Interferon-γ (IFN-γ), a cytokine with neuromodulatory properties, has been shown to enhance inhibitory transmission. Because early inhibitory neurotransmission sculpts functional neuronal circuits, its developmental alteration may have grave consequences. Here, we investigated the acute effects of IFN-γ on γ-amino-butyric acid (GABA)ergic currents in layer 5 pyramidal neurons of the somatosensory cortex of rats at the end of the first postnatal week, a period of GABA-dependent cortical maturation. IFN-γ acutely increased the frequency and amplitude of spontaneous/miniature inhibitory postsynaptic currents (s/mIPSC), and this could not be reversed within 30 min. Neither the increase in amplitude nor frequency of IPSCs was due to upregulated interneuron excitability as revealed by current clamp recordings of layer 5 interneurons labeled with VGAT-Venus in transgenic rats. As we previously reported in more mature animals, IPSC amplitude increase upon IFN-γ activity was dependent on postsynaptic protein kinase C (PKC), indicating a similar activating mechanism. Unlike augmented IPSC amplitude, however, we did not consistently observe an increased IPSC frequency in our previous studies on more mature animals. Focusing on increased IPSC frequency, we have now identified a different activating mechanism—one that is independent of postsynaptic PKC but is dependent on inducible nitric oxide synthase (iNOS) and soluble guanylate cyclase (sGC). In addition, IFN-γ shifted short-term synaptic plasticity toward facilitation as revealed by a paired-pulse paradigm. The latter change in presynaptic function was not reproduced by the application of a nitric oxide donor. Functionally, IFN-γ-mediated alterations in GABAergic transmission overall constrained early neocortical activity in a partly nitric oxide–dependent manner as revealed by microelectrode array field recordings in brain slices analyzed with a spike-sorting algorithm. In summary, with IFN-γ-induced, NO-dependent augmentation of spontaneous GABA release, we have here identified a mechanism by which inflammation in the central nervous system (CNS) plausibly modulates neuronal development. IFN-γ, the only type-II IFN, is a pleiotropic inflammatory cytokine and contributes to immune defense against viruses, bacteria, and tumors . As a paBeyond its canonical role in controlling intracellular pathogens, a growing body of evidence suggests a direct impact of IFN-γ on the CNS. The absence of IFN-γ leads to neurodevelopmental abnormalities  that difIh, but unlike type-I IFNs (A channel number in a protein kinase C (PKC)-dependent manner  activation  during ongoing GABAergic synaptogenesis in rats . We mainE. coli-derived recombinant IFN-γ  was reconstituted in double distilled water, aliquoted to 1.0 e5 IU, and stored at −20°C. Aliquots were thawed directly prior to usage. Final concentration for all experiments was 1,000 IU ml–1.Chinese hamster ovary or For all experiments, Wistar pups of either sex were used on postnatal days 6 and 7 (P6–7). All experiments were carried out in agreement with the European Communities Council Directive of 22 September 2010 (2010/63/EU) under the licenses T 0212/14, T-CH 0034/20 for wild-type rats, and T0215/11 for transgenic rats. For some experiments, we used W-Tg(Slc32a1-YFP*)1Yyan vesicular GABA transporter (VGAT)-Venus transgenic rats that express the yellow fluorescent protein Venus in VGAT expressing neurons, developed by 2; 5% CO2) 2°C sucrose artificial cerebrospinal fluid (sACSF) containing (in mM): 85 NaCl, 2.5 KCl, 1 NaH2PO4, 7 MgCl2, 26 NaHCO3, 50 sucrose, 10 D(+)-glucose , and 0.5 CaCl2 . 300 μM thick coronary slice containing somatosensory cortex were cut using a VT1200S vibratome . Slices were recovered for 30 min in 34°C sACSF and were subsequently held at room temperature in modified ACSF containing (in mM): 92 NaCl, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 25 D(+)-glucose (Carl Roth), 5 sodium ascorbate, 20 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES), 3 sodium pyruvate, 2 MgSO4, 2 CaCl2 (Merck), and 2 thiourea  until recording. All variations of ACSF were continuously carbogenated.As generally described in 2PO4, 1.3 MgCl2, 26 NaHCO3, 10 D(+)-glucose (Carl Roth), and 2.5 CaCl2 (Merck) at 32 ± 2°C. Slices were visualized using infrared differential interference contrast (DIC) microscopy with a Zeiss Axioskop 2 FS plus . Glass micropipettes (2–5 MΩ) were pulled from lead  or borosilicate  glass capillaries using a P-97 micropipette puller . For IPSC recordings, 6-cyano-7-nitroquinoxaline-2,3-dione  and D(-)-2-Amino-5-phosphonopentanoic acid   were added to the perfusate to block glutamatergic ion currents and pipettes were filled with a high chloride internal solution containing (in mM): 140 CsCl , 4 NaCl, 1 MgCl2 (Carl Roth), 10 HEPES, 0.1 ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 0.3 GTP, 2 Mg2+–ATP (Merck), , and 5 mM QX-314 (Merck) to improve the signal-to-noise ratio. Calphostin C  was dissolved in DMSO at 1 mM and aliquoted to working sizes. Prior to respective experiments, Calphostin C was added to the pipette solution to a final concentration of 100 nM. When Calphostin C was used, slice and pipette were continuously exposed to full-spectrum white light (S-Nitroso-N-acetylpenicillamine (SNAP) , 1H-oxadiazoloquinoxalin-1-one (ODQ) (Tocris), tetrodotoxin (TTX) (Tocris), and N-[3(Aminomethyl)benzyl] acetamidine dihydrochloride (1400W) . ODQ was dissolved in dimethylsulfoxide (DMSO) at 100 mM and aliquoted to working sizes. Total DMSO in perfusate never exceeded 0.1%. All other reagents and drugs, unless specified otherwise, were dissolved and aliquoted in double distilled water.Slices were transferred to a submerged type recording chamber continuously perfused with carbogenated ACSF containing (in mM): 119 NaCl, 2.5 KCl, 1 NaHte light . Other d2, 2 Mg2+-ATP, 0.3 GTP, 10 KCl (Merck), and 1 CaCl2 (Carl Roth). After establishing whole-cell configuration, pipette solution was allowed to diffuse into the cell, and blockers were bath applied for at least 5 min before recording started. Spontaneous/miniature inhibitory postsynaptic current (s/mIPSC) recordings were excluded from analysis when series resistance (Rs) changed by more than 25%, initial IPSC frequency was below 10 events min–1 (0.167 events s–1), holding current . Spontaneous and miniature IPSCs were analyzed using Synaptosoft MiniAnalysis Software . Negative deflections deviating from baseline by more than 3 RMS (root mean square) noise were counted as IPSCs. Upon manual review, erroneously detected events were removed and missed events were added. For every data point, events from two consecutive minutes of recordings were averaged. For washout experiments, slices were incubated in ACSF containing IFN-γ, CNQX (20 μM), and DAP-5 (25 μM) at 34°C for 30 min. IPSCs were recorded under continuous perfusion of the same solution until stable recording conditions were achieved. Then, IFN-γ was removed from the perfusion. Electrically evoked orthodromic IPSCs have been rarely demonstrated before P14 . For every datapoint, 20 traces were analyzed by dividing the amplitude of the second-evoked IPSC through the amplitude of the first. Of 20 ratios, the mean ratio was calculated for comparison. Current-clamp recordings were analyzed using FitMaster software for spike detection and counting. Calculations requiring polynomial fitting were performed using OriginPro 2022 . Neurons recorded in voltage clamp had a mean capacitance of Cbefore = 118.0 ± 3.4 pF that slightly decreased to Cafter = 112.0 ± 3.2 pF  over the course of voltage clamp recordings.For current-clamp recordings, internal solution contained (in mM) 120 K-Gluconate, 11 EGTA, 10 HEPES, 10 phosphocreatine, 1 MgClfore P14 . In thisfore P14 . To avoivia fluorescence. To validate correct cell selection, in a subset of recordings, Nε -(+)-Biotinyl-L-lysine (biocytin)  was added to the pipette solution to a final concentration of 1%. After recording, slices were fixed in cold phosphate-buffered saline with 4% paraformaldehyde for 1 h. Then, slices were washed in 0.1 M phosphate buffer and subsequently incubated in 4°C phosphate buffer with 0.3% Triton-X (Sigma-Aldrich), 0.05% NaN3 (Merck), and fluorescent-conjugated streptavidin  for 48 h. Following another washing step, slices were mounted and imaged using a laser scanning confocal microscope .For cell selection, pyramidal neurons were identified by their typical morphology in DIC microscopy, VGAT expressing interneurons could be identified –1), maintained at 30°C by the Pt100 Temperature Controller System (MCS). In all recordings, the K+-channel blocker 4-Aminopyridine  was used to enhance activity. Recordings were started after 5 min of stabilization and lasted 30 min, under control and test condition, respectively. Electrical activity was recorded by the MEA 2100-120 system  and sampled at 10 kHz using Multi Channel Experimenter software (MCS). Given the electrode distance of 200 μm, it is unlikely but possible that events cross the detection threshold on multiple channels. Event sorting was performed with the spyKing circus algorithm , consisting of 60 electrodes in an 8 × 8 layout with electrode diameters of 10 μM, and 200 μM spacing , Reutlingen, Germany). Slices, prepared as described above, were placed in the recording chamber, so that somatosensory cortex covered the electrodes. The recording chamber was briefly drained to ensure contact between slice surface and electrodes. Slices were secured by steel anchors  and were continually perfused with carbogenated ACSF (4–6 ml minlgorithm  to avoidp ≤ 0.05), the Wilcoxon signed-rank test for paired data was used to test for statistical significance. Probability distributions were tested for equality using the Kolmogorov–Smirnov test. Due to low event counts, data for all recordings under a certain experimental condition were pooled for probability distributions. Total number of events analyzed, number of recordings, and number of animals are given in the respective figure legends. When normal distribution could not be rejected, Student’s t-test or paired Student’s t-test was used, respectively. The null hypothesis was rejected for p ≤ 0.05. All data are given in dvariable = mean ± standard error of the mean , unless otherwise specified.The study was designed to enable longitudinal pairwise comparison. Note that a direct comparison of IPSC frequencies between different neurons is hampered by the huge variance of the frequency of GABAergic inputs at this developmental stage that might be due to staggered maturation, and many other factors . Images of biocytin-filled neurons were adjusted  with Fiji software . Final a–1) increased the mean amplitude of sIPSCs at P6–7 from Ictrl = 25.6 ± 2.2 pA to IIFN–γ = 36.1 ± 6.7 pA . We then addressed whether these relatively acute effects ceased upon removal of IFN-γ. We found that, when preincubated with IFN-γ for at least 30 min, sIPSC frequency and amplitude remained stable for at least 30 min during washout  and amplitude , remained comparable , rheobase, lowest current capable of eliciting a single action potential, Irheobase ctrl = 83.7 ± 4.9 pA vs. Irheobase IFN–γ = 86.3 ± 9.7 pA , slope of the frequency–current relationship FI-slopectrl = 358.0 ± 44.5 Hz nA–1 vs. FI-slopeIFN–γ = 362.2 ± 40.9 Hz nA–1  and frequency at 2 × rheobase f2xRB ctrl = 28.8 ± 3.7 Hz vs. f2xRB IFN–γ = 31.1 ± 4.0 Hz . Notably, FI-slope and firing frequency at 2 × rheobase are parameters that reflect a neuron’s input–output relationship  and amplitude increased from Ictrl = 23.5 ± 2.8 pA to IIFN–γ = 25.7 ± 3.4 pA  . However, the cumulative probability distribution reveals a slight shift toward shorter rise times  . It rema, N = 8) , give inse times . BecauseTaken together, we have found that the IFN-γ-induced increase in sIPSC frequency appears independent of and cannot be attributed to an increase in layer 5 interneuron excitability or presynaptic firing.A receptors via a PKC-dependent mechanism .We have previously reported that IFN-γ augments GABAergic currents by promoting postsynaptic membrane association of GABAechanism  in late These findings suggest that the observed IFN-γ-induced IPSC amplitude increase at the end of the first postnatal week is both postsynaptic and PKC mediated, as previously described for older stages . In addiIFN-γ induces nitric oxide (NO) production . NO is afctrl = 2.79 ± 0.36 events s–1 to fSNAP 3.32 ± 0.43 events s–1 , while sIPSC amplitude remained comparable .To test whether NO is sufficient to induce an increase in inhibitory release at P6–7, we recorded sIPSCs and added SNAP (300 μM), an NO releasing agent widely used to study the impact of NO on neurophysiology . Within channels . In our via binding to its direct receptor sGC . The IFN-γ induced increase in sIPSC amplitudes from IODQ ctrl = 25.5 ± 2.2 to IODQ IFN–γ = 30.8 ± 2.6 pA , however, persisted under ODQ application  , whereas sIPSC frequency remained comparable  . To assee (iNOS) . We obsef 7.3 μM , concentf 7.3 μM .In summary, these data indicate that IFN-γ increases presynaptic GABA release in an iNOS and sGC-dependent fashion.PPR100msctrl = 0.9 ± 0.1 to PPR100msIFN–γ = 1.3 ± 0.1, p = 0.005, paired t-test, n = 9, N = 6 and from PPR50msctrl = 1.0 ± 0.2 to PPR50msIFN–γ = 1.3 ± 0.2, p = 0.02, paired t-test, n = 9, N = 6; r = −0.46, p = 0.04. Using normalized amplitudes, we then pooled all experiments in each group. Pooled analysis showed no correlation between first and second eIPSC amplitudes . Subsequently, only four recordings matched our usual Rs constraints for absolute amplitude comparison in long-term recordings. Amplitudes remained comparable in a preliminary analysis of absolute amplitude values of evoked potentials in these , we slightly released the restrictions on Rin these . This isin these . MoreovePPR100msctrl = 1.0 ± 0.1 vs. PPR100msSNAP = 1.0 ± 0.1, p = 0.8, paired t-test, n = 9, N = 3 and PPR50msctrl = 0.9 ± 0.1 vs. PPR50msSNAP = 0.9 ± 0.1, p = 0.8, paired t-test, n = 9, N = 3; To test whether NO might contribute to IFN-γ-induced paired-pulse facilitation, we recorded another series using the paired-pulse paradigm and observed whether NO donor SNAP has an effect on PPR. SNAP (300 μM), applied for 5 min, did not alter the PPR at 100 or 50 ms ISI   to the effect. Additionally, IFN-γ alters presynaptic function by shifting paired-pulse ratios toward facilitation. In contrast to the effect of IFN-γ on IPSCs, the effect on short-term synaptic plasticity was not dependent on NO. Functionally, IFN-γ constrains network activity in a GABA- and partially NO-dependent manner.Combining measurements at the single cell and network level, we herein have found that, during a period of GABA-dependent cortical maturation, IFN-γ acutely increases the frequency and amplitude of GABAergic IPSCs on neocortical pyramidal neurons. This effect was not reversible within 30 min. The increase in IPSC frequency is not a result of enhanced interneuron excitability, since interneuronal sub- and suprathreshold excitability parameters remained stable, and elevated IPSC frequency was still present under action potential suppression. We have identified iNOS and sGC as mediators of increased IPSC frequency upon IFN-γ, plausibly acting through elevated levels of NO. Notably, manipulations of the iNOS–NO–sGC pathway revealed a differential effect on frequency and amplitude, i.e., a specificity of NO for the putatively pre-synaptically mediated frequency increase. Together with the apparent separation of the IFN-γ effects on the frequency and amplitude of IPSCs, the cessation of IPSC amplitude increases when PKC was blocked postsynaptically argues against an increased GABAA receptor number as an underlying mechanism mainly by peak-scaled non-stationary noise analysis  may elevate IPSC frequency Ca2+ independently  in the boutons of early postnatal interneurons  signaling during cortical development can lead to persisting changes . BalanceThe neuroimmune interaction we describe here might have clinical implications as altered GABAergic transmission is associated with neurodevelopmental disorders . Even inOur findings shed light on the acute effects of IFN-γ on early GABAergic transmission thus providing a mechanism for how early inflammation could interfere with cortical development. This might have far-reaching consequences because functional disturbances in early microcircuits lead to aberrant cortical wiring at later stages that may cause neurological or psychiatric conditions .The original contributions presented in this study are included in the article/The animal study was reviewed and approved by the European Communities Council Directive of September 22nd, 2010 (2010/63/EU) under the licenses T 0212/14, T-CH 0034/20 for wildtype rats and T0215/11 for transgenic rats.post hoc stainings. AF supervised, analyzed, and contributed to MEA recordings. GJ, EB, and US contributed to patch clamp recordings. All authors contributed to the article and approved the submitted version.ND and US conceptualized the study and wrote the manuscript. ND performed and analyzed patch clamp recordings and


Banisteriopsis caapi is used to prepare the psychoactive beverage ayahuasca, and both have therapeutic potential for the treatment of many central nervous system (CNS) conditions. This study aimed to isolate new bioactive compounds from B. caapi extract and evaluate their biological activity, and that of the known β-carboline components of the plant , in BV-2 microglial cells, the in vivo activation of which is implicated in the physiopathology of CNS disorders. B. caapi extract was fractionated using semipreparative liquid chromatography (HPLC-DAD) and the exact masses ([M + H]+m/z) of the compounds in the 5 isolated fractions were determined by high-resolution LC-MS/MS: F1 (174.0918 and 233.1289), F2 (353.1722), F3 (304.3001), F4 (188.1081), and F5 (205.0785). Harmine (75.5–302 µM) significantly decreased cell viability after 2 h of treatment and increased the number of necrotic cells and production of reactive oxygen species at equal or lower concentrations after 24 h. F4 did not impact viability but was also cytotoxic after 24 h. Most treatments reduced proinflammatory cytokine production , especially harmaline and F5 at 2.5 µM and higher concentrations, tetrahydroharmine (9.3 µM and higher), and F5 (10.7 µM and higher). The results suggest that the compounds found in B. caapi extract have anti-inflammatory potential that could be explored for the development of treatments for neurodegenerative diseases. Psychotria viridis and Banisteriopsis caapi. Its use by Amazonian natives dates to ancient times and, more recently, it has been used by Christian and shamanic groups [B. caapi to treat central nervous system (CNS) disorders, including depression and posttraumatic stress disorder [B. caapi play a major role in the biological activity of the beverage. They inhibit DYRK1A  in cultured neurons, an enzyme that is involved in the pathophysiology of several neurodegenerative diseases [Ayahuasca is a beverage with psychoactive and medicinal properties prepared mainly by decoction of the plants c groups . Variousdisorder ,4,5,6,7,disorder ,10,11,12disorder ,14,15, adisorder . The β-cdiseases ,17; incrdiseases ,19; and diseases .B. caapi are harmine, harmaline, and tetrahydroharmine (THH)  . Other mB. caapi . DespiteOne of the many cell types in the brain are the nervous system-specific immune cells known as microglia, which work as tissue-resident macrophages . MicroglActivated microglia are traditionally categorized into two groups based on the displayed phenotype: classically activated M1 and alternatively activated M2. The M1 phenotype is characterized by proinflammatory activity due to the production of mediators, such as interleukin (IL)-6, IL-1β, tumor necrosis factor (TNF), and interferon gamma (IFN-γ). M2 microglia are regarded as anti-inflammatory, based on the secretion of anti-inflammatory factors, including IL-4, IL-13, IL-10, and transforming growth factor (TGF)-β; they contribute to recovery after injury and suppress the proinflammatory response ,31. ThusB. caapi and its components have been demonstrated, more studies are necessary to elucidate the mechanisms underlying such effects. In this study, the cytotoxicity and anti-inflammatory activity of B. caapi extract, selected ß-carbolines , and new plant components were investigated using BV-2 microglial cells.Although some neuromodulator properties of Analytical standards of harmine (98% purity) and harmaline (95% purity) were obtained from Sigma-Aldrich  and tetrahydroharmine  from LGC Standards . Methanol, acetonitrile, and acetic acid, all HPLC grade, were purchased from Merck ; analytical-grade ammonium acetate was purchased from Neon ; and formic acid was purchased from Sigma-Aldrich .Banisteriopsis caapi sample  acetic acid (B). The gradient was applied as follows: 14 to 22% B 0–15 min; 22 to 25% B 15–20 min; 25 to 100% B 20–21 min; 100 to 14% B 21–26 min; 14% B 26–40 min at a flow rate of 4 mL/min. The injection volume was 1000 µL and detection was set at 230 nm. This wavelength was chosen based on the highest absorption shown by all peaks of interest. Peak fractions were collected in test tubes using a Gilson (FC-203B) fraction collector.A g/g THH; ) was colCollected fractions were evaporated in a Centrivap Vacuum Concentrator System  and lyophilized . The dry fractions were resuspended in methanol with 0.1% formic acid and subsequently analyzed by high-resolution mass spectrometry  coupled with a TripleTOF 5600+  for exact mass and fragmentation pattern determination.B. caapi extract at 10.24 mg/mL were prepared by resuspension in ultrapure water. Stock solutions of standards of harmine, harmaline, and THH were prepared in MilliQ® water at 5.12 mg/mL. All stock solutions were then diluted in Dulbecco’s Modified Eagle Medium  in serial dilutions to be used as treatment for cell assays. In all experiments, 4 µL of the treatment solution were used.Stock solutions of isolated fractions and dry v/v) fetal bovine serum, 1% (v/v) antibiotics  , and 1% (v/v) non-essential amino acids  at 37 °C and 5% CO2 in a humidified atmosphere . Cells were detached with trypsin , transferred to a sterile conical tube, centrifuged at 200× g for 10 min, and suspended in prepared medium at an appropriate density for each experiment. Cell viability was assessed with trypan blue in PBS .BV-2 microglial cells were obtained from Rio de Janeiro Cell Bank . Cells were cultured in 50 mL cell culture flasks  in DMEM supplemented with 10% , which determines the number of viable cells in culture based on quantitation of the ATP. Cells were transferred to 96-well cell culture plates  at a density of 50,000 cells/well in 50 µL of the cell suspension and treated with 4 to 512 µg/mL of B. caapi extract, fractions F1, F2 (11.4 to 1454 µM), F3 (13.2 to 1690 µM), F4 (21.4 to 2738 µM), and F5 (19.6 to 2510 µM). This concentration range was defined based on a previous test  that showed that ayahuasca decreased BV-2 cell viability at concentrations higher than 4 µg/mL. Harmine, harmaline, and THH were tested at concentrations ranging from 9 to 302 µM, which is comparable to the concentration ranges tested using other cell lines [2 at 37 °C and left for 30 min at room temperature prior to the addition of 50 µL of the reagent from the CellTiter-Glo® kit. The plate content was mixed in an orbital shaker for 2 min to induce cellular lysis and then left to stabilize for 10 min at room temperature before luminescence reading in a SpectraMax® Plus 384 luminometer from Molecular Devices .The viability of the BV-2 cells after treatment was assayed by CellTiter-Gloll lines ,34. Cell2, and the plates washed with phosphate-buffered saline with pH of 7.2 (PBS) at 37 °C. Cells were treated with F2 (5.7 to 727 µM), F4 (10.7 to 1369 µM), and harmine (2.4 to 75.5 µM). DMEM served as a negative control and dimethyl sulfoxide (DMSO) 10% (v/v) was used as a positive control. Plates were incubated for 24 h at 37 °C with 5% CO2 in a humidified atmosphere and then washed with PBS prior to the addition of 50 µL of FITC anexin V/PI. Cell suspension was analyzed by flow cytometry , with a total of 10,000 events per sample, using FACSDiva software v 7.0 . Mean fluorescence intensity (MFI) was obtained by processing data in FlowJoTM software v 10.6.1. The concentrations tested in this assay were defined based on the results of the viability test (2 h incubation) and longer incubation time (24 h).Apoptosis/necrosis detection was carried out using an FITC Annexin V/PI kit from BD Biosciences . Cells (50 µL) were plated at a density of 100,000 cells/well in DMEM, incubated for 2 h at 37 °C with 5% CO2DCFDA) from Invitrogen . Cells were plated, treated (1 to 32 µM) as described for the apoptosis/necrosis assay, washed twice with PBS, and incubated with 200 µL of 10 µM carboxy-H2DCFDA diluted in DMEM. Treatment concentrations ranged from 2.8 to 90.9 µM for F2, from 5.3 to 171 µM for F4, and from 4.7 to 151 µM for harmine. The fluorescence intensity of carboxy-H2DFCDA was assessed by flow cytometry as described in Reactive oxygen species (ROS) were assessed using 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate . DMEM served as a control. After incubation, the cell culture supernatant was collected and stored at −80 °C until analyses. The test assay was performed according to the manufacturer’s specifications. Briefly, samples were incubated for 2 h with antibody-coated beads (capture beads) and detection antibody (detection reagent). After incubation, the plate was washed once and the complex (capture beads-cytokine-detection antibody) was suspended with kit buffer assay. Flow cytometry was used to evaluate 2500 events/samples using FACSDiva software v7.0  and data were processed by FCAP Array software V. 3.0 . A standard curve for each cytokine (20 to 5000 pg/mL) was prepared and used for quantitative analysis.Cytokine production was assessed using a Cytometric Bead Array Mouse Th1/Th2/Th17 Cytokine Kit from BD Biosciences . Cells were plated as described for viability and incubated for 2 h at 37 °C and 5% COp-value < 0.05 was deemed as statistically significant and is indicated in the figures and tables by an asterisk. p-values < 0.01 and <0.001 are indicated by 2 and 3 asterisks, respectively.The data are reported as the mean and standard error of the mean (SEM) of at least 3 and a maximum of 6 independent experiments. Experimental differences were tested for statistical significance using one-way analysis of variance (ANOVA) using GraphPad Prism 5 software . A B. caapi methanolic extract using a semi-preparative column yielded several peaks, with high absorbance observed at 230 nm (+) of m/z 174.0923 and 233.1294. The other fractions were F2 (m/z 353.1721), F3 (m/z 304.3012), F4 (m/z 188.1082), and F5 (m/z 205.0799). To the best of our knowledge, the compounds found in these fractions have not previously been described in B. caapi samples. A tentative elucidation of the compound structures was performed by nuclear magnetic resonance (NMR), but the spectra were not conclusive due to the low amount obtained during the purification process.HPLC-DAD analysis of the t 230 nm . Fractiom/z 579.2425), epicatechin (m/z 291.0868) [m/z 245.0923), first reported by Hashimoto and Kawanishi [B. caapi (BC) extract (mixture of many compounds) and F1 (mixture of two compounds).The exact masses and fragmentation patterns of three collected fractions already described in the literature were putatively identified as procyanidin (91.0868)  and harmawanishi . The ideB. caapi extract, isolated fractions (F1 to F5), and the β-carbolines . Treatment with B. caapi, F3, F4, and F5 did not have any significant impact on cell viability , then decreasing it at 16 µg/mL (75.5 µM) and further decreasing it at the 2 highest doses (151 and 302 µM) . HarmaliB. caapi extract, its fractions, and β-carbolines therein might exert their effects is through the modulation of reactive species’ steady state levels.The viability of immortalized rat mesencephalic dopaminergic neurons was shown to decrease significantly after treatment with harmine and THH at 10 µM, and with harmaline at 80 µM . These cA significant increase in ROS production was found 24 h after treatment with F4 at all concentrations tested (2.7 to 342 µM) and for harmine from 4.7 to 75.5 µM, reaching control levels at 151 µM . This is+ T lymphocytes, regulates T cell development and homeostasis, with immunostimulatory or immunoinhibitory activity depending on the target cell [Most treatments displayed an anti-inflammatory pattern on BV-2 cells, with a reduction in the release of proinflammatory cytokines  and an increase in the release of IL-4 or IL-10, which are anti-inflammatory, probably due to a change in the cell phenotype to an M2 pattern ,31. IL-2get cell ,42. Seveget cell ,44. For B. caapi crude extract (BC), and with fractions F1 and F2 are shown in The results of the cytokine release in BV-2 cells treated with The results for the treatments with F3, F4, F5, and β-carbolines are shown in IL-6 production was impacted by F4 at concentrations equal or higher than 10.7 µM (except at 171 µM), and at the intermediate doses, IL-6 levels were below the detection limit, also indicating a U-shaped dose response pattern. Treatment with F5 significantly decreased the production of all proinflammatory cytokines by BV-2 cells at low concentrations, starting at 2.5 µM for IL-17A. As the F5 concentration increased, the concentration of these cytokines decreased to levels that could not be detected .Harmine did not exert much of an effect on most cytokines at the tested concentrations, except for a gradual significant decrease in TNF release at the highest doses (18.9 to 75.5 µM) and IL-2 at 18.9 and 75.5 µM . In RAW Harmaline significantly reduced the release of all proinflammatory cytokines, except IL-6. The reduction in IL-17A, IFN-γ, and TNF levels occurred at the three highest doses while IL-2 production was not detected at all tested concentrations. Harmaline was the only treatment that decreased the release of the anti-inflammatory cytokine IL-10 but only at 18.9 µM. Finally, THH treatment significantly decreased IL-6, TNF, and IFN-γ production at 18.5 µM µg/mL or higher while it increased that of IL-4 at 2.3 µM, although this anti-inflammatory effect was not maintained at higher doses .B. caapi extract. These components might help explain the anti-inflammatory effects of ayahuasca or the plant observed in vivo and in vitro [Noteworthy, isolated fractions F4 and F5 exerted a more prominent anti-inflammatory effect on BV-2 cells than harmine or the in vitro ,50,51. Iin vitro .The role of excessive microglial activation accompanied by the chronic release of cytokines and other cytotoxic factors in neurodegenerative diseases has been widely documented ,29,30. PB. caapi extract have a cytotoxic effect at high concentrations but also exert an important anti-inflammatory effect at low levels in microglial BV-2 cells by decreasing proinflammatory cytokine release. This effect was mainly found for harmaline and THH and the fractions F4 and F5, indicating the importance of the new components in the overall therapeutical potential of ayahuasca for the treatment of many central nervous system conditions described in various studies. Future work should focus on isolating a larger amount of the compounds in the identified fractions to completely elucidate their chemical structures.This study showed that selected β-carbolines and some of the new components, which had not been previously described, found in B. caapi in modulating the release of pro- and anti-inflammatory cytokines by microglia cells. Such activity warrants further investigation using other cell lines and the measurement of other cytokines involved in inflammatory events.To the best of our knowledge, this is the first study to report the effects of substances present in


Overall, our study suggested beneficial effects on lipid metabolism by reduced energy intake, modulation of biomarkers such as PDE activity and improved DNA integrity associated with PRB consumption.Polyphenols are a diverse and widely distributed class of secondary metabolites, which possess numerous beneficial properties including a modulation of glucose and lipid metabolism. This placebo-controlled human intervention study was performed to explore effects of polyphenol-rich beverage (PRB) uptake on lipid metabolism, as well as DNA integrity. In this case, 36 healthy men were randomly divided to consume either 750 mL of a PRB  or a placebo drink daily for eight weeks. Only PRB consumption was found to decrease fat and protein intakes significantly compared to the preceding one-week washout period. During the intervention with PRB an increased fat-free mass was shown after four weeks, whereas a significant elevation in body weight and leptin was observed in placebo group. Blood lipids were not significantly altered after PRB consumption, while triglyceride levels increased after placebo drink intake. In platelets, a significant inhibition of phosphodiesterase (PDE) activity was observed, more pronounced in test group. Consuming the PRB decreased total DNA strand breaks in whole blood as well as H Since the prevalence of overweight and obesity in humans has risen in recent years and increased the risk for diseases such as type 2 diabetes, cardiovascular diseases, and cancer, it has become a major health problem worldwide -cAMP). The enzyme reaction was stopped on ice after 20 min with 250 µL ZnSO4 (0.266 mM) and -5′-AMP was precipitated by adding 250 µL Ba(OH)2 (0.266 mM). Tubes were centrifuged  and then 450 µL of the supernatant was mixed with 3.5 mL of a scintillation cocktail, and the resulting radioactivity was measured using a liquid scintillation counter . Typing for PDE isoenzyme activity in platelet-rich plasma from the intervention study subjects was achieved with the aid of specific inhibitors, namely 50 µM erythro-9-(2-hydroxy-3-nonyl)adenine , 10 µM Milrinone (PDE 3), 50 µM Rolipram (PDE 4), and 10 µM Zaprinast (PDE 5) [−1 mg−1 protein). Therefore, protein concentrations of the samples were determined according to the method of Bradford using bovine serum albumin as a standard [For cAMP-PDE activity assays, isolated platelets were resuspended in Tris buffer  ,32. For standard .Plasma leptin and GLP-1 levels were determined using commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kits . All analyses were carried out according to the manufacturer’s protocols.15N2-uric acid as an internal standard (IS) according to the method published by Kim et al. with slight modifications [−1 for uric acid and 1600 µg mL−1 for IS and stored at −18 °C. The quantification of uric acid in plasma was carried out by a calibration curve using concentrations from 2 µg mL−1 to 100 µg mL−1, with a final concentration of 40 µg mL−1 IS. The calibration curves were plotted as concentration vs. peak area ratio of the analytes and the IS, yielding a correlation coefficient of R2 > 0.99. Signal-to-noise ratios were defined as 3:1 relative to the LOD and 10:1 relative to the LOQ. LOD was determined as 0.2 µg mL−1 and LOQ as 0.4 µg mL−1. Intra-day repeatability was assessed by ten replicate analyses of one standard concentration plus fixed concentration of IS. Inter-day reproducibility was obtained by analysis of one concentration level of the analyte plus fixed concentration of IS for five days in a row. The coefficient of variation was 3.4% for intra-day and 4.6% for inter-day experiments. Recovery of 94 ± 8% was determined by spiking plasma with 8, 10, and 12 µg mL−1 of uric acid. The IS (40 µg mL−1) was added to 200 µL plasma sample. After 3 h equilibration at 4 °C, 40 μL of trichloroacetic acid (10%) was added for protein precipitation and the sample was centrifuged for 1 min at 20 °C and 17,000× g. The supernatant was filtered through a 0.2 µm nylon filter by centrifugation for 5 min at 17,000× g. The filtrate was further analyzed by HPLC-ESI-MS/MS.The uric acid content in the plasma samples was measured using HPLC-ESI-MS/MS with isotope labelled 1,3-ications . The sto−1. The injection volume was 20 μL. The gradient was as follows: 0–3.8 min, 5% B; 3.8–6.7 min, 5–35% B; 6.7–7.6 min, 35%B; 7.6–8.0 min, 35–95% B; 8.0–9.8 min, 95% B; next, reversed to the original composition of 5% B over 0.5 min, after which it was kept constant for 1.8 min to re-equilibrate the column. The ESI source was operated in negative ion mode (−4000 V), and nitrogen was used as nebulizer gas (45 psi), heater gas , curtain gas (30 psi), and collision gas (4 psi). Uric acid as well as the isotopically labelled IS were detected in multiple reaction monitoring (MRM) mode. The parent ion of uric acid was m/z 167.0 and monitored MRM ions were m/z 123.9 and 95.8. In case of IS the parent ion of 1,3-15N2-uric acid was m/z 169.0 and monitored MRM ions were m/z 124.9 and 96.9. The substance-specific parameters of individual compounds are listed in Analysis of the samples was performed with an HPLC Agilent 1100 Series equipped with a degasser (G1322A), quaternary pump (G1311A), autosampler (G1313A), column oven (G1316A)  and coupled an API 2000 triple-quadrupole mass spectrometer  with ESI source. Liquid chromatography analyses were performed in a gradient elution mode using Phenomenex Luna 5 μm C18(2) 100Å (250 mm × 4.6 mm) column  coupled with a Phenomenex Luna C8(2), 5 μm particle size guard column. The mobile phase used included 5 mM ammonium acetate/0.1% acetic acid (A) and acetonitrile/0.1% formic acid (B). The mobile phase flow was 0.9 mL minAlkaline single-cell gel electrophoresis (comet assay in whole blood with and without FPG solution) was performed according to Collins et al.  with sli2O2 (50 µM) on ice for 5 min, according to previous publications [For the comet assay in PBLs, 2 × 50,000 freshly isolated PBLs were centrifuged, and the pellet was mixed with low-melting agarose. Next, the slides were exposed to Hications . After t−1), were applied to a solid-phase extraction cartridge , preconditioned with methanol (4 mL) and equilibrated with water/formic acid . Elution was performed with methanol/formic acid , eluates were concentrated using a vacuum centrifuge . The residue was resuspended in 200 µL of water/formic acid/acetonitrile . After centrifugation , 20 µL of the supernatant was analyzed with HPLC-ESI-MS/MS using an Agilent HPLC system  coupled to an API 3200 MS . HPLC chromatographic separation was carried out with a Luna 5 µm C18(2) 100Å (250 mm × 4.6 mm) column  coupled with a Phenomenex Luna C18(2), 5 μm particle size guard column. The HPLC parameters were as follows: flow 0.7 mL min−1; solvent A ; solvent B . The gradient was comprised of 0–0.7 min, 2% B; 0.7–14.9 min, 2–14% B; 14.9–28.6 min, 14% B; 28.6–35.7 min, 14–15% B; 35.7–39.3 min, 15–19% B; 39.3–46.4 min, 19–20% B; 46.4–46.5 min, 20–99% B; linear step to 99% B for 4.5 min; and return to initial condition of 2% B for 0.1 min (equilibration time: 16.9 min). The ESI-MS instrument-parameters were carried out in positive mode: curtain gas (30 psi), ion spray (5000 V), temperature (450 °C), gas 1 (50 psi), and gas 2 (40 psi). The substance-specific parameters of individual compounds and transitions are listed in −1. Cyanidin-3-galactoside was used as qualifier and a LOD of 0.05 µg mL−1 was determined.Compliance to the study was ascertained by monitoring urinary excretion of anthocyanins in all volunteers. The anthocyanin content of acidified spot urine samples was determined according to Mueller et al. with slight modifications . Urine sThe results of tested parameters were presented as the mean and standard deviation (SD). Statistical analyses were conducted using the Analysis Tool Excel of Microsoft 365 Apps for Enterprise , Origin 2020  and R 2.13.1 . The Anderson-Darling test was used for the analysis of normal distribution. The statistical significance of differences in parameters between the study phases within each respective group were analyzed with a one-sided paired t-test  or a one-sided paired Wilcoxon’s signed rank test . The statistical significance of differences in parameters between the test group and placebo group were analyzed by either a two-sample t-test  or a Mann-Whitney U-test . Spearman correlation coefficients were calculated based on the changes of the washout period to both the intervention period after four and after eight weeks. Significant correlations were determined by means of t-test.


Al addition progressively decreases the hydrogen storage capacity but also destabilizes fcc dihydride formation for alloys with x ≤ 0.10. Among the different compositions, the most promising alloy was found to be that with x = 0.05 Al content that exhibited high initial storage capacity (2.96 wt.%), a less stable hydride (ΔH = −52 kJ/mol H2 and ΔS = −141 J/K∙mol H2), better desorption properties (desorption onset temperature around 100 °C) and enhanced reversible capacity during cycling (2.83 wt.%) compared to the ternary TiVNb. In situ and ex situ synchrotron X-ray powder diffraction, together with thermal desorption experiments, showed improved desorption properties with Al addition, together with a two-step reaction with hydrogen. These findings highlight the use of small quantities of lightweight Al in refractory multi-principal element alloys as a promising approach for enhancing the solid-state hydrogen storage performance of bcc-type alloys.The effect of Al addition on the structure, microstructure and hydrogen storage properties of the ternary TiVNb alloy was investigated from small amounts to equimolar composition. Al These properties make them good candidates for the investment of research effort to overcome their limitations and achieve efficient hydrogen storage systems which can operate in near ambient conditions bcc Ti0.30 X = Ta ), withou94 wt.%) . Ferraz t (wt.%) . Finally.7 wt.%) . FurtherNb)0.90) . Despitex(TiVNb)1−x with x = 0.05, 0.175 and 0.25, synthetized by arc melting. Our objective was to vary the Al content to tune the microstructure, physicochemical, and hydrogen sorption properties. For this purpose, laboratory and large-scale experimental facilities were combined to clarify the effect of the amount of Al introduced into TiVNb. Well-known laboratory techniques, as well as ex situ and in situ synchrotron X-ray powder diffraction, were carried out and the results compared to our previous results for ternary TiVNb and quaternary Al0.10(TiVNb)0.90 alloys [Consequently, further research is needed to increase knowledge of the hydrogen storage properties of lightweight HEAs with a view to achieving high hydrogen capacity, fast hydrogen absorption/desorption kinetics close to standard temperature and pressure (STP) conditions, favorable thermodynamic properties, and high reversible hydrogen capacity during cycling . Based ox(TiVNb)1−x  compositions were synthetized using an electric arc melting method from bulk pieces of Ti , V , Nb  and Al . Due to the high melting points of Ti, V and Nb (2468 °C for Nb > 1902 °C for V > 1660 °C for Ti) these three metals were pre-alloyed in an electric arc furnace under Ar atmosphere (around 300 mbar) and remelted 16 times by flipping the ingot between each melting to achieve good homogeneity. Due to its low melting point (660 °C) and to avoid loss of material, Al was added to the pre-alloyed TiVNb and melted under the same conditions as above with eight repetitions. The mass loss during the synthesis was less than 1% over the total mass of alloy (3 g).The Al−5 mbar) overnight. Two types of measurements were carried out: absorption kinetics at 25 °C and pressure-composition isotherms (PCIs) for a range of temperatures. For the absorption kinetics, the sample holder was placed in a water bath under isothermal conditions (25 °C) and a final equilibrium pressure (Peq) of around 38 ± 5 bar of H2. For acquisition of the PCIs, small doses of hydrogen were used up to around 47 bar. A resistive furnace was used for the acquisition of PCIs at high temperatures (above 25 °C). The mass of the samples was measured carefully before hydrogenation to calculate the storage capacity using the real gas state equation for H2 in GASPAK Version 3.32 (Horizon Technologies). For the absorption/desorption cycling experiments, absorption was performed at 25 °C and a final Peq around 32 ± 4 bar H2, while desorption was performed by heating at 410 °C under secondary vacuum (~1 × 10−5 mbar) for 12 h.The hydrogenation process was carried out using a Sievert apparatus, which consisted of a home-made manual manometric device with thermostatically calibrated volumes. A stainless-steel sample holder was filled with about 400–500 mg of sample cut into small pieces, sealed using metal gaskets to prevent gas leakage and connected to the Sievert apparatus. Before starting the hydrogenation process, an activation procedure was carried out by exposing the sample at 410 °C under dynamic vacuum . In addition, both ex situ and in situ structural characterizations were carried out by synchrotron X-ray powder diffraction (SR-XRD) at the Cristal beamline of the SOLEIL facility. The intermediate hydrides of TiVNb and Al0.05(TiVNb)0.95 compositions were placed into 0.2 mm diameter borosilicate capillaries and characterized by ex situ SR-XRD in the scanning range 10° to 60° (2θ) with the wavelength λ = 0.72907 Å. In situ SR-XRD measurements during hydrogen desorption were carried out for Al0.05(TiVNb)0.95 and TiVNb by applying a constant heating ramp with 2 °C/min from 25 °C up to 450 °C under dynamic secondary vacuum. Quartz capillaries with 1.2 and 1.0 mm diameter were used for Al0.05(TiVNb)0.95 and TiVNb, respectively. To reduce the X-ray absorbance, the powdered samples were mixed with fumed silica at a mass ratio of 1:2, respectively. The in situ SR-XRD patterns were recorded every 4 °C in the scanning range 10° to 60° (2θ) with the wavelength λ = 0.67156 Å. The phase distribution and related lattice parameters were determined from ex situ and in situ SR-XRD patterns using the Rietveld refinement method implemented in Fullprof software (Thompson-Cox-Hastings pseudo-Voigt function for the peak shape).The crystalline structure of the samples was investigated by laboratory X-ray powder diffraction (XRD) using a D8 Advance Bruker diffractometer  using a Zeiss Merlin instrument equipped with an EDX (energy-dispersive X-ray spectroscopy) detector from Oxford Instruments. The powder samples were embedded in an epoxy resin and bulk samples were fixed with MCP 70 alloy, then finely polished and finally coated with a 1.5–2.5 nm Pd layer. For elemental analysis, an accelerated electron voltage of 10 keV was used and the element quantification was performed using Al(K), Ti(K), V(K) and Nb(L) signals. The analyses were carried out for the as-cast and after cycling samples.−6 mbar). The desorption profile  was recorded by heating the sample at a constant rate of 5 °C/min up to 450 °C. The hydrogen desorption properties were analyzed by thermal desorption spectroscopy (TDS) using a home-made instrument with a quadrupole mass spectrometer QMS, as described previously . The prox(TiVNb)1−x alloys with x = 0, 0.05, 0.10, 0.175 and 0.25, are listed in 0.10(TiVNb)0.90 (x = 0.10) alloys from reference [The physicochemical and empirical parameters, such as the lattice distortion  and therTi = 1.46 Å > rNb = 1.43 Å > rAl = 1.43 Å > rV = 1.39 Å [xAly series, where Al atomic size was very similar to that of the other alloying elements [The lattice distortion (δ) decreased with increase in Al content , which w= 1.39 Å ), exceptelements . 0.5NbTa0.5TiZr and AlMo0.5NbTa0.5TiZr with densities of 8.23 g/cm3 and 7.40 g/cm3, respectively [In the same way, the parameter VEC and the bulk density (ρ) were reduced with increasing Al content , with thectively . bcc solid solutions are expected: δ < 6.6% [For all the compositions, δ and VEC were in the range where single-phase δ < 6.6%  and VEC δ < 6.6% .x(TiVNb)1−x alloys with x = 0 [bcc structure, as can be seen in the XRD patterns in bcc lattice parameters, as determined by the Rietveld analysis, are listed in 0.05(TiVNb)0.95, Al0.175(TiVNb)0.825 and Al0.25(TiVNb)0.75 compositions are given in All as-cast Alth x = 0 , 0.05, 0th x = 0 , 0.175 abcc lattice with unit cell parameters of 3.18 ± 0.03 Å [0.25(TiVNb)0.75 in In this series of alloys, only the equimolar composition AlTiVNb has previously been reported by Stepanov et al.  and Yurc± 0.03 Å  and 3.18± 0.03 Å . These fbcc phases in HEAs/MPEAs containing refractory metals [bcc solid solutions [It is of note that Al has been reported to favor the stabilization of y metals ,27 despiy metals . It was olutions .bcc lattice parameters of the series Alx(TiVNb)1−x steadily decreased with increasing Al content, as shown in xNbTiMoV alloys [rTi = 1.46 Å > rNb = 1.43 Å > rAl = 1.43 Å > rV = 1.39 Å. Thus, the number of atoms with intermediate sizes increased at the expense of both the largest and the smallest atoms. This behavior has been reported in previous investigations on refractory alloys containing Al [The V alloys . The redining Al ,17.Nb > 1902 °CV >1660 °CTi > 660 °CAl), explaining why it was found in the dendritic microstructures. Interestingly, the difference in the Nb concentration between the dendritic and the interdendritric zones tended to lessen with increasing Al content, which suggests that the presence of Al helps to increase the overall chemical homogeneity of these alloys. The microstructure and chemical distribution of the as-synthesized compositions with Al are detailed in 2 pressure at 25 °C for the activated Alx(TiVNb)1−x alloys with x = 0.05, 0.175 and 0.25. The maximum capacities were: 0.69H/M (1.25 wt.%), 0.81H/M (1.41 wt.%) and 1.84H/M (2.96 wt.%) for Al0.25(TiVNb)0.75, Al0.175(TiVNb)0.825 and Al0.05(TiVNb)0.95, respectively. The capacities obtained under these conditions and the related XRD patterns for the whole series are plotted in 100−xAlx [First, absorption kinetics were recorded under 38 ± 5 bar H100−xAlx , which w0.05(TiVNb)0.95 alloy has one of the highest reported capacities (2.96 wt.%) among several Al containing alloys, such as, Mg35Al15Ti25V10Zn15 (2.7 wt.%) [0.10Ti0.30V0.25Zr0.10Nb0.25 (2.9 wt.%) [90Al10 (1.4 wt.%) and (ZrTiVFe)80Al20 (1.16 wt.%) [fcc dihydride 0.90 (x = 0.10) [bcc hydrides were observed (fcc Al0.05(TiVNb)0.95H1.84 is given in bcc hydride phases  , Al0.10T.9 wt.%) , (ZrTiVF = 0.10) . Howeverobserved b. The laxpansion . Interesxpansion . This mi0.10(TiVNb)0.90 alloys have previously been reported [0.05(TiVNb)0.95 recorded at several temperatures (25–175 °C) are provided in this section, whereas the discussion of thermodynamics will extend to previous results. Hydrogen absorption PCI curves for ternary TiVNb and quaternary Alreported . Thus, o0.05(TiVNb)0.95 revealed the existence of two plateaus 0.95 have been determined from the Van’t Hoff plots in 0.10(TiVNb)0.90 are also plotted, based on reference information in [2) and entropies (∆S in J/K·mol H2) of dihydride formation for x = 0 [The thermodynamic properties of the dihydride formation in Alation in . The entor x = 0 , 0.05 anor x = 0  are listor x = 0  with ver2 for x = 0, 0.05 and 0.10, respectively. It is of note that a similar thermodynamic destabilization as observed for the addition of x = 0.05 Al content was achieved by the addition of x = 0.25 Cr in Crx(TiVNb)1−x (equimolar composition) [The increase in Al content in TiVNb led to a progressive decrease in the experimental enthalpy of dihydride formation from −67 to −52 and −49 kJ/mol Hosition) , suggest0.05(TiVNb)0.95 were less sloped than for the ternary pristine alloy, as reported previously [Furthermore, the plateaus of the Aleviously . Thus, t0.05(TiVNb)0.95 was investigated by ex situ SR-XRD. The hydrogenation process was stopped at several hydrogen concentrations. The SR-XRD patterns of the intermediate hydrides with uptakes = 0.5, 0.9 and 1.3 H/M, the dihydride (1.8 H/M), as well as the fully desorbed alloy, are displayed in The two-step transformation induced by hydrogen absorption in Al0.05(TiVNb)0.95H0.5 consisted of a mixture of two phases: a bcc solid solution with hydrogen with a slightly enlarged lattice parameter  and an fcc dihydride (afcc = 4.392 Å). At full hydrogenation (1.8 H/M) the material was fully transformed into an fcc dihydride with afcc = 4.412 Å 0.825H0.81 showed a main desorption event with a maximum desorption peak at 276 °C, whereas the Al0.25(TiVNb)0.75H0.69 revealed two main desorption peaks with maxima at 305 °C and 375 °C (The desorption properties of the hydrides were determined by thermo-desorption spectroscopy (TDS) with a heating rate of 5 °C/min. The TDS profile of Ald 375 °C a. 0.05(TiVNb)0.95H1.84 (0.10(TiVNb)0.90H1.6 [, though the range of desorption occurred at a lower temperature for the alloy with higher Al content. The desorption onset temperature for the fcc-forming hydride materials  was found to decrease with increasing Al content, as depicted in 0.30V0.25Zr0.10Nb0.25X0.10 HEAs [The TDS profile of Al.95H1.84 b showed 0.90H1.6 , though .10 HEAs . bcc lattice with a lattice parameter close to the as-cast material (0.25(TiVNb)0.75 after desorption showed a bcc structure with a very strong preferential orientation along the (200) direction 0.95H1.84 and (TiVNb)H2.0 are plotted in 0.05(TiVNb)0.95H1.84 and (TiVNb)H2.0 are shown in To better understand the effect of Al on the desorption process and the related crystalline phases’ evolution, in situ SR-XRD measurements for Alfcc dihydride Al0.05(TiVNb)0.95H1.84  has been observed previously by in situ neutron diffraction of Al0.10(TiVNb)0.90D1.66 during deuterium desorption [bcc phase strongly increased at the expense of the fcc dihydride  Å  Å at 450 °C was larger than 3.206(1) Å obtained for the desorbed phase at room temperature  Å , which wperature . Moreoveperature . However2 0.95 alloy. Although initially the fcc lattice parameter increased with temperature, starting from 200 °C, it decreased, with this phase completely disappearing at around 350 °C. The bcc lattice parameter steeply decreased up to 350 °C and approached an almost stable value at higher temperature. Comparable to the Al0.05(TiVNb)0.95 alloy, the bcc lattice parameter at 450 °C was 3.234(1) Å 0.95 alloy.The in situ SR-XRD profile from the desorption of the ternary (TiVNb)H2 b showed 234(1) Å , which wat 25 °C  but sligat 25 °C . This subcc phase ⇄ bcc monohydride ⇄ fcc dihydride, as demonstrated by the PCI curves and the ex situ SR-XRD measurement, the question arises: why was the transition from the bcc monohydride to the desorbed bcc phase not visible from the in situ SR-XRD? Presently, the origin of this effect is not completely certain; however, it might be attributed to the fact that this transition is more difficult to detect in situ as it involves a very small displacement/rearrangement of atoms among the same bcc crystal structures, as was previously suggested for Ti0.30V0.25Cr0.10Zr0.10Nb0.25 [If both alloys undergo a reversible two-step reaction: 0.05(TiVNb)0.95 and the ternary TiVNb was the desorption that occurred at lower temperature for the Al-containing alloy compared to the pristine material. Moreover, both the fcc dihydride and the bcc desorbed phases coexisted within a large temperature range for Al0.05(TiVNb)0.95, as was also observed for Al0.10(TiVNb)0.90 during the in situ neutron diffraction reported in our previous work [The most important difference between the in situ SR-XRD experiments on Alous work , whereas0.05(TiVNb)0.95 was submitted to 10 cycles of hydrogen absorption/desorption to evaluate the reversible storage capacity, the crystalline structure stability, and the effect on the reaction kinetics. The cycling process consisted of complete hydrogen absorption with a final Peq around 32 bar at 25 °C, followed by complete desorption at 410 °C under a secondary vacuum for 12 h. The kinetics were recorded during the absorption of hydrogen for each cycle 0.95 in terms of wt.% (2.83 wt.%) was higher than both values for Al0.10(TiVNb)0.90 (2.55 wt.%) and TiVNb (2.8 wt.%) that we reported previously [0.05(TiVNb)0.95 alloy lost around 4.5% of the initial capacity until stabilization occurred (from 2.96 wt.% to 2.83 wt.%), which was higher than the 1.8% loss observed for Al0.10(TiVNb)0.90, but smaller than the 14 % loss for TiVNb. This suggests that Al addition in TiVNb positively affected the reversible capacity when limited Al amounts were used. An optimum capacity was found for x = 0.05 Al content in TiVNb with very good cycling stability and one of the best stable capacities reported so far. The improvement in the stability of the reversible capacity and the gravimetric capacity of hydrogen storage by Al addition as a lightweight element was in very good agreement with the behaviors reported for Ti-V-Zr-Nb with 10 at.% Al [The capacity slightly decreased from 1.84 H/M to 1.80 H/M after the first cycle and stabilized at 1.76 H/M at the 10th cycle. Interestingly, the stable capacity of Aleviously , as show at.% Al . 0.05(TiVNb)0.95H1.76 hydride phase was analyzed by XRD 0.90 and TiVNb [0.05(TiVNb)0.95H1.76 after 10 cycles is shown in After the cycling process, the Ald by XRD . This dend TiVNb . The RieA simple comparison among these three compositions suggests that, not only the addition of non-absorbing lightweight elements in refractory MPEAs is important, but also that the composition must be carefully tuned to optimize both the structure/microstructure of the alloys and their hydrogen sorption performances. x(TiVNb)1−x alloys with x = 0–0.25 were prepared by arc melting as single-phase bcc materials with a decreasing lattice parameter by increasing the Al amount. They showed good chemical homogeneity with the typical Nb-rich dendritic microstructure of refractory HEAs. The effect of Al addition on the structure, microstructure, and hydrogen sorption properties of a ternary TiVNb alloy was studied from low Al content up to equimolar composition. Albcc solid solutions after hydrogen absorption with small hydrogen uptakes , whereas the alloys with low Al content (x = 0.05 and 0.10) absorbed large amounts of hydrogen-forming fcc hydrides. Moreover, a strong thermodynamic destabilization of the hydride formation was observed with increasing Al content. The enthalpy of hydride formation reduced from −67 kJ/mol H2 for the pristine TiVNb alloy to −52 and −49 kJ/mol H2 for x = 0.05 and 0.10 Al quantities, respectively. Interestingly, the desorption onset temperatures from the fcc dihydride forming materials  showed a strong decrease with increasing Al content, indicating thermal destabilization of the hydrides by Al addition. The alloys absorbed hydrogen quickly at room temperature with the storage capacity showing a strong dependence on the Al content. Under the experimental conditions used, the richest Al alloys (x = 0.175 and 0.25) formed 0.05(TiVNb)0.95 alloy underwent a reversible two-step reaction from the initial bcc to an intermediate bcc monohydride and, finally, to an fcc dihydride, as confirmed by the two plateaus observed in the PCIs and the ex situ synchrotron XRD. Moreover, a flattening of the plateaus by Al addition was observed which was an important factor in the reduction in errors in the calculation of the thermodynamic properties by the Van’t Hoff equation. Intriguingly, during hydrogen desorption, only one phase transition from the fcc dihydride to the bcc desorbed state was detectable by in situ SR-XRD for both Al0.05(TiVNb)0.95H1.84 and TiVNb. The expected two-step reaction fcc dihydride → bcc monohydride → bcc desorbed phase was not noticeable, though complete desorption was confirmed by structural analysis. One plausible explanation might be that the bcc monohydride → bcc desorbed phase transition was more difficult to detect since it involved a very small rearrangement of atoms within the same bcc crystalline structure. However, the in situ SR-XRD clearly demonstrated the coexistence of an initial fcc dihydride with the bcc desorbed phase in a wide temperature range for Al0.05(TiVNb)0.95, in contrast with the thermal behavior of TiVNb. The Al0.05(TiVNb)0.95 alloy possessed better cycling properties than both the TiVNb and Al0.10(TiVNb)0.90 alloys, with a stable reversible capacity of 1.76 H/M (2.83 wt.%), which is one of the best in the recent literature of MPEAs designed for solid-state hydrogen storage.Finally, the Al


Supernatant and cells were evaluated at 0, 2, 4, and 8 h post asbestos exposure for reactive oxygen species (ROS) generation, DNA damage (oxidized guanine), inflammasome activation (caspase-1 activity) and associated pro-inflammatory cytokine release , and markers of oxidative stress  and 8-iso-prostaglandin F2a (8-iso-PGF2α). Asbestos induced a time-dependent ROS increase that was significantly (p < 0.0001) reduced (29.4%) by LGM2605 treatment. LGM2605 pretreatment also reduced levels of asbestos-induced DNA damage by 73.6% ± 1.0%. Although levels of inflammasome-activated cytokines, IL-1β and IL-18, reached 29.2 pg/mL ± 0.7 pg/mL and 43.9 pg/mL ± 0.8 pg/mL, respectively, LGM2605 treatment significantly (p < 0.0001) reduced cytokine levels comparable to baseline (non-asbestos exposed) values . Furthermore, levels of IL-6 and TNFα in asbestos-exposed mesothelial cells were high , while remaining undetectable with LGM2605 pretreatment. HMGB1  release was reduced 75.3% ± 0.4% by LGM2605. Levels of MDA and 8-iso-PGF2α, markers of oxidative cell injury, were significantly (p < 0.001) reduced by 80.5% ± 0.1% and 76.6% ± 0.3%, respectively. LGM2605, given preventively, reduced ROS generation, DNA damage, and inflammasome-activated cytokine release and key inflammatory mediators implicated in asbestos-induced malignant transformation of normal mesothelial cells.Although alveolar macrophages play a critical role in malignant transformation of mesothelial cells following asbestos exposure, inflammatory and oxidative processes continue to occur in the mesothelial cells lining the pleura that may contribute to the carcinogenic process. Malignant transformation of mesothelial cells following asbestos exposure occurs over several decades; however, amelioration of DNA damage, inflammation, and cell injury may impede the carcinogenic process. We have shown in an in vitro model of asbestos-induced macrophage activation that synthetic secoisolariciresinol diglucoside (LGM2605), given preventively, reduced inflammatory cascades and oxidative/nitrosative cell damage. Therefore, it was hypothesized that LGM2605 could also be effective in reducing asbestos-induced activation and the damage of pleural mesothelial cells. LGM2605 treatment (50 µM) of huma n pleural mesothelial cells was initiated 4 h prior to exposure to asbestos (crocidolite, 20 µg/cm Asbestos refers to a group of naturally occurring hydrated fibrous silicate fibers used in commercial and industrial applications. Preclinical studies in animal exposure models, as well as clinical data, have confirmed that asbestos fiber inhalation can lead to chronic lung conditions, such as pulmonary fibrosis. In addition, asbestos exposure can lead to neoplastic diseases such as malignant mesothelioma (MM) and lung cancer ,2. MM isMany western countries have restricted asbestos use; however, it is still used in many countries around the world, such as India, Russia, and China. Brazil is the world’s 3rd leading producer of asbestos despite the 2017 ban and, in fact, it was responsible for 96% of asbestos imports into the United States from 2015–2018. Despite an estimated decrease of asbestos mineral consumption from 2 million tons in 2010 to 1 million tons in 2019, a number of asbestos-containing products  continue to be manufactured and sold globally. There will, thus, likely be a dramatic increase in MM cases in developing countries, such as India, where the use of asbestos has increased with few precautions taken. However, even in the developed world, important ongoing and persistent exposures still exist. This includes environmental exposures, as occurs in older buildings and schools, and occupational exposures that expose workers to pre-existing asbestos . Furthermore, there are also environmental and domestic exposures from the disturbance of naturally occurring asbestos (NOA). For example, there is an increased risk of MM in areas where mining or asbestos factories have closed. The Libby Asbestos Superfund Site in Libby, Montana, and the BoRit and Ambler Asbestos Piles Superfund Sites in Ambler, Pennsylvania, are classic examples [There is, thus, an unmet need to develop a chemopreventive agent effective against asbestos-induced MM in high-risk populations.A major issue in the link between asbestos and cancer is that inhaled asbestos fibers  can persist in the lung for very long periods of time, resulting in continuous damage, even after removal from the exposure. Because of this long latency period (often up to 30–50 years), individuals exposed in the past remain at an increased risk of MM and other cancers throughout their lives, presumably due to continuous, persistent inflammation . AlthougChronic inflammation induced by asbestos exposure is associated with the development and progression of MM. Reducing tissue inflammation and oxidative damage may potentially prevent MM. Currently, it is thought that retained asbestos fibers stimulate both lung phagocytic cells (macrophages) and pleural mesothelial cells to produce reactive oxygen and nitrogen species and inflammatory cytokines that ultimately lead to permanent genetic changes and malignant transformation of the mesothelial cells ,9,10,11.Our group has found that flaxseed and its lignan compounds are non-toxic, but effective, multifunctional anti-inflammatory and antioxidant agents that may be useful for lung cancer chemoprevention in cancer cells and a mouse model of chemical carcinogen-induced lung cancer ,13,14. WRecent studies have shown in an in vitro model of asbestos-induced macrophage activation that LGM2605, given preventively, reduced inflammatory cascades and oxidative/nitrosative cell damage . Althoug2), and cell culture medium and mesothelial cells were collected at 0, 2, 4, and 8 h after asbestos exposure  increase in DCF fluorescence at 0.5, 1, and 2 h post exposure (p < 0.05) reduced ROS generation at 2 h post asbestos by 36.2% and 71.1%, respectively  at 0, 0.5, and 2 h post asbestos exposure. Exposure to asbestos led to a significant (exposure a. While ectively b. Importp < 0.0001) elevations in ROS generation at 4, 6, and 8 h post asbestos. LGM2605 led to marked reductions in intracellular ROS. The significant (p < 0.05) decrease in asbestos-induced ROS generation with 50 µM LGM2605 treatment was observed through 8 h post asbestos exposure (p < 0.0001) increased following asbestos exposure, LGM2605 reduced ROS levels by 43.4%, 40.8%, and 28.5% at 4, 6, and 8 h post asbestos, respectively.We then determined the kinetics of acute asbestos-induced ROS generation in human mesothelial cells following exposure to crocidolite asbestos over an 8 h period using immunofluorescence and the oxidative stress sensitive dye, CellROX™ green reagent c  elevated following asbestos exposure, from 0.43 µM at baseline to 21.54 µM at 8 h following asbestos exposure. LGM2605 pretreatment (50 µM) significantly (p < 0.0001) reduced MDA release by 65.0% at 8 h after asbestos exposure (iso-PGF2α was significantly (p < 0.0001) elevated following asbestos exposure from 49.1 pg/mL at baseline to 418.9 pg/mL at 8 h following asbestos exposure. LGM2605 pretreatment (50 µM) significantly (p < 0.0001) reduced 8-iso-PGF2α release by 92.4% at 8 h after asbestos exposure (p < 0.05) reduced levels of 8-iso-PGF2α release at 2, 4, and 8 h.MDA release, a marker of lipid peroxidation, was significantly (exposure a. 8-iso-exposure b. Comparp < 0.0001) elevated in cells exposed to asbestos (p < 0.05) further increase in nuclear Nrf2 levels at 2 and 4 h post asbestos exposure, with a 7.86-fold increase relative to asbestos-only exposure at 4 h. Treatment with LGM2605-only (50 µM) led to modest increases in nuclear Nrf2 relative to untreated, unexposed (CTL) mesothelial cells at 2 and 4 h (p < 0.01).We have previously observed an increase in nuclear factor E2-related factor 2 (Nrf2) signaling and endogenous antioxidant enzyme response with LGM2605 treatment (50 µM) in asbestos-exposed peritoneal macrophages ,17. We, asbestos c. Pretrep < 0.0001) elevated, reaching concentrations of 37.7 µM, 65.8 µM, and 109.5 µM at 2, 4, and 8 h, respectively, following asbestos exposure (p < 0.0001) reduced the concentration of total nitrates and nitrites by 72.9%, 75.2%, and 80.5% at 2, 4, and 8 h post asbestos exposure, respectively. Treatment with LGM2605-only (50 µM) had no significant effect.In addition to harmful ROS, asbestos exposure generates toxic reactive nitrogen species (RNS) that contributes to inflammation and cellular damage . As a maexposure a. LGM260p < 0.0001) elevated following asbestos exposure, peaking at 2 h post asbestos (p < 0.0001) decreased levels of nuclear NF-κB by 64.1%, 68.7%, and 90.8% at 2, 4, and 8 h post asbestos exposure, respectively. By 8 h post asbestos exposure, no statistically significant difference was observed between levels of nuclear NF-κB among untreated, unexposed mesothelial cells and cells exposed to asbestos and treated with 50 µM LGM2605.Activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is responsible for activation of inducible nitric oxide synthase (iNOS), which leads to the generation of nitric oxide, nitrates, and nitrites. Levels of nuclear NF-κB were significantly (asbestos b. Treatmp < 0.05) reduced caspase-1 activity by 51.4%, 46.5%, and 56.5% at 2, 4, and 8 h post asbestos exposure, respectively. Treatment with LGM2605-only (50 µM) had no significant effect.Mesothelial cell exposure to crocidolite asbestos fibers induces the activation of the NLRP3 inflammasome . The actp < 0.0001) elevated at 2 h post asbestos exposure (4.60 pg/mL ± 0.30 pg/mL) and continued to increase at 4 h (12.03 pg/mL ± 0.60 pg/mL) and 8 h (29.23 pg/mL ± 0.72 pg/mL) (p < 0.0001) reduced IL-1β release by 75.1%, 89.4%, and 92.4% at 2, 4, and 8 h post asbestos exposure, respectively. Levels of IL-18 were also significantly (p < 0.0001) elevated at 2 h post asbestos exposure (5.50 pg/mL ± 0.46 pg/mL) and continued to increase at 4 h (13.82 pg/mL ± 0.07 pg/mL) and 8 h (43.90 pg/mL ± 0.79 pg/mL) (p < 0.0001) reduced IL-18 release by 73.0%, 78.2%, and 89.6% at 2, 4, and 8 h post asbestos exposure, respectively.Levels of IL-1β were significantly (2 pg/mL) b. 50 µM 9 pg/mL) c. 50 µM p < 0.0001) elevated at 2 h post asbestos exposure (18.50 pg/mL ± 0.43 pg/mL) and continued to increase at 4 h (119.66 pg/mL ± 2.62 pg/mL) and 8 h (289.05 pg/mL ± 2.94 pg/mL)  a. Levelsp < 0.0001) elevated at 2 h post asbestos exposure (72.09 pg/mL ± 0.08 pg/mL) and continued to increase at 4 h (337.13 pg/mL ± 13.44 pg/mL) and 8 h (511.32 pg/mL ± 10.19 pg/mL)  b. Levelsp < 0.0001) elevated at 2 h post asbestos exposure (18.36 ng/mL ± 0.16 ng/mL) and continued to increase at 4 h (27.54 ng/mL ± 0.41 ng/mL) and 8 h (58.09 ng/mL ± 0.02 ng/mL) (p < 0.0001) reduced HMGB1 release by 85.2%, 86.9%, and 89.2% at 2, 4, and 8 h post asbestos exposure, respectively.Levels of HMGB1, a critical damage signaling molecule and proinflammatory mediator, were significantly (2 ng/mL) c. Treatmp < 0.01) elevated at 2 h and 6 h following asbestos exposure (p < 0.0001) elevated at 2 h post asbestos exposure (33.18 pg/mL ± 1.45 pg/mL) and continued to increase at 4 h (52.66 pg/mL ± 0.27 pg/mL) and 8 h (120.98 pg/mL ± 1.56 pg/mL) (p < 0.001) reduced levels of oxidized guanine species at 0, 2, 4, and 8 h. 50 µM LGM2605, administered 4 h prior to asbestos exposure, significantly (p < 0.0001) reduced the level of oxidized guanine species by 90.9%, 85.3%, and 84.4% at 2, 4, and 8 h post asbestos exposure, respectively.DNA damage following asbestos exposure was evaluated in mesothelial cells by determining levels of γH2AX a,b, oxidexposure a. Treatmexposure b. Levels6 pg/mL) c. Comparp < 0.0001) elevated at 4 and 8 h. LGM2605 pretreatment (50 µM) significantly (p < 0.0001) reduced asbestos-induced DNA SSBs by 94.5%, 97.4%, and 91.8% at 2, 4, and 8 h post asbestos exposure, respectively (p < 0.0001) reduced levels of oxidized guanine species as compared to untreated control cells, no significant differences in DNA SSBs were observed.Asbestos-induced DNA single-strand breaks (SSBs) were determined using the alkaline comet assay. Mean tail moment peaked at 2 h following asbestos exposure and remained significantly (ectively d. While 2) and supernatant and cells were evaluated at 0, 2, 4, and 8 h post asbestos exposure. Pretreatment with LGM2605 reduced asbestos-induced ROS and DNA damage, as determined by the oxidized guanine species, comet assay analysis, and γH2AX levels. In addition, markers of oxidative and nitrosative stress  were reduced, while promoting activation of Nrf2 signaling. Furthermore, asbestos induced inflammation (NF-κB nuclear translocation and caspase-1 activity) and associated proinflammatory cytokine  release was ameliorated by the action of LGM2605. Thus, our findings, using a human mesothelial cell asbestos exposure model system, provide novel evidence that LGM2605 may be a good candidate for the chemoprevention of asbestos-induced malignancy.In this study, LGM2605 treatment (50 µM) of human pleural mesothelial cells was initiated 4 h prior to exposure to asbestos  sounds simple, several issues need to be addressed for the identification of an appropriate chemopreventive agent. First, the molecular mechanisms by which carcinogens induce cancer are complex , outlineIn the current study, treatment with LGM2605 was initiated 4 h prior to asbestos exposure. While the usefulness of chemopreventive strategies in asbestos-induced MM would most likely be initiated post asbestos exposure, the opportunity for intervention is broad given the long latency from time of exposure to detectable/symptomatic MM. Thus, a safe, non-toxic agent that can be administered during this period may disrupt the carcinogenic process across the entire spectrum of malignant transformation, with the ultimate goal of delaying, or even preventing MM. This is particularly pertinent to asbestos exposure, where persistent asbestos fibers induce continued inflammation and oxidative stress. Additionally, the presence of NAO fibers in communities and the fact that occupations, such as construction workers and firefighters, remain provide opportunities for pretreatment where anticipated asbestos exposures exist.The current study is strengthened by the use of non-malignant human pleural mesothelial cells, which is the cell type that undergoes malignant transformation leading to MM formation following asbestos exposure. Additionally, our findings of LGM2605 treatment, which has been shown to be safe and non-toxic, of mesothelial cells supports our previously published findings in other in vitro and in vivo models of asbestos exposure. The present data in human mesothelial cells underscore the chemopreventive properties of LGM2605 against asbestos-induced MM formation. Importantly, LGM2605 achieved a downregulation of molecular signatures, such as HMGB1 and IL-1β release, that give rise to the formation of MM following asbestos exposure. These results highlight LGM2605 as a promising chemopreventive agent for the inhibition of human mesothelial cell damage produced by asbestos.6 cells per well) and allowed to adhere to the bottom of the wells for 24 h. They were then used to determine the effects of LGM2605 in preventing crocidolite asbestos-induced generation of ROS and RNS, NLRP3 inflammasome activation, proinflammatory cytokine secretion, and asbestos-induced genotoxicity. Treatment groups were as follows: untreated control (CTL), 50 µM LGM2605-only (LGM2605), 20 µg/cm2 asbestos-only (ASB), and 20 µg/cm2 asbestos and 50 µM LGM2605 (ASB + LGM2605).Mesothelial cells were derived from a pleural effusion fluid obtained from a single donor congestive heart failure patient as previously described . Approva2 based on our previous studies in murine peritoneal macrophages  asbestos fibers as previously described ,17,18. Bages see  [15,17,1S,S)-SDG (the major isomer in whole grain flaxseed) and -SDG (the minor isomer in whole grain flaxseed) was synthesized from vanillin via secoisolariciresinol and a glucosyl donor (perbenzoyl-protected trichloroacetimidate under the influence of TMSOTf) through a concise route that involved chromatographic separation of diastereomeric diglucoside derivatives. LGM2605 was reconstituted to a stock concentration of 10 mM, and cells were exposed to 50 µM LGM2605 4 h prior to asbestos exposure  has been previously described . Brieflysure see . The 50 ®, ThermoFisher Scientific, Waltham, MA, USA) as previously described [4 cells/well) and exposed to 20 µg/cm2 sterile crocidolite asbestos fibers and treated with LGM2605. Initial studies were conducted to determine the dose–response relationship between asbestos-induced ROS generation and various concentrations of LGM2605 treatment  at 0, 0.5, and 2 h post asbestos exposure. The fluorescence intensity was measured on a SpectraMax i3x Multi-Mode microplate reader  using an excitation wavelength in the range of 492–495 nm and a fluorescence emission detection at 517–527 nm.ROS generation following asbestos exposure was determined using the cell-permeant fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA)  as previously described [2) and evaluated at 0, 2, 4, 6, and 8 h post asbestos exposure. Mesothelial cells were incubated with 5 μM CellROX® Green Reagent  for 20 min at 37 °C after which cells were washed with phenol red-free RPMI and imaged at λex 488 nm on a Nikon TMD fluorescence microscope  equipped with a Hamamatsu ORCA-100 camera . All fluorescent cell images were acquired at the same magnification (10× lens), exposure (200 ms exposure), and acquisition settings using the MetaMorph software . The fluorescent images of cells were processed and quantitated for CellROX® Green Reagent fluorescence by the use of ImageJ software . For each experimental condition, 3–4 fields were imaged for n = 3 independent experiments. The intensity of cells in each field was integrated and normalized to area to obtain the total fluorescence intensity, and expressed as arbitrary fluorescence intensity units (AFU). Scale bar = 15 μM.Subsequent studies evaluated the ability of 50 µM LGM2605 to reduce asbestos-induced ROS generation through 8 h post asbestos exposure using CellROXescribed . Human p6 cells/well) and exposed to 50 µM synthetic SDG (LGM2605) 4 h prior to exposure to sterile crocidolite asbestos fibers (20 µg/cm2). The levels of MDA were determined in the cell culture medium at 0, 2, 4, and 8 h post asbestos exposure. Specifically, levels of thiobarbituric acid reactive substances (TBARS) were quantified by measuring the fluorescence of malondialdehyde-thiobarbituric acid (MDA-TBA) adducts in cell culture medium samples. According to the manufacturer’s instructions, MDA-TBA adducts were formed via acid hydrolysis at 100 °C and measured fluorometrically using a SpectraMax i3x Multi-Mode microplate reader  with an excitation wavelength of 530 nm and an emission wavelength of 550 nm. Levels of lipid peroxidation in cell culture medium samples are reported as the concentration (µM) of MDA.Malondialdehyde (MDA), an indicator of oxidative stress, was measured in the cell culture medium using a commercially available kit  according to the manufacturer’s protocol. Human pleural mesothelial cells were plated in a 6-well plate  kit  according to the manufacturer’s protocol. Human pleural mesothelial cells were plated in a 6-well plate (2 × 106 cells/well) and exposed to 50 µM synthetic SDG (LGM2605) 4 h prior to exposure to sterile crocidolite asbestos fibers (20 µg/cm2). The levels of 8-iso-PGF2α were determined in the cell culture medium at 0, 2, 4, and 8 h post asbestos exposure. Cell culture medium samples were run undiluted and the data are reported as the concentration (pg/mL) of 8-iso-PGF2α in the cell culture medium.Levels of 8-Levels of total nitrates and nitrites, metabolites of nitric oxide, in the culture medium were determined using a nitrate/nitrite colorimetric assay kit  according to the manufacturer’s protocol. The assay kit quantifies levels of total nitrates/nitrites (stable breakdown products of nitric oxide) by first converting nitrates to nitrites using nitrate reductase and then measuring total nitrites by adding Greiss Reagent to the reaction mixture, which produces a purple azo compound in the presence of nitrites that can be measured spectrophotometrically. The absorbance of the azo chromophore was measured at 540 nm measured using a SpectraMax i3x Multi-Mode microplate reader . Cell culture medium samples were run undiluted and the data are reported as the concentration (µM) of total nitrate/nitrites in the cell culture medium.450) to the nuclear extract protein concentration (μg). Absorbance measurements were obtained using a SpectraMax i3x Multi-Mode microplate reader .The presence of nuclear factor E2-related factor 2 (Nrf2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 subunit was determined in nuclear extracts isolated from human mesothelial cells exposed to asbestos and harvested at 0, 2, 4, and 8 h post asbestos exposure. Cytoplasmic and nuclear extracts were prepared using a commercially available nuclear extraction kit . Transcription factor assay kits  were used to detect nuclear Nrf2 and NF-κB transcription factors, as previously described ,18. The Caspase-1  activity was determined using a commercially available fluorometric assay kit , as previously performed . BrieflyLevels of proinflammatory cytokines, IL-1β, IL-6, IL-18, TNFα, and HMGB1 were determined in the cell culture medium at multiple time points post asbestos exposure  using ELISA. Samples were run undiluted in triplicate and assays were performed according to manufacturer’s instructions. Levels of IL-1β, IL-6, IL-18, and TNFα are reported as picograms per milliliter (pg/mL) of culture medium, and levels of HMGB1 released into the culture medium are reported as nanograms per milliliter (ng/mL). Human ELISA kits  were purchased from BD Biosciences , MBL International  (human IL-18 ELISA Kit), and Chondrex, Inc.  (HMGB1 Detection Kit). Absorbance measurements were obtained using a SpectraMax i3x Multi-Mode microplate reader .2), and cells were evaluated at 0, 2, and 6 h post asbestos exposure. Mesothelial cells were fixed, permeabilized, and immunostained using anti-γH2AX (1:200) as primary antibody followed by secondary antibody conjugated to Alexa488 (green). Fluorescence imaging was performed at λ ex 488 nm on a Nikon TMD fluorescence microscope equipped with a Hamamatsu ORCA-100 camera. All fluorescent cell images were acquired at the same magnification (10× lens), exposure (250 ms exposure), and acquisition settings using the MetaMorph software . The fluorescent images of cells were processed and quantitated the use of ImageJ software . For each experimental condition, 3–4 fields were imaged for n = 3 independent experiments. The intensity of cells in each field was integrated and normalized to area to obtain the total fluorescence intensity, and expressed as arbitrary fluorescence intensity units (AFU). Scale bar = 15 μM.Human pleural mesothelial cells were exposed to LGM2605 (50 µM) 4 h prior to asbestos challenge (20 µg/cm6 cells/well) and exposed to 50 µM synthetic SDG (LGM2605) 4 h prior to exposure to sterile crocidolite asbestos fibers (20 µg/cm2). The levels of oxidized guanine species were determined in the cell culture medium at 0, 2, 4, and 8 h post asbestos exposure. The absorbance was measured at 410 nm (OD410) using a SpectraMax i3x Multi-Mode microplate reader . Cell culture medium samples were run undiluted and the data are reported as the concentration (pg/mL) of oxidized guanine species in the cell culture medium.Levels of markers of oxidative DNA and RNA damage, 8-hydroxy-2′-deoxyguanosine from DNA, 8-hydroxyguanosine from RNA, and 8-hydroxyguanine from either DNA or RNA, were determined in the cell culture medium using an DNA/RNA Oxidative Damage ELISA kit  according to the manufacturer’s protocol. Human pleural mesothelial cells were plated in a 6-well plate (2 × 102). As previously described [5 cells/mL in 1× PBS) were mixed with LMAgarose®  and immediately pipetted onto CometSlides™. Cells were then lysed (4 °C for 30 min) and kept in the dark for unwinding (RT). Electrophoresis was done in a horizontal electrophoresis unit at 18 volts (200 Amp) for 25 min. Slides were washed twice with distilled water, fixed in 70% ethanol and dried at 45 °C. DNA was stained by SYBR green . At least 100 cells were scored per group. Visual analysis of cells and comet tail length was measured using Comet Image Analysis software . Images were captured on an Olympus IX51 fluorescence microscope using a monochrome CCD FireWire camera with a 40× objective lens.Exponentially growing human pleural mesothelial cells were cultured in 6-well plates and exposed to 50 µM LGM2605 4 h prior to exposure to sterile crocidolite asbestos fibers 20 µg/cm µg/cm2. www.graphpad.com. Results are reported as the mean ± the standard error of the mean (SEM) from three separate experiments. Statistically significant differences were determined with p-value < 0.05. Asterisks shown in figures indicate significant differences between unexposed, control (CTL) and asbestos-exposed (ASB) groups at each respective time point: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. A # sign shown in figures indicates significant differences between asbestos-exposed (ASB) and asbestos-exposed and LGM2605-treated (ASB + LGM2605) human pleural mesothelial cells at each respective time point: # p < 0.05, ## p < 0.01, ### p < 0.001, and #### p < 0.0001.All data were analyzed using two-way analysis of variance (ANOVA) to test for the main effects of time and treatment, and the interaction between these variables, on study outcome measures. If the overall F-test was statistically significant, Tukey’s HSD post hoc tests were conducted to determine significant differences between treatment groups  within each respective time point. Statistically significant differences were determined using GraphPad Prism version 7.00 for Windows, GraphPad Software, La Jolla, CA, USA, In summary, pretreatment of normal human pleural mesothelial cells with LGM2605 reduced asbestos-induced ROS generation, inflammasome activation and associated cytokine release, and key inflammatory mediators implicated in asbestos-induced malignant transformation of mesothelial cells, while reducing oxidative/nitrosative genotoxic cell damage. We believe that LGM2605 will be an excellent, safe agent for chemopreventive intervention in the context of asbestos exposures.


N-acetylcysteine (a precursor of antioxidant glutathione) on exercise-related muscle ionic shifts. We assigned 20 recreationally-active men to 6 weeks of high-intensity exercise training, comprising three weekly sessions of 4–10 × 20-s all-out bouts interspersed by 2 min recovery , or habitual lifestyle maintenance (n = 10). Before and after SET, we measured ionic shifts across the working muscle, using leg arteriovenous balance technique, during one-legged knee-extensor exercise to exhaustion with and without N-acetylcysteine infusion. Furthermore, we sampled vastus lateralis muscle biopsies for analyses of metabolites, mitochondrial respiratory function, and proteins regulating ion transport and antioxidant defense. SET lowered exercise-related H+, K+, lactate−, and Na+ shifts and enhanced exercise performance by ≈45%. While N-acetylcysteine did not affect exercise-related ionic shifts before SET, it lowered H+, HCO3−, and Na+ shifts after SET. SET enhanced muscle mitochondrial respiratory capacity and augmented the abundance of Na+/K+-ATPase subunits (α1 and β1), ATP-sensitive K+ channel subunit (Kir6.2), and monocarboxylate transporter-1, as well as superoxide dismutase-2 and glutathione peroxidase-1. Collectively, these findings demonstrate that high-intensity exercise training not only induces multiple adaptations that enhance the ability to counter exercise-related ionic shifts but also potentiates the effect of N-acetylcysteine on ionic shifts during exercise.This study investigated whether high-intensity exercise training alters the effect of  At rest and during knee-extensor exercise at 30% and 50% of Wmax, duplicate samples were drawn and the mean of the two samples was used for data analysis.Arterial and venous blood samples were drawn in heparinised syringes and were immediately analysed on a blood gas analyser  for plasma . Thereafter, samples were rotated for 1 h at 4 °C followed by centrifugation at 17,500× g for 20 min at 4 °C. The supernatant (lysate) was collected and a bovine serum albumin (BSA) standard kit  assayed in triplicate was used to determine total protein concentration in each sample. Then, lysate samples were diluted in double-distilled H2O and 6 × Laemmli buffer  to reach equal protein concentrations. Equal amounts of protein (range: 6–12 µg) were loaded in each well of 4–15% precast gels . Samples from the same leg of the same subject were loaded on the same gel, with the samples from pre and post being loaded in adjacent wells. To allow gel-to-gel comparisons, the same pool of a mixed human muscle standard lysate was loaded in three different wells per gel and the mean intensity of these samples was used for normalisation, as previously described ) was determined in dry weight (d.w.) muscle tissue (~2.0 mg). Determination was made by extraction in 1.5 M perchloric acid, neutralisation to pH 7.0 with 2.2 M KHCO3, followed by fluorometric analyses of the supernatant as previously described . A reagent solution was created, which consisted of 1.5 M glycylglycine, 1.5 mM NAD+, and 10.2 mM glutamic acid, and stock solution of 4.0 mM lactate−  was used to create a standard curve. The microplate reading was set at excitation at 355 nm with emission set at 460 nm  which was read before and 60 min after glutamic-pyruvic transaminase  and LDH  was added to the samples.The muscle lactate3− physiochemical buffer capacity was calculated from moles of H+ required to change pH from 7.1 to 6.5 and is expressed as mmol H+·kg d.w.−1·pH−1  during submaximal exercise. Specifically, after the training intervention, arterial plasma [lactate−] was 0.6 mmol·L−1  lower at 50% Wmax in SETSaline, while it was 0.6 mmol·L−1  and 1.1 mmol·L−1  lower at 50 and 80% Wmax, respectively, in SETNAC  and 1.0 mmol·L−1  lower at 30 and 50% Wmax, respectively, after the training intervention in SETSaline, while it was 0.5 , 1.1 , and 1.5 mmol·L−1  lower at 30, 50, and 80% Wmax, respectively, after the training intervention in SETNAC. While arterial and venous plasma [lactate−] at exhaustion did not change with SET, lactate− levels were greater in recovery after the training intervention in both SETSaline and SETNAC (p < 0.001).SET lowered arterial and venous plasma [lactaten SETNAC . Venous −] difference was 0.2 , 0.4 , and 0.1 mmol·L−1  lower at 30, 50, and 80% Wmax, respectively, after training in SETSaline, with corresponding venous-arterial differences being 0.3 , 0.5 , and 0.4 mmol·L−1  lower at 30, 50, and 80% Wmax, respectively, after training in SETNAC. At exhaustion, the venous-arterial plasma [lactate−] difference was not significantly affected by training in SETSaline (p = 0.109) and SETNAC (p = 0.052). In recovery, the venous-arterial plasma [lactate−] difference was higher after the training intervention in SETSaline, but only in the immediate recovery (+0.5 min) for SETNAC.The venous-arterial plasma  during submaximal exercise was not affected by the training intervention in either SETSaline or SETNAC, while the mean venous plasma [HCO3−] during submaximal exercise was non-significantly lower after training in SETSaline (p = 0.091) and SETNAC (p = 0.072) . Specifically, mean arterial plasma [HCO3−] was lower after training in SETSaline  but not in SETNAC (p = 0.841), and mean venous plasma [HCO3−] was 1.7 , 1.5 , 2.1 , and 1.4 mmol·L−1  lower (p < 0.001) at 0.5, 1, 2, and 4 min into recovery, respectively, after training in SETSaline, with no changes observed in SETNAC.The arterial plasma [HCO= 0.072) . In reco3−] difference during submaximal exercise was non-significantly lower and lower after training in SETSaline (p = 0.072) and SETNAC (p < 0.001), respectively. The venous-arterial plasma [HCO3−] difference was 0.9 mmol·L−1  lower at 80% Wmax after training in SETSaline, while it was 0.8 mmol·L−1  and 0.8 mmol·L−1  lower at 50 and 80% Wmax, respectively, after training in SETNAC.The mean venous-arterial plasma [HCO+] during submaximal exercise was non-significantly lower after training in both SETSaline (p = 0.052) and SETNAC (p = 0.063)  and 0.1 mmol·L−1  at 30 and 50% Wmax, respectively, after training in SETSaline, while it was non-significantly lower by 0.3 mmol·L−1  at 80% Wmax after training in SETNAC. The mean venous plasma [K+] during submaximal exercise was non-significantly lower and lower after training in SETSaline (p = 0.091) and SETNAC (p = 0.001), respectively. Specifically, venous plasma [K+] was 0.2 mmol·L−1  lower at 50% Wmax after training in SETSaline, and it was 0.3 mmol·L−1  lower at 80% Wmax after training in SETNAC. At exhaustion, venous plasma [K+] was non-significantly higher by 0.3 mmol·L−1  after training in SETSaline, with no training-induced changes in SETNAC (p = 0.371). The rate of decline in venous plasma [K+] from exhaustion and 0.5 min into recovery increased by 0.3 mmol·L−1  with training in SETSaline, whereas it did not change in SETNAC (p = 0.120). No changes were observed in venous plasma [K+] during recovery.The mean arterial plasma [K= 0.063) . Specifi+] difference during submaximal exercise was lower after training in SETNAC only (p = 0.024). The venous-arterial plasma [K+] difference was 0.1 mmol·L−1  and 0.1 mmol·L−1  lower at 30 and 50% Wmax, respectively, after training in SETNAC. However, at exhaustion, the venous-arterial plasma [K+] difference was 0.3 mmol·L−1  higher after training in SETSaline, but was unchanged in SETNAC (p = 0.319). The rate of decline in the venous-arterial plasma [K+] difference from exhaustion and 0.5 min into recovery was 0.2 mmol·L−1  greater after training in SETSaline, with no training-induced change observed in SETNAC (p = 0.537). No changes were observed in the venous-arterial plasma [K+] difference during recovery.The mean venous-arterial plasma  during submaximal exercise did not change with training in either SETSaline (p = 0.529) or SETNAC (p = 0.104). However, arterial plasma [Na+] was 1.1 mmol·L−1  lower at 80% Wmax after training in SETNAC , with mean venous plasma [Na+] being unaffected by training in SETSaline (p = 0.474), but being 1.6 mmol·L−1  and 2.3 mmol·L−1  lower at 50 and 80% Wmax, respectively, after training in SETNAC.The mean arterial plasma [Nan SETNAC . No othe+] difference during submaximal exercise was lower after training in both SETSaline (p = 0.009) and SETNAC (p < 0.001). Specifically, the venous-arterial plasma [Na+] difference at 80% Wmax was non-significantly lower by 0.7 mmol·L−1  after training in SETSaline, whereas it was 1.6 mmol·L−1  and 1.3 mmol·L−1  lower at 50 and 80% Wmax, respectively, after training in SETNAC. At exhaustion, the venous-arterial plasma [Na+] difference was 0.7 mmol·L−1  lower after training in SETSaline, while it was 1 mmol·L−1  higher 2 min into recovery.The mean venous-arterial plasma  and muscle pH at exhaustion were not affected by training in SETSaline, whereas they were 25 mmol·kg d.w.−1  lower and 0.19 mmol·kg d.w.−1  higher, respectively, in SETNAC  lower after training in SETNAC, but not in SETSaline (p = 0.295)  and 57 nmol·L−1  lower after training in SETSaline and SETNAC, respectively (−] gradient and peak net leg lactate− release (p = 0.115) as well as the muscle [H+] gradient and peak net leg H+ release (p = 0.098) were not affected by training in SETSaline. However, in SETNAC, the training intervention changed the relationship between the muscle [lactate−] gradient and peak net leg lactate− release (p = 0.003), as well as the muscle [H+] gradient and peak net leg H+ release (p = 0.020), so that a lower muscle concentration gradient resulted in a greater peak net leg release  and 146 ± 13 and 142 ± 12 mmol H+·kg d.w.−1·pH−1 in SETNAC  before and after the training intervention, respectively.Muscle  observed during submaximal exercise after SET. Indeed, a lower relative exercise intensity after SET would reduce motor unit firing frequency and the amount of K+ released from the contracting fibres. Furthermore, the ability to counter K+ shifts during submaximal exercise may be related to improved metabolic stability, as indicated by lowered lactate− and H+ shifts, since metabolic perturbations such as lowered muscle adenosine nucleotide content and pH can exacerbate K+ efflux by increasing Kir6.2 opening probability  and [H+] at exhaustion was of a lower magnitude after SET only in the leg receiving N-acetylcysteine infusion, suggesting that accumulation of myocellular lactate− and H+ per se does not cause fatigue during intense exercise, as also previously suggested [+ reuptake capacity was likely of major importance, as excessive extracellular K+ accumulation can cause sarcolemma inexcitability, thereby contributing to muscle fatigue [As expected, SET markedly improved intense exercise performance, as measured by time to exhaustion during incremental knee-extensor exercise . Howeveruggested . Convers fatigue ,2,3,4,5.N-acetylcysteine with respect to its action on exercise-related ionic shifts in young males. This supports a training dependency for N-acetylcysteine responsiveness. In addition, the present results indicate that SET enhances the ability to counter ionic shifts during exercise via a multitude of factors not only related to upregulation of muscle ion handling proteins but also antioxidant enzymes and greater mitochondrial respiratory capacity of the trained muscle.This study demonstrates that a period of SET potentiates the effect