Symphyocladia latiuscula (Harvey) Yamada, is a good source of bromophenols with numerous biological activities. This study aims to characterize the anti-diabetic potential of 2,3,6-tribromo-4,5-dihydroxybenzyl derivatives isolated from S. latiuscula via their inhibition of tyrosine phosphatase 1B (PTP1B) and α-glucosidase. Additionally, this study uses in silico modeling and glucose uptake potential analysis in insulin-resistant (IR) HepG2 cells to reveal the mechanism of anti-diabetic activity. This bioassay-guided isolation led to the discovery of three potent bromophenols that act against PTP1B and α-glucosidase: 2,3,6-tribromo-4,5-dihydroxybenzyl alcohol (1), 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether (2), and bis- (3). All compounds inhibited the target enzymes by 50% at concentrations below 10 μM. The activity of 1 and 2 was comparable to ursolic acid ; however, 3 was more potent  against PTP1B. Interestingly, the activity of 1–3 against α-glucosidase was 30–110 times higher than acarbose . Again, 3 was the most potent α-glucosidase inhibitor . Similarly, 1–3 showed concentration-dependent glucose uptake in insulin-resistant HepG2 cells and downregulated PTP1B expression. Enzyme kinetics revealed different modes of inhibition. In silico molecular docking simulations demonstrated the importance of the 7–OH group for H-bond formation and bromine/phenyl ring number for halogen-bond interactions. These results suggest that bromophenols from S. latiuscula, especially highly brominated 3, are inhibitors of PTP1B and α-glucosidase, enhance insulin sensitivity and glucose uptake, and may represent a novel class of anti-diabetic drugs.The marine alga,  Diabetes is a group of metabolic diseases characterized by high blood sugar levels. As the disease progresses, it can lead to blurred vision, cardiovascular diseases, kidney and/or other organ damage and dysfunction, and even depression . DiabeteThe marine environment is a rich source of structurally diverse and unique compounds. Approximately 5000 halogenated compounds have been identified in marine sources thus far. In response to various ecological pressures, marine organisms such as algae, sponges, and corals produce a wide range of biologically active secondary metabolites, 45% of which are brome-metabolites due to the high bromine concentration in sea water ,9. NaturSymphyocladia latiuscula (Harvey) Yamada has been reported to be a good source of bromophenols with numerous biological activities including antibacterial -2-deoxyglucose (2-NBDG) glucose uptake assays in insulin-resistant HepG2 cells and PTP1B protein expression using the western blot technique.Halogenation is a traditional tool in drug optimization and is an important factor in drug discovery and development for four main reasons: (1) the degree of halogenation influences lipophilicity and cell membrane solubility; (2) halogenation improves membrane binding, permeation, and diffusion; (3) halogenation increases the half-life of pharmaceutical drugs; and (4) halogenation enhances blood-brain barrier permeability and improves central nervous system delivery ,15,16,17acterial , antiviracterial , antifunacterial , anticanacterial , free raacterial , aldose acterial , α-glucoacterial , and othacterial ,26,27. BSymphyocladia latiuscula was the evaluation of PTP1B and α-glucosidase inhibition potentials of methanol extract and fractions obtained by MeOH extract partition with different solvents using pNPP and pNPG as substrates, respectively. Enzyme inhibition levels are expressed as mean IC50 values with standard deviation. As shown in 50 values of 9.01 ± 0.33 and 91.58 ± 9.66 μg/mL, respectively. The CH2Cl2, EtOAc and n-BuOH solvent fractions all inhibited the activity of both the enzymes with more promising results. The EtOAc fraction showed particularly strong inhibition of both the PTP1B and α-glucosidase enzymes with IC50 values of 2.79 ± 0.11 and 6.71 ± 0.15 μg/mL, respectively. Because this latter fraction gave the most promising inhibition, further research was carried out to characterize potential anti-diabetic phytochemicals in the EtOAc fraction.The initial step in evaluating anti-diabetic secondary metabolites from 1–3  showed that the activity of bromophenols was comparable with ursolic acid. Bromophenol 3 had an IC50 value of 5.29 ± 0.08 μM, making it the most active among the tested compounds, followed by 1  and 2 . Similarly, the α-glucosidase inhibition assay was validated with acarbose  as a reference drug. The tested bromophenols showed a 30–110-fold increase in α-glucosidase inhibition activity compared to acarbose. The relative α-glucosidase enzyme inhibition of the three bromophenols was similar to that of PTP1B enzyme inhibition: 3 was the most active with an IC50 value 1.92 ± 0.02 μM, followed by 1  and 2 .Three bromophenols were isolated from the active EtOAc fraction by using open Si gel column chromatography and purified via series of multiple Reverse Phase column chromatography. The PTP1B and α-glucosidase inhibitory activities of bromophenols 1–3  are presIn order to discern the mode of PTP1B and α-glucosidase inhibition by bromophenols, a kinetic study was performed at different substrate concentrations for both enzymes.1 and 2 appeared to be mixed-type inhibitors for the PTP1B enzyme  with Ki values of 1.19 for compound 1 and 2.40 μM for compound 2. In contrast, compound 3 appeared to be a competitive inhibitor with an inhibition constant of 2.25 μM. In competitive inhibition mode (y-intercept (constant Vmax) but different x-intercepts . Similarly, 1 and 2 were both non-competitive inhibitors of α-glucosidase  with dissociation constants of 1.92 and 5.54 μM, respectively (2-carboxyphenyl)amino]-1-naphthyl}-L-alanyl) amino]pentyl}oxy)-2-naphthoic acid (compound A) as a catalytic inhibitor and 3--2-ethyl-benzofuran-6-sulfonic acid (4-sulfamoyl-phenyl)-amide (compound B) as an allosteric inhibitor. Simulation results are presented in 1 and 2 bound to both the catalytic and allosteric pockets of PTP1B with similar binding energies , forming various H-bond interactions and halogen-bond interactions via bromine atoms. The PTP1B-1 inhibitor complex at the allosteric pocket had a −5.83 kcal/mol binding energy with three hydrogen bonds interacting with amino acid residues Ala189, Glu276, and Gly277. Beside this, other interactions with Arg221, Trp179, Gln266, Thr263, and Gly183 were observed for both inhibitors 1 and 2. The 7-OH group of 1 displayed additional H-bond interactions with Phe182 and Lys116 at a distance of 2.0 and 2.2 Å in a catalytic pocket, respectively. However, H-bond interactions with these two residues were not observed for inhibitor 2  was investigated via molecular docking simulation . The simibitor 2 A,B. At ted lines D,E. The d Lys116 C. In add1 inhibitor complex at the catalytic pocket showed four H-bond interactions  at a distance of 2.0 to 2.2 Å. In addition, bromine atoms at C2, C3, and C6 positions were involved in multiple interactions with Thr263, Val184, Gln266, Gly183, Arg221, Phe182, Lys116, and Trp176. Similarly, PTP1B-2 inhibitor complex displayed three H-bond interactions with Arg221, Gln266, and Gly183 at a distance of 1.9 to 2.1 Å. Three bromine atoms of 2 showed multiple interactions with Arg211, Gln266, Trp179, Thr283, Asp265, Asp181, Lys116, Phe182, and Val184. The catalytic inhibitor, compound A, formed a complex with PTP1B at the catalytic site forming four H-bond interactions with Asp48, Lys116, Lys120, and Asp181 along with other non-polar interactions with Ala217, Tyr46, Met258, and Gln262. Common interacting residues for inhibitors  at the catalytic pocket involved Asp181 and Lys116, which were determining residues for catalytic inhibition.The PTP1B-Saccharomyces cerevisiae co-crystallized with maltose (PDB ID: 3A4A) was used as the α-glucosidase protein because it showed 85% similarity to yeast α-glucosidase (MAL12) through homology modeling +m/z 752.5364 calculated for C14H879Br6O5, m/z 754.5344 calculated for C14H879Br581BrO5, m/z 756.5324 calculated for C14H879Br481Br2O5, m/z 758.5304 calculated for C14H879Br381Br3O5, m/z 760.5284 calculated for C14H879Br281Br4O5, m/z 762.5266 calculated for C14H879Br81Br5O5, and m/z 764.5250 calculated for C14H881Br6O5.Bis-  from triplicate experiments.The potential for MeOH extract, solvent-soluble fractions, and isolated compounds to inhibit PTP1B enzyme activity was evaluated using the ubstrate . Experimubstrate . UrsolicpNPG at 405 nm using a microplate spectrophotometer (Molecular Devices). Acarbose was used as a reference compound.The α-glucosidase inhibition assay procedure, previously reported by Li et al. , was folpNPP for PTP1B and 1.25, 2.5, or 5 mM pNPG for α-glucosidase) on Dixon plots . Lineweaver–Burk plots for inhibition of PTP1B were obtained in the presence of various concentrations of the test compounds . Similarly, for the inhibition of α-glucosidase, the test concentrations used were 0, 2, 3, and 5 μM for 1; 0, 1.5, 7.5, and 15 μM for 2; and 0, 0.125, 0.5, and 2 μM for 3. The enzymatic procedure consisted of the same aforementioned assay method. The inhibition constants (Ki) were determined by interpreting Dixon plots, where the value of the x-intercept indicated the value of −Ki [The modes of enzyme inhibition were evaluated by monitoring the effects of different concentrations of the substrates (2-carboxyphenyl)amino]-1-naphthyl}-L-alanyl)-amino]pentyl}oxy)-2-naphthoic acid (known as ‘compound A’ in our study) were obtained from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank website [Z)-3-butylidenephthalide (BIP) (known as ‘Compound D’ in our study) were obtained from the RCSB Protein Data Bank website [1–3 were constructed using Chem3D Pro v12.0 with pH adjusted to 7 using MarvinSketch . A Lamarckian genetic algorithm (GA) method was used for docking simulations. Gasteiger charges were added by default, the rotatable bonds were set with AutoDockTools (ADT), and all torsions were allowed to rotate. The docking simulation was conducted with 15 independent GAs with the default parameters. Results were analyzed and visualized using University of California, San Francisco (UCSF)’s Chimera tool (http://www.cgl.ucsf.edu/chimera/).The complex structure of PTP1B with its selective allosteric inhibitor 3--2-ethyl-benzofuran-6-sulfonic acid (4-sulfamoyl-phenyl)-amide (known as ‘compound B’ in our study) (PDB ID: 1T49) and the 3D structure of the selective catalytic inhibitor 3- cells obtained from the American Type Culture Collection  were maintained in 10% FBS MEM at 37 °C in a humidified atmosphere with 5% COTT assay . FollowiTT assay , an insuTT assay .The relative percent glucose uptake upon sample treatment in IR-HepG2 cells was evaluated by a flourimetric method using the fluorescent D-glucose analog 2-NBDG and measured on a fluorescence microplate reader  at 485 nm excitation and 528 nm emission wavelengths. Detailed experimental conditions and procedures are reported in our previous publication .1–3 or rosiglitazone (10 μM) in 24-well plates for 24 h followed by stimulation with 100 nM insulin diluted in SFMEM for 30 min. Cells were then washed twice with ice cold PBS, collected, and lysed with sample buffer. Protein was quantified by the modified Bradford protein assay kit using bovine serum albumin (BSA) as a standard, electrophoresed on sodium dodecyl sulfate-polyacrylamide gels , and then transferred to polyvinylidene difluoride (PVDF) membranes  using the Pierce G2 Fast Blotter  at 100 V for 60 min in a standard semi-dry system. Membranes were blocked in blocking buffer and incubated overnight with primary antibodies at 4 °C on a shaker, followed by incubation with the appropriate secondary antibodies for 2 h at room temperature. The membranes were washed three times with tris-buffered saline with tween (TBST) and the protein bands were visualized with the Supersignal West Pico Chemiluminescence Substrate  on X-ray films  and quantified using CS analyzer software . Detailed laboratory procedures and sample buffer and antibody solution compositions are reported in our previous paper [Insulin-resistant HepG2 cells were exposed to a range of concentrations of us paper .t-test  were used to determine the statistical significance. Values of p < 0.05, 0.01, and 0.001 were considered significant. All results are presented as the mean ± standard deviation (SD) of triplicate experiments.One-way analysis of variance (ANOVA) and Student’s S. latiuscula (Harvey) Yamada: 2,3,6-tribromo-4,5-dihydroxybenzyl alcohol (1), 2,3,6-tribromo-4,5-dihydroxybenzyl methyl ether (2), and bis- (3). Notably, these bromophenols enhanced insulin sensitivity in insulin-resistant HepG2 cells and aided glucose uptake by downregulating PTP1B expression. Structural insights into the PTP1B and α-glucosidase enzymes inhibition revealed the importance of bromine and phenolic group number. In addition, molecular docking simulation revealed the mechanisms by which H-bonds and other interactions between the active ligands and enzymes are critical for inhibition. Bromophenols from S. latiuscula may represent a novel class of anti-diabetic drugs. However, in vivo study is of the utmost importance in confirming these findings, and further studies on the molecular mechanisms of insulin signaling in HepG2 cells is underway.In summary, bioassay-guided isolation led to the discovery of three potent dual PTP1B and α-glucosidase inhibitors from a red alga,


The presence of longitudinal ridges documented in long runout landslides across our solar system is commonly associated with the existence of a basal layer of ice. However, their development, the link between their occurrence and the emplacement mechanisms of long runout landslides, and the necessity of a basal ice layer remain poorly understood. Here, we analyse the morphometry of longitudinal ridges of a martian landslide and show that the wavelength of the ridges is 2–3 times the average thickness of the landslide deposit, a unique scaling relationship previously reported in ice-free rapid granular flow experiments. We recognize en-echelon features that we interpret as kinematic indicators, congruent with experimentally-measured transverse velocity gradient. We suggest that longitudinal ridges should not be considered as unequivocal evidence for presence of ice, rather as inevitable features of rapid granular sliding material, that originate from a mechanical instability once a kinematic threshold is surpassed. The occurrence of longitudinal ridges on large landslide masses on planetary bodies is enabled by long runout distances, which have so far been attributed to the presence of ice. The authors here present a challenging model based on mechanical instabilities within the flow, suggesting that ice is not needed. The emplacement dynamics of long-runout landslides is a subject of ongoing debate9, and various mechanisms have been proposed for the reduction of basal friction, which appears to be a condition necessary for controlling the ability of long-runout landslides to travel even more than ten times their vertical drop over nearly flat surfaces10.Distinctive longitudinal raised ridges are common in large-scale mass movements across the surfaces of planetary bodies, but their formation mechanism is poorly understood. These longitudinal ridges have been identified in impact ejecta on every planetary surface with sufficient image resolution11. Consequently, their morphological similarity to those observed in martian landslides has been used as an analogy to infer the presence of an icy substrate at the time of landslide emplacement13 on Mars. It has been proposed that presence of ice on the surface reduces the frictional coefficient, permitting the splitting of the deposit as a consequence of either lateral spreading13 or the dominant role of inertia over lateral spreading forces12, leading to the formation of ridges and troughs perpendicular to the spreading direction. The presence of ice is also suggested to explain the development of longitudinal grooves in martian double-layered ejecta craters14, which also show morphological similarity to the longitudinal ridges associated with landslides, although other mechanisms are not excluded15. However, longitudinal ridges have been observed also in landslides on the Moon18, which is considered to be free of ice throughout its geological history.Longitudinal ridges and furrows are seen ubiquitously on the surface of long-runout landslides on Mars. On Earth, such features are commonly found in landslides emplaced on glaciers, amongst which is the iconic Sherman Glacier landslide19 suggested the possibility that longitudinal ridges and furrows of large planetary rock avalanches may be related to fluid-like instabilities forming convection cells that result in longitudinal stripes parallel to the flow direction. This was based on previous experimental and theoretical work21 demonstrating turbulent flow processes that are well known to occur in fluid mechanics. The experimental work and stability analysis on rapid granular flows conducted by Forterre and Pouliquen21 showed for the first time the spontaneous formation of longitudinal vortices that manifest as superficial longitudinal ridges and furrows, which initiation depends on the velocity of the flow, roughness of the basal surface, and the consequent strong shear at the base of the flow. Importantly, Forterre and Pouliquen20 demonstrated a clear morphological scaling relationship in that the wavelength of the ridges scales with the thickness of the flow by a factor of ~2–3 and that the pattern drifts in the transverse direction. Further experiments and numerical modelling by Borzsonyi et al.19 confirmed that the longitudinal pattern naturally arises from mechanical instabilities within rapid granular flows. While demonstrated experimentally, such scaling relationship have yet to be demonstrated in any planetary landslide.Borzsonyi et al.22 and HiRISE23 cameras) in order to analyse a giant martian landslide with some of the best defined longitudinal ridges , the ridges are the faster-flowing region19. In both regimes, the faster-flowing regions are denser, i.e., their solid fraction is greater than the slower-flowing features. As a consequence, when the flow ceases, inversion of topography is expected in the dilute regime. Here, the ridges, being less dense, are expected to deflate and sink. The ridges become furrows, and the fast-flowing dense furrows become ridges. The en-echelon features that we report in Fig. We also identified s-shaped en-echelon features superposed on longitudinal ridges that we interpret as kinetic indicators Fig. . Laborat25 suggesting that a dilute layer of highly agitated particles at the bottom of the flow can explain the apparent low coefficient of friction in long-runout landslides and that such a layer should form naturally in rapid granular flows. As the formation of longitudinal vortices also seems to be a natural development of rapid granular flows that involves a low-density layer at the bottom of the flow21, it is not unreasonable to suggest that longitudinal ridges and furrows are inevitable features that originate from a mechanical instability once certain threshold conditions within a flow moving over a rough surface are passed.A self-lubrication mechanism for long-runout landslides has been proposed by Campbell20 relies on the inversion of the density profile, with denser granular packing closer to the top of the slide, and on a continuous energy supply to the system, in the form of shear forces, so to maintain the collisional regime and, therefore, to prevent the granular temperature to decay rapidly due to the inelastic nature of particle collisions25. The instability derives from the inversion of density profile, due to an increase of granular temperature27 at the base of the flow by collision and shear between particles and the rough substrate. Such particle interactions may well contribute to acoustic fluidisation28 processes within the landslide that would lead to the dynamic reduction of viscosity and explain the long-runout distance. Several other dynamic weakening mechanisms have also been proposed to explain long run-outs, such as flash heating3, frictional melting29, and thermal decomposition33 . Such mechanisms rely on the generation of heat at the base of the landslide, which would contribute to the energy supply at the base of the system.The mechanism responsible for the formation of longitudinal vortices proposed by Forterre & Pouliquen20. Although not ruling out the presence of ice and other mineralogical facies as a key factor, our results suggest that the origin of longitudinal ridges does not necessarily depend on the presence of an icy substrate.From the results of our morphometric analysis of longitudinal ridges in a giant martian landslide appears the similarity of the scaling relationship between the wavelength of longitudinal ridges and the thickness of the landslide seen in nature and in experiments of self-forming ice-free ridges. Moreover, scaling analysis of the martian landslide studied here indicates that its emplacement velocity could have been as high as tens to hundreds of m/s (see the Methods section). It follows that longitudinal ridges that characterise long-runout terrestrial and planetary landslides may be the expression of an instability that emerges within the flowing mass once a velocity threshold is surpassed, as observed in laboratory experiments on rapid granular flowsOur results and observations represent the first field evidence of experimental and numerical studies that show the spontaneous development of longitudinal ridges in ice-free rapid granular flows, and support the existence of a convincing fundamental process responsible for the origin of such morphologies, challenging existing analogies to terrestrial landslides on ice that have been proposed to explain their widespread occurrence throughout the Solar System. If indeed the scaling relations that we identify indicate that the martian landslide that we study developed longitudinal ridges through an instability that emerges from the physics of fast-flowing dry grains, then we should expect such morphological features throughout the  Solar System, regardless of the availability of other lubrication mechanism, such as ice layers.22 and HiRISE23 cameras) provided by the NASA Planetary Data System. This study has made use of the USGS Integrated Software for Imagers and Spectrometers (ISIS)34 package to pre-process CTX stereo-pair images  was conducted using Mars Reconnaissance Orbiter imagery , the higher elevations of the blue lines 1 and 2 are plausible. However, we are also expecting a random natural roughness of the valley floor at various scales so that also blue lines 3 and 4 appear plausible, as their maximum offset (blue line 3) is ~80 m, which is within a reasonable magnitude of topographic variation .We use these profiles to evaluate the errors on the deposit thickness, and we do so by accounting for the standard deviation of the elevation in the topographic profiles (Supplementary Table 19 identified a range of scaled downstream surface velocity.The experimental work of Börzsönyi et al.u is scaled with g is the gravitational acceleration and d is the grain size, providing the normalised downstream surface velocity The flow velocity 19, we consider 2, and the largest grain sizes recognisable on the surface deposit of the martian landslide, d = 1–20 m (Supplementary Fig. From the data in Fig. Supplementary InformationPeer Review File


Context: Friedelin is a triterpenoid with several biological activities. However, the affects of Friedelin on the activity of human liver cytochrome P450 (CYP) enzymes remains unclear.Objective: This study investigates the inhibitory effects of Friedelin on the major human liver CYP isoforms .Materials and methods: First, the inhibitory effects of Friedelin (100 μM) on the eight human liver CYP isoforms were investigated in vitro using human liver microsomes (HLMs), and then enzyme inhibition, kinetic studies, and time-dependent inhibition studies were conducted to investigate the IC50, Ki and Kinact/KI values of Friedelin.Results: The results indicate that Friedelin inhibited the activity of CYP3A4 and 2E1, with the IC50 values of 10.79 and 22.54 μM, respectively, but other CYP isoforms were not affected. Enzyme kinetic studies showed that Friedelin is not only a noncompetitive inhibitor of CYP3A4, but also a competitive inhibitor of CYP2E1, with Ki values of 6.16 and 18.02 μM, respectively. In addition, Friedelin is a time-dependent inhibitor of CYP3A4 with Kinact/Ki value of 4.84 nM/min.Discussion and conclusion: The in vitro studies of Friedelin with CYP isoforms suggested that Friedelin has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP3A4 and 2E1. Further clinical studies are needed to evaluate the significance of this interaction. Maytenus ilicifolia (Mart.) ex. Reissek (Celastraceae) and has several biological activities such as antioxidant, in vitro-cytotoxic, antiobesity, anti-inflammatory and antiulcer (Shimizu and Tomoo Friedelin is a triterpenoid isolated from the leaves of Cytochrome P450 (CYP) enzymes are important phase I enzymes in the biotransformation of xenobiotics, and most of the CYP enzymes can be inhibited or induced by a variety of drugs and chemicals that can give rise to toxicity or treatment failure , phenacetin (CYP1A2), coumarin (CYP2A6), chlorzoxazone (CYP2E1), dextromethorphan (CYP2D6), diclofenac (CYP2C9), S-mephenytoin (CYP2C19) and paclitaxel (CYP2C8) were used as probe substrates to determine the effects of Friedelin on eight CYP enzymes. Also, enzyme kinetic studies were conducted to determine the inhibition mode of Friedelin on CYP enzymes.This study investigates the effects of Friedelin on eight major CYP isoforms in human liver microsomes (HLMs). d-Glucose-6-phosphate, glucose-6-phosphate dehydrogenase, corticosterone (≥98%), NADP+, phenacetin (≥98%), acetaminophen (≥98%), 4-hydroxymephenytoin (≥98%), 7-hydroxycoumarin (≥98%), 4′-hydroxydiclofenac (≥98%), sulfaphenazole (≥98%), quinidine (≥98%), tranylcypromine (≥98%), chlorzoxazone (≥98%), 6-hydroxychlorzoxazone (≥98%), paclitaxel (≥98%), 6β-hydroxytestosterone (≥98%), clomethiazole (≥98%), and furafylline (≥98%) were obtained from Sigma Chemical Co. Montelukast (≥98%) was obtained from Beijing Aleznova Pharmaceutical . Coumarin (≥98%), diclofenac (≥98%), dextromethorphan (≥98%), and ketoconazole (≥98%) were purchased from ICN Biomedicals. Pooled HLMs were purchased from BD Biosciences Discovery Labware. All other reagents and solvents were of analytical reagent grade.Friedelin (≥98%) and testosterone (≥98%) were obtained from the National Institute for the Control of Pharmaceutical and Biological Products . O-demethylation for CYP1A2, coumarin 7-hydroxylation for CYP2A6, chlorzoxazone 6-hydroxylation for CYP2E1, dextromethorphan O-demethylation for CYP2D6, diclofenac 4′-hydroxylation for CYP2C9, S-mephenytoin 4-hydroxylation for CYP2C19 and paclitaxel 6α-hydroxylation for CYP2C8. All incubations were performed in triplicate, and the mean values were utilized. The typical incubation systems contained 100 mM potassium phosphate buffer (pH 7.4), NADPH generating system , the appropriate concentration of HLMs, a corresponding probe substrate and Friedelin (or positive inhibitor for different probe reactions) in a final volume of 200 μL.As shown in The concentration of Friedelin was 100 μM, and the positive inhibitor concentrations were as follows: 1 μM ketoconazole for CYP3A4, 10 μM furafylline for CYP1A2, 10 μM tranylcypromine for CYP2A6, 50 μM clomethiazole for CYP2E1, 10 μM quinidine for CYP2D6, 10 μM sulfaphenazole for CYP2C9, 50 μM tranylcypromine for CYP2C19, 5 μM montelukast for CYP2C8. Probe substrates, positive inhibitors (except for dextromethorphan and quinidine which were dissolved in water) and Friedelin were dissolved in methanol, with a final concentration of 1% (v/v) and 1% neat methanol was added to the incubations without inhibitor. The final microsomal protein concentration and incubation times for the different probe reactions are shown in 50). Ki values were obtained by incubating various concentrations of different probe substrates  in the presence of 0–50 μM Friedelin.Friedelin (100 μM) was used to initially screen for its direct inhibitory effects toward different human CYP isoforms. For the CYP isoforms whose activities were strongly inhibited, secondary studies were performed to obtain the half inhibition concentration , the reactions were terminated by adding a 100 μL acetonitrile internal standard mix and then placed on ice; the corresponding metabolites were determined by HPLC.To determine whether Friedelin could inhibit the activity of CYP3A4 and 2E1 in a time-dependent manner, Friedelin (20 μM) was preincubated with HLMs (1 mg/mL) in the presence of an NADPH-generating system for 30 min at 37 °C. After incubation, an aliquot (20 μL) was transferred to another incubation tube  containing an NADPH-generating system and probe substrates whose final concentrations were approximate to Ki and kinact values for the inactivation of CYP3A4, the incubations were conducted using higher probe substrate concentrations  and various concentrations of Friedelin (0–50 μM) after different preincubation times (0–30 min), with a two-step incubation scheme, as described above.To determine the Vmax and Km. Inhibition data from the experiments that were conducted using multiple compound concentrations were represented by Dixon plots, and inhibition constant (Ki) values were calculated using nonlinear regression according to the following equation:I is the concentration of the compound, Ki is the inhibition constant, S is the concentration of the substrate, and Km is the substrate concentration at half the maximum velocity (Vmax) of the reaction. The mechanism of the inhibition was inspected using the Lineweaver–Burk plots and the enzyme inhibition models. The data comparison was performed using Student’s t test and performed using IBM SPSS statistics 20 (SPSS Inc.).The enzyme kinetic parameters for the probe reaction were estimated from the best fit line using least-squares linear regression of the inverse substrate concentration versus the inverse velocity (Lineweaver–Burk plots), and the mean values were used to calculate To investigate whether Friedelin affects the catalytic activity of CYP enzymes, the probe reaction assays were conducted with varying concentrations of Friedelin. Specific inhibitors of CYP3A4, 1A2, 2A6, 2E1, 2D6, 2C9, 2C19 and 2C8 were used as positive controls. As shown in 50 values of 10.79 and 22.54 μM, respectively.The enzyme-inhibition study showed that inhibition of CYP3A4 and 2E1 by Friedelin was concentration-dependent, with ICKi values of Friedelin on CYP3A4  over the in vivo concentration of the inhibitor (Ito et al. in vivo system studies are needed to identify the interactions of Friedelin with CYP isoform in humans.As we know, in vitro metabolism of drugs that are substrates of CYP3A4 and 2E1, and therefore, herb–drug interaction might occur when Friedelin is co-administered with the substrates of the CYP3A4 and 2E1. However, there is little data available for the in vivo pharmacokinetic data of Friedelin, and therefore, the herb-drug interaction between Friedelin and other drugs is still difficult to predict. Due to the pharmacological activities of Friedelin, further pharmacokinetic studies should be conducted.The results of this study indicate that Friedelin may influence the in vitro. The results showed that Friedelin could inhibit the activity of CYP3A4 and 2E1, while the activity of other CYP enzymes was not affected. Therefore, to avoid adverse herb-drug interactions, caution should be exercised when Friedelin is co-administered with other drugs metabolized by CYP3A4 and 2E1.In conclusion, the effects of Friedelin on the activity of the major CYP enzymes were investigated


Pyrosequencing generated 273,102 reads for the five triplicate ice samples, which corresponded to 3,464 operational taxonomic units (OTUs). The distribution of the bacterial phyla in the perennial cave ice varied with age, organic content, and light exposure. Proteobacteria dominated the 1 and 900 years old organic rich ice deposits, while Actinobacteria was mostly found in 900 years old ice strata, and Firmicutes was best represented in 400 years old ice. Cyanobacteria and Chlorobi representatives were identified mainly from the ice block surface samples exposed to sunlight. Archaea was observed only in older ice strata, with a high incidence of Crenarchaeota and Thaumarchaeaota in the 400 years old ice, while Euryarchaeota dominated the 900 years old ice layers, with Methanomicrobia representing the predominant taxa. A large percentage (55.7%) of 16S rRNA gene amplicons corresponded to unidentified OTUs at genus or higher taxa levels, suggesting a greater undiscovered bacterial diversity in this glacial underground habitat. The prokaryotes distribution across the cave ice block revealed the presence of 99 phylotypes specific for different ice layers, in addition to the shared microbial community. Ice geochemistry represented an important factor that explained the microbial taxa distribution in the cave ice block, while the total organic carbon content had a direct impact on the cell density of the ice microcosm. Both bacterial and archaeal community structures appeared to be affected by climate variations during the ice formation, highlighting the cave ice microbiome as a source of putative paleoclimatic biomarkers. This report constitutes the first high-throughput sequencing study of the cave ice microbiome and its distribution across the perennial underground glacier of an alpine ice cave.Ice entrenched microcosm represents a vast reservoir of novel species and a proxy for past climate reconstitution. Among glacial ecosystems, ice caves represent one of the scarcely investigated frozen habitats. To characterize the microbial diversity of perennial ice from karst ecosystems, Roche 454 sequencing of 16S rRNA gene amplicons from the underground ice block of Scarisoara Ice Cave (Romania) was applied. The temporal distribution of bacterial and archaeal community structures from newly formed, 400, and 900 years old ice layers was surveyed and analyzed in relation with the age and geochemical composition of the ice substrate. The microbial content of cave ice layers varied from 3.3 10 In contrast to these icy environments, there is a limited information on the microbial communities from perennial ice accumulations in caves (hereafter “ice caves”)6. These ice bodies form throughout the slow freezing of dripping and inflowing water, and/or snow diagenesis. The combination of climatic conditions, cave morphology, and feedback mechanisms7 allows for the preservation of ice throughout the year and genesis of millennia-old cave glaciers. The microbial community from such secluded frozen habitats could constitute reservoirs of novel cold-adapted microorganisms10. Moreover, while perennial cave ice could represent paleoclimatic archives16, the ongoing climatic changes during cave ice formation are expected to affect the microbial community structure entrapped in various cave ice layers, providing putative biomarkers for the past climate variations. Few ice-contained bacterial species were isolated from Austrian alpine ice caves17, and from the rock-ice interface in a lava tube from Oregon Cascades, USA18. Three Antarctic fumarolic ice caves near Mt. Erebus were recently explored, and the diversity of the bacterial19 and fungal20 communities from sub-ice sediments was determined. A recent survey on ice deposits and sediments on Hawaiian lava tubes revealed the presence of a diverse bacterial community and the occurrence of Euryarchaea in such cryogenic formation21.Ice habitats, encompassing Arctic and Antarctic ice-sheets, ice shelves and mountain glaciers, unraveled the presence of complex and unique microbiomes26. This limestone cave, located at 1165 m asl in the Apuseni Mountains, Romania , hosts the largest  and oldest  cave ice block in the world28. The 22.5 m thick27 underground ice block was formed by the freezing of a shallow pond formed on top of the existing ice block, resulting in a structure of alternating layers of clear ice and organic sediment-rich strata30. The origin of the accumulated supraglacial pond is in the drips that reach the cave after percolating through the thin (~30 cm) soil and highly fissured limestone (~15 m thick) above the cave. A small proportion of water also originated from direct precipitation and snow melting at the bottom of the entrance shaft. The ice strata vary between 1–15 cm in thickness27, and includes both organic  and inorganic material derived from the surface and carried inside the cave by both dripping and inflowing water15. The presence of ice leads to a distinctive underground climate, with air temperatures dropping below 0 °C from late November to early spring (March), and being maintained at 0 °C throughout the remaining of the year, as the heat delivered to the cave by conduction  and dripping water “being consumed” by the slow melting of the ice30. The ice block from Scarisoara Ice Cave yielded a wealth of information on the variability of climate and vegetation during the past ~10,000 years27. These studies have shown that during the past ~1,000 years (the timeframe of the present study), climate in the area has changed from warm and wet during the Medieval Warm Period (800–1250 AD) to cold and dry, but with frequent heavy rains, during the Little Ice Age (1250–1850 AD)27.One ice cave study model on ice genesis and dynamics, paleoclimate significance and microbiome of underground perennial ice accumulations is Scarisoara Ice Cave31 and from the ice layers up to 900 years old belonging to the perennial ice block33. Cultured and uncultured fungi were also identified by Denaturating gradient gel electrophoresis (DGGE) analysis in newly formed, 400 and 900 years old cave ice deposits34. In this study, we assessed the diversity of uncultured bacterial and archaeal communities across the perennial cave ice block using 454-pyrosequencing, and compared the microbial communities trapped in the cave ice layers of different geochemical signatures formed during various climate-associated periods of the last millennium. This is the first report on the prokaryotic community structure in a cave ice block chronosequence.The presence of cultured bacteria was reported from the ice stalagmites of the Little Reservation area of this cave4 to 7.5 105 cell mL−1. The highest cell content (7.5 × 105 cells mL−1) was found in the recently formed ice sample 1-S. The cell density decreased with both the age and organic sediment content of the ice deposits. Thus, organic sediment-rich samples 1-S, 400-O (5.1 × 105 cells mL−1) and 900-O (1.1 × 105 cells mL−1) showed a linear decline of cell content with the age of ice sediment  and 900-I (0.3 × 105 cells mL−1) contained 3-fold fewer cells than the corresponding organic-rich ice deposits of same age, indicating a similar age-dependent decline rate of cell density in this habitat. This data confirmed a temporal dependence of microbial cell density in Scarisoara cave perennial ice. Similar tendency of total cell counts and live/dead ratio has been previously observed for the permafrost microcosms formed during the last decade35, and 27 kyr to 33 kys BP36. The viable cell density of each ice sample (Table 5 cells mL−1) and 400-O (3.4 × 105 cells mL−1), and 5-fold lower in older ice 900-O (0.8 × 105 cells mL−1). The clear-ice samples 1-L (1.9 × 105 cells mL−1) and 900-I (0.2 × 105 cells mL−1) contained less viable cell as compared to the corresponding sediment-rich strata of the same age, 1-S (2-fold) and 900-O (3-fold), respectively. The cell viability in this habitat ranged from 68–78% for most of the ice layers, with a lower value (59%) for 1-S sample. Active microorganisms, presenting not only basal metabolic functions but also cell growth and division, were found in permanent frozen environments41. However, limitations of the well-established staining cytometry technique due to the transient permeability of the cell membrane after physical and chemical stress exposures, recently evidenced by Davey and Hexley42, suggest that the actual number of viable cells in cave ice deposits could be higher. Such temporary cell membrane damage might occur during ice thawing, aggregate disruption by sonication, and HD-staining processes prior to flow cytometry measurements. Therefore, the lower live/dead cell ratio values obtained for the recently formed light exposed ice layer 1-S could be due to a higher content of less adapted microorganism to life in frozen habitats subjected to thaw stress.Total microbial cell density of Scarisoara ice samples measured by flow cytometry Table  ranged fQuality filtration of the raw pyrosequencing 16S rRNA gene amplicons led to a total number of 273,102 reads for the 15 samples, corresponding to 3,464 OTUs. The number of OTUs found in 1-S, 1-L, 400-O, 900-O and 900-I ice samples varied in the 1,736–2,574 interval, with higher values found in the organic rich samples 400-O and 900-O Table . PrincipVENN diagram of the OTUs distribution in the five cave ice layers Fig.  revealedBacterial OTUs accounted for 94.9%-99.03% of the ice-contained phylotypes , while a 2-fold lower content was present in 1-L and 400-O samples, and 20-fold lower in both 900-O and 900-I samples. Moreover, Actinobacteria, which appeared to dominate the 900-years old strata was highly abundant in the organic rich layers 1-S (4.5 × 104 cell mL−1), 400-O (8.5 × 104 cell mL−1) and 900-O (6.5 × 104 cell mL−1), while the clear ice strata of same age 1-L and 900-I showed a lower cell density by 2-fold and 6-fold, respectively. Firmicutes prevailed in 400-O (27 × 104 cell mL−1), in accordance with the highest relative content value for this phylum , while in 1-L the estimated density of Cyanobacteria was 15-fold lower  and 1-L (39.8%) from the ice block surface, while only small fractions were identified in 900-O (4.3%), and in both 400-O and 900-I ice strata 0.2–0.3%)  in 400-O.Among the 27 assigned genera of the cave ice microbiome, a high relative content of ely Fig. . Cryobacata Fig. . Among iLeptolyngbya, Pseudanabaena and Phormidium species were highly represented in 1-S and 900-O ice, while Acaryochloris, Chroococcidiopsis and Gloeobacter had a lower incidence in both 900 years old ice strata. At higher taxa levels, Nostocaceae and Chamaesiphononaceae families, as well as SM1D11 and YS2 orders, were scarcely found in all ice layers. The cave ice microbiome also revealed the presence of three Chlorobi OTUs, dominated by Chlorobiaceae, a green-sulfur bacterial family of obligate anaerobic photoautotrophs44. Unidentified Cytophagales/green sulfur bacterium OPB5645 and uncultured SJA-28 clade46 were scarcely present in both 400 and 900 years old ice strata. In addition, phototrophic purple nonsulfur bacteria belonging to genera Rhodoplanes and Rhodoferax48 showed a slightly increased relative abundance in the old ice strata, suggesting that bacterial communities from 400 and 900 years old ice were capable of maintaining photosynthetic activities in the presence of very low light intensity49.The cave ice phototrophic community comprised 13 cyanobacteria phylotypes. At genus level, Gemmata and Planctomyces OTUs were assigned at genus level. These anammox bacteria that are present in cold environments50 occurred mainly in 900 years old cave ice, in accordance with their specific anoxic metabolism.Moreover, ten Planctomycetes phylotypes belonging to Phycisphaerae, ODP123, Planctomycetia, and vadinHA49 classes were present in all cave ice samples, representing 0.36% of identified prokaryotic taxa. Among these, Archaeal taxa (7 OTUs) were identified only in older cave ice strata 400-O, 900-O and 900-I Fig. . The relCandidatus Nitrososphaera species, and Methanomicrobiales order OTUs (6–21%), besides a scarce presence of Methanobacterium spp. (8%) and Methanomassiliicoccaceae (class Thermoplasmata) (3–15%). Novel Methanobacterium species were found in permanently frozen habitats such as Arctic51 and Siberian52 permafrost. The main archaeal groups in 900-O ice layer were Methanomicrobiales representatives, encompassing Methanolinea species (17–29%), and unidentified OTUs at higher taxonomic ranks (42–62%). Candidatus Nitrososphaera species belonging to Nitrosphaerales order were present in two replicates with a wide relative abundance variation (29–73%). Meanwhile, the 900-I clear ice was dominated by the strictly carbon dioxide reducing methanogens belonging to Methanomicrobiales (95%), with a high relative content of Methanolinea species (16–50%). Representatives of this genus were found in other cold environments including sediments from the permanent frozen lake Fryxell 53 and permafrost from the Tibetan Plateau54. Thermoplasmata species were also present in all the 900-I sample replicates, represented by the Methanomassiliicoccaceae family (16–18%).The relative abundance of different archaeal phylotypes Fig.  highlighR = 0.7256, P < 0.001).A statistical ANOSIM analysis indicated a significant difference between the ice samples  period, and 900-years old ice, corresponding to Medieval Warm Period (MWP)32.The sunlight-exposed ice from the top of the cave ice block (sample 1-S) was characterized by a high content of phototrophs belonging to Chlorobi and Cyanobacteria phyla. The non-specific 16S rRNA gene amplification of algal and plant chloroplast from this sample sustained the high presence of phototrophs in recent ice formed near the cave entrance, as observed in the supraglacial pond formed during summer time in the corresponding small area directly exposed to sunlightMethanobacterium (Euryarchaeota) revealed the presence of anaerobic hydrogenotrophic methanogens in old ice strata62. OTUs belonging to MCG, a predominant archaeal group in anoxic environments, mainly found in deep marine habitats63 with possible significant roles in the global biogeochemical cycles, were also highly present (40–69%) in LIA ice layer.Specific bacterial and archaeal phylotypes were found in 400 (cold and dry LIA period) and 900 years old (warm and wet MWP period) cave ice samples Fig. , constitKoribacter spp. (Acidobacteria) are among soil acidophiles growing at low temperatures that metabolize carbon monoxide in the presence of high Fe concentration64. In Scarisoara cave ice, Firmicutes OTUs belonging to the genus Dethiobacter were among the specific 400-O ice microbiota, representing obligate anaerobic chemolithoautotrophs capable of reducing sulphur compounds65. This distribution preference is in accordance with the higher iron content of this ice layer as compared to the 900-O (by 1.5-fold) and 900-I ice (4-fold) strata, respectively (to be published elsewhere). The Alphaproteobacteria phylotypes uniquely found in LIA ice layer belonged to the nitrogen-fixing Pleomorphomonas genus (Methylocystaceae), while Betaproteobacteria was represented by the denitrifying Sterolibacterium species (Rhodocyclaceae). Meanwhile, the ice deposits 900-O and 900-I formed during MWP contained a wide variety of Actinobacteria represented by 10 genera: Actinomyces, Nesterenkonia, Pillimelia, Nocardia, Friedmanniella, Actinomadura, Williamsia, Euzebya, Rubrobacter, and Patulibacter. Playing an important role in the soil development and biogeochemical cycling, Actinobacteria survive in extreme environments, being a key component of frozen habitats such as permafrost, glacier forefields, ice cores and cryoconites66. Moreover, Deltaproteobacteria were mainly found in MWP formed ice, including Plesiocystis and the propionate-oxidizing anaerobe Smithella species, specific for methanogenic environments67.19 indicated a large variation of phyla composition, two of the sites (Harry’s Dream and Hubert’s Nightmare) showing an important relative abundance of Proteobacteria (17–22%). A high representation of Actinobacteria was found in the lava cave characterized by lower temperatures and slightly higher pH, similar to the 900-years old ice layers from Scarisoara Ice Cave. In contrast, Verrucomicrobia phylum, also majorly found (15%) in this Antarctic cave, showed a higher occurrence (7.2%) in the recently formed cave ice sample 1-S characterized by neutral pH. Moreover, Bacteroidetes, representing 22% of Hubert’s Nightmare ice cave sediments, was mostly present in the 1-L and 400-O ice samples. Chloroflexi is one of the dominant taxa (22–66%) in two of the Antarctic caves, but only scarcely represented (1.5%) in Scarisoara ice block where was mostly found in 400 years old ice (8.9%). Meanwhile, a high relative content (26%) of Cyanobacteria populated the light-exposed sediments in Harry’s Dream cave, similar to recently formed ice 1-S from Scarisoara (32%), indicating the flourishing of phototrophic microbiota in various icy habitats exposed to sunlight and their lower conservation in perennial ice after 400 and 900 years. However, while Antarctic caves are not ice caves sensu stricto that constitute “rock caves hosting perennial accumulations of ice”25, but caves carved in glaciers formed by snow diagenesis, differences in deposition mechanisms most likely have a major impact on the ice-embedded micriobial community structure. Recent investigation of Hawaiian lava tubes21 highlighted the presence of various bacterial and archaeal communities in silica and calcite deposits and ice pond water. The ice bacterial community was dominated by Proteobacteria phylum (39%), similar to the recent ice deposits of Scarisoara Ice Cave, in addition to Bacteroidetes (18%), Verrucomicrobia (8%) and candidate division OD1 (7%) also spread in the 1-S, 1-L and 400-O alpine cave ice samples. Meanwhile, the mineral samples of Hawaiian volcanic caves had a high relative abundance (86%) of Actinobacteria, similar to 900-years old ice layers of the alpine ice block, and Euryarchaeota OTUs were also found in the volcanic ice cave, but very scarcely represented (<1%) unlike the MWP-formed ice layers of Scarisoara cave. However, in spite of some commonly found taxa in these two types of caves, a wider diversity and a particular composition and distribution were characterizing the prokaryotic community across the perennial ice block of Scarisoara Ice Cave. This could be partly due to the less abundant soil cover above the Hawaiian caves, as compared to that above Scarisoara cave, where a 20–50 cm thick podosol developed under mixed spruce and birch forest mantles the cap rock above the cave.In comparison with the ice microbiomes from lava caves, our current data revealed a unique bacterial community structure in the perennial ice formed during the last millennium in Scarisoara Ice Cave. Sediments from Antarctic volcanic ice caves in the vicinity of Mt. Erebus Antarctica30, evaporation of the formed supraglacial lake prior to annual freezing is not expected to play a decisive role in ice layer thickness. Therefore, ice layers formed during the dryer LIA period (sample 400-O) showed a higher (by 3.5-fold) microbial cell density relative to that of the wetter MWP period (sample 900-O), in accordance with differences in the precipitation regime. Such correlation was also observed in the glacier ice cores from Tibetan Plateau, where a higher microbial abundance was reported for dryer regions, while lower abundance occurred in areas with high precipitations affected by the Indian Monsoon35. Also, Antarctic ice core formed during the last 68 kyr showed higher cell density in strata formed during the colder and dryer Last Glacial Maximum period68.In addition, ice deposition process (thickness of ice strata) could be influenced by climate variations during ice formation, the environmental parameters (temperature and precipitation) being in direct relation with the thickness of the ice strata, thus affecting the microbial cell density in successively formed ice strata. Taking into consideration that the temperature in the glacial part of the cave showed little variations around 0 °C68, suggesting a more complex input of various modeling factors of the microbial community structure in Scarisoara cave ice block.Meanwhile, the prokaryotic diversity (Shannon index) from the ice cave showed only a 10% decrease between LIA and MWP formed ice, unlike the lower prokaryotic variability during dryer periods in both Tibetan and Polar glacier ice cores15, the source of microorganisms in the perennial cave ice is expected to be mainly the surrounding soils and karst/ground water. In Scarisoara ice, the pyrosequencing data showed that the most abundant prokaryotic taxa from each ice sample  was found across the ice block, with higher values in 900-years old ice strata formed during MWP interval as compared to that of LIA-formed ice. Electrical conductivity (EC) varied in from 15 µS cm−1 to 124.2 µS cm−1, showing a decrease with the age of ice. Total organic carbon (TOC) content ranged in the 3.03–33.38-mg L−1 interval, with higher values for 1-S (33.38 mg L−1) and 400-O (29.96 mg L−1) samples, and reduced concentrations for the recently formed clear ice and 900 years old ice samples by 3-fold (1-L), 5-fold (900-O) and 10-fold (900-I), respectively. The highest total nitrogen (TN) concentrations were also found in 1-S and 400-O samples (2.15–2.23 mg L−1), while clear ice 1-L (0.53 mg L−1) and both 900 years old ice samples 900-O and 900-I (0.62–0.64 mg L−1) showed reduced nitrogen content by 4 and 5 fold, respectively.The geochemistry of the 1-S, 1-L, 400-O, 900-O and 900-I cell-free melted ice samples (using 0.22 µm membranes) was previously determined  than the corresponding clear ice layer 900-I. Also, the MWP formed ice (900-O) contained 14% less OTUs than the LIA-formed ice layer (400-O), partially correlated with the decrease in TOC (by 20%) between these ice strata.69. The constant below-freezing temperature (<0 °C) in the vicinity of the ice block from Scarisoara cave30 could favor metabolic activity in the formed ice liquid-water veins, as reported for temperate glacier ice70. Thus, the putative cell division occurring in the cave ice for the last millennium could have an impact on the cell abundance and community structure of recently-formed and older ice strata, considering the neutral to slightly alkaline pH of all ice samples, suitable for active bacteria occurrence72. Taking into account the total and viable microbial cell density from each ice strata  microbiome were of 1.5 × 10−5 g C (g C)−1 h−1  and 2.2 × 10−5 g C (g C)−1 h−1 (viable biomass), while no metabolism  or very reduced (5.7 × 10−7 g C (g C)−1 h−1 for viable biomass) was estimated for the microbiome within the 900-O organic rich ice layer. At temperatures around freezing point (0 °C) that characterize Scarisoara cave ice block30, these values correspond to metabolic rates for maintenance of cellular functions in clear-ice (900-I), and for survival in organic rich ice (900-O), based on Price and Sowers study on temperature-dependence metabolic rates of microbiomes from various cold environments69. In this case, differences between metabolic rates could be explained by the presence of a more abundant heterotrophic community in organic-rich strata (400-O and 900-O) as compared to that from clear ice (900-I), thus recycling organic carbon at a higher rate. However, these hypothetical metabolic rates sustaining the presence of active microbial cells in this habitat remain to be confirmed by determining the actual microbial contribution to the ice carbon content through direct measurements of the microbiome activity. Meanwhile, active bacteria presence across a 25-m deep ice core from Scarisoara cave was confirmed, based on Illumina sequencing of RNA transcripts (to be published elsewhere), sustaining the contribution of post-depositional processes that are modeling the bacterial community structure across the cave ice block.The post-depositional processes following the formation of ice layers could also be considered as a modeling factor of the cave ice bacterial profileta Table  and the ta Table , the estThe current data on Scarisoara Ice Cave microbiome underscored a highly diverse bacterial community throughout the cave ice block, and the occurrence of various archaeal taxa in old ice layers formed during 400 and 900 years BP, revealing large dissimilarities in the ice-embedded microbial profiles that varied with the age, organic content and light exposure of the ice. Both heterotrophic and autotrophic prokaryotes were identified in recent and old ice deposits, with the putative presence of various anaerobic microbial groups. In addition, climate variations recorded during ice formation within LIA and MWP time-intervals appeared to have an impact on both major bacterial and archaeal phyla, with distinct prokaryotes identified up to genera level that might serve as putative climate proxies. Extended investigations on the total and active microbial communities from Scarisoara cave ice block accumulated during the last 13,000 years, currently underway, will contribute to unravel the role of cave ice microbiome in shaping this frozen habitat in relation with the substrate geochemistry and climate variations, and to provide novel psychrophilic strains of high applicative potential in bionanotechnologies.14C-dated to 1-year old, 400-years old and 900-years old ice deposits33. The 400 and 900 years old ice layers correspond to Little Ice Age (LIA) and Medieval Ice Period (MWP) climate periods, respectively. Recent ice samples 1-S and 1-L were collected from direct (1-S) and indirect (1-L) sunlight-exposed areas of the ice block surface located in the Great Hall zone of the cave    to determine the live/dead cell content75. The number of total and dead cells contained in 90 μl treated ice sample was measured immediately after labeling. The density of living cells was calculated by subtracting the number of HD-labeled (dead) cells from the total number of SG-labeled microorganisms. Cell density was expressed as number of cells contained in 1 ml of melted ice. Unstained cells were used as negative controls.The cell density of total and viable microorganisms contained in ice samples was measured by flow cytometry, using a Gallios flow cytometer . Ice samples (1 ml) were thawed at 4 °C under sterile conditions, and the cellular aggregates were dispersed in the presence of 0.1% Tween 80  after two cycles of incubation for 10 min in an ultrasonic bath Sonorex Digital 10 P  at 5% power, followed by 1 min vortexing. The resulted suspension was labeled with 1 × SybrGreen I (SG)  for total cell counting33. All these steps were carried out in a Class II Type A2 ASAL900 Atlantic laminar flow hood , under aseptic conditions, to avoid contamination. 16S rRNA gene libraries of ice microbiota were obtained in triplicate by 454 pyrosequencing for the selected ice samples. A first amplification step was carried out in 20 μl mixture containing 5 x Phusion HF buffer , 0.2 mM dNTP mixture, 0.4 U Phusion Hot Start II DNA Polymerase , 0.5 µM of 341 F (5′-CCTACGGGAGGCAGCAG-3′) and 806 R (5′-GGACTACYVGGGTATCTAAT-3′) prokaryotic primers flanking the V3-V4 hypervariable regions76, and 1 µl of 10 x diluted DNA template (11 ng). PCR amplification started with incubation at 98 °C for 30 s, followed by 30 cycles of 98 °C for 5 s, 56 °C for 20 s and 72 °C for 20 s, with a final extension at 72 °C for 5 min. The PCR products were migrated on a 1.25% (w/v) agarose gel, extracted and purified using Montage DNA Gel Extraction Kit . In a second step, 1 µl of purified PCR-product was amplified using the reaction mixture of the previous PCR step in the presence of MID-barcoded 341F and 806R primers, a generous gift from Dr. Carsten S. Jacobsen , by following the same reaction protocol but with a reduced number (15) of cycles. The amplicons were migrated and purified as described for the first amplification step, and the DNA fragments were quantified using a Qubit fluorometer . Equal amounts (100 ng) of each DNA sample were mixed up, and the 454 pyrosequencing of the pooled amplicons was carried out on a Genome Sequencer FLX Titanium Pico TiterPlate using a GS FLX Titanium Sequencing Kit XLR70 .Microbial cells were collected from melted ice samples (500 ml) at 4 °C by filtration on 0.22 µm MF-Millipore sterile membrane filters . Before melting, ice samples were subjected to superficial layer removal (~1 cm) to eliminate possible contamination. Genomic DNA was extracted using DNeasy Blood and Tissue kit  by a modified protocol in the presence of mutanolysin77. Demultiplexing of the reads using the Roche 454 GS FLX Titanium platform implied a minimum and maximum read length of 200 and 1,000 bp, respectively, allowing 6 ambiguous bases per read, with a minimum mean Phred quality score of 25. After sequence denoising using denoise_wrapper.py software78, the OTU selection was carried out by USEARCH clustering method79 at 97% similarity for removing chimeras and singletons80, against the Greengenes 13_08 reference database82. Sequences were aligned with PyNAST83 against the QIIME core set of aligned sequences, and the taxonomy was determined with UCLUST consensus taxonomy assigner77. The read counts were rarefied to equal depth to the level of the sample with the lowest reads. Statistical analysis using ANOSIM84 was also conducted in QIIME.The raw pyrosequencing dataset was analyzed  using QIIME85. Beta Diversity in ice samples was examined using permutational multivariate analyses of variance (PERMANOVA)86. PERMANOVA was conducted using model with Type III sum of squares and 9999 permutations on resemblance matrix constructed using a Bray Curtis similarity measure for Phyla composition, on fourth-root transformed. Principal coordinate analysis (PCO)86 performed on the resemblance matrix was used to visualize structure within different years of ice samples, with vectors for the geochemical data and Phyla that were correlated (Pearson’s r) with the ordination structure. The PCO biplot was generated using PRIMER software, by overlapping the separately generated plots for environmental and phyla data. PERMANOVA analyses and ordinations were made using PRIMER (version 6.1.16). The VENN diagram was generated using the R free environment for statistical computing and graphics software87.Diversity analyses were carried out using R package vegan: Community Ecology packageSupplementary material


To establish a strategy for identifying protein-N-myristoylation-dependent phosphorylation of cellular proteins, Phos-tag SDS-PAGE was performed on wild-type (WT) and nonmyristoylated mutant (G2A-mutant) FMNL2 and FMNL3, phosphorylated N-myristoylated model proteins expressed in HEK293 cells. The difference in the banding pattern in Phos-tag SDS-PAGE between the WT and G2A-mutant FMNL2 indicated the presence of N-myristoylation-dependent phosphorylation sites in FMNL2. Phos-tag SDS-PAGE of FMNL2 mutants in which the putative phosphorylation sites listed in PhosphoSitePlus (an online database of phosphorylation sites) were changed to Ala revealed that Ser-171 and Ser-1072 are N-myristoylation-dependent phosphorylation sites in FMNL2. Similar experiments with FMNL3 demonstrated that N-myristoylation-dependent phosphorylation occurs at a single Ser residue at position 174, which is a Ser residue conserved between FMNL2 and FMNL3, corresponding to Ser-171 in FMNL2. The facts that phosphorylation of Ser-1072 in FMNL2 has been shown to play a critical role in integrin β1 internalization mediated by FMNL2 and that Ser-171 in FMNL2 and Ser-174 in FMNL3 are novel putative phosphorylation sites conserved between FMNL2 and FMNL3 indicate that the strategy used in this study is a useful tool for identifying and characterizing physiologically important phosphorylation reactions occurring on N-myristoylated proteins. In geIn many cases, the functions of these N-myristoylated proteins are regulated by reversible protein–membrane interactions mediated by protein N-myristoylation, as in the case of G protein α-subunits and the Src family of tyrosine kinases ,4–6. ForIn this study, to establish a strategy for identifying protein N-myristoylation-dependent phosphorylation of cellular proteins, we performed Phos-tag SDS-PAGE analysis of wild-type (WT) and nonmyristoylated mutant (G2A-mutant) FMNL2 and FMNL3, which are phosphorylated N-myristoylated model proteins expressed in HEK293 cells ,16. As aH-1,2,3-triazol-4-yl)methyl]amine (TBTA) were obtained from Sigma . Protein G-HRP conjugate was sourced from Bio-Rad . HRP-conjugated anti-mouse IgG was purchased from Cell Signaling Technology . The other reagents used were obtained from Wako Pure Chemical Industries, Ltd.  or Daiichi Pure Chemicals Co., Ltd. , and were of analytical or DNA grade.Human cDNAs coding for FMNL2 (Q96PY5-3) and FMNL3 (Q8IVF7-3) were purchased from Promega . ECL Prime Western Blotting Detection Reagent was sourced from GE Healthcare . The dye terminator cycle sequencing kit, Lipofectamine LTX with PLUS reagent and Hoechst 33342 were obtained from Life Technologies Corp. . Anti-FLAG monoclonal antibody and anti-mouse IgG-FITC antibody were obtained from Sigma . Tetradec-13-ynoic acid (Alk-Myr) was sourced from Cayman Chemical Co. . 5-TAMRA Azide (Az-TAMRA) was purchased from Click Chemistry Tools . Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and tris on ice for 20 min.The metabolic labeling of cells with myristic acid analogue (Alk-Myr) was performed as described previously . HEK293 4·5H2O (50 mM)] in a total reaction volume of 50 μL for 1 h at room temperature -2-(hydroxymethyl)propane-1,3-diol hydrochloride  and a stacking gel (1.8 mL) consisting of 4% w/v polyacrylamide and 357 mM Bis-Tris–HCl buffer (pH 6.8) as a neutral phosphate-affinity SDS-PAGE system  for phoseviously .To confirm that protein N-myristoylation occurred on FMNL2 and FMNL3, cellular metabolic labeling experiments were performed by using cDNA encoding C-terminally FLAG-tagged FMNL2 and FMNL3. For this analysis, a nonmyristoylated G2A-mutant in which Gly-2 was replaced with Ala was used and its susceptibility to protein N-myristoylation was compared with that of WT protein. As shown in We next tested the phosphorylation of FMNL2-FLAG and FMNL3-FLAG expressed in HEK293 cells. Both FMNL2-FLAG  and FMNL3-FLAG  expressed in HEK293 cells were analyzed by SDS-PAGE followed by western blotting analysis using anti-FLAG antibody . In the http://www.phosphosite.org/homeAction), which is an online database provided by Cell Signaling Technology, Inc.  that lists the locations of phosphorylation sites, as described previously [ cDNA. Phos-tag SDS-PAGE followed by western blotting analysis using anti-FLAG antibody revealed that the phosphorylated species corresponding to the upper band (P) disappeared in the S174A mutant in the same manner as in the G2A mutant, as shown in Phosphorylation of Ser1072 in FMNL2 has been shown to play a critical role in stimuli-induced integrin β1 internalization mediated by FMNL2 . Therefo2–Trp–Ser–Arg–Ser(P)–Ile–Glu–Asp–Leu–COOH) were exactly the same, as shown in When amino acid sequences around the N-myristoylation-dependent phosphorylation sites identified in FMNL2 and FMNL3 were compared, it was found that the amino acid sequence surrounding the phosphorylation sites in the N-terminal GBD/FH3 domain were highly conserved between FMNL2 and FMNL3. In fact, the sequences of eight amino acids around the phosphorylated Ser residue , many proteins in various signal transduction pathways have been shown to be N-myristoylated –6,12–14.https://www.uniprot.org) revealed that FMNL2 and FMNL3 have two and three isoforms, respectively. In these isoforms, the N-myristoylation motif at the N-terminus is highly conserved and differences in amino acid sequence are observed mainly in about 30 amino acids in the C-terminus. Since isoform 2 of FMNL2 and isoform 3 of FMNL3 have been used in most studies on FMNL2 and FMNL3, we used these isoforms in the present study.In this study, FMNL2 and FMNL3, members of the formin family of proteins, were used as models for phosphorylated N-myristoylated proteins. Formin family proteins have been found to promote the formation of actin networks and to be involved in the regulation of cellular actin-dependent processes such as cell adhesion, migration, division, morphogenesis, or intracellular trafficking of protein –30. FormWith respect to protein N-myristoylation, we have demonstrated that FMNL2 and FMNL3 are N-myristoylated and that protein N-myristoylation is required for plasma membrane localization and for cellular morphological changes induced by overexpression of FMNL2 and FMNL3 ,35. WithWhen Phos-tag SDS-PAGE was performed on WT and nonmyristoylated-mutant (G2A-mutant) FMNL2 and FMNL3, significant differences in the banding patterns of WT and G2A-mutant FMNL2 and FMNL3 were observed , indicathttp://www.phosphosite.org) [We next tried to identify the N-myristoylation-dependent phosphorylation sites in FMNL2 and FMNL3 by using our recently established Phos-tag-based strategy for identifying specific phosphorylation sites from existing information on the location of phosphorylation sites (phosphoproteomic data) deposited in the database PhosphoSitePlus (ite.org) . Phos-taAs previously described, phosphorylation of Ser-1072 in FMNL2 by PKCα has been reported to play critical role in integrin β1 internalization mediated by FMNL2 . The amiRegarding the conserved N-terminal phosphorylation sites of FMNL2 and FMNL3, no direct experimental results indicating that these amino acids undergo phosphorylation has been reported. However, proteomic analyses have identified a phosphorylation site for Ser-171 in FMNL2 . In addiIn the present study, we have shown that an electrophoretic analysis by Phos-tag SDS-PAGE of the WT protein and its nonmyristoylated G2A-mutant, expressed in mammalian cells, is an efficient strategy for identifying and characterizing the physiologically important phosphorylation reaction of the N-myristoylated protein. The advantages of the use of this strategy are, first, the use of MS is not required; secondly, specific phosphorylation sites can be determined by utilizing phosphoproteomic data; and, thirdly, the cellular physiological role of protein phosphorylation at a distinct position can be studied by using phosphorylation-deficient mutants. To confirm the efficacy of this strategy in studies on the N-myristoylation-dependent phosphorylation reaction, we are currently studying the phosphorylation reactions of many N-myristoylated kinases and kinase substrates.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 FigThe merged images of phase contrast images and fluorescence microscopic images of COS-1 cells transfected with cDNA coding for wild type and G2A-mutants of FMNL2-FLAG and FMNL3-FLAG presented in (TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.


The average values of GI50 or LC50 of the heterocyclic quinones correlated significantly with their reduction potentials. These results suggested that reduction-oxidation potentials could be a useful method for the discovery of novel antitumor agents.To search for possible anti-tumor agents or anti-tumor promoters among natural or synthetic products, we used cyclic voltammetry to determine the reduction-oxidation potentials of heterocyclic quinones in phosphate buffer at pH 7.2. We determined the growth inhibitory- and cytotoxic activities of 12 heterocyclic quinone anti-tumor agent candidates against a panel of 39 human cancer cell lines (JFCR39). The average concentrations of the heterocyclic quinones required for 50% growth inhibition (GI HR-EI-MS m/z: 199.0281 ; 1H-NMR  δ: 7.05 , 7.87 , 8.03 , 9.10 , 9.43 .5-Methyl-4H-thienocarbazole-4,10(5H)-dione (11). HR-EI-MS m/z: 267.0349 ; 1H-NMR  δ: 4.19 , 7.41 , 7.55 , 8.34 . et al. /[C – T0]), while (ii) when T < T0, cell growth (%) = 100 × ([T – T0]/T0). GI50 was calculated as 100 × ([T – T0]/[C – T0]) = 50. The LC50, an index of cytotoxic effect, was determined as the concentration of the compound at which 100 × (T – T0)/T0 = –50. The mean graph was produced by computer processing of the GI50 (LC50) values as described [-4, 10-5, 10-6, 10-7, and 10-8 M) and a negative control. All assays were performed in duplicate. Mean graphs, which show differential growth inhibition of each drug against the cell line panel, was generated based on calculations using a set of GI50 values [et al. [i-xm)(yi-ym))/(Σ(xi-xm)2Σ(yi-ym)2)1/2, in which xi and yi are log GI50 values for compounds A and Bagainst each cell line and xm and ym are the mean values of xi and yi, respectively. We verified the accuracy of measured data by checking the dose response curves of reference control chemicals, such as mitomycin-C, paclitaxel, and SN-38, in every experiment.Inhibition experiments were performed to assess the sensitivity of cells to various chemicals as described by Yamori et al. . Growth et al. ,28,29. Aescribed . For eac0 values . To anal2 , Ag/AgCl (saturated NaCl) electrodes, and platinum coil electrodes were used as the working, reference, and counter electrodes, respectively. Before recording each voltammogram, the working electrode was pretreated as previously described [2 gas prior to voltammetric measurements. The electrolytic cell was water-jacketed to maintain a constant temperature of 25 ± 0.1 ºC. Cyclic voltammetric measurements were performed on a conventional three-electrode system using a laboratory-constructed microcomputer-controlled system in which the working electrode potential was controlled by a potentiostat . Plastic-formed-carbon (PFC) electrodes with a surface area of 0.071 cmescribed . AliquotCorrelations of the electrochemical and electronic parameters with the cytotoxic activities of heterocyclic quinones were determined using Pearson's correlation coefficient.P and LUMO energy were also useful parameters to predict the cytotoxic activity. In our previous study, the first reduction potentials also correlated with the inhibitory effects of anthraquinone derivatives on EBV-EA activation; both the number of hydroxy groups and LUMO were useful parameters to predict inhibitory activity [in vitro screening. We have determined the growth inhibitory- and cytotoxic activities of 12 heterocyclic quinone anti-tumor agent candidates against a panel of 39 human cancer cell lines (JFCR39). The first reduction potentials, determined at a physiological pH (7.2), correlated with the cytotoxic activities against JFCR 39 of the heterocyclic quinones. In addition, log activity . From th