP_003403396.2) with high level homology to the CYP9Q sub-family were identified in the public protein database  and forms part of the same genomic locus. Sequencing of both the genomic and reversed transcribed mRNA (cDNA) amplicons further confirmed that this is a novel, full-length P450 ORF. No premature stop codons were found in either sequence suggesting that the gene is not a pseudogene and is likely to be functional. The protein sequence of the novel gene shares 62.6% and 61.5% identity with CYP9Q4 and CYP9Q5 respectively and 55.4% identity with A. mellifera CYP9Q3  in an insect cell line and microsomal preparations were incubated with each insecticide. Analysis of the resulting metabolites, by liquid chromatography tandem mass spectrometry (LC-MS/MS), revealed that recombinant CYP9Q6 readily metabolises both compounds in the presence of NADPH  and B. terrestris (CYP9Q5) which metabolise neonicotinoids with much lower efficiency, if at all, have the conserved threonine XT motif of the I-Helix, known as the oxygen binding motif, which is part of a substrate recognition site 4 . In CYP9hreonine . Furthera lugens . In thisa lugens . Finallysistance . Moleculsistance .Fig. 4PrMyzus persicae CYP6CY3  are current employees of Bayer AG, manufacturer of neonicotinoid insecticides. The study was partially funded by Bayer AG.


Lamium album L.  in providing relief from pain in rheumatism as well as vaginal and cervical inflammation was described. The aim of the study was to screen for the anti-inflammatory bioactivity of compounds isolated from aqueous-methanolic extract of Lamium album herb in human neutrophils (PMNs). The effect of the compounds on the inhibition of selected inflammatory markers released by neutrophils, such as cytokines , was studied. The molecular masses and the purity of compounds were determined using high-performance liquid chromatography coupled with diode array detection and mass spectrometry (HPLC-DAD-MSn). The level of cytokines production after incubation with the compounds (1–25 µM) was measured by ELISA. Two derivatives of quercetin, not previously described, were isolated in this study. Phenylpropanoids (verbascoside and phlinoside D), as well as iridoids , and flavonoids revealed to be more significant inhibitors of IL-8 secretion than TNF-α. The compounds at a concentration of 25 µM, except for shanzhiside methyl ester (6), inhibited secretion of IL-8 in the range from 29.1 to 50.0%. In conclusion, L. album might be a valuable source of bioactive compounds and may provide constituents to limit noninfectious inflammation associated with the aforementioned diseases.The traditional role of  Lamium album L.  in providing pain relief from rheumatism as well as vaginal and cervical inflammations was described pyranoid skeleton (iridane skeleton). The majority of iridoids occur as glucosides. The glycosidic linkage between the hydroxyl group of the anomeric carbon of D-glucose and the hydroxyl group in the Ctianales .cis-acteoside, and lamalboside (lamiuside A) were primarily reported in Lamium album L. − (m/z 623) in the negative ESI mode, whereas the main ions in the MS2 pattern in negative ESI mode were at m/z 461 and m/z 315. The typical ion m/z 461 was detected in phenylpropanoids such as verbascoside and echinacoside, fragmentation was due to the loss of a caffeoyl unit (∆m = 162) in the MS2 pattern in negative ESI mode. Subsequent loss of rhamnose residue (∆m = 146) provides the ion at m/z 315 in negative ionization mode − (m/z 769) in negative ESI mode, wheras the major MS2 ions in the negative ionization mode were [M − H − 132]− (m/z 637), [M − H − 162]− (m/z 607), and [M − H − 176]− (m/z 593) corresponding to the neutral losses of pentose, caffeoyl, and feruloyl residues, respectively. Compound 7 was finally identified as phlinoside D by comparing its 1H and 13C NMR spectra with the spectral data of phlinosides available in the literature [3 (Rt = 32.8 min) was [M − H]− (m/z 785), and the MS2 fragmentation pattern showed signals at m/z 623 [M − H − 162]−, m/z 609 [M − H − 176]−, and m/z 591 [M − H − 194]− due to the neutral losses of caffeoyl, feruloyl, and ferulic acid residues, respectively, in the negative ionization mode. These ions have been previously assigned to phenylpropanoid-like compounds. However, the MS2 fragmentation pattern of compound 3 as well as 8 (Rt = 30.7 min) showed signals at m/z 301 in the negative ionization mode, which is likely to be the ion of quercetin. The main ion of compound 8 in the MS spectrum was [M − H]− (m/z 931), and fragmentary ions showed signals at m/z 785 [M − H − 146]−, m/z 609 [M − H − 146 − 176]−, and m/z 301 [M − H − 146 − 176 − 162 − 146]−. All MS data were compared with previous reports of similar structures -β-glucopyranoside. The structure of compound 8 was quercetin 3-O-α-rhamnopyranosyl-(1→2)[(4′′′′-O-E-feruloyl)-α-rhamnopyranosyl-(1→6)]-β-glucopyranoside. Compounds 3 and 8 were identified as new compounds, which have not been described to date. The detailed 1H and 13C spectral data of compounds 3 and 8 have been provided in 5 (Rt = 10.1 min) and 6 (Rt = 14.8 min) to the class of iridoids. In addition, the compounds 5 and 6 were characterized by [M + HCOOH − H]− ions at m/z 467 and 451 in the negative ionization mode, respectively. The major ions from the MS2 fragmentation pattern were [M − H]− (m/z 421) and [M − H]− (m/z 405), respectively. In addition, the fragmentary peak m/z 259 of compound 5 was observed due to the direct loss of anhydroglucose (Δm = 162). On the other hand, fragmentary ions at m/z 283 [M − 122]− and m/z 225 [M − 162 − 18]− of compound 6 were formed due to the loss of the 4-carbonylethylen piran ring and anhydroglucose along with dehydration − m/z 4 and [M −1H NMR spectra with the spectral data available in the literature.In addition to MS data, the structures of compounds were confirmed by comparing their The isolated compounds in the concentration range of 1 to 25 µM did not influence PMNs viability . The popL. album were active inhibitors of inflammatory mediator secretion, in particular of chemokine secretion, such as IL-8 , 7 (53.7 ± 4.5%), and 3 (56.0 ± 8.1%) at the concentration of 25 µM compared with nontreated control cells (97.6 ± 5.0%). On the other hand, a score of IL-8 release by cells treated with verbascoside (4) and compound 8 was less relevant 60.8 ± 5.1% and 65.9 ± 4.9% of the control, respectively. The identified iridoid (5) expressed activity similar to verbascoside (4), 60.1 ± 5.1% of control. Compound 1 showed less relevant activity (70.9 ± 5.5% compared to LPS-treated cells) at the highest concentration. It is worth to note that compounds 1 and 2 displayed concentration-dependent activity. However, none of these compounds reached activity comparable with the positive control, dexamethasone .Most of the compounds isolated from the herb of  as IL-8 . All com2, 3, 4, 5, and 7 at the concentration of 25 µM significantly inhibited the secretion of cytokine TNF-α (5) and phlinoside D (7) at the concentration of 25 µM released 62.8 ± 7.2% and 66.0 ± 14.6% of TNF-α, respectively, compared to control cells (100.0 ± 7.3%). In addition, astragalin (2) and verbascoside (4) treated cells produced 71.8 ± 8.4% and 68.9 ± 7.6% of TNF-α, respectively, as well as the concentration-dependent activity observed in the case of astragalin (2) and verbascoside (4). The percentage of TNF-α secretion by cells treated with LPS and dexamethasone as the positive control was 18.5 ± 3.7% compared to LPS-treated cells.Compounds ne TNF-α . The cel2), at the concentration of 25 µM, significantly inhibited the oxidative burst in stimulated PMNs (3 and 8). The cells treated with compounds 3 and 8 generated 16.9 ± 1.2% and 10.0 ± 2.6% ROS, respectively, compared to control cells (104.5 ± 8.3%). These results were similar to the results obtained for quercetin (8.8 ± 1.8%) used as a positive control. Phlinoside D (21.0 ± 3.4% compared to f-MLP-treated cells) inhibited ROS production even more significantly than verbascoside (42.6 ± 4.2% compared to f-MLP treated cells). The percentage of ROS production by cells treated by iridoids such as lamalbid (5) and shanzhiside methyl ester (6) as well as the apigenin derivative (1) was 68.6 ± 5.2%, 45.7 ± 1.4%, and 42.6 ± 5.1%, respectively, compared to f-MLP-treated cells.All tested compounds, except for astragalin -glucoside (1), lamalbid (5), and phlinoside D (7), have been isolated previously; their biological activity has never been established according to our knowledge. Moreover, two new constituents of L. album herb, such as feruloyl glycosides of quercetin (3 and 8), have been isolated for the first time from the plant material, despite that their isomers are already known [In the presented study, the role of compounds isolated from extract of dy known . It is wNeutrophils, through the generation of oxidative burst and release of other antimicrobial substances, constitute the first line of defense against pathogens . Neverth2O2-nontreatement IL-8 secretion, but it was able to inhibit IL-8 secretion as well as NF-ĸB translocation in human colon adenocarcinoma in conditions of oxidative stress [L. album. In contrary to verbascoside, phlinoside D is characterized by the presence of feruloyl moiety instead of caffeoyl residue. We have previously established that trans-lamiuside E, which also possess two methoxyl groups in its structure, significantly inhibited IL-8 secretion. On the other hand, we made a conclusion that methoxy-phenylpropanoids might exert less relevant antioxidant activity [∙O2− production [L. album in the prevention of chronic diseases, along with its potential dietary supplementation, should not be neglected. On the other hand, the compounds, which were isolated for the first time and identified as quercetin feruloyl-glycosides, were compounds 3 and 8. In our study, it was shown that quercetin derivatives attenuated the secretion of inflammatory mediators, in particular IL-8, in PMNs. Additionally, they were the most active antioxidants of all tested compounds. The feruloyl moiety in the structures of these compounds, as well as phlinoside D, might contribute to their activity. Furthermore, the presence of quercetin core may enhance the antioxidant properties of compounds 3 and 8 as well as regulate IL-8 secretion in this pathway.One of the most relevant known phenylpropanoid glycosides is verbascoside (acteoside). In vitro and in vivo studies, including the intestinal inflammation model, demonstrating the antioxidant and anti-inflammatory activities of verbascoside have been previously reported. It was suggested that verbascoside shows direct and indirect antioxidant activity. Additionally, it decreases NOS activity and reduces NF-ĸB activation and nuclear translocation leading to the decrease of concentration of pro-inflammatory mediators ,34. Verbe stress . It is we stress . Howeveractivity . In the oduction . Therefo−1 (corresponding to 296 µM), inhibited ROS generation by 57%, but did not affect iNOS and NF-ĸB activity [Many bioactivities, including antioxidant, anti-inflammatory, immunomodulating, and antimicrobial, were assigned to the iridoids ,37,38,39activity . In our 6). On the other hand, their contribution is likely to support the activity of other constituents of L. album in term of its properties.Last but not least, the activity of identified iridoids should not be neglected. Due to their different biosynthetic pathway their structures are not closely related with phenylpropanoids or flavonoids. Hydroxyl groups linked with a cyclopentan ring are found in their structures. However, we are not able to point out the direct structure–activity relationship of iridoids. Their antioxidant activity does not seem to affect cytokine secretion as it was noted in the case of shaznhiside methyl ester (3), diethyl ether (DE), ethyl acetate (EA), ethanol, and methanol (MeOH) for extraction were obtained from POCH. HEPES, l-glutamine, luminol, and formyl-met-leu-phenylalanine were purchased from Sigma-Aldrich . Lipopolysaccharide (LPS from Escherichia coli 0111:B4) was purchased from Merck . Human ELISA sets (IL-8 and TNF-α) and propidium iodide were obtained from BD Biosciences. Phosphate buffered saline (PBS) was purchased from Gibco . Penicillin–streptomycin were obtained from PAA. Hanks’ balanced salt solution (HBSS), RPMI 1640 medium, and fetal bovine serum (FBS), as well as formic acid eluent additive for UHPLC-MS, were purchased from Sigma-Aldrich . Water was obtained using water purification system MILLIPORE Simfilter Simplicity UV.Acetonitrile (MeCN) of gradient grade for UHPLC, butanol (BuOH), chloroform . A specimen (No FW25_20140425_LA) of aerial parts is available in the herbarium of the Department of Pharmacognosy and Molecular Basis of Phytotherapy, Medical University of Warsaw. The plant material   in an ultrasonic bath. The organic solvent was evaporated under reduced pressure at 40 °C. Next, the aqueous methanol residue was partitioned between chloroform (5 × 1 L), diethyl ether (3 × 1 L), ethyl acetate (5 × 1 L), and n-butanol saturated with water (5 × 1 L), every time in ratio 1:1 of residue and solvent. The obtained fractions, such as DE (2.5 g), EA (7.4 g), and BuOH (47.3 g) were evaporated to dryness under vacuum and fractionated as follows. DE and EA residue was fractionated over silica gel  using a CHCl3 gradient in EtOAc (100–0%), and a CHCl3 gradient in MeOH (0–100%) as an eluent, respectively. BuOH residue was fractioned over DIAION (6 × 20 cm) using a MeOH gradient in water (20–100%) as an eluent. The obtained fractions were pooled into six (DE1-DE6), nine (EA1-EA9), and five (BuOH1-BuOH5) main fractions based on their TLC profile , derivatized with 1% Natural Product Reagent .Isolation of compounds: 1370 g of powdered herb of 18—5 mm, 150 mm × 21.2 mm, Agilent , 280 nm, flow 20 mL·min−1, mobile phase: 0.1% HCOOH in water (A) and 0.1% HCOOH in acetonitrile (B); elution program: 0% B–40% B (0–60 min), 40% B–50% B (60–65 min). Fractions were collected based on UV–Vis chromatograms to give pure compounds 1 .Fraction DE6 (640 mg) was subjected to preparative HPLC  and 3 . Fraction EA8 (1.1 g) was subjected to column chromatography on Sephadex LH-20 (2.5 cm × 130 cm) eluted with MeOH  to give 144 fractions combined to 11 main fractions EA81-EA811 based on TLC profile , derivatized with 1% Natural Product Reagent, Carl Roth). Fraction EA85 (195.8 mg) was subjected to preparative HPLC (instrumentation and gradient elution as described above). Fractions were collected based on UV–Vis chromatograms to yield pure compound 4 .Fraction EA6 (1.2 g) was subjected to preparative HPLC (instrumentation and gradient elution as described above). Fractions were collected based on UV–Vis chromatograms to give pure compounds v/v), to give each 144 fractions combined to six (BuOH21-BuOH26) and seven (BuOH41-BuOH47) main fractions, respectively, based on TLC profile , derivatized with 1% Natural Product Reagent, Carl Roth, Karlsruhe, Germany). Next, fraction BuOH21 (530 mg) was subjected to preparative HPLC (instrumentation and gradient elution as described above). Fractions were collected based on UV–Vis chromatograms to yield pure compound 5 . Fractions BuOH41 (2.0 g), BuOH42 (0.4 g), and BuOH43 (0.9 g) were subjected to column chromatography on Sephadex LH-20 (2.5 cm × 130 cm) eluted with MeOH . Finally, using preparative HPLC (instrumentation and gradient elution as described above) pure compounds were obtained as follows, from fraction BuOH41B (944 mg): 6 , from fraction BuOH42C (1 g): compound 7 , and from fraction BuOH43C (160 mg): compound 8 .Fractions BuOH2 (4.0 g) and BuOH4 (9.4 g) were subjected to column chromatography on Sephadex LH-20 (2.5 cm × 130 cm), eluted with MeOH  and Bruker AVANCE 500 MHz spectrometers  (for compounds 3 and 8). Structures of the isolated compounds have been determined based on UV–Vis, MS, and n analysis was performed on a UHPLC-3000 RS system  with DAD detection and an AmaZon SL ion trap mass spectrometer with ESI interface . Separation was performed on a Zorbax SB-C18 column . The column temperature was 25 °C. For preliminary phytochemical analysis of extracts the mobile phase (A) was 0.1% HCOOH in water and the mobile phase (B) was 0.1% HCOOH in acetonitrile, as well as a linear gradient system was used: 0–60 min 5–60% B. The flow rate was 0.2 mL·min−1. The column was equilibrated for 10 min between injections. UV spectra were recorded over a range of 200 to 450 nm, chromatograms were acquired at 240, 280, 325, and 350 nm. The LC eluate was introduced directly into the ESI interface without splitting. The nebulizer pressure was 40 psi; dry gas flow 9 L·min−1; dry temperature 300 °C; and capillary voltage 4.5 kV. Analysis was carried out using scan from m/z 200 to 2,200. Compounds were analyzed in negative ion mode. The MS2 fragmentation was obtained for the most abundant ion at the time.HPLC-DAD-MSBuffy coats were obtained from healthy adult volunteers (<35 years old) from the Warsaw Blood Donation Centre. Donors were recognized as healthy according to medical history and routine laboratory test. Donors declared that they were nonsmokers and were not taking medication. They were clinically confirmed to be healthy, and a routine laboratory test showed values within normal range. Neutrophils were isolated by dextran sedimentation and centrifugation in a Ficoll Hypaque gradient . Erythrocytes were removed by hypotonic lysis. The purity of neutrophils preparation was >97%. Following isolation, the cells were suspended in an appropriate medium, such as RPMI 1640 medium and were maintained at 4 °C before use .6 in RPMI 1640 medium) with the tested compounds at concentration range from 1 to 25 µM they were centrifuged at 2000 rpm for 10 min at 4 °C. PMNs were washed twice with PBS and resuspended in 500 μL of PBS containing 5 μL of propidium iodide  and left for 15 min in the dark. The cells were analyzed by flow cytometry FACSCalibur (Becton Dickinson), and data from 10,000 events were recorded. Cells that displayed high permeability to propidium iodide were expressed as a percentage of PI (+) cells. The results of the viability were acquired according to an equation: 100% − PI (+) cells%. TritonX was used as a positive control.Neutrophils cytotoxicity was also assessed by flow cytometry with the propidium iodide assay. After 24-h incubation of neutrophils (2 × 106) were cultured in a 24-well plate in RPMI 1640 medium with 10% FBS, 10 mM HEPES, and 2 mM l-glutamine for 24 h at 37 °C with 5% CO2 in the absence or presence of compounds at concentrations of 25 µM added 30 min before the stimulation with LPS . The TNF-α and IL-8 released into cell supernatants was measured by enzyme-linked immunosorbent assay (ELISA) following the indications of the manufacturer. The effect on TNF-α and IL-8 production was calculated as the percentage of released agent in comparison with stimulated control without tested extract. Dexamethasone (Dex) was used as a positive control.Neutrophils (2 × 105) was incubated with 50 μL of compounds at concentrations 25 µM, and luminol (20 mM) in a 96-well plate. The ROS production was initiated by the addition of f-MLP (0.1 μg·mL−1) to obtain a total volume of 200 μL per well. Changes in chemiluminescence were measured over a 40 min period at intervals of 2 min using the microplate reader . Background chemiluminescence produced by nonstimulated cells was also determined. The percentage of inhibition was calculated in comparison to the control without tested extract in the maximum of luminescence. Quercetin was used as a positive control.The ROS production by f-MLP -stimulated neutrophils was determined using luminol-dependent chemiluminescence, respectively. Following isolation, cells were resuspended in HBSS. Cell suspension .The results were expressed as a means ± S.E.M. Statistical significance of differences between means was established by ANOVA with Tukey’s L. album revealed that phenylpropanoid glycosides along with iridoids and flavonoids might be the valuable bioactive compounds present in this species. In particular, the feruloyl and caffeoyl moieties, as well as quercetin core in the described structures, are likely to affect the signaling pathways of the inflammatory response. Thus, the antioxidant activity of these compounds probably influence cytokines secretion to some extent. Our study allowed us to make the conclusion that L. album might be a valuable source of bioactive compounds and might provide the constituents required to limit noninfectious inflammation associated with diseases such as cardiovascular disorders and diabetes type 2. Additionally, the synergistic or additive activity of the constituents of this plant material should not be excluded. However, further investigation is required to support the traditional and nutritional significance of L. album.In conclusion, the screening of activity of compounds isolated from herb of


Glioblastoma multiform (GBM) tumors are very heterogeneous, organized in a hierarchical pattern, including cancer stem cells (CSC), and are responsible for development, maintenance, and cancer relapse. Therefore, it is relevant to establish new GBM cell lines with CSC characteristics to develop new treatments. A new human GBM cell line, named R2J, was established from the cerebro-spinal fluid (CSF) of a patient affected by GBM with leptomeningeal metastasis. R2J cells exhibits an abnormal karyotype and form self-renewable spheres in a serum-free medium. Original tumor, R2J, cultured in monolayer (2D) and in spheres showed a persistence expression of CD44, CD56 (except in monolayer), EGFR, Ki67, Nestin, and vimentin. The R2J cell line is tumorigenic and possesses CSC properties. We tested in vitro the anticancer effects of sodium selenite (SS) compared to temozolomide TMZ. SS was absorbed by R2J cells, was cytotoxic, induced an oxidative stress, and arrested cell growth in G2M before inducing both necrosis and apoptosis via caspase-3. SS also modified dimethyl-histone-3-lysine-9 (H3K9m2) levels and decreased histone deacetylase (HDAC) activity, suggesting anti-invasiveness potential. This study highlights the value of this new GBM cell line for preclinical modeling of clinically relevant, patient specific GBM and opens a therapeutic window to test SS to target resistant and recurrent GBM. Glioblastoma represents the most common type of primary tumors of the central nervous system (CNS) and has a poor prognosis . Recent Recent findings have identified a subpopulation of stem-like cells within tumors, known as cancer stem cells (CSC), exhibiting characteristics of both stem cells and cancer cells. In addition to self-renewal and differentiation capacities, CSCs have the ability to seed tumors when transplanted into the animal host ,8. ConceEpigenetic modifications are implied in GBM progression . HistoneTemozolomide (TMZ) is the standard of care for GBM, but it still has limitations and unsatisfactory outcomes, particularly in patients with tumor cells expressing the O(6)-methyguanine-DNA-methyltransferase (MGMT) ,23. MoreIn this way, we study sodium selenite’s (SS) anticancer properties. We have previously showed that the chemical form and doses influenced its ways of actions ,25 and wThe susceptibility of the cultures to the effects of SS depends on the cell type. Indeed, several authors showed that SS was preferentially toxic to malignant glioma cells over normal astrocytes. For example, Rooprai et al.  found anThis last point is of great relevance for GBM therapy as crossing the blood brain barrier (BBB) is one of the challenge of new drugs: SS crosses the BBB .https://clinicaltrials.gov/ct2/show/study/NCT01959438), is ongoing. The phase I has allowed determination of the maximal tolerated dose (MTD) as 10.2 mg/m² (phase I). The aim of the phase II, which is in progress, is to use MTD and to study responses, if any, in malignant tumor and in treatment resistant tumors.Anti-cancer properties of sodium selenite in vitro are so encouraging that SS deserves to be deeply studied for chemotherapeutic responses, before being given up. The clinical trial, SECAR , 10% of the cells were round, giant (30–50 µm), and multinucleated (2–5 nuclei), and less than 20% of the cells exhibited fibroblastic- or glial-like morphology c. CytospR2J cells were able to form colonies with a PE = 12.4% ± 3.1 (PE = (counted colonies/cells plated) × 100).n = 3 independent experiments).When R2J are plated in a cell repellent 10 cm-dish, small spheres containing 10–20 cells were formed 5–6 days after the plating. The sphere diameter increased each day and tumor spheres can be passaged every week for many generations in fresh medium without serum d. The peCompared to the original tumor, R2J cells in culture (2D and spheres) lost the GFAP and CD56 expressions (only 2D) whereas Ki67, vimentin and nestin expressions were conserved as well as mesenchymal shift markers, such as CD44 , Table 1Comparing 2D vs. spheres, it appears that only olig2 and CD56 were expressed in spheres. E-Cad transcript was tardily detected in RT-q-PCR (Ct = 37.1 ± 0.9) and the protein was not detected . Concernn = three independent experiments). U251 cell line was used as a negative control for MGMT status (no Ct) and T98G was used as a positive control with Ct = 26.11 ± 0.04 (n = three independent experiments).R2J cells expressed MGMT transcript  with a cycle threshold (Ct) value=34.8 ± 4.1  and in spheres  were tumor bearing  vs. 3.7% ± 1.3 at 10 µM (p < 0.005 vs. 2.5 µM) for n = three independent experiments.Se was measured by Inductively Coupled Plasma Mass Spectrometry ICP-MS both in lysates and medium in R2J-2D cells treated with SS. The quantity of Se absorbed significantly increased with the SS concentration added. Indeed, at 2.5 µM, the percentage of Se measured vs. Se added was 0.6% ± 0.2 vs. 2.8% ± 0.7 at 5 µM (p < 0.0001 vs. 2.5 µM) and vs. 79.9% ± 7.0 at 10 µM (p < 0.0001 vs. 2.5 µM) for n = three independent experiments. It means that a loss of Se depended on the concentration added.Se recovery was 102.7% ± 1.1 at 2.5 µM vs. 83.2%±4.1 at 5 µM  and significantly at 5 µM (p = 0.0318)  b.Oxidative stress was evaluated by quantifying thiol groups concentration in R2J and U251 cells, grown in the monolayer, after 24 h of SS treatment. SS significantly decreased thiol groups in both cell lines whereas TMZ did not a.Western-blots showed that SS at 2 µM and TMZ at 100 µM repressed autophagy with an increase of p62 and LC3-II both in R2J and in U251 cells and induced a reticulum stress with an increase of Grp78 only in R2J cells b. It shoDespite the great efforts made to fight against GBM and the advent of TMZ as the first line CT agent available for long term application, the prognosis of patient suffering for GBM remains poor . One proIn vitro tests are essential to raise these questions with the development of new cancer cell lines with CSC characteristics. Their individual phenotypic and/or genotypic characteristics provides a valuable means for this search ,35,36. TThere is a diversity of cellular phenotypes within the R2J-2D cell population that may reflect mixtures of progenitors, differentiated cells, and presumed cancer stem cells. Indeed, our data supports that R2J cells are derived from a tumor containing multipotent neural stem cell-like cells where a population of CSC persists. Indeed, R2J cells expressed Nestin and CD44, which is different from other usual reported GBM MGMTt+ cell lines, like T98G . R2J celThe most prevalent form of glioma is referred to as astrocytoma, based on the predominance of GFAP+ astrocytes-like cells within the tumor mass. GFAP is then used as an astrocytic lineage-specific marker . The oriProliferation may be altered by cellular signaling pathways modifications related to growth factor receptor expressions ,3. EGFR,The xenograft of R2J cells cultivated in 2D or in spheres resulted in the formation of a detectable tumor within 14 days post implantation into the striatum of nude mice. These results allowed conclusions on the tumorigenicity of this cell line and on its CSC properties . Indeed,As a first conclusion, although numerous GBM cell lines have been published, few of them have been established from tumor cells expressing MGMT transcript and having resistance to TMZ. Indeed, T98G is p53 mutation, has a positive MGMT status, and is resistant to TMZ . Only U2In this line, as we reported SS-anticancer effects in GBM cell lines cultivated in 2D  and in cSS was cytotoxic in the micromolar range and much more toxic than TMZ, for which the IC50 was not reached at a higher dose and after long term treatment. This is consistent with the fact that the patient did not respond to TMZ that may be correlated with a MGMT transcript positive expression. Similar results were obtained in the T98G cell line, also positive for MGMT transcript expression , which eR2J cells absorbed SS, and an uncomplete recovery suggest the generation of volatiles Se metabolites, as reported in prostate, colon, and Jurkat cell lines . The SS SS metabolism generated Se intermediates, which are also responsible for SS toxicity, such as selenide, selenocystine, and elemental Se . OxidatiTo sustain an oxidative stress way of action and subsequent pathways, we showed that SS depleted thiol group concentration, in both R2J (p53 wild–type) and U251 (p53 mutant) cells, contrary to TMZ. Indeed, reactive oxygen species ROS production is dependent on the reduction of selenite to selenide through the consumption of thiols . It was Epigenetic regulation of chromatin organization is a key regulator of gene expression that can significantly impact tumor biology and tumor response to therapies. In many tumor types, mutated or modified epigenetic regulators have been clearly associated to tumorigenesis because of their effects on gene expression (reviewed in ), and thIn October 2009, the patient  felt faint and lost consciousness. Magnetic resonance imaging (MRI) showed a right tempo extensive process compatible with a GBM. In November 2009, the tumor was resected and histological analysis confirmed the diagnosis of GBM with a leptomeningeal extension . After tInstead, the patient received weekly intrathecal injections of Trastuzumab because CSF glioma cells overexpressed HER2. In May 2010, symptoms and the leptomeningeal extension decreased, but the patient presented a local tumor progression on the MRI. So, the patient was treated with radiotherapy plus concomitant TMZ. In August 2010, because of tumor progression and leptomeningeal carcinomatosis progression, no more treatment was compatible with the patient’s health condition and he received standard palliative care. The patient died in October 2010.The tumor cells were isolated from the CSF of the patient (November 2009). The analysis of CSF showed numerous tumor cells with a high mitotic index, indicating a leptomeningeal carcinomatosis. These cells were cultured and the derived R2J cell line was successfully cryopreserved at different passages.The primary biopsy  was processed for histopathologic study using standard methods and sections were stained with hematoxylin and eosin safran (HES).R2J cells from the CSF were isolated by cytocentrifugation  and stained using May Grunwald Giemsa (MGG) using a Zeiss microscope . Pictures were taken using a Canon Power Shot S50 digital camera .U251 cells were purchased from the American Type Culture Collection ATCC. They were cultured in DMEM medium containing 10% fetal calf serum (FCS) supplemented with penicillin (100 IU/mL), streptomycin  and L-Glutamine (2mM) , in humidified normoxia incubator. U251 were harvested using 0.5%-Trypsin-EDTA (10×) . U251 was chosen due to its MGMT negative and p53 mutant status.2, 5% CO2, 37 °C). R2J were harvested as described for U251.R2J were cultured in RPMI 1640 medium enriched as described for U251, in humidified hypoxia incubator  coated with PolyHema  in DMEM-F12 medium  without serum containing 20 ng/mL human EGF, FGF-2, and 1× NeuroBrew-21 , and PS (as above) in hypoxia. To conform with already published methods, the cells were plated at low density (10 per µL) to allow clonal sphere formation ,54.PolyHema was prepared at 10 mg/mL in ethanol 95% and sterilized by filtration on a Stericup . To coat the 10 cm Petri-dish, 4mL of this solution was applied. The dishes were put in a dry incubator overnight, at 60 °C to dry the PolyHema.Embedded paraffin tumor was analyzed in 2009 for CD56, Ki67, GFAP, NeuN, NF70, and synaptophysin, according to the current procedure of the laboratory. The Bouin’s fluid fixative used at this time is not compatible with the current methods of the laboratory and the immuno-histo staining obtained for some markers  was unsatisfactory except for EGFR, CD44, CD34, and Nestin, performed after 2009.® plus kit  and Vector novaRED™ substrate kit for peroxidase or Ventana Benchmark® XT platform . Only nestin was revealed using Fast Red Bond Polymer Red Refine Detection .R2J cells cultured in 2D or in spheres were fixed in formol 4%, 30 min before being included in HistogelTM  and paraffin embedded. HES and IHC analyses were performed from 3 µm paraffin sections using a HistostainCD-56 (#MAB24081) and Olig2 (#AF2418) antibodies are from R&D Systems , MIB1/KI67 (#M7240), p53 (#M7001) and Vimentin (#M0725) were from Dako , GFAP , EGFR (#28-0005), E-Cadherin (#18-0223), and MDM2 (#33-7100) were from Zymed Lab, , IDH1 , Nestin (#MAB5326), and NeuN (#MAB377) were from Zymed Lab , ATRX (#HPA001906) and CD44 (#144M-95) were from Sigma Aldrich, P16 , Neurofilament 70-200 , and CD34 .R2J-2D cells were seeded at 6000 cell/cm² in 10 cm Petri-dishes 48 h before treatment with SS (Sigma) for 24 h or 48 h, after which cells and medium were recovered, centrifuged , and cells were rinsed twice with PBS1X.Apoptosis and the cell-cycle were evaluated with the FITC-Annexin V Apoptosis Detection kit and the Cycle Test Plus DNA reagent kit  following the manufacturer’s instructions. The analysis of cells in the subG1 phase was used to determine DNA fragmentation.6 cells were fixed 30 min in PFA 1% on ice and then permeabilized with 0.1% Triton × 100 in PBS, 30 min on ice. R2J cells were washed in PBS/BSA 1% twice before adding 1 µL of the rabbit monoclonal antibody, anti-H3K9m2 , 45 min. The H3K9m2 level was revealed using a (FITC)-conjugated F(ab)′2 fragment goat anti-rabbit IgG(H+L) (Beckman Coulter), 30 min, 4 °C.For dimethyl-Histone H3-Lysine-9 (H3K9m2) analysis, 10Cell fluorescence was detected with FACSCantoII (Becton Dickinson) and analysed with FACS Diva Software.The chromosomes were analyzed according to the International System for Human Cytogenetic Nomenclature. Culture was exposed to 10 µg/mL colchicine for 60 min. Slides were processed for R-binding using heat and the Giemsa-RHG method .An LDA was performed to quantify in R2J cell lines, the frequency of tumor spheres initiation cells (TS-ICs). R2J parental cells were calibrated to 1000 cells/mL (corresponding to 200 cells/200 µL/well) and then diluted into gradients of cell titers at 500, 250, 125, 62, 100, 50, 25, and 12.5 /mL and transferred into the wells of the 96-well microplate. Clonal spheres  were counted under an inverted microscope , at the end of two weeks. The results are expressed as the percentage of TS-ICs to examine the efficacy of clonogenesis .5) or spheres (2 × 105 or 1000) were implanted into the striatum of 4–6 week male athymicNudeFoxn1nu mice  to analyze the tumor initiating capacity. Experimental details are available in All animal protocols were approved by the GIN Animal Care and Use Committee (number 2017121517085709). R2J-2D cells . Acquisition duration was 9 min 20 s. Tumor volume was calculated by adding each tumor surface × slice thickness.MRI sessions were performed at 4.7 T  using an actively decoupled cross-coil setup . All MR experiments were performed under anaesthesia: 5% isoflurane for induction and 2% for maintenance in air. Mice were maintained at 37.0 °C and breath rate was cared for 60 breath/min throughout the acquisition. After shimming, MRI sequences were designed for anatomical T2-weighted (T2W) images acquisition using a spin-echo MRI sequence  20 × 20 mmR2J-2D cells were seeded at 6000 cell/cm² in 10 cm Petri-dishes, 24 h before SS supplementation .78Se/71Ga and an external calibration curve  allowed the determination of the Se concentration. The results (mean ± SD) were calculated in nmoles of Se per gram of protein and expressed as a percentage of the Se measured vs. Se added. The Se recovery was calculated by adding the quantity of Se both measured in the medium and in the lysates and compared to the Se quantity added (referred as 100%).Se concentration was determined both in cell lysates and medium using an ICP-MS  equipped with Collision Cell interference elimination Technology (CCT) after dilution in 1% nitric acid containing Gallium (Ga) as an internal standard. The gas introduced in the CTT was a mix of He and H2. The isotope ratio 5 cells/well were seeded in 96-wells plate 24h before the addition of SS. The results (mean ± SD) are reported as the cell survival percentage versus the control (not treated). IC50 (concentrations inducing 50% cell death) values were determined by curve-fitting .A MTT -2,5-Diphenyltetrazolium Bromide) assay (Sigma) was performed to determine R2J-2D cell survival after SS treatment at 2.5 to 12 µM for 24 h, 48 h, or 72 h. 1 × 10In parallel, TMZ  was dissolved in dimethyl sulfoxide (DMSO) and R2J cells were treated for 24 h, 48 h, and 72 h with 1, 10, 100, 500, and 1000 µM of TMZ. Cells were also treated with DMSO alone at the corresponding volume. A long term treatment was also performed by exposing R2J-2D cells to 400 µM TMZ for 72 h followed by a wash-out of 72 h. Results are expressed as the percentage of cell survival versus control (DMSO concentrations used were not cytotoxic).2 incubator, 37 °C, 20 min. After staining, cells were installed on the microscope incubation chamber with a controlled atmosphere at 37 °C and 5% CO2 . Images were collected with a Leica TCS SP2 AOBS  inverted laser scanning confocal microscope equipped with an ×63 oil immersion objective (HCX PL APO 63.0× 1.40). Laser excitation and emission (adjusted with AOBS) were, respectively, 351–364/425–485 nm for Hoechst, 488/500–540 nm for FDA, and 543/600–650 nm for PI. Confocal pinhole (Airy units) was 1 for all channels. Each experiment was performed on a randomly chosen field. Raw image merging was obtained by LCS Lite software .R2J-2D cells were set on a Lab-Tek-Chamber Slide system  and were incubated with 5 µM of Fluorescein Diacetate , 1µM Hoechst 33342 , and 10mg/mL Propidium Iodide (PI) (Interchim) in a 5% COThe caspase-3 Fluorometric Assay  was used. R2J-2D cells were harvested and lysed into 50 μL of chilled Cell Lysis Buffer at 4 °C, 10 min, and centrifuged for 10 min, 10000 g at 4 °C. Protein concentration was determined (see below) and 50µL of supernatant containing 50 µg to 200 µg of protein was mixed with 50 µL of 2× reaction buffer and 5 μL of DEVD-AFC substrate (1 mM), in a 96-wells plate. Reaction was achieved for 1h at 37 °C. The plate was read on a fluorometer (Ex/Em = 400/505 nm). Caspase-3 activity is expressed in AU/g prot.R2J-2D cells were treated for 24 h, 48 h, and 72 h with 2.5 and 5 µM SS and harvested. Pellets were rinsed twice in PBS and lysed in 400 µL sterile water, 10 min. HDAC activity was investigated in cell lysates from 50 µg total protein using a Fluorometric Assay  as described by the manufacturer. HDAC activity is expressed in relative fluorescence units (RFU) per µg protein.Total RNA extraction was performed with an RNeasy Mini Kit, following the manufacturer’s recommendations, with RNase-free DNase I treatment . cDNA was reverse transcribed from 1 µg of total RNA with the SuperScriptIII First-Strand Synthesis (Life Technologies) followed by RNaseH (1 µL) treatment. Real time PCR was conducted using the QuantiTect SYBR Green RT-PCR kit (Qiagen) and the Stratagene 3005MxPro . The primers (sequences in Gene expression was quantified using the comparative threshold cycle (Ct) method  with a nThiol groups were determined by a colorimetric method using the reducing properties of the SH groups as previously described . The thiU251 and R2J, after 72 h treatment followed by 72 h of wash-out with TMZ or SS or chloroquine , were harvested in protein extraction buffer . After pipetting up and down with a P1000, supernatants were collected after centrifugation at 10,000 g for 10 min, at 4 °C. Protein concentration was measured by the BCA method. Proteins  were treated with SDS-PAGE sample buffer , boiled for 5 min at 95 °C, and loaded on 15%  and 8% (for Grp78) acrylamide/bisacrylamide gels. Proteins were transferred onto nitrocellulose membranes, blocked with 5% bovine serum albumin containing 0.5% Tween (Euromedex) (PBST) for 1 h at room temperature, and probed overnight at 4 °C with the following primary antibodies diluted in 5% bovine serum albumin-PBST: 1:1000 rabbit anti-p62 antibody (Sigma-Aldrich), 1:200 goat anti-Grp78 antibody , 1:1000 anti-LC3-II antibody, 1:1000 rabbit anti-phospho-H2Ax antibody , and 1:5000 mouse anti-alpha tubulin (Sigma-Aldrich). Membranes were washed with PBST, incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (from Jackson Immunoresearch), and visualized using electrochemiluminescence (ECL) western blotting substrate (ThermoFisher Scientific) on a Chemidoc imaging system .The concentration of protein in cell lysates (obtained as mentioned above) was determined using the BCA assay  as previously described . The abs® . The sources of variation for multiple comparisons were assessed by analysis of variance ANOVA. The differences were considered statistically significant at p < 0.05 versus the control.Results are expressed as means ± SD for the number of experiments indicated. All statistical analysis of data was computed using StatViewIn conclusion, we described a newly established glioblastoma cell line (R2J), from a CSF from a patient affected by a metastatic GBM. R2J were MGMT+ and resistant to TMZ. In the presence of FGFb and EGF, the R2J forming spheres were phenocopied on 2D cultured R2J cells except for CD56, a neuronal marker, suggesting that R2J may differentiate into neuronal-like cells. The cell line could form a tumor when intracranially implanted into nude mice even at 1000 spheres, providing conclusions on their CSC properties. In vitro, SS could exert its anti-cancer properties in R2J cells, and is thus relevant for testing in this new GBM bearing mouse model.


We demonstrated the strong mutual influence of the chemical compositions of magnetic fillers (Ba(Fe1−xGax)12O19 0.01 < x < 0.1 solid solutions), and the main magnetic  and microwave (resonant frequency and amplitude) characteristics of functional composites with 30 wt.% of hexaferrite. The paper presents a correlation between the chemical compositions of composites and amplitude–frequency characteristics. Increase of Ga-content from x = 0 to 0.1 in Ba(Fe1−xGax)12O19/epoxy composites leads to increase of the resonant frequency from 51 to 54 GHz and absorption amplitude from −1.5 to −10.5 dB/mm. The ability to control the electromagnetic properties in these types of composites opens great prospects for their practical applications due to high absorption efficiency, and lower cost in comparison with pure ceramics oxides.The paper describes preparation features of functional composites based on ferrites, such as “Ba(Fe Barium hexaferrite BaFe12O19 (BaM) is the first compound of this type which has been studied in detail and is now widely used. It has a high value of saturation magnetization (Ms) of 72 emu/g (1 emu/g = 1 (A × m2)/kg) and a high Curie temperature of 450 °C. BaM shows large magnetocrystalline anisotropy, about 17 kOe along the c-axis [c-axis, and therefore, these materials can be used as microwave EMR absorbers [3+, In3+, Ga3+, Co2+ and Sc3+ makes it possible to adapt the magnetic characteristics, and consequently, to control the range of operating frequencies up to 70 GHz [1−xDIx)12O19 demonstrate, depending on the substitution type and ratio, a change in the frequency and maximum absorption (associated with the NFMR) owing to a corresponding change in magnetocrystalline anisotropy (MCA).One of the major tasks in creating modern mobile communication devices is the development of new materials which can work in a wide up to 100 GHz) frequency range in switches, circulators, phase shifters, transceivers, antennas and effective electromagnetic radiation (EMR) absorbers, which improve the electromagnetic compatibilities of devices 00 GHz fr,3,4,5. Mbsorbers ,12,13. Do 70 GHz ,19,20. Ao 70 GHz , the tra1−xDIx)12O19 powders into the polymer matrix opens up new application possibilities of such composites [The inclusion of Ba12O19/epoxy) composites in the frequency range up to 67 GHz. Moreover, the correlation between the concentration x of Ga diamagnetic ions (0 < x < 0.1) and the electromagnetic properties of the composites was established.Previously, we demonstrated results about the magnetic and microwave properties of the Ga-substituted hexaferrites as bulk (ceramics). That study focused on principally new objects—composites based on Ga hexaferrite powders dispersed in epoxy. We examined the electromagnetic properties and microwave absorbing characteristics in Ga-substituted barium hexaferrite with epoxy (Ba(Fe1−xGax)12O19 0 < x < 0.1 powders we investigated were obtained from high purity oxides Fe2O3, Ga2O3 and carbonate BaCO3 via two-step solid state reactions. The precursor manufacturer was Xiamen Ditai Chemicals Co., Ltd . The oxides and carbonate were mixed in the design ratio. Then, the annealing was performed for 6 h at 1200 °C in air. The final synthesis was carried out during 6 h at 1300 °C in air. The samples were slowly cooled after the synthesis (100 °C/h) [The Ba(Fe00 °C/h) .1−xGax)12O19/epoxy CMs with 30 wt.% of Ba(Fe1−xGax)12O19 were obtained by mixing in solution with additional sonication. The manufacturing process was as follows. At first, an appropriate amount of L285 epoxy resin was pre-dissolved in acetone. Further, BaFe12−xGaxO19 was introduced into solution and sonicated in a BAKU ultrasonic bath for 1 h with 40 kHz frequency and 50 W power. Finally, curing agent H285 was added (40% by weight of L285). To complete the polymerization, one day after the CMs were prepared, they were heated for 5 h at a temperature gradually increased from 40 to 80 °C.Epoxy-based composites (CMs) containing the Ga-substituted hexaferrites in low-viscosity epoxy  with an appropriate cross-linking agent (hardener) H285, were produced for this study. Namely, Ba(Fe1−xGax)12O19 filler powders and the samples of CMs were investigated by X-ray diffraction, which was carried out using a DRON-4-07 X-ray diffractometer  with Co Kα filtered radiation (λ = 1.7902 A) at room temperature.The crystal structures of Ba(Fe1−xGax)12O19 powders and Ba(Fe1−xGax)12O19/epoxy composites was characterized by Mira 3 Tescan scanning electron microscope (SEM) .The morphology of Ba(Fe11) and transmission coefficient (S21) of each composite was converted into material shielding efficiency (SET), reflection coefficient (SER) and coefficient of absorption (SEA) according to the equations:Keysight PNA N5227A vector network analyzer  was used to determine the electromagnetic parameters of the composites in the frequency range of 1–67 GHz by the transmission line method. Full, two-port calibration was initially performed on the test setup to remove errors due to the directivity, source match, load match, isolation and frequency response of each of the forward and reverse measurements. The coaxial measuring cell  had the 3.The magnetic properties (field dependencies of the magnetization) were determined using a universal cryogenic high-field measuring system (Liquid Helium Free High Field Measuring System (B14T) by Cryogenic Ltd., London, UK) at the temperature of 300 K in external magnetic fields up to 5 T (field magnetization curve). The size of each sample for magnetic measurements was 5.2 × 2.6 × 2.6 mm1−xGax)12O19 (0 ≤ x ≤ 0.1) powders measured at the room temperature. The calculated positions of reflections for BaFe12O19 , it is visible from the XRD pattern . It is the closest angle to the position calculated for (112) reflection of Fe2O3, but szs strongly biased in comparison with it. Intense reflection is also observed at 2θ = 43.440, which does not coincide with the available data calculated for BaFe12O19. Perhaps the reflection at 43.44° is a (222) reflection for Fe3O4, but this is doubtful, since the most intense line for Fe3O4 should be (311) at 2θ = 41.46°, and this was not observed for our sample. The reflection with the position of 2θ = 67.59° can be identified as (511) for Fe3O4, but its intensity is also lower than the expected tabulated values. Therefore, both Fe3O4 and Fe2O3 phases in the studied sample, if any, were estimated to have a small amount.Some fairly intense reflections do not correspond to the data calculated for barium hexaferrite. One of the most intense reflections of that sample was observed at 2θ = 38.40° (between (107) and (114) BaFe1−xGax)12O19 (0.01 ≤ x ≤ 0.1), it is clear from the XRD patterns  reflection of BaFe12O19 phase in Ga-substituted samples became the most intense instead of the (114) reflection of unsubstituted BaFe12O19 (see data in 12O19 (between (107) and (114) BaFe12O19 reflections) was not observed for all Ga-substituted samples.As for the Ga-substituted samples of barium hexaferrite Ba and (114) reflections, which correspond to the inclined 1−xGax)12O19 (0 ≤ x ≤ 0.1) was determined using the well-known Scherrer formula from the line broadening of the diffraction profile of the strongest peaks of (107) and (114) planes:Co = 1.790263 Å), h1/2 = Full width at half maximum—FWHM (in radians) and θ is the position of the reflection (in radians). The average size of the crystallites in the Ba(Fe1−xGax)12O19 (0 ≤ x ≤ 0.1) powders (using the FWHM of (107) and (114) reflection) was calculated by Equation (4). The values are presented in The average crystallite size in Ba(Fe1−xGax)12O19 (0 ≤ x ≤ 0.1) was estimated to be about 33–37 nm and it depended on the content x of Ga in Ba(Fe1−xGax)12O19 (0 < x < 0.1) (see 1−xGax)12O19 (0 ≤ x ≤ 0.1) crystallites were single-domain which correlates well with data for BaM are 460 nm [1−xGax)12O19 (x = 0.1)/epoxy composite is shown in 1−xGax)12O19 (x = 0.1) which means that this composite contained crystalline barium hexaferrite. The preponderance of crystalline peaks of BaM was attributed to its encapsulation by epoxy resin. The relative intensity of 30 wt.% Ba(Fe1−xGax)12O19/epoxy composite is weakened compared to pure BaM powder. Again, using the above Scherrer formula with the line broadening of the diffraction profile of the strongest peaks of the planes (107) and (114), the average crystallite size of 30 wt.% Ba(Fe1−xGax)12O19/epoxy composite was estimated at about 35–36 nm which is consistent with the data obtained for Ba(Fe1−xGax)12O19 (0.01 ≤ x ≤ 0.1) powders.The average crystallite size of Ba(Fe0.1) see . Therefoe 460 nm . The XRD1−xGax)12O19 (0.01 ≤ x ≤ 0.1) fillers. The samples are densely packed polycrystals (>98%).x = 0.01, and 52.4% of the crystallites were from 0.740 μm to 0.860 μm. Grains with sizes smaller than 0.170 μm or larger than 1.400 μm were not detected. The precise value of the average crystallite size of 〈D〉 ≈ 0.873 μm for x = 0.01 was obtained from quantitative stereological analysis. The average crystallite size increased to ≈ 950 nm with an increase in the substitution coefficient to x = 0.1 [The change in grain size is more significant for samples with low Ga concentration. The crystallites combine and form an entire ceramic. A certain dispersion of particle sizes was characteristic for all samples. This is in good agreement with our previous data: the grain size variation interval was between 0.223 and 1.279 μm for  x = 0.1 .1−xGax)12O19/epoxy (x = 0.1) composite at different magnifications. It can be seen that individual particles and aggregates of x = 0.1 are coated with epoxy in different positions. As shown in x = 0.1 and their aggregates were found in the epoxy matrix. The aggregates are 10–20 μm in size, but there are many globules with a typical size of about 50 μm. These globules mainly have a regular spherical form. SEM analysis shows a slightly disturbed uniform distribution of ferrite globules in the polymer matrix of 30 wt.% Ba(Fe1−xGax)12O19 (x = 0.1)/epoxy composite samples.1−xGax)12O19 (0 ≤ x ≤ 0.1). Here, we present the magnetic properties of epoxy-based composites, namely, 30 wt.% Ba(Fe1−xGax)12O19/epoxy (0 ≤ x ≤ 0.1) with random distribution of the filler. Typical M–H field magnetization curves of the composites at room temperature are shown in S), residual magnetization (Mr), saturation magnetic field (Hsat) and coercivity (HC) are given in We discussed in ,32 the m1−xGax)12O19/epoxy composites shows clear hysteresis behavior. Such magnetization field dependence is a characteristic for all investigated 30 wt.% Ba(Fe1−xGax)12O19/epoxy composites as a consequence of magnetic response of the Ba(Fe1−xGax)12O19 component. It is assumed that the total magnetization of the composite is formed only due to Ba(Fe1−xGax)12O19 filler, as epoxy is non-magnetic. As can be seen from the data in C increases monotonically from 0.060 to 0.166 T with an increase of Ga content in the filler while the saturation magnetization MS decreases with Ga content increase (S was observed for 30 wt.% Ba(Fe1−xGax)12O19/epoxy composite. One can see that MS changes from 20.31 to 14.70 emu/g for composites with different contents of Ga (0 ≤ x ≤ 0.1) in the filler. The values of HC, Mr and MS almost coincide for the magnetic moment measured in both orientations of 30 wt.% Ba(Fe1−xGax)12O19/epoxy composites samples. It is assumed that small deviations of the measured magnetic moment in this case indicate heterogeneity of the filler distribution in the epoxy matrix. The dependencies of Ha (x), HC (x) and MS (x) of Ba(Fe1−xGax)12O19 (0 ≤ x ≤ 0.1) powders and 30 wt.% Ba(Fe1−xGax)12O19/epoxy composites (0 ≤ x ≤ 0.1) are presented for comparison in The magnetization field dependence for 30 wt.% Ba decreases with increasing Ga content x in the filler for both materials investigated, but the value of MS is higher for Ba(Fe1−xGax)12O19/epoxy (0 ≤ x ≤ 0.1) composites. As it was shown in our previous paper [1−xGax)12O19 (0 ≤ x ≤ 0.1) polycrystalline samples indicates a decrease in the maximum magnetic energy with an increase in the concentration of Ga3+ cations and the absence of abrupt anomalies. It means a decrease in deviations from the linear dependence of magnetic energy with increasing substitution ratio, which testifies to the hypothesis of a statistical distribution of Ga3+ cations between different nonequivalent crystallographic positions in the M-type barium hexaferrite structure. The polymer coating on magnetic particles obviously affects the contributions of the surface anisotropy, shape anisotropy and interface anisotropy to the total anisotropy [1−xGax)12O19/epoxy composites are higher than the corresponding parameters of pure Ba(Fe1−xGax)12O19 (0 ≤ x ≤ 0.1) polycrystalline samples, while the shape of MS (x) dependencies remains unchanged. C of Ba(Fe1−xGax)12O19 polycrystals with low content of Ga (x = 0.01) notably decreases from 2.1 kOe to 0.6 kOe when they are embedded into the polymer matrix of the composite. A monotonic increase in HC is observed with increasing content of Ga in Ba(Fe1−xGax)12O19/epoxy composites.As seen in us paper , such beisotropy ,37. The 1−xGax)12O19/epoxy composites is relatively small, which may be due to the prevalence of domain rotation in the high-field region. The relationship between M and H in this region is called the “law of approach to saturation” and is usually written as [S is the saturation magnetization of the domains. The term ΘH represents the field-induced increase in the saturation magnetization of the domains, or forced magnetization; this term is usually small at temperatures well below the Curie point and may often be neglected. Constant A is generally interpreted as a result of inclusions and/or microstress, and B is due to magnetocrystalline anisotropy. The magnetization data in a field range of about 3 kOe were plotted against 1/H2 in [The magnetization-field dependence for 30 wt.% Ba12O19/epoxy composites (0 ≤ x ≤ 0.1) were calculated using Equations (7) and (8), and the results are presented in The anisotropy field H1−xGax)12O19/epoxy composites (0 ≤ x ≤ 0.1) were recorded in millimeter wave range and are shown in T) are quite small up to frequencies of 40–45 GHz.Transmittance spectra of 30 wt.% Ba12O19/epoxy composites (0 ≤ x ≤ 0.1) was the absorption of electromagnetic waves in the materials. The most intensive EMR absorption was observed in 40–50 GHz frequency range for all the above-mentioned composite samples. The NFMR in barium hexaferrite powders provides high EMR absorption in the indicated frequency range. The transmission value at the global minimum of microwave transmission spectrum determines the resonant transmission Ares. The frequency in the global minimum of microwave transmission spectrum determines the resonant transmission frequency fres. The global minimum width, measured at Ares/2, i.e., half of the resonant transmission value, determines the width of the absorption band Wres—the bandwidth. One can see in x. The NFMR frequency fres was measured at the half of the bandwidth Wres/2 and the results are presented in res in Ba(Fe1−xGax)12O19 ferrite filler. fres in unsubstituted BaFe12O19 is near 50 GHz and can be calculated as [As seen in lated as :fres = γres for 30 wt.% Ba(Fe1−xGax)12O19/epoxy composites (0 ≤ x ≤ 0.1) was evaluated and the values are also presented in res values determined from DC magnetization measurements are in a rather good agreement with fres values determined by microwave measurements.Here γ is the gyromagnetic ratio. Using the above-stated experimental results on the anisotropy field and saturation magnetization, the NFMR frequency f1−xGax)12O19 (0 ≤ x ≤ 0.1) powders (see 1−xGax)12O19 (0 ≤ x ≤ 0.1) powders is non-monotonic and demonstrates the minimum at x = 0.05. As one can see, encapsulation of Ba(Fe1−xGax)12O19 particles in epoxy leads to the resonance frequency increase for 30 wt.% Ba(Fe1−xGax)12O19/epoxy composites in comparison with pure Ba(Fe1−xGax)12O19 (0 ≤ x ≤ 0.1) powders and it essentially changes the shape of fres(x) dependencies.As it was shown in our previous publications ,32, the ders see . Howeverres values in 30 wt.% Ba(Fe1−xGax)12O19/epoxy composites (0.01 ≤ x ≤ 0.1) in comparison with fres in Ba(Fe1−xGax)12O19 (0 ≤ x ≤ 0.1) powders could be explained using the results of magnetic measurements.Higher f12O19/epoxy polymer composite magnetic properties has shown [res towards higher frequencies.As our previous research on BaFeas shown , encapsuA is in direct proportion to the sample thickness t and can be expressed by the following equation [T is total electrical conductivity which is composed of frequency dependent and independent components; µ is the permeability.It is known that the magnitude of the absorption coefficient SEequation :SEA = −81−xGax)12O19/epoxy composites (0 ≤ x ≤ 0.1), the adjusted value of the absorption coefficient SEA/t was introduced, and the frequency dependencies of SEA/t are presented in So, in order to evaluate the influence of the Ga content in hexaferrite fillers on the absorption spectra of 30 wt.% Ba12O19. These dependencies are presented in These data were used for Ares decreases monotonically with an increase in the substituent concentration of Ga3+. It is obvious that peculiar properties of Ba(Fe1−xGax)12O19 determined the frequency dependence of the EMR absorption process in the Ba(Fe1−xGax)12O19/epoxy composites.As shown in a is the crystalline anisotropy contribution and△Hp is the porosity induced line broadening contribution. Karim et al. in [a and relates to the crystalline anisotropy. Parameter △Hp1.5(4πMS)P accounts for porosity (P) induced linewidth broadening contributions [In polycrystalline ferrites, the total NFMR linewidth △H depends crucially on the superposition of intrinsic and extrinsic contributions :△H = △Hit al. in  speculatibutions ,44.res broadening in pure Ba(Fe1−xGax)12O19 powders with different contents of Ga is played by static inhomogeneities, such as impurity cations, which lead to increases in the magnetocrystalline anisotropy; and Wres values increase monotonically, increasing the substitution ratios. As the samples were obtained at the same time and using identical technology, they had identical morphologies in terms of their crystallites. However, it can be supposed that P does not appreciably differ in the samples with different x and the changes of P are negligible. Ares and Wres increases monotonically with an increase in x in the 30 wt.% Ba(Fe1−xGax)12O19/epoxy composites. However, the values of ΔH were greater than those reported for BaFe12−xGaxO19, which can be explained by a random distribution of the anisotropy axes in the crystallites and by an increase of porosity and the random orientations of crystallites themselves [res variation from point to point within the material, in turn, broadening the resonance line.The main role in Wemselves ,47,48,491−xGax)12O19 (0.01 ≤ x ≤ 0.1) grains using the Scherrer formula showed that the Ba(Fe1−xGax)12O19 crystallites we investigated (not grains) are single domain, and the crystallite size remains unchanged in composites with Ba(Fe1−xGax)12O19 filler. Encapsulation of Ga-substituted barium hexaferrite particles in the epoxy matrix allowed us to prepare microwave absorbing composites. The magnetization-field dependence measurements showed that coercive force HC of Ba(Fe1−xGax)12O19/epoxy composites increases monotonically from 0.060 to 0.166 T, while the saturation magnetization MS decreases with increasing Ga content in filler. The values of magnetic parameters of Ba(Fe1−xGax)12O19/epoxy composites were higher than that of pure Ba(Fe1−xGax)12O19 polycrystalline samples, while the shape of MS (x) dependencies remained unchanged. Calculation of anisotropy field Ha and anisotropy constant K1 for 30 wt.% Ba(Fe1−xGax)12O19/epoxy composites was carried out. Studies of Ba(Fe1−xGax)12O19/epoxy composites with different substitution ratio of gallium filler (0.01 ≤ x ≤ 0.1) showed a noticeable effect of Ga content on the microwave characteristics. The most intensive EMR absorption was observed in the 49–54 GHz frequency range for all composites tested, which was attributed to the effect of NFMR in Ba(Fe1−xGax)12O19 hexaferrites. Higher resonance frequencies of 30 wt.% Ba(Fe1−xGax)12O19/epoxy composites (0.01 ≤ x ≤ 0.1) in comparison with resonance frequencies of Ba(Fe1−xGax)12O19 (0.01 ≤ x ≤ 0.1) powders could be explained by a decrease in the saturation magnetization of the magnetic particles due to their encapsulation in the epoxy which affects the contributions of the surface anisotropy, the shape anisotropy and the interface anisotropy to the net anisotropy. The absorption band Wres decreases monotonically with an increase in Ga concentration in the hexaferrite filler while the resonant amplitude Ares increases.Ga-substituted barium hexaferrite powders were synthesized via two-step solid state reactions and characterized by XRD and SEM methods. Calculations of the crystallite size of Ba(Fe