A or GABAB receptors, with effects prevented by selective GABA receptor antagonists. GABA agonists slightly modified the frequency sensitivity of DA release during short stimulus trains. GABA agonists also suppressed DA release evoked by optogenetic stimulation of DA axons. Furthermore, antagonists of GABAA and GABAB receptors together, or GABAB receptors alone, significantly enhanced DA release evoked by either optogenetic or electrical stimuli. These results indicate that striatal GABA can inhibit DA release through GABAA and GABAB receptors and that these actions are not mediated by cholinergic circuits. Furthermore, these data reveal that there is a tonic inhibition of DA release by striatal GABA operating through predominantly GABAB receptors.Nigrostriatal dopamine (DA) is critical to action selection and learning. Axonal DA release is locally influenced by striatal neurotransmitters. Striatal neurons are principally GABAergic projection neurons and interneurons, and a small minority of other neurons are cholinergic interneurons (ChIs). ChIs strongly gate striatal DA release via nicotinic receptors (nAChRs) identified on DA axons. Striatal GABA is thought to modulate DA, but GABA receptors have not been documented conclusively on DA axons. However, ChIs express GABA receptors and are therefore candidates for potential mediators of GABA regulation of DA. We addressed whether striatal GABA and its receptors can modulate DA release directly, independently from ChI regulation, by detecting DA in striatal slices from male mice using fast-scan cyclic voltammetry in the absence of nAChR activation. DA release evoked by single electrical pulses in the presence of the nAChR antagonist dihydro-β-erythroidine was reduced by GABA or agonists of GABASIGNIFICANCE STATEMENT The principal inhibitory transmitter in the mammalian striatum, GABA, is thought to modulate striatal dopamine (DA) release, but definitive evidence for GABA receptors on DA axons is lacking. Striatal cholinergic interneurons regulate DA release via axonal nicotinic receptors (nAChRs) and also express GABA receptors, but they have not been eliminated as potentially critical mediators of DA regulation by GABA. Here, we found that GABAA and GABAB receptors inhibit DA release without requiring cholinergic interneurons. Furthermore, ambient levels of GABA inhibited DA release predominantly through GABAB receptors. These findings provide further support for direct inhibition of DA release by GABA receptors and reveal that striatal GABA operates a tonic inhibition on DA output that could critically influence striatal output. DA outpm in rat . Cholinem in rat . ChIs mam in rat , with thm in rat . Furtherm in rat . StriataA receptor, or bicuculline, a GABAA antagonist, respectively decreased and increased extracellular dopamine in intact rats measured by microdialysis  and also when DA release was evoked by targeted activation of DA axons using optogenetics when DA release is not under nAChR control  or DAT-Cre mice injected with an adeno-associated virus encoding Cre-dependent ChR2. For experiments with light activation, DAT-Cre mice were bred from homozygotes for DAT-internal ribosome entry site (IRES)-Cre, obtained from Jackson Laboratories (B6.SJL-Slc6a3tm1.1(cre)Bkmn /J, stock 006660) (RRID:IMSR_JAX:006660). P25–P35 DAT-Cre mice were anesthetized with isoflurane, placed in a small animal stereotaxic frame (David Kopf Instruments), and injected with an adeno-associated virus  encoding Cre-dependent ChR2 (AAV5-EF1α-DIO-hChR2(H134R)-eYFP) (RRID:SCR_002448). A total volume of 1 μl of virus solution was injected bilaterally (500 nl per hemisphere/injection) into substantia nigra pars compacta . Virus solution was injected at an infusion rate of 50 nl/min with a 32 gauge Hamilton syringe and withdrawn 5–10 min after the end of injection. Virus-injected mice were used for experiments >4 weeks after viral injection.Animals were either wild-type C57BL6 male mice at postnatal day 35 (P35) to P70 (RRID:m): 120 NaCl, 20 NaHCO3, 6.7 HEPES acid, 5 KCl, 3.3 HEPES salt, 2 CaCl2, 2 MgSO4, 1.2 KH2PO4, and 10 glucose. Slices were then maintained in HEPES-buffered artificial CSF at room temperature for at least 1 h before recording. All procedures were performed according to institutional guidelines and conformed to the UK Animals (Scientific Procedures) Act of 1986.Wild-type C57BL6 animals (P35–P70) or DAT-Cre mice (P53–P63) were decapitated after cervical dislocation and the brains were extracted. Coronal slices, 300 μm thick, were prepared in ice-cold HEPES-buffered artificial CSF (aCSF) containing the following (in m2/ 5% CO2 at 31–32°C containing the following (in mm): 124 NaCl, 26 NaHCO3, 3.8 KCl, 2.4 CaCl2, 1.3 MgSO4, 1.3 KH2PO4, and 10 glucose. All experiments with electrical stimulation were conducted in the presence of 1 μm DHβE to prevent the effects of nAChR activation on DA release (o) was monitored using FCV with 7-μm-diameter carbon fiber microelectrodes (tip length 50–100 μm) and a Millar voltammeter  as described previously  and locally written Excel macros.Evoked DA release was detected in acute slices using fast-scan cyclic voltammetry (FCV). Slices were superfused with a bicarbonate-buffered aCSF saturated with 95% O release . Extraceeviously . In briem DHβE, which prevents nAChR activation. Stimulation intensity was set to 80% maximal [DA]o: ∼0.6 mA. Applied stimuli were single 200 μs pulses (1p) or five pulses (5p) at 5, 25, and 100 Hz. Mean peak [DA]o evoked by 1p was equivalent to that of a 1 Hz train; 1p is used in frequency comparison to indicate maximum 1 Hz data. Electrical stimulations were repeated at 2.5 min intervals, which allowed stable release to be sustained over several hours. Mean [DA]o evoked by a single electrical pulse in control conditions (in the presence of DHβE) across release sites was 1.13 ± 0.06 μm.Recordings were obtained from the dorsal striatum. Electrical stimulation was delivered by a local bipolar concentric electrode . All experiments with electrical stimulation were conducted in the presence of 1 μo. During recordings, slices were visualized on an upright microscope (Olympus BX50WI) with fluorescence optics for visualizing eYFP. Mean [DA]o evoked by a single light pulse in control conditions was 0.90 ± 0.09 μm.Light stimulation was delivered by an LED system . DA release was evoked with full-field 470 nm blue light and a pulse duration of 2 ms. Experimental stimulation intensity was determined by delivering a light pulse sufficient to drive ∼50% maximal [DA]n” refers to the number of experiments. Each experiment was performed at a single recording site in one brain slice. For each experiment at a given recording site, data for each variable were obtained in at least triplicate before averaging to obtain the value for that individual parameter. The number of animals in each dataset was ≥ 3. Data are expressed as extracellular concentration of dopamine ([DA]o) or as [DA]o normalized to mean peak [DA]o evoked by single pulses in control conditions. In all cases, [DA]o displayed typical kinetics to peak and decay, indicative of good slice quality and were obtained from recording sites that maintained sufficiently stable levels of release over time. No data were excluded after acquisition.Data are represented as means ± SEM, and “o evoked by 5p/1p were obtained by dividing each 5p-evoked [DA]o value with an average 1p-evoked [DA]o value in the same condition at that recording site. In graphs representing peak [DA]o evoked by 1p over time, missing data points were time points when data were acquired using different stimulation parameters interspersed among 1p stimulations. Comparisons for statistical significance were assessed by one- or two-way ANOVA, paired t tests, or Mann–Whitney U tests where data were not normally distributed using GraphPad Prism (RRID:SCR_002798).Data acquired immediately before drug application were used as predrug control data and were compared with data acquired after drug effects had equilibrated after ∼10–20 min of application. Ratios for [DA]DHβE, saclofen, and bicuculline were obtained from Tocris Bioscience. Baclofen, picrotoxin, and GABA were obtained from Sigma-Aldrich. Muscimol and CGP 55845 hydrochloride were obtained from Abcam. Stock aliquots of drugs were prepared at 1000–10,000× final concentrations in deionized water, aqueous acid (baclofen), or DMSO (picrotoxin) and stored at −20°C. DHβE was present throughout all experiments with electrical stimulation.m) to inhibit nAChRs as described previously (m to 1 μm) on [DA]o evoked by a single electrical pulse. DHβE concentration-dependently inhibited evoked [DA]o  and 1 μm DHβE was supramaximal for inhibition of [DA]o.We assessed whether GABA can modulate striatal DA release in the absence of nAChR activity using the antagonist DHβE (1 μeviously . We firsm). Bath application of GABA  concentration dependently reduced DA release evoked by single electrical pulses by ∼15–50%  = 48.32, p < 0.0001, 100 μm GABA, U = 0, p = 0.016, n = 7; U = 6, 5 mm, p = 0.0079, n = 5, Mann–Whitney tests), indicating that GABA is capable of inhibiting DA release independently from any effects on ChI input to nAChRs.GABA was applied to striatal slices in the presence of the nAChR antagonist DHβE (1 μA or GABAB agonists. Activation of GABAA receptors with muscimol (20 μm) suppressed DA release evoked by single electrical pulses by ∼30%  and this effect was prevented by prior application of GABAA channel blocker picrotoxin (100 μM), confirming an effect via GABAA receptors . Activation of GABAB receptors with baclofen (10 μm) also suppressed DA release evoked by single electrical pulses by ∼25%  and this effect was confirmed to be due to GABAB receptors by prior application of the GABAB antagonist saclofen (100 μM), which prevented the effect of baclofen .To identify which GABA receptors can inhibit DA release, we tested the effect of GABAo evoked by 5-pulse trains at all frequencies tested  = 67.55; p = 0.0025, frequency effect: F = 259.6, p < 0.0001, n = 7) and, furthermore, there was a significant interaction between stimulation frequency and drug effect  = 5.485, p = 0.0025), which was borne out by an increase in the 5p/1p ratio examined at 100 Hz  = 3.46, p = 0.014). In other words, activation of GABAA receptors can marginally promote the contrast in DA signals released by different firing patterns or rate.We investigated whether GABA receptor activation inhibited DA release during trains of stimuli and across a range of frequencies. Muscimol significantly decreased [DA]o  = 6.271, drug effect: p = 0.016; frequency effect: F = 144.5, p < 0.0001, n = 7). We did not detect a significant interaction between stimulation frequency and drug effect  = 0.040, p = 0.99); however, the 5p/1p ratio for [DA]o evoked at 100 Hz was slightly increased with baclofen  = 3.272, p = 0.017), suggesting that GABAB receptors only marginally change the contrast in DA signals released by different activity.Baclofen significantly decreased evoked [DA]A agonist muscimol or the GABAB agonist baclofen suppressed DA release evoked by single blue light pulses , indicating that GABA receptor agonists do not require coincident activation of another striatal input to suppress DA release. We also confirmed that, as with electrical stimulation, the effects of muscimol and baclofen were prevented by prior application of antagonists for respectively GABAA  or GABAB receptors .To determine whether the inhibition of DA release by GABA receptors depended on the coactivation during electrical stimulation of some other local neuron type or input, we used an optogenetic approach to selectively activate DA axons only. We expressed ChR2-eYFP in DA neurons and axons in DAT-Cre mice using an established viral approach as described previously . Either Some striatal GABA neurons are tonically active and it has been reported that there is an ambient GABA tone in striatum . We therA and GABAB antagonists bicuculline (10 μm) and CGP 55845 (2 μm), respectively, significantly enhanced [DA]o evoked by single light pulses in DAT-Cre ChR2-expressing mice by ∼20% , indicating a tonic inhibition of DA release by endogenous striatal GABA in the absence of other stimuli. We investigated which receptors could mediate inhibition of DA release by endogenous GABA using electrical stimulation in wild-type mice (in the presence of DHβE). GABAA antagonist bicuculline appeared to slightly elevate, but did not significantly increase, evoked [DA]o  = 1.58, p = 0.215; effects of frequency: F = 128.4, p < 0.0001, n = 5) or interact with stimulation frequency  = 0.115, p = 0.95) or the 5p:1p ratio  = 1.217, p = 0.263, paired t test). The GABAB antagonist saclofen significantly increased evoked [DA]o  = 4.87, p = 0.0371; effects of frequency, F = 57.8, p < 0.0001). There was no significant interaction with stimulation frequency  = 0.205, p = 0.89) or 5p:1p ratio  = 1.49, p = 0.233, paired t test).We found that coapplication of GABAA receptors, and GABAB receptors in dorsal striatum inhibit DA release and that these actions are not mediated via regulation of striatal ACh acting at nAChRs. By eliminating ChIs as a necessary site of action of GABA, a direct action on DA axons becomes plausible as a site for GABA action on DA transmission. Furthermore, we show that GABA receptor antagonists increase DA release evoked by single optogenetic stimuli, revealing that there is tonic inhibition of DA release by endogenous striatal GABA arising from a tonically active source or ambient level.Here, we show that GABA, GABAA and GABAB receptor ligands can inhibit DA release whether evoked electrically in the presence of nAChR antagonist or optogenetically by targeted activation of ChR2-expressing DA axons in DAT-Cre mice. Light-activated DA release is not under tonic control by striatal nAChRs in these stimulation conditions  that evoked DA release should not be under the control of any GABA that might be coreleased by this stimulus. Mesostriatal DA neurons can apparently synthesize, store, and corelease GABA , which carations . Our finAn ambient GABA tone that can act on DA axons to limit DA output should not be entirely surprising. At least one type of GABAergic interneuron, the LTS interneuron, is capable of autonomous firing in striatal slices . FurtherA and GABAB receptors and these are certainly functional in somatodendritic compartments (B receptors have been immunocytochemically detected in striatal neuropil, consistent with localization of these receptors on DA axons (A receptors have not yet been shown on DA axons. It can be difficult to verify the ultrastructural location of membrane bound receptors using classic immunocytochemical methods, which depend on appropriate fixation methods and conditions for antibody penetration; absence of proof is not proof of absence.The site of GABA receptor localization remains undefined. DA neurons in substantia nigra express GABAartments , but theDA axons , but GABA receptors are acting indirectly through an intermediary GABA circuit that modulates DA via downstream GABAB receptors, then GABAA receptor agonists would be expected to inhibit the intermediary GABA circuit and therefore increase DA release, which is the opposite of what was seen here. The remaining candidate locations for the GABA receptors that regulate DA are therefore either an undisclosed neuron or other cell type with currently unknown actions on DA or, more parsimoniously, DA axons themselves. Responses to GABA have been described for axons of several other neurons (A receptors can reduce axonal spike amplitude and propagation and promote spike failures (B receptors inhibit voltage-gated Ca2+ channels (Key candidates for alternative sites of action can however be eliminated. Here, we exclude actions of GABA receptors on ChIs acting through downstream nAChRs. Furthermore, GABAergic interneurons are unlikely as intermediaries: for example, if GABA neurons . For exafailures , whereaschannels . SimilarA and GABAB receptors can gate DA output, but that there is a tonic inhibition by endogenous GABA through an apparent ambient GABA tone. In addition to directly regulating striatal output, striatal GABA tone might therefore also govern striatal integration via dampening DA output. We exclude cholinergic interneurons acting through nAChRs on DA axons as intermediaries for GABA regulation of DA, thereby adding support to the hypothesis that GABA is acting directly on DA axons. With the assistance of new genetic tagging and imaging tools, future studies should revisit the potential localization of GABAA and GABAB receptors to DA axons.In conclusion, we show here not only that GABA


To address these questions, recordings were obtained with 2 mm Ca2+ and 0 mm Na+ in the bath and 120 mm Cs+ in the pipettes from myocytes isolated from adult mouse interventricular septum (IVS), left ventricular (LV) endocardium, apex, and epicardium and from human LV endocardium and epicardium. On membrane depolarizations from a holding potential of −100 mV, ICa(TTX) was identified in mouse IVS and LV endocardial myocytes and in human LV endocardial myocytes, whereas only TTX-sensitive outward Cs+/K+ currents were observed in mouse LV apex and epicardial myocytes and human LV epicardial myocytes. The inward Ca2+, but not the outward Cs+/K+, currents were blocked by mm concentrations of MTSEA, a selective blocker of cardiac Nav1.5-encoded Na+ channels. In addition, in Nav1.5-expressing tsA-201 cells, ICa(TTX) was observed in 3 (of 20) cells, and TTX-sensitive outward Cs+/K+ currents were observed in the other (17) cells. The time- and voltage-dependent properties of the TTX-sensitive inward Ca2+ and outward Cs+/K+ currents recorded in Nav1.5-expressing tsA-201 were indistinguishable from native currents in mouse and human cardiac myocytes. Overall, the results presented here suggest marked regional, cell type-specific, differences in the relative ion selectivity, and likely the molecular architecture, of native SCN5A-/Scn5a- (Nav1.5-) encoded cardiac Na+ channels in mouse and human ventricles.Tetrodotoxin (TTX) sensitive inward Ca All data are presented as means ± SEM. The statistical significance of observed differences was evaluated using a paired (compared in the same cell) two-tailed student’s t test, one-way or two-way ANOVA, as indicated in the text or figure legends; P values ≤ 0.05 were considered statistically significant.Electrophysiological data were compiled and analyzed using Clampfit 10.3 (Molecular Devices) and GraphPad (Prism). Peak inward/outward current amplitudes were measured in each cell at various test potentials and normalized to the whole-cell membrane capacitance (in the same cell); current densities (pA/pF) are reported. The decay phases of currents  and −50 mV (□) in this cell, are plotted as a function of the test potential in Whole-cell voltage-clamp recordings, obtained from isolated mouse interventricular septum (IVS) myocytes with 2 m current –30, now or ICa,L ,31. The  −100 mV , panel c2+ current in mouse IVS myocytes is selectively blocked . As illustrated in μm TTX eliminated the low threshold, rapidly activating and inactivating component of the inward Ca2+ current without measurably affecting the slowly activating and inactivating Ca2+ current, ICa,L. The current-voltage relation for ICa,L (μm TTX (▲), is plotted in C. Offline digital subtraction of the inward currents evoked from a HP of −100 mV in the presence of TTX  [Additional experiments revealed that the rapidly activating and inactivating component of the inward Ca blocked ) by μM cor ICa,L , panel be of TTX , panel be of TTX , panel aTTX  ,10,12,202+ channels [2+ current component evoked from a holding potential of −100 mV in isolated mouse IVS myocytes is not affected by the selective T-type Ca2+ channel blocker Ni2+ .Although low threshold, “T” type, Cachannels  have prechannels , additioker Ni2+ ) at 100 erapamil  in a dosICa(TTX) was identified in all mouse IVS myocytes (n = 20) examined, although there was, as is illustrated in +) currents were observed at −25 mV (Using the protocol illustrated in t −25 mV ). The met −25 mV ; at morem) MTSEA, a selective inhibitor of Nav1.5-encoded cardiac Na+ channels [ICa(TTX) in mouse IVS myocytes. As illustrated in the representative recordings obtained from a mouse IVS cell presented in 2+ currents were recorded under control conditions with 2 mm Ca2+and 0 mm Na+ in the bath and 120 mm Cs+ in the recording pipette (left panel). When the bath solution was switched to one containing 2 mm MTSEA, the currents were eliminated  Na+ in the bath, much larger amplitude inward (Na+) currents were observed  and after (o) the application of 2 mm MTSEA are presented in When recordings were obtained from isolated adult mouse IVS myocytes with increased , LV epicardium  and LV apex  are presented in Additional experiments were completed to quantify the expression of TTX-sensitive inward/outward currents in myocytes isolated from different regions of adult mouse left ventricles (LV). As in the experiments on mouse IVS myocytes, voltage-activated inward/outward currents, evoked from a HP of −100 mV in response to 300 ms test potentials between −90 mV to −25 mV (in 5 mV increments) were recorded with 2 m+ currents were recorded in cells from all three regions of the adult mouse LV, outward currents were larger in LV epi and LV apex myocytes, and inward ICa(TTX) was only evident in recordings from LV endo cells (ICa(TTX) density (at −40 mV) in LV endo cells of −0.9 ± 0.5 pA/pF (n = 11), however, was much lower than the mean + SEM peak ICa(TTX) density (at −40 mV) of (−2.9 ± 0.5 pA/pF (n = 20) measured in IVS myocytes. Similar results were obtained when 120 mm K+ replaced the Cs+ in the recording pipettes (data not shown). These combined observations suggested the interesting hypothesis and that inward TTX-sensitive Ca2+ currents might be revealed in mouse LV apex (and LV epi) myocytes if the outward Cs+/K+ currents were blocked. To test this hypothesis, additional experiments were conducted with NMDG+, which does not permeate voltage-gated Na+ channels [+/K+, in the recording pipettes. As illustrated in 2+ currents were recorded from LV apex and, as expected, from IVS myocytes with NMDG+-containing pipette solution (see: Methods). In addition, although the mean ± SEM amplitudes/densities of the currents are different, the voltage-dependent properties of ICa(TTX) recorded in IVS and LV apex myocytes with the NMDG+-containing pipette solution are indistinguishable .2+ and Na+ on the time and voltage-dependent properties of TTX-sensitive inward currents in mouse IVS myocytes. Currents, evoked during voltage steps to test potentials between −90 mV and −25 mV from a HP of −100 mV and from a HP of −50 mV, were recorded in isolated adult mouse IVS myocytes with 2 mm Ca2+/0 mm Na+ in the bath and 120 mm Cs+ in the recording pipettes. The bath solution was then changed sequentially to one containing 2 mm Ca2+/10 mm Na+, followed by 2 mm Ca2+/20 mm Na+ and, finally, 0 mm Ca2+/20 mm Na+, and the voltage-clamp paradigms were repeated. The rapidly activating and inactivating inward currents under each recording condition, isolated by off-line digital subtraction of the currents evoked from −50 mV from those evoked from −100 mV, are presented in m Na+ (and 2 mm Ca2+) to the bath  further increased the peak inward current (2+ (replaced with 2 mm Mg2+) from the bath solution, inward current amplitudes were increased (m Ca2+/20 mm Na+ (m Ca2+/0 mm Na+ (°), 2 mm Ca2+/20 mm Na+ (●) and 0 mm Ca2+/20 mm Na+ (■) in the bath were determined and are presented in m Ca2+/20 mm Na+ (■), compared with 2 mm Ca2+/20 mm Na+ (●). The normalized voltage-dependences of activation of the inward currents recorded with 2 mm Ca2+/20 mm Na+ (•) and 0 mm Ca2+/20 mm Na+ (■) in the bath, each fitted with a single Boltzmann, are presented in Increasing the Na current , panel cncreased , panel d0 mm Na+ , panel cSCN5A (Nav1.5) and enhanced green fluorescent protein (eGFP). Using voltage-clamp paradigms similar to those used in the experiments on mouse ventricular myocytes described above, currents, evoked during voltage steps to test potentials between −90 mV and −25 mV from a HP of −100 mV and from a HP of −50 mV, were recorded from eGFP-positive tsA-201 cells with 2 mm Ca2+/0 mm Na+ in the bath and 120 mm Cs+ or 145 mm K+ in the recording pipettes. The bath solution was then changed sequentially to 2 mm Ca2+/10 mm Na+, followed by 2 mm Ca2+/20 mm Na+ and, finally, 0 mm Ca2+/20 mm Na+, and the voltage-clamp paradigms were repeated. The currents obtained with offline digital subtraction of the currents evoked from −50 mV from those evoked from −100 mV were analyzed.Additional experiments were conducted to detail the properties of the TTX-sensitive currents expressed in tsA-201 cells transiently transfected with a plasmid encoding human + (K+) were detected  in the bath (m Na+ (and 2 mm Ca2+) in the bath (2+ was removed (and replaced with 2 mm Mg2+) from the bath (m Ca2+/0 mm Na+ (o), 2 mm Ca2+/20 mm Na+ (●) and 0 mm Ca2+/20 mm Na+ (■) in the bath are presented in + in the bath, but were best described by the sum of two exponentials with 20 mm Na+ (and either 2 mm Ca2+ or 0 mm Ca2+) in the bath , compared with 2 mm Ca2+/20 mm Na+ (●), in the bath.In three (of the 20) eGFP-positive tsA-201 cells from which recordings were obtained using the protocols described above, inward currents were observed -c), wherthe bath , panel bthe bath , panel cthe bath , panel dm Ca2+/0 mm Na+ in the bath and 145 mm K+ (m Cs+ (not shown) in the recording pipettes. With increased Na+  in the bath, the outward currents progressively decreased and inward Na+ currents were recorded (2+ (replaced with Mg2+) from the bath further increased the inward current amplitudes (+ (o), and inward Na+  currents are plotted as a function of test potential in + currents recorded in 20 mm Na+ and 2 mm Ca2+ (●) and 20 mm Na+ and 0 Ca2+ (■), fitted with single Boltzmanns, are presented in 2+ and outward Cs+/K+ currents in Nav1.5-expressing tsA-201 cells were blocked by MTSEA (data not shown).In most (17/20 cells) tsA-201 cells expressing Nav1.5, only outward currents were observed in the presence of 2 m45 mm K+ , panel arecorded , panels plitudes , panel dm Ca2+ and 0 mm Na+ in the bath and 120 mm Cs+ in the recording pipettes. Currents, evoked from a HP of −100 mV in response to 300 ms test potentials between −90 mV to −25 mV (in 5 mV increments), were recorded in the absence and in the presence of 10 µM TTX, and the TTX-sensitive currents, obtained by offline digital subtractions of the records before and after exposure to TTX, were analyzed. As illust-rated in the representative records shown in n = 16) and LV Epi  are presented in n = 16) and LV Epi  myocytes are plotted as a function of test potential in Whole-cell recordings were obtained from myocytes isolated from the endocardial (LV Endo) and epicardial (LV Epi) surfaces of human LV free wall (see Methods) with 2 m2+ currents and outward Cs+/K+ currents in myocytes isolated from adult mouse interventricular septum, LV apex, LV endocardial and LV endocardial myocytes, as well as in LV endocardial and epicardial myocytes isolated from non-failing human hearts, although marked differences in the relative amplitudes of the currents were observed in cells isolated from different regions of the LV in both mouse and human. The rapidly activating and inactivating, TTX-sensitive inward Ca2+ currents in adult mouse IVS myocytes were unaffected by Ni2+ and by verapamil , and that the pharmacological and the time- and voltage-dependent properties of the heterologously expressed Nav1.5-encoded currents were indistinguishable from those of native ventricular (TTX-sensitive inward and outward) currents in mouse and human ventricular myocytes. Also, similar to the findings in mouse ventricular myocytes, the TTX-sensitive inward Ca2+ and outward Cs+/K+ currents in Nav1.5-expressing tsA-201 cells were blocked by MTSEA. The simplest interpretation of these combined results is that both the inward Ca2+ currents and the outward Cs+/K+ currents recorded in mouse and human ventricular myocytes when extracellular Na+ is removed reflect currents through Nav1.5-encoded Na+ channels.Additional experiments revealed that TTX-sensitive inward Ca2+ and outward Cs+/K+ currents in adult mouse ventricles. ICa(TTX), for example, was identified in all myocytes isolated from the mouse IVS, although peak inward Ca2+ current amplitudes/densities were quite variable among (mouse IVS) cells. Heterogeneities in I(TTX)Ca densities have also been reported in human atrial myocytes [+) currents were also observed at the more positive test potentials. Heterogeneous expression of ICa(TTX) and TTX-sensitive outward (Cs+/K+) currents was also evident in mouse LV apex and LV free wall endocardial and epicardial myocytes, as well as in human LV endocardial and epicardial myocytes. These combined observations are quite different from the findings of Alvarez and colleagues [ICa(TTX) in different regions of infarcted rat left ventricles, suggesting species differences in the expression of TTX-sensitive currents. Alternatively, it is possible that the homogeneous expression of ICa(TTX) reported by Alvarez and colleagues [Marked regional differences were observed in the expression of TTX-sensitive inward Camyocytes . In addilleagues  who repolleagues  reflectsICa(TTX) reflects inward Ca2+ flux through Nav channels encoded by the predominate Nav α subunit expressed in the heart, Nav1.5 [ICa(TTX), however, are distinct from TTX-sensitive cardiac Nav currents [+ and/or K+ [INa in adult rat ventricular myocytes, had no effect on ICa(TTX) [ICa(TTX) and TTX-sensitive outward Cs+/K+ currents.The sensitivity to the membrane (holding) potential and to TTX led to suggestions that , Nav1.5 . The timcurrents ,4,6. In nd/or K+ ,10. InteICa(TTX) , observa2+ currents and outward Cs+/K+ currents in tsA-201 cells transiently transfected with a cDNA construct encoding human SCN5A (Nav1.5). Similar to the observations in mouse and human ventricular myocytes, these experiments also revealed TTX-sensitive inward Ca2+ currents in a subset of the transiently transfected tsA-201 cells and outward Cs+/K+ currents in the others. The observed regional differences in the functional expression of TTX-sensitive inward Ca2+ currents and outward Cs+/K+ currents in mouse and human ventricular myocytes suggest that native Nav1.5-encoded cardiac Na+ channels are molecularly heterogeneous. Interestingly, it was recently reported that there are marked differences in Scn5a transcript expression levels and functional Nav current densities in adult mouse left and right ventricular endocardium and epicardium [Scn5a/SCN5A transcripts [+ and Ca2+ channels [+ channels in mouse and human ventricles.The experiments here, however, revealed TTX-sensitive inward Caicardium . Cell tynscripts , as has channels –41, as wchannels ,43 and Tchannels . Regionachannels  and/or ochannels ,47, as wchannels  and/or ochannels –47, coul2+ currents and outward Cs+/K+ currents suggest that there are (at least) two molecularly distinct types of Scn5a-/SCN5A-encoded channels in both mouse and human LV myocytes. The molecular determinants of these channels may also be distinct from the Nav1.5-encoded channels that mediate inward Na+ flux and control cardiac myocyte action potential generation and propagation that display low Ca2+ and Cs+/K+ permeability [ICa(TTX) activates at potentials negative to the activation of INa, these results suggest a possible role for Ca2+ entry through ICa(TTX) channels in regulating myocardial excitability and rhythmicity, particularly in the subthreshold range of membrane potentials. If there are changes in the relative expression and/or the distribution of ICa(TTX) channels under pathophysiological conditions [The observed regional differences in the functional expression of TTX-sensitive inward Caeability ,14. Togenditions , these c


Polyglycerol-based thermoresponsive nanogels (tNGs) have been shown to have excellent skin hydration properties and to be valuable delivery systems for sustained release of drugs into skin. In this study, we compared the skin penetration of tacrolimus formulated in tNGs with a commercial 0.1% tacrolimus ointment. The penetration of the drug was investigated in ex vivo abdominal and breast skin, while different methods for skin barrier disruption were investigated to improve skin permeability or simulate inflammatory conditions with compromised skin barrier. The amount of penetrated tacrolimus was measured in skin extracts by liquid chromatography tandem-mass spectrometry (LC-MS/MS), whereas the inflammatory markers IL-6 and IL-8 were detected by enzyme-linked immunosorbent assay (ELISA). Higher amounts of tacrolimus penetrated in breast as compared to abdominal skin or in barrier-disrupted as compared to intact skin, confirming that the stratum corneum is the main barrier for tacrolimus skin penetration. The anti-proliferative effect of the penetrated drug was measured in skin tissue/Jurkat cells co-cultures. Interestingly, tNGs exhibited similar anti-proliferative effects as the 0.1% tacrolimus ointment. We conclude that polyglycerol-based nanogels represent an interesting alternative to paraffin-based formulations for the treatment of inflammatory skin conditions. Tacrolimus, also known as FK 506, is an immunosuppressive drug acting predominantly via inhibition of T cell proliferation. This macrolide molecule builds a complex with the FK binding protein, which in turn binds to calcineurin and inhibits the dephosphorylation of the nuclear factor of activated T cells (NFATs) and its translocation to the nucleus ,2,3. As ®) consists of a paraffin-based ointment containing 0.03% or 0.1% tacrolimus in hard, liquid and white soft paraffin. In addition to insufficient drug penetration rates, mis-sensations such as skin stinging, burning and pruritus at the application site frequently lead to treatment discontinuation non-4-yn-9-ylmethyl (4-nitrophenyl) carbonate was purchased from Synaffix . The cyanine dye indodicarbocyanine (IDCC) was purchased from Lumiprobe GmbH .Commercially available chemicals from standardized sources were used as delivered. Solvents were purchased as reagent grade and distilled if necessary. Anhydrous solvents were either purchased as ultra-dry solvent (Acros OrganicsN-isopropylacrylamide) (pNIPAM)-based nanogels (NG-pNIPAM), NIPAM (66 mg), acrylated dendritic polyglycerol (dPG-Ac10%) (33 mg), sodium dodecyl sulfate (SDS) (1.8 mg), and ammonium persulfate (APS) (2.8 mg) were dissolved in 5 mL of distilled water. Argon was bubbled into the reaction mixture for 15 min, which was followed by stirring under argon atmosphere for another 15 min. The reaction mixture was transferred into a hot bath at 68 °C and polymerization was activated after 5 min with the addition of a catalytic amount of N,N,N′,N′-tetramethylethane-1,2-diamine . The mixture was stirred at 500 rpm for at least 4 h, prior to purification by dialysis. For the synthesis of fluorescently labeled nanogels, a mixture of unlabeled dPG-Ac10% (30 mg) and indodicarbocyanine (IDCC)-labelled dPG-Ac10% (3 mg) was used.The tNGs were synthesized according to previously reported methods. For detailed synthesis and characterization description, refer to the following publications ,26,28. B8%) (10 mg) and di-azide functionalized tPG (tPG-(N3)2) (20 mg) were mixed in 1 mL of dimethylformamide (DMF), cooled in an ice bath and injected with a syringe into 20 mL of water at 45 °C. The mixture was stirred for 3 h and the unreacted alkynes were quenched with azidopropanol or alternatively with IDCC-N3.For the synthesis of tPG-based nanogels (NG-tPG), dPG functionalized with -bicyclo[6.1.0]non-4-yn-9-ylmethyl carbonate  were added to a suspension of tacrolimus (5 mg) in 2 mL of MilliQ water. The nanogels were let to swell, followed by sonication in an ice water bath for 30 min. The suspension was stirred overnight at 25 °C and the encapsulated fraction was separated from the free drug by filtration with a 0.45 µm regenerated cellulose syringe filter. Following, 100 µL of the encapsulated fraction were lyophilized for determination of the total concentration, while the encapsulation capacity was determined by LC-MS/MS measurements. The nanogels with the encapsulated drugs were stored at 4 °C. The characterization of the investigated nanogels is reported in 2 were marked. In order to induce skin barrier disruption, three methods were used: tape stripping 50 times (50 × TS), laser poration (LP), and sodium lauryl sulfate (SLS). TS was performed with adhesive, polyethylene tape . For LP, a P.L.E.A.S.E® Professional system  was used with the following setting: delivered energy 73.1 J/cm2, pulse length 125 μs, repetition rate 300 Hz, 10 pulses per pore, treated surface 10 × 10 mm, pore density 8%. For SLS treatment, 20 µL/cm2 of 5 % SLS (w/v) in deionized distilled water were applied on a filter paper disc  and incubated for 4 h at 37 °C. Thereafter, skin was carefully cleaned with a paper towel.Breast and abdominal skin were obtained after informed consent from healthy donors undergoing plastic surgery. The study was conducted after approval by the Ethics Committee of the Charité–Universitätsmedizin Berlin  and in accordance with the Declaration of Helsinki guidelines. The excised skin was used between 3 and 6 h after surgery and was examined to exclude injured parts, including macroscopic skin surface damages but also deeper structural defects like stretch marks. Subcutaneous fat tissue was partially removed (approximately 0.5 cm was kept) and skin was stretched and fixed on a Styrofoam block using needles. Skin areas of 2 cm®, manufactured by LEO Laboratories Ltd., Dublin, Ireland). Safety margins of at least 0.5 cm were left. Controls consisted of skin treated with 20 µL/cm2 of sterile 0.9% NaCl solution. Samples and controls were placed in humid chambers (plastic boxes with lid filled with humidified towels) and incubated for 24 h at 37 °C, 5% CO2 and 100% humidity. After incubation, non-penetrated material was removed with a paper towel, the untreated safety margins were cut and the treated tissue blocks were plunge frozen in liquid nitrogen and stored at −80°C for further processing.A total amount of 5 µg tacrolimus per square centimeter was applied either incorporated in an aqueous suspension of nanogels or as 0.1% marketed formulation  with a microtome : the first 100 µm corresponding roughly to epidermis and the remaining 900 µm to dermis. The sections were put in 500 µL of extraction buffer , homogenized  for 10 s, and incubated on ice for 45 min. Then, samples were sonicated at 4 °C for 10 min, vortexed and centrifuged for 5 min at 450× 13C1,D4]tacrolimus , vortexed, and centrifuged at 9300× g for 3 min (4 °C). Analyses were conducted with an Agilent 1260 Infinity LC system coupled to an Agilent 6490 triple quadrupole-mass spectrometer  interfaced with an electrospray ion source operating in the positive ion mode (ESI+). Chromatographic separation was carried out using an Agilent Zorbax SB-C18 column . Aqueous ammonium formate  and methanol  were used as eluents A and B, respectively. Samples (5 µL) were injected into a mobile phase consisting of 90% eluent A. Tacrolimus and its internal standard tacrolimus were eluted from the column, which was tempered at 30 °C, with a 4-min linear gradient to and a subsequent isocratic stage for 5 min at 2:98 (v:v) eluent A/B at a flow rate of 0.35 mL/min. Tacrolimus and tacrolimus co-eluted from the separation column at 5.3 min. The total run time for one analysis was 13 min, including re-equilibration of the LC system. The following ion source parameters were taken from Koster et al. tacrolimus was carried out using the multiple reaction monitoring (MRM) approach. Ammonium adducts [M+NH4]+ were selected as precursor ions by the first quadrupole. The following mass transitions were recorded (optimized collision energies in parentheses): tacrolimus: m/z 821.5 → 786.5 (16 eV), m/z 821.5 → 768.5 (20 eV), m/z 821.5 → 576.2 (24 eV); tacrolimus: m/z 826.5 → 791.5 (16 eV), m/z 826.5 → 773.6 (20 eV), m/z 826.5 → 581.4 (24 eV). Thereby, the loss of two hydroxyl groups and ammonium from the precursor ion, represented by m/z 821.5 → 768.5 for tacrolimus and m/z 826.5 → 773.6 for tacrolimus, was used for quantification. The dwell time for each of the six mass transitions recorded was 150 ms.Skin extracts were thawed at room temperature, spiked with stable-isotope labeled internal standard [r et al. , who use® Multimode plate reader .Human IL-6 and IL-8 were investigated using ELISA kits  Invitrogen Corporation, Carlsbad, CA, USA) following the manufacturer instructions. The amounts of cytokines were normalized to total protein content measured with Pierce 660 nm Protein Assay . Absorbance was measured with EnSpireSkin samples treated with fluorescent nanogels were placed in tissue freezing medium  and plunge-frozen in liquid nitrogen. Cryosections of 5 μm thickness were prepared and observed by means of a fluorescence microscope . The following filter combinations were used: bright pass = 545–580 nm, long pass > 610 nm for IDCC (red) and bright pass = 470–490 nm, long pass > 550 nm for FL. Pictures (magnification of 200×) of at least 20 randomly chosen skin sections per donor and skin sample were taken, and the mean fluorescence intensity of areas in the SC, viable epidermis, and dermis was calculated using the ImageJ software (Version 1.47).2 in PBS). Dermis tissue was digested by incubation for 2 h at 37 °C with an enzyme cocktail made of 0.6 g/mL collagenase II , 0.3 g/mL hyaluronidase , and 1 μg/mL DNase . Enzymes were stopped with RPMI-1640 cell culture medium  containing 10% fetal calf serum  and cells were harvested by repeated pipetting, filtering through a 70 µm cell strainer  and washing twice with PBS . After centrifugation at 300× g for 10 min, cells were fixed with 4% paraformaldehyde  and stored at 4 °C until analysis by flow cytometry . At least 20,000 events were collected in the selected gate. The software FCS Express  was used for data analysis. Pictures of isolated cells were taken by means of a confocal laser microscope .After incubation with nanogels, skin samples were cut in small pieces (0.2 × 0.2 cm) and incubated overnight at 4 °C in 2.4 U/mL dispase  in order to detach epidermis from dermis. The epidermis sheets were incubated for 10 min at 37 °C in 5 mL of trypsin solution  supplemented with 10% fetal calf serum, 100 µg/mL streptomycin, and 100 I.E./mL penicillin. Non-adherent cells were collected and red blood cells were lysed with 1% Triton X-100 in PBS for 10 min on ice. Cells were then washed with PBS and incubated with carboxyfluorescein succinimide ester  for 20 min at room temperature. Cells were washed with 1% bovine serum albumin and re-suspended in supplemented RPMI 1640 medium. Cells were left untreated, incubated with IL-2 (2.5 µg/mL) only, or incubated with IL-2 and tacrolimus in 10% ethanol or tacrolimus-loaded NG-tPG nanogels . Cell proliferation was measured after 5 days by flow cytometry. T cells were gated in forward vs. side scatter dot plots and at least 10,000 events were collected.™, Corning, New York, NY, USA). These were placed in a 6-well plate with 2 mL supplemented RPMI 1640 medium and 106 Jurkat cells (clone E6.1) per well. Jurkat cells had previously been stained with CFSE as described above. Skin was maintained at the air-liquid interface and co-cultured with Jurkat cells for 2 or 5 days . 500 µL of fresh medium were replaced every 2 days. Jurkat cells were collected and analyzed by flow cytometry.After topical application of tacrolimus formulations, skin samples were transferred on 8 µm-pore inserts  in ex vivo human skin. Different methods  were applied to simulate a barrier dysfunction and improve drug penetration. Laser poration (at the used energy) induces a local removal of stratum corneum (micropores), whereas tape stripping reduces the thickness of the stratum corneum. Both methods result in a partial removal of both lipids and corneocytes ,31. On tLow penetration of tacrolimus was measured in intact abdominal skin. Pretreatment with TS had no significant effects on drug penetration, independently on the tested formulation . Little These results suggest that breast skin is more permeable than abdominal skin, probably due to a thinner SC and a higher density of hair follicles, which have been shown to contribute to the skin permeation of topically applied substances ,34,35. TWith regard to tacrolimus penetration in breast skin, TS pretreatment resulted in a marked increase of tacrolimus penetration for both ointment and nanogel formulations  .The degree of skin barrier disruption was assessed by measuring the SC thickness in images of skin sections from different regions of the sample. In These results clearly show that, when skin barrier is compromised, nanogels can penetrate to viable skin layers, be taken-up by epidermal and dermal cells and that uptake in dendritic cells is favored in pro-inflammatory environment. This observation may be of special advantage for the treatment of inflammatory skin diseases where, besides T cells, dendritic cells are key targets of therapeutics like tacrolimus. ,6.As outlined in the previous sections, assessment of a potential benefit of enhanced tacrolimus penetration on the cellular level is limited by its irritative effects on keratinocytes and fibroblasts. Given that immune cells are main therapeutic targets, we created an experimental set-up of skin tissue/T cell co-cultures to measure the effects of penetrated tacrolimus on T cell proliferation ,10. FirsApproximately 20% of cells were stimulated to proliferate after addition of IL-2. Both tacrolimus in solution and loaded on NG-tPG reproducibly reduced the percentage of proliferating cells b,c. TacrAs next step, we cultured ex vivo skin at the air/liquid interface with Jurkat cells using a trans-well set-up a.2 of 0.1% tacrolimus ointment, corresponding to 10 and 20 µg/cm2 of tacrolimus, respectively (2 (We hypothesized that, when tacrolimus is applied topically on skin, the penetrated drug would reach the medium compartment and affect the proliferation of Jurkat cells. In fact, all treated samples had higher MFI than the controls, as shown in the representative histogram in ectively c. When tively (2 d,e, a clively (2 e. IntereThis In this work, we show that tacrolimus formulated as ointment or nanogel suspension penetrates skin with different efficiency in dependence on SC thickness and integrity. Irritation effects of tacrolimus ointment and nanogel formulations, reflected by the released inflammatory cytokines IL-6 and IL-8, were more pronounced in barrier-disrupted and immuno-activated skin. Extensive barrier disruption by mechanical removal of SC resulted also in enhanced penetration of topically applied materials, including deformable macromolecules like nanogels, with consequent interaction and uptake by skin immune active cells. These results support the key role of the SC as barrier for drug and nanocarrier penetration. Our results further demonstrate the critical balance of penetration enhancement and potential increase of side effects. Slow release systems as observed herein already addresses this aspect. The slow drug release and delivery combined with SC hydration and special interaction with skin cells in inflamed skin may explain the good performance of nanogels with respect to tacrolimus ointment. Thus, we conclude that tNGs represent an attractive alternative water-based formulation for the topical delivery of high molecular weight, poorly water-soluble drugs like tacrolimus.


Chaetomium thermophilum. The gene was heterologously expressed in Escherichia coli as a His6-tagged fusion protein and characterized for function and structure. The enzyme exhibits dual cofactor specificity for NADPH and NADH and prefers D-xylose over other pentoses and investigated hexoses. A homology model based on a XR from Candida tenuis was generated and the architecture of the cofactor binding site was investigated in detail. Despite the outstanding thermophilicity of its host the enzyme is, however, not thermostable.While in search of an enzyme for the conversion of xylose to xylitol at elevated temperatures, a xylose reductase (XR) gene was identified in the genome of the thermophilic fungus  Chaetomium thermophilum is a thermophilic fungus of the phylum Ascomycota. It is one of the most thermophile Eukaryotes, growing optimally at temperatures of 50–55 °C on rotting organic matter [Neurospora crassa or Aspergillus sp. The complete nuclear and mitochondrial genomes of C. thermophilum are sequenced and the nuclear genome is estimated to be 28.3 Mbp with 7227 open reading frames while the mitochondrial genome is 127 kbp with 15 protein coding genes [c matter . The orgC. thermophilum have been deposited to this date; a number that has been rapidly increasing in recent years compared to only 19 entries until the year 2013 [Due to the host’s outstanding thermostability, it is a promising source of stable enzymes. In total, 191 Protein Data Bank (PDB) entries for d-xylose to the sugar alcohol xylitol. This reaction marks the initial step of the fungal xylose catabolism, eventually leading to the pentose phosphate pathway via oxidation of xylitol to d-xylulose by the enzyme xylitol dehydrogenase. Generally, wild type XRs strongly favor NADPH as cofactor, while dual cofactor specificity for NADH and NADPH was only observed in some cases [+-dependent xylitol dehydrogenases [8-barrel fold composed of eight alpha-helices running antiparallel to eight beta sheets in the center of the barrel [The enzyme D-xylose reductase (EC 1.1.1.307) belongs to the aldose reductase family. It is commonly found in filamentous fungi and yeasts, and catalyzes the reduction of me cases ,5,6,7. Ome cases . More reogenases ,13,14,15e barrel . Commonle barrel .XRs are of significant industrial interest due to their importance for the utilization of hemicellulosic biomass as an energy and carbon source for ethanol production in yeasts ,19, as wN. crassa [Candida tenuis [A considerable number of different XRs has been investigated with regard to stability and reactivity and mutated variants have been produced with the aim of increased activity and shift in cofactor preference, with examples in references ,11,13,15. crassa , and 50 a tenuis .C. thermophilum, with the aim to heterologously express, purify and characterize a novel XR.In order to find an XR with high activity at elevated temperatures, we chose to investigate the genome of one of the most thermostable Eukaryotes, namely After 34 h total fermentation time, 230 g/L wet cell weight (corresponding to 62 g/L dry cell weight (DCW)) were harvested. Crude extract derived from 230 g wet cell weight contained a total activity of 360 U, resulting in a titer of 360 U/L or 5.8 U/g DCW. The space time yield (volumetric productivity) equals 10.6 U/L/h.CtXR was purified to near homogeneity and the apparent molecular mass (Mw), estimated with SDS-PAGE to be approximately 41 kDa, agreed with the theoretical Mw of 39,244 Da. As shown in CtXR during purification was significant. Mass overload during immobilized metal affinity chromatography (IMAC) caused product loss in the flow through. Poor product retention during spin filtration and cutting of product peak during size exclusion chromatography (SEC), to increase purity, led to further product loss. This results in a total recovery of only 3% but a high purification factor of 25. Product size and purity of the various fractions can be seen in the SDS-PAGE image in Recombinant His6-tagged CtXR was kinetically characterized at 30 and 55 °C with the substrate D-xylose in limiting concentrations while NADPH was provided in non-limiting concentrations (cat increases from 30 to 55 °C while there is only little change in KM. (cat) increases, the apparent affinity towards the substrate only decreases slightly with rising temperature.trations . kcat ine in KM. . This meCtXR in a citrate-disodium phosphate buffer at pH 7.0 , while others are converted slightly or to no extent .Relative activity compared to -mannose . The enzM, kcat and catalytic efficiency for d-xylose, l-arabinose, d-galactose and d-glucose  of CtXR was determined at 30, 55, 65 and 75 °C. Enzyme inactivation was assumed to follow first order kinetics. After plotting the natural logarithm of the specific activity against the incubation time, the decay rate λ was determined as the slope of a linear fit during the degradation phase  or substrate (d-xylose) could not be confirmed in vitro (data not shown).The half-life  provides a reliable prediction of the CtXR with a global model quality estimation (GMQE) score of 0.80, and QMEAN Z-score (reliability) of −0.73.The initial homology model generated by SWISS-MODEL  with CaXde 1SM9, ; 52.7% sCtXR are identical in sequence to that of CaXR (PDB code 1MI3). Structural discrepancies are confined to the adenosine-binding site (adenine ring and ribose moieties) where amino-acid replacements in the loop comprising residues 276–283 (same numbering in CtXR and CaXR) are predicted to affect the precise details of adenosine binding, redox properties, and the relative preference for NAD(H) versus NADP(H). Notable CaXR-to-CtXR replacements include N276T, L277R, R280I and Q283S  binding due to the replacements of Leu277 and Gln280 by Arg and Ser in CtXR, respectively, are less obvious from the homology model.Comparing the modeled NAD(H) complex of CtXR A with thXR (CaXR B indicatCtXR with bound NADP(H) was based on details of NADP(H) binding in the crystal structure of pig aldehyde reductase SsADH (PDB code 1HQT). While CtXR and SsADH display more pronounced overall differences at the sequence level, the cofactor-binding site appears sufficiently similar to justify using SsADH as template for prediction of NADP(H) binding in CtXR. In CtXR, the ribose 2′-phosphate group of NADP(H) would receive stabilization through the donation of a hydrogen bond from Thr276 Og1 and ionic interactions with the guanido group of Arg277, and possibly Lys274 depending on its precise side-chain conformation  of CtXR are about average, whereas KM, (Xylose) is the lowest of the reported XRs  ratio of 0.013 [Comparison with other XRs in the literature shows that krted XRs . The pH . crassa  or C. tropicalis ). Like m. crassa , while ao of 1.4  and an Xof 0.013 .d-lyxose from Carbosynth ; D-arabinose from TCI Europe ; NADPH, as tetrasodium salt, and all other chemicals were ordered from Carl Roth .NADH, as disodium salt hydrate, L-xylose and Bradford reagent were ordered from Sigma-Aldrich ; C. thermophilum DSM 1495 by homology search and the construct was ordered from genscript , codon optimized for Escherichia coli (wild type and codon optimized nucleotide sequences as well as the amino acid sequence of CtXR can be found as E. coli BL21(DE3) using a heat shock protocol: 2 µL of plasmid (160 ng/µL) were added to 50 µL cell suspension. After 30 min incubation on ice, heat shock was performed at 42 °C for 45 s. Cells were transferred back on ice for 2 min and subsequently diluted with 950 µL SOC medium. After 1 h at 37 °C cells were plated on LB-agar plates containing 100 mg/L ampicillin for picking transformants after overnight incubation at 37 °C . Correct insertion and integration were verified by sequencing .The XR gene was identified in the genome of −1) to increase cell density to 35 g/L DCW and finally an induced fed-batch phase , performed at 25 °C to increase protein solubility. Production of the recombinant protein was induced by aseptic addition of 0.1 mM IPTG .The protein was produced via fed-batch fermentation in a 15 L Sartorius Cplus bioreactor with 8 L working volume . The preculture was grown in a shake flask for 20 h at 37 °C and 230 rpm in 500 mL of a medium according to DeLisa et al.  containig, 4 °C, 30 min) 14 h after induction at a final cell density of approximately 62 g/L DCW. The cell pellet was stored at −20 °C for later use.Cells were harvested by centrifugation (4500× 2) was measured with a dissolved oxygen electrode Visiferm DO425  and dO2 levels were maintained above 30% by aerating with 7.5 L/min pressurized air. Air was substituted with pure oxygen if necessary. pH was monitored with an Easyferm electrode (Hamilton) and kept at 7.2 by addition of NH4OH (12.5% v/v) via the pump module of the bioreactor. Feed was supplied via a Preciflow peristaltic pump  following a feed-forward controlled exponential feeding strategy. Mixing was performed at 1400 rpm. CO2 and O2 content in the offgas were analyzed with a DASGIP GA, . Process parameters were adjusted and recorded via the process information management system Lucullus .Dissolved oxygen  to a total volume of 500 mL. Cell disruption was carried out with a homogenizer .230 g frozen biomass were thawed and resuspended in a loading buffer , resulting in 400 mL cell free crude extract.w/v glycerol, pH 6.5). Protein concentration was monitored in the flowthrough at 280 nm and the target protein was collected manually in a single fraction of 8 mL.For protein purification an Äkta Pure25  was used. IMAC was used as the first purification step, followed by size exclusion chromatography. 365 mL crude extract was loaded on a His Trap FF Crude column (5 mL column volume (CV); GE Healthcare) with a flow rate of 15 cm/h. After washing with 2 CVs with loading buffer, elution was carried out with a linear gradient over 6 CVs . Fractions of 1 mL were collected.After concentration to a volume of 2 mL with centrifugal filters with a 10 kDa cut off , the protein was purified via size exclusion chromatography (SEC). 2 mL protein solution were loaded on a high load superdex 75 pg column  with a flow rate of 30 cm/h  were loaded onto 4–15% Mini-PROTEAN TGX precast gels . Gels were run for 45 min at 160 V and stained with Coomassie Sensitive stain , 100 mL/L ethanol, 23.5 mL/L orthophosphoric acid, 0.2 g/L Coomassie blue G250) over night, washed with water and analyzed with Gel Doc XR system and ImageLab software .For evaluation of purification steps, enzyme activity (U/mL) and protein concentration (mg/mL) in crude extract, IMAC eluate, spin filter retentate and collected SEC fraction were determined and the respective specific activities (U/mg) were determined. By comparing the specific activity of the crude extract to those of the fractions the purification factor (PF) was calculated Equation (1).To quantify the loss of target protein during the purification, the recovery of the catalytic activity was calculated according to Equation (2).Protein concentration was measured photometrically  using the Bradford Coomassie Blue assay with bovine serum albumin as standard. When necessary, samples were diluted with their respective buffers.340 = 6.22 mM−1·cm−1) in duplicates. The activity was calculated according to Equations 3 and 4 . The mean specific activity was plotted against the substrate concentration (7 to 8 data pairs) and by fitting a Michaelis-Menten curve to the data points, Vmax and KM were determined using the program SigmaPlot 12.5 . The standard errors for KM and kcat specified in the Results section were calculated per default, with SigmaPlot describing the quality of the fit. kcat and the catalytic efficiency (kcat/KM) were calculated according to Equations 5 and 6. For the calculation of the confidence interval for kcat/KM both the errors from kcat and KM were considered using the formula for the propagation of uncertainty, as shown in Equation (7).Enzyme activity, proportional to the conversion of the cofactors NADPH or NADH, was determined photometrically following the decrease of absorption at 340 nm ; Vtotal: Assay volume (mL); ε: Extinction coefficient of NAD(P)H at 340 nm (6.22 L·mmolNAD(P)H−1·cm−1); Venzyme: Volume of enzyme solution (mLXR); d: Path length (1 cm); Volumetric activity: Activity per mL of enzyme solution (µmolNAD(P)H·min−1·mLXR−1 = U/mL); cenzyme: Concentration of enzyme solution (mgXR/mLXR); Specific activity: Activity per mg of enzyme (µmolNAD(P)H·min−1·mgXR−1 = U/mg); Vmax: Maximum enzyme activity (molNAD(P)H·s−1·mgXR−1); Menzyme: Molecular mass of enzyme ; kcat: Turnover number (molNAD(P)H·molXR−1·s−1); KM: Apparent affinity constant (Msubstrate); kcat/KM: Catalytic efficiency (molNAD(P)H·molXR−1·Msubstrate−1·s−1); σcatalytic efficiency: confidence interval of kcat/KM (molNAD(P)H·molXR−1·Msubstrate−1·s−1); SE: standard error (of kcat (molNAD(P)H·molXR−1·s−1) or KM (Msubstrate)).ΔAbs: Change in absorption . Depending on the investigated cofactor and substrate the enzyme concentration in the assay ranged from 0.564 to 45.2 µg/mL. One unit of activity was defined as the amount of enzyme catalyzing the oxidation of 1 μmol NAD(P)H/min. The kinetic parameters KM, kcat and catalytic efficiency were determined at 30 and at 55 °C (the highest temperature at which a characterization was justified due to the enzyme´s moderate thermostability). d-Xylose (0.001–0.5 M) was used as substrate and NADPH as cofactor (0.4 mM). To ensure that the change in absorption was not caused by thermal degradation of NAD(P)H, activity assays containing no CtXR were performed as blanks. In accordance with the literature [The purified enzyme was incubated in 50 mM bis-tris buffer pH 6.5 containing different amounts of cofactor and terature , thermald-xylose and 0.4 mM NADPH.Activity of samples derived during protein purification was measured at 30 °C in 50 mM bis-tris buffer pH 6.5 containing 0.5 M d-xylose (0.5 M) as substrate. Measurements were performed in triplicates. Additionally, the pH optimum was determined in 50 mM citrate and phosphate buffers.pH range was determined by measuring the enzymatic activity at 30 °C in a citric acid-disodium phosphate buffer (McIlvaine buffer)   and NADH (0.01–0.8 mM) in a 50 mM bis-tris buffer, pH 6.5, at 65 °C.Kinetic parameters Kd-xylose, l-xylose, d-arabinose, l-arabinose, d-ribose, d-lyxose, d-glucose, d-galactose and d-mannose. Measurements for the determination of the relative activity were performed in triplicates using 0.5 M substrate concentration and 0.4 mM NADPH as cofactor in a 50 mM bis-tris buffer, pH 6.5, at 30 °C. Additionally, kinetic parameters KM, kcat and catalytic efficiency were determined for the substrates d-xylose, l-arabinose, d-galactose and d-glucose (each 0.001–0.5 M) using NADPH as cofactor (0.4 mM).We determined relative activities for the substrates d-xylose (0.5 M) as substrate. For the calculation of the temperature stability of the enzyme, data acquired during the first ~15 s of incubation were omitted, since this was the time required to fully heat the test tubes (including 0.5 mL sample volume) on the thermoblock. The half-life (T1/2) of CtXR was determined at 30, 55, 65 and 75 °C according to Equation (8).The enzyme solution  was incubated at 30, 55, 65 and 75 °C. At different time points aliquots were drawn and the catalytic activity was immediately determined at 30 °C. NADPH (0.4 mM) was used as cofactor and −1); T1/2: Half-life (min)λ: Decay rate (minCtXR was produced using the SWISS-MODEL server (https://swissmodel.expasy.org) [COOT (version 0.8.9.1) [CtXR in complex with either NAD or NADP were generated manually by careful inspection of NAD binding in xylose reductase from Candida tenuis   that was0.8.9.1) . Based ode 1MI3, ; 1SM9, [de 1MI3, ) and NADde 1HQT; ).C. thermophilum was chosen for this study, since we were looking for a thermostable enzyme for the conversion of d-xylose to xylitol for the utilization in high temperature processes operating at around 75 °C. The enzyme exhibits dual cofactor specificity for NADPH (KM = 35.8 ± 7.6 mM) and NADH (KM = 121 ± 12 mM), has reduced activity on l-arabinose and residual activity on d-galactose and d-glucose, and optimal activity between pH 6.0 and 7.0. Unfortunately, with a half-life of only several seconds at 75 °C, the investigated CtXR cannot be used in the desired high temperature environments. Under thermophilic conditions XRs from N. crassa and C. tropicalis, described by Woodyer et al. [cat/KM values compared to CtXR and can potentially be deployed in more thermophilic processes due to higher stability.The particular xylose reductase from r et al.  and Zhanr et al. , respect


Plants were divided into the following three treatment groups: Group A, watered normally (control); Group B, not watered for 6 days (drought treatment); and Group C, not watered for 5 days and then rewatered for 1 day (recovery treatment). A total of 5014 proteins were detected, including 4932  that were matched to known proteins in a BLAST search. We detected 218, 107, and 348 proteins that were upregulated in Group B compared with Group A, Group C compared with Group A, and Group B compared with Group C, respectively. Additionally, 306, 145, and 231 downregulated proteins were detected during the same comparisons. Seventy differentially expressed proteins were analyzed and classified into 10 categories: photosynthesis, sulfur metabolism, amino sugar and nucleotide sugar metabolism, oxidative phosphorylation, ribosome, fatty acid elongation, thiamine metabolism, tryptophan metabolism, plant–pathogen interaction, and propanoate. Kenaf adapted to stress mainly by improving the metabolism of ATP, regulating photosynthesis according to light intensity, promoting the synthesis of osmoregulators, strengthening ion transport signal transmission, and promoting metabolism and cell stability. This is the first study to examine changes in protein expression in kenaf plants exposed to drought stress. Our results identified key drought‐responsive genes and proteins and may provide useful genetic information for improving kenaf stress resistance.The molecular mechanisms that underlie drought stress responses in kenaf, an important crop for the production of natural fibers, are poorly understood. To address this issue, we describe here the first  Seedlings were grown in pots , each containing the same amount  of red soil mixed with humus (1 : 1). All pots were incubated in a greenhouse with a 16‐h light : 8‐h dark (20 ± 1 °C) photoperiod and 65–70% relative humidity.Defang Li  donated the kenaf ; Group B, not watered for 6 days (drought treatment); and Group C, not watered for 5 days and then rewatered for 1 day (recovery treatment). Plant C received 200 mL of water at 8:00 am per day. All stress experiments were conducted in a greenhouse under a 16‐/8‐h (light/dark) photoperiod and a photon flux density of 380 umol·mThe physiological index (data not shown) was evaluated at eight critical time points . Drought stress most directly affected plant morphology. Thus, morphological traits  tags 113 and 114. Additionally, three drought‐treated H368 samples were labeled with iTRAQ tags 115, 116, and 117, while three rewatered H368 samples were labeled with iTRAQ tags 118, 119, and 121.For each sample, 100 μg protein was digested overnight with trypsin  at 37 °C m ammonium formate and 2% ACN, pH 10) and loaded onto a Gemini‐NX column . The peptides were eluted with Buffer A for 10 min and then with 5–30% Buffer B  for 15 min. The column was then treated with 30–80% Buffer B for 3 min. All flow rates were 1 mL·min−1.The Shimadzu LC‐20AB HPLC Pump System was used for high‐pH reversed‐phase chromatography. The iTRAQ‐labeled peptide mixtures were reconstituted in 0.5 mL Buffer A  at 3 μL·min−1 for 4 min. The peptides were eluted onto an analytical C18 column with an inner diameter of 75 μm. The column was then treated with a linear gradient of 8–25% Running Buffer B (ACN and 0.1% FA) for 45 min, a linear gradient up to 80% over 10 min, and then a 5‐min hold at 80%. The flow rate at all steps was 300 nL·min−1.Each fraction was resuspended in Buffer A (5% ACN and 0.1% FA) and centrifuged at 18 000 × Data were acquired using the TripleTOF 5600 System under the following conditions: ion spray voltage, 2.3 kV; ion source gas, 6 PSI; curtain gas, 30 PSI; and interface heater temperature, 150 °C. For the MS scans, the m/z scan range was 350–1800 Da.P‐value ≤ 0.05. A t‐test was applied to analyze the relative quantities of the three replicates. Up‐ or downregulated proteins were identified based on a P‐value < 0.05 with a twofold cutoff.The original MS/MS data file (*.wiff) was used to search a database of transcript sequences (data not shown) using the Protein Pilot 5.0 program (AB Sciex), with the Sample Type search parameter set at iTRAQ 8‐plex (labeled peptide). Iodoacetamide was used to alkylate cysteine residues of samples that were digested with trypsin. Proteins were considered identified if the Unused ProtScore was > 1.3 (corresponding to a peptide confidence ≥ 95%). Meanwhile, the threshold used to detect significantly enriched proteins was based on a Gossypium (72.47%), Theobroma (16.32%), Solanum, Citrus, Jatropha, and Populus (3.97%)  represents percentages of proteins identified, and y‐axis (right) represents the protein number. The ten most significantly enriched terms in level 4 GO hierarchy are shown in Figs Proteins affected by drought conditions were functionally analyzed. All identified proteins were functionally annotated with Gene Ontology (GO) terms from the following three main categories P‐value are shown on the right side of column in Figs Enriched KEGG pathways are clustered into the metabolism subcategories, the number of involved proteins in a specific pathway and corresponding P < 0.1 for decreased accumulation, 17 proteins were differentially accumulated and six proteins were downregulated in Group B vs Group A. Additionally, 34 proteins were differentially accumulated and one protein was downregulated in Group C vs Group A. With a fold‐change cutoff of 4 for increased accumulation and b < 0.25 for decreased accumulation, nine proteins were differentially accumulated and 13 proteins were downregulated in Group C vs Group B. Genes gi|823175577 and gi|224088250 were detected in the Group B vs Group A and Group C vs Group B comparisons. Furthermore, genes gi|728833390, gi|728833158, gi|823256519, gi|823189918, gi|403391437, gi|728844267, gi|321272239, and gi|823263864 were detected in the Group B vs Group A and Group C vs Group A comparisons. The data suggested that these eight proteins play an important role in the process of kenaf drought stress treatment and rehydration treatment.The expression levels of 70 differentially expressed proteins Data  were exaThe functions of differentially expressed proteins in response to drought stress were further analyzed. And 70 proteins were grouped into BP, CC, and MF categories. Diverse BP, CC, and MF subcategories were detected, including 30 important functional groups Fig. . The pre2‐alleviated Cd resistance is associated with activated sulfur metabolism Functional analyses were conducted based on the following four factors: fold changes in gene/protein abundance, protein–protein interactions, KEGG pathway enrichment, and BP protein enrichment. A network model was generated with the Cytoscape web application Fig.  2, with An effect that may occur during abiotic stress condition exposure is the participation of amino acids metabolism Plant growth, development, and stress responses require a considerable amount of energy in the form of ATP Photosynthesis is sensitive to drought and other stresses  This article was written, guided, and amended preliminarily by XA. XA and GJ contributed reagents and materials. XA, GJ, JZ, XL, CC, WL, GM, LJ, LD, XS, WW, and GZ participated in data collection and amendment. All authors read and approved the final manuscript.Data S1. Proteins identified by BLAST.Click here for additional data file.Data S2. The detected proteins belonged to diverse BP, CC, and MF subcategories in Group B vs Group A.Click here for additional data file.Data S3. The detected proteins belonged to diverse BP, CC, and MF subcategories in Group C vs Group A.Click here for additional data file.Data S4. The detected proteins belonged to diverse BP, CC, and MF subcategories in Group C vs Group B.Click here for additional data file.Data S5. The expression levels of 70 differentially expressed proteins.Click here for additional data file.Data S6. 70 proteins were grouped into BP, CC, and MF categories.Click here for additional data file.


Phaeodactylum tricornutum, could have beneficial health effects. Therefore, we investigated the anti-inflammatory, antioxidative and antiproliferative effects of fucoxanthin derived from this diatom in vitro. The effects of purified fucoxanthin on metabolic activity were assessed in blood mononuclear cells and different cell lines. In cell lines, caspase 3/7 activity was also analyzed. Nitrogen monoxide release and mRNA-expression of proinflammatory cytokines were measured. For antioxidant assays, cell free assays were conducted. Additionally, the antioxidant effect in neutrophils was quantified and glutathione was determined in HeLa cells. The results show that neither did fucoxanthin have anti-inflammatory properties nor did it exert cytotoxic effects on mononuclear cells. However, the metabolic activity of cell lines was decreased up to 58% and fucoxanthin increased the caspase 3/7 activity up to 4.6-fold. Additionally, dose-dependent antioxidant effects were detected, resulting in a 63% decrease in chemiluminescence in blood neutrophils and a 3.3-fold increase in the ratio of reduced to oxidized glutathione. Our studies show that fucoxanthin possesses antiproliferative and antioxidant activities in vitro. Hence, this carotenoid or the whole microalgae P. tricornutum could be considered as a food or nutraceutical in human nutrition, showcasing beneficial health effects.Microalgae contain a multitude of nutrients and can be grown sustainably. Fucoxanthin, a carotenoid from  Microalgae are microscopic small unicellular organisms that are abundant in various habitats around the globe. They can be cultured in open ponds or photobioreactors without the use of arable land and thus represent a promising sustainable alternative to plant-based proteins. They do not only produce high amounts of proteins but are also a good source for fatty acids, and vitamins, which can provide health promoting effects in a human diet. Hence, they can serve as novel functional foods or be incorporated into existing food products ,2.Phaeodactylum tricornutum exists in three different morphotypes and its genome has been already fully sequenced [P. tricornutum, although this microalga was shown to have a high content of fucoxanthin between 16.5 to 26.1 mg per gram dry matter (dm) [P. tricornutum might be a suitable source for the commercial production of fucoxanthin.The unicellular pennate diatom equenced . The diaequenced ,5,6,7,8.equenced ,10. It wequenced ,13,14,15ter (dm) . Thus, dter (dm) . It is ater (dm) ,20,21. HP. tricornutum might be used as a new nutraceutical, if it also exhibits the health beneficial effects that were already shown for the carotenoid derived from macroalgae. The prevalence of diseases linked to oxidative stress and inflammation is constantly increasing in developed countries ,23,24. TP. tricornutum UTEX 640 was cultivated in 180 L Flat-Panel-Airlift photobioreactors in an outdoor pilot scale plant located at the Fraunhofer Center for Chemical-Biotechnological Processes CBP in Leuna, Germany. Modified Mann and Myers medium was used as culture medium as described in Meiser et al. [w/w) (HPLC). It was compared to a commercial fucoxanthin standard  using UHPLC-MS.r et al. ,27. The r et al. . Brieflyr et al. ). SubseqIt was compared to a commercial fucoxanthin standard  using UHPLC-MS.Fucoxanthin was quantified using the HPLC method described by Gille et al.  with sliP. tricornutum was analyzed and compared to the commercial standard by UHPLC-DAD  using a Zorbax Eclipse Plus C18 (2.1 × 50 mm) column with a particle size of 1.8 µm. Mobile phase A contained water with 0.1% formic acid and mobile phase B consisted of methanol with 0.1% formic acid. The gradient used is shown in m/z at a temperature of 275 °C and −9.0 V. The m/z values . Isolation of polymorphonuclear leukocytes (PML) and peripheral blood mononuclear cells (PBMCs) via dextran sedimentation and density gradient centrifugation was conducted as previously described by El Benna & Dang (2007)  and Neum5 cells per well) and RAW 264.7 cells (1 × 104 cells per well) were incubated with fucoxanthin , β-carotene (1 µg/mL), dimethyl sulfoxide  as solvent control or DMSO (5%) as positive control for 24 h. Subsequent, MTT assay was conducted as previously described [Metabolic activity was assessed using the tetrazolium dye 3--2,5-diphenyltetrazolium bromide (MTT). PBMCs (3 × 10escribed . Nitric oxide (NO) production in RAW 264.7 cells was measured using the Griess assay. Therefore, 5 × 105 cells were treated with fucoxanthin (0.1 and 1 µg/mL) or β-carotene (1 µg/mL) and stimulated with lipopolysaccharide  for 24 h. The level of NO was measured using the Griess reaction, as previously described .The effects of fucoxanthin on the LPS-induced mRNA expression of the cytokines interleukin- (IL-)1β, IL-6, tumor necrosis factor α (TNF-α) and the enzyme cyclooxygenase-2 (COX-2) in PBMCs was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cells were incubated with fucoxanthin , β-carotene (1 µg/mL) or DMSO (0.1%) as solvent control for 24 h. Cells were stimulated with LPS (1 µg/mL) for 6 h. The mRNA expression was measured as formerly described .Total phenolics content (TPC) was determined using the Folin-Ciocalteu method  with min2+ per gram dm.The ferric reducing antioxidant power (FRAP) assay was performed in accordance to the method of Benzie and Strain . An indiFor the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay a calibration curve was obtained by using DPPH concentrations in the range of 0-100 µM. A DPPH solution was freshly prepared in methanol. 150 µL DPPH (0.1 MM) were mixed with 50 µL fucoxanthin , 150 µL methanol instead of DPPH were used for blank measurement. The percentage inhibition was calculated using the following formula:The half maximal inhibitory concentration (IC50) was calculated by linear regression, plotting the percentage inhibition against the different extract concentrations.The glutathione (GSH) to glutathione disulfide (GSSG) ratio as a marker for oxidative stress was determined using the GSH/GSSG-Glo™ Assay . 2 × 104 HeLa cells were incubated with fucoxanthin , β-carotene (1 µg/mL), DMSO (0.1%) as solvent control or menadione (40 µM) as control for 24 h.2′,7′-dichlorofluorescin (DCF) fluorescence and ROS production using luminol chemiluminescence were measured in human PMLs. For DCF fluorescence 100 µL of freshly drawn blood were incubated with 2′,7′-dichlorofluorescin diacetate (20 µM) at 37 °C for 15 min. Lipopolysaccharide (10 ng/mL) and fucoxanthin  or β-carotene (1 µg/mL) were added for 1 h at 37 °C. Cells were then stimulated with N-formylmethionyl-leucyl-phenylalanine . The reaction was stopped after 5 min by placing the samples on ice. After red blood cell lysis using the BD FACS lysing solution according to the manufacturer’s instructions, DCF fluorescence was measured with a BD FACS Canto II . The percentage of fluorescent PMLs was calculated using the BD FACS Diva Software.In luminol assays, PMLs were incubated with fucoxanthin , β-carotene (1 µg/mL), DMSO (0.1%) as solvent control or menadione (40 µM) as control. The assay was conducted as previously described  and cellMTT assays were conducted with Caco-2, HeLa and Hep G2 cells to assess cytotoxic activity of fucoxanthin on cancer cells. Cells were incubated with fucoxanthin , β-carotene (1 µg/mL), DMSO (0.1%) or staurosporine (1 µM) for 48 h, as described elsewhere . For apop < 0.05) between groups. The equality of variances was tested utilizing Levene’s test. For equal variances, Tukey’s HSD post hoc test was used; for unequal variances, Dunnett’s T3 post hoc test3. Data are expressed as mean ± standard error of the mean (SEM) in graphs or ± standard deviation (SD) in tables. Graphs were generated using GraphPad Prism 5 , statistics were done using IBM SPSS Statistics 25 , graphs. Normal distribution was tested using the Shapiro–Wilk test. One-way analysis of variance (ANOVA) was used to evaluate statistic significant differences . Fucoxanthin in the highest concentration (50 µg/mL) increased the ratio significantly (Neither fucoxanthin nor β-carotene showed effects on the DCF fluorescence of PMLs (data not shown). Menadione decreased the GSH/GSSG ratio. However, according to statistical analysis this change was not significant  supports these findings . AuthorsHaematococcus pluvialis which is already successfully commercialized [Fucoxanthin showed strong antioxidant effects in cell-free and cell-based assays. The IC50 concentration of fucoxanthin with 201 µg/mL in the DPPH assay was higher than that reported by Sachindra et al. (2007) . The higcialized ,43. BothIn the DCF fluorescence assay, neither fucoxanthin nor β-carotene showed antioxidant effects while the luminol assay revealed dose-dependent antioxidant properties of fucoxanthin. The DCF assay is used for the intracellular detection of ROS, especially H2O2 . Luminol2O2 and superoxide in the carcinoma cells triggers the apoptosis. This is, however, inconsistent with the antioxidant effects of the carotenoid that was shown in this study. On the other hand, Kotake-Nara et al. (2005) reported that the induced apoptosis is not accompanied by the production of ROS and caused by the loss of mitochondrial membrane potential [Although fucoxanthin had no effect on the metabolic activity of human blood cells, a dose-dependent influence on different carcinoma cell lines was shown. For Caco-2 cells and different cell lines, this was already shown previously ,51,52. Totential . It is aotential . ROS migotential . The rolotential ,55. P. tricornutum. Fucoxanthin was able to inhibit the oxidative burst in human PMLs, scavenge radicals and increase the GSH to GSSG ratio. Additionally, the metabolic activity was decreased, and apoptosis increased by the carotenoid. This leads to the conclusion that fucoxanthin or the whole microalgae biomass, including fucoxanthin in high amounts, could be considered in nutrition in order to ameliorate the effects of diseases linked to oxidative stress. Additionally, fucoxanthin could help to support traditional cancer treatment because of its beneficial health effects. Human trials are needed in future to further support these suggestions. In summary, we were able to show antioxidant and antiproliferative but no anti-inflammatory effects of the carotenoid fucoxanthin extracted from the microalgae


Supplemental Digital Content is available in the text Preoperative prognostic nutritional index (PNI) has been proven to be associated with clinical outcomes in patients with malignancies. However, data regarding the role of PNI in human glioblastomas (GBMs) is lacking. We, therefore, aimed to investigate the association between PNI and clinical parameters and survival in GBM patients.9/L). SPSS 22.0, GraphPad Prism 5, and X tile were the primary tools used for data analysis, figuring drawing, and calculating optimal cutoffs, respectively.This retrospective analysis included 300 GBM patients who were surgically treated at our institute from 2008 to 2017. PNI was calculated as albumin (g/L) + 5×total lymphocyte count -1R132H mutations or predicted survival in GBM patients without such mutations. Univariate analysis showed that a high preoperative Karnofsky performance score, gross total resection, completed chemoradiotherapy, IDH-1R132H mutations, and higher PNI levels were associated with favorable outcomes. Multivariate analysis showed that only completed chemoradiotherapy and IDH-1R132H mutations were independent prognostic factors.Mean albumin value, lymphocyte count, and PNI were 42.13 ± 4.43 g/L, 1.73 ± 0.71 × 10Our results indicated that PNI is associated with age and sex in GBM patients but fails to provide independent prognostic values. While molecular pathology results have served as predictive factors, blood markers related to inflammation, coagulation, and albumin,7 have also been used to predict GBM patients’ survival. Additionally, the prognostic nutritional index (PNI), calculated by combining albumin levels and lymphocyte counts, has been shown to be another predictive prognostic factor for cancers.–10Central nervous system (CNS) tumors are relatively rare neoplasms, with an estimated 23,800 new cases and 16,700 existing cases reported in 2017.,7 Moreover, survival rate has been reported to be reduced in GBM patients who are either under- or overweight. Xu et al and Zhou et al observed that PNI was an independent prognostic marker in GBM patients. However, inadequate data have been reported regarding the association between PNI and molecular subtypes of GBMs. Therefore, retrospectively investigated the prognostic value of PNIs in GBM patients, including a consideration of IDH status.Several studies have indicated that nutrition status is a key factor influencing GBM patients’ survival. Increased plasma albumin levels have been correlated with favorable clinical outcomes in patients with GBMs.22.1 and provided written informed consent. The mutation of IDH-1R132H was identified by immunohistochemistry (IHC) according to our previous report. Adjuvant chemoradiotherapy was performed according to the Stupp protocol. The adjuvant therapy was considered complete when patients had received chemoradiotherapy plus an additional 2 cycles that included temozolomide. PNI was calculated as the total of albumin (g/L) plus 5× total lymphocyte count (109/L). We excluded patients who had been diagnosed with diabetes, metabolism disease, hypertension, heart disease, autoimmune disease, or infection within the previous 3 months, as in a previous study. We calculated overall survival (OS) in months, and defined it as the period from operation to death or censorship. Our follow-up ended in December 2017, and no patients were lost. The ethics committee of Sanbo Brain Hospital approved all study procedures.This retrospective study included 300 patients who underwent surgery at Sanbo Brain Hospital, Beijing, China from 2008 to 2015. All the patients were diagnosed with GBMs according to pathology results2.2 software systems were used to analyze data, draw figures, and find the optimal cutoff for PNI in predicting OS, respectively. We used the Student t test when comparing PNI values between 2 groups. The correlation between PNI and age was analyzed with the Spearman test. Survival analysis was applied using the Kaplan–Meier method and log-rank test. Cox proportional hazards models were adopted for the calculation of the hazard ratios (HRs) of death in GBM patients with regard to univariable or multivariable analysis. A P-value of <.05 was considered statistically significant.SPSS 22.0 , GraphPad Prism 5 , and X title 33.1R132H mutations were detected in 25% (75/300) of patients. Preoperatively, the mean serum albumin level and lymphocyte count were 42.13 ± 4.43 g/L (range: 25.7–52.7 g/L) and 1.73 ± 0.71 × 109/L (range: 0.28–5.22 × 109/L), respectively. The mean PNI level was calculated as 50.80 ± 6.01 (range: 34.2–76.2).We included 300 GBM patients in our study; their clinicopathological data are presented in Table 3.2t tests (Table P = .002) and men (P = .038). Thus, PNI was significantly associated with age in GBM patients . No differences were found in PNI levels among KPS, tumor type, or IDH-1R132H status. Furthermore, PNI levels did not differ among patients who had GTR or had completed chemoradiotherapy.We compared preoperative PNI levels with clinicopathological factors using independent ts Table . Interes3.3P = .031, Fig. R132H, a PNI of ≥44 was associated with a favorable outcome, although it failed to achieve significance    of 13.83 months  in patients with PNI ≥44 was significantly greater than the mOS of 9.87 months  observed in patients with PNI <44  versus those aged ≥60 years  , female sex, and better OS. However, this conclusion needs to be confirmed in a large prospective study. Moreover, we suggest that PNI could be introduced into clinical practice as a prognostic marker, as well as a therapeutic target, to improve outcomes of GBM patients.Conceptualization: Changxiang Yan.Data curation: Jinduo Ding, Kun Yao, Pengfei Wang.Formal analysis: Jinduo Ding.Funding acquisition: Changxiang Yan.Investigation: Kun Yao.Methodology: Jinduo Ding, Kun Yao, Pengfei Wang.Resources: Changxiang Yan.Software: Pengfei Wang.Supervision: Changxiang Yan.Writing – original draft: Jinduo Ding, Pengfei Wang.Writing – review & editing: Pengfei Wang, Changxiang Yan.


In non-NF2-mutated IVMs, previously reported genetic alterations including TRAF7, AKT1, SMO, KLF4, PIK3CA, and TERT were lacking, suggesting alternative genes in the pathogenesis of non-NF2 IVMs. In silico analysis revealed possible damaging mutations of APC, GABRA6, GSE1, KDR, and two SMO missense mutations differing from previously reported ones. Interestingly, all WHO°II IVMs (n = 3) harbored SMARCB1 and SMARCA4 mutations, indicating a role of the SWI/SNF chromatin remodeling complex in aggressive IVMs.Intraventricular meningiomas (IVMs) account for less than 5% of all intracranial meningiomas; hence their molecular phenotype remains unknown. In this study, we were interested whether genetic alterations in IVMs differ from meningiomas in other locations and analyzed our institutional series with respect to clinical and molecular characteristics. A total of 25 patients with surgical removal of an IVM at our department between 1986 and 2018 were identified from our institutional database. Median progression-free survival (PFS) was 79 months (range of 2–319 months) and PFS at 5 years was 86%. Corresponding tumor tissue was available for 18 patients including one matching recurrence and was subjected to targeted panel sequencing of 130 selected genes frequently mutated in brain cancers by applying a custom hybrid capture approach on a NextSeq500 instrument. Loss of chromosome 22q and 1p occurred frequently in 89 and 44% of cases. Deleterious NF2 mutations were found in 44% of IVMs (The online version of this article (10.1186/s40478-019-0793-4) contains supplementary material, which is available to authorized users. Intraventricular meningiomas (IVMs) are rare tumors; they account for only 0.5–5% of all intracranial meningiomas and up to 14% of all intraventricular tumors , 26, 35.In meningiomas, there is increasing knowledge about their molecular phenotype. The most common genetic alteration found in meningiomas involves the tumor suppressor neurofibromatosis gene 2 (NF2) on chromosome (chr) 22q. Loss of heterozygosity (LOH) at this chromosomal region is typically detected in 40–80% of sporadic meningiomas , 31, 34.Recent studies revealed the association of meningioma location and distinct mutations. For instance, NF2/chr22loss meningioma meningiomas typically originate from the lateral regions and posterior fossa, while the vast majority of non-NF2 meningiomas commonly harbor location-specific mutations in SMO (olfactory groove), KLF4/TRAF7 , and AKT1/TRAF7 (anterior skull base) , 9, 20. Volume = (Width^2 x Length)/2. Progression-free survival (PFS) was defined as the interval from 1st surgery until last MRI scan while follow-up was defined as the interval from 1st surgery until last patient contact. The extent of resection (EOR) was determined on the basis of surgical reports or early postoperative MRI scans if available. EOR was categorized in gross total resection  or subtotal resection  according to Simpson’s grading. Fresh tumor material obtained intraoperatively was immediately snap-frozen and stored at − 80 °C until further processing. Histological diagnosis and grading of IVMs was based on the World Health Organization Classification of Central Nervous System Tumors in use at the time of 1st surgery. Only tissue samples with a vital tumor cell content > 60% as determined on hematoxylin and eosin stained slides by a board-certified neuropathologist  were processed further. DNA was extracted from tumor tissues using the AllPrep Kit  according to the manufacturer’s instructions. DNA amount was quantified by NanoDrop ND-1000 spectrophotometer  and then stored at − 80 °C until further analysis. The institutional review board at Heidelberg Medical Faculty approved this study. Written informed consent was retrieved from all patients.Our institutional database was screened for patients with surgical resection of an IVM at the Department of Neurosurgery at the University Hospital Heidelberg, Germany between 1986 and 2018. Demographic, tumor-related , treatment-related and outcome data were collected retrospectively from medical charts’ review and magnetic resonance imaging (MRI) studies. Tumor size was calculated by MRI scans with the formula IVM tissue samples were profiled by targeted panel sequencing. The panel contained 130 genes reported to be frequently mutated in brain tumors including meningiomas as described previously . The panRaw data were processed as described before . BrieflyTo identify novel, not previously reported mutations in IVMs, we used previously published in silico algorithms including SIFT, PhyloP, PolyPhen2, LIB-LRT, and MutationTaster to predict the impact of missense mutations on the protein function . As puren = 12, 48%), followed by the fibroblastic subtype encountered in 7 cases (28%)  compared to the right lateral ventricle . Only one tumor underwent STR and in one IVM, the EOR was unknown. Two patients with meningioma WHO°II received postoperative radiotherapy. The median follow-up was 13 months (range 0.7 to 399 months). Meanwhile, radiographic tumor recurrence/progression was observed in five patients . Median PFS was 79 months (range 2–319 months). PFS at 5 years among all tumors was 86%   and were analyzed by targeted panel sequencing as described before [From our clinical cohort of 25 IVM patients, corresponding tumor samples were available for 18 patients including one matching recurrent WHO°II tumor (d before . This pan = 8/18). In detail, in four cases the NF2 gene was disrupted by frameshift mutations , in three cases we discovered a stop-gain mutation  and in one case a mutation causing a splicing error of the NF2 gene was detected  , BRCA1 , KMT2D , CDKN2A , and CDKN2C . NTRK2 (neurotropic receptor tyrosine kinase 2) encodes a neurotrophin receptor, which is known to play a role in gliomagenesis [Subsequently, we analyzed our targeted gene panel for other frequent genetic alterations Fig. . Apart fagenesis . Howeveragenesis . KMT2D pagenesis . We founagenesis . Howevern = 1 WHO°I IVM) is associated with epilepsy, but, until now, has not been linked to cancer [By using in silico algorithms including SIFT, PhyloP, PolyPhen2, LIB-LRT, and MutationTaster, we were able to identify five possible pathogenic mutations: APC, GABRA6, GSE1, KDR, and SMO Table . In one o cancer . GSE1 , but also in 33% (n = 5/15) of WHO°I IVMs.Copy number profiles were calculated from the low-resolution the sequencing array data. This method was previously described and showed high concordance compared to 450 k methylation data, but may differ from high-resolution techniques such as array comparative genomic hybridization , 50. Evan = 8/18) by frameshift mutations (n = 4/18), stop-gain mutations (n = 3/18) and splicing error (n = 1/18), which is in accordance with the literature as the primary inactivated tumor suppressor in meningiomas [Next, we focused on the most prevalent genetic alterations reported in meningiomas so far, including NF2, SMARCB1, as well as TRAF7, AKT1, SMO, KLF4, PIK3CA, and TERT in non-NF2 mutated meningiomas , 20. NF2ingiomas , 9, 28. ingiomas . Due to ingiomas . The SMOingiomas . Functioingiomas . In one ingiomas , 52. Moringiomas . This isingiomas . Even moingiomas . Taken tingiomas , 44.A number of studies highlight the importance of the Wnt signaling pathway in meningioma . APC plaIn conclusion, NF2 mutations were the most frequent genetic alteration in our cohort of IVMs, whereas other common genetic alterations previously reported in WHO°I meningiomas of distinct tumor locations were not found. However, members of the chromatin remodeling complex SWI/SNF SMARCB1 and SMARCA4 may play a role in the pathogenesis of aggressive IVMs.Additional file 1:Sequencing data of IVMs. (XLSX 1955 kb)