Kd) range was characterized using Isothermal Titration Calorimetry (ITC). The RSV Kd value accounted to 6.6 µM in case of BRD4(1). Molecular docking proposed the binding mode of RSV against BRD4(1) mimicking the acetyl-lysine interactions. All these results suggest that RSV can also recognize epigenetic readers domains by interacting with BET bromodomains.The chemopreventive and anticancer effects of resveratrol (RSV) are widely reported in the literature. Specifically, mechanisms involving epigenetic regulation are promising targets to regulate tumor development. Bromodomains act as epigenetic readers by recognizing lysine acetylation on histone tails and boosting gene expression in order to regulate tissue-specific transcription. In this work, we showed that RSV is a pan-BET inhibitor. Using Differential Scanning Fluorimetry (DSF), we showed that RSV at 100 µM increased the melting temperature (∆Tm) of BET bromodomains by around 2.0 °C. The micromolar dissociation constant ( Vitis venifera species. This compound has received worldwide attention due to its role in regulating multiple transduction pathways of several diseases, including cancer, cardiovascular, neurodegerative, and inflammatory diseases [Resveratrol  is a naturally occurring polyphenol found in apples, peanuts, plums, berries, and fruit seeds of diseases .For cancer, RSV interferes in the different stages of tumors development, such as initiation, promotion, and progression . The rolAmong the multiple effects of RSV against cancer, those involving the regulation of epigenetic mechanisms seem to be promising to regulate tumor development . RSV is Bromodomains (BRDs) are epigenetics “readers” because they recognize lysine acetylation in histone tails and other nuclear proteins to boost gene expression, which has been involved in the development of many diseases, including cancer. Currently, 61 bromodomains present in 46 proteins have been reported . CrystalKd) for BRD4. Moreover, the binding mode of RSV into Kac of BRD4(1) was proposed using molecular docking.In order to evaluate the ability of the dietary compound RSV to inhibit BET-BRDs, we performed here the screening against BRD2, BRD3, BRD4, and BRDT using Differential Scanning Fluorimetry (DSF). Isothermal Titration Calorimetry (ITC) was used to determine the binding constant (E. coli BL21 (DE3) cells, which were kept on ice for 30 min. For this solution, heat shock was performed at 42 °C for 40 s, and then it was kept in on ice. Luria-Bertani (LB) Broth  medium was added (100 µL) and all cells were grown at 37 °C for 1 h. After, cells were selected on plates enriched by agar containing kanamycin (50 µg/mL)  and chloramphenicol (34 µg/mL) . Then, 50 mL of 2× LB medium concentration with kanamycin (50 µg/mL)  and chloramphenicol (34 µg/mL)  were inoculated with the selected cells. The cells were grown overnight .Plasmid vectors coding bromodomains (BRD2(1), BRD3(1), BRD4(1), BRDT(1) and BRD4(2)) were added into Eppendorf tube  containing chemically competent Terrific broth medium (TB medium) was prepared in advance (4× concentrated and autoclaved). This medium was diluted in water (1 L) and the medium containing the cells and kanamycin (50 µg/mL)  was pipetted at a volume of 10 mL. The cells were grown up to 1.5 of Density Optical (DO) . The shaker was set up at 18 °C, and the cells were grown up to 3.0 DO (180 RPM) and then induced overnight with 500 µM of Isopropyl β-D-1-thiogalactopyranoside (IPTG) . After, the cells were harvested at 8.700 g using centrifuge Sorval LyNX 6000-Thermo  and stored at −20 °C.2+ beads (5 mL) were added to a glass column (3 cm diameter), washed with water (50 mL) and followed by equilibration using lysis buffer (50mL). The cellular lysate containing protein was eluted through stationary phase chromatography using imidazole in lysis buffer . This procedure was performed at 4 °C and purity fractions were analyzed by SDS-PAGE [The harvested cells were resuspended in 30 mL lysis buffer  and lysed using a cell sonicator . After that, 0.15% of Polyethyleneimine (PEI) (Fluka (Honeywell), Morris Plains, NJ, USA) were added and the cell debris was centrifuged . CoSDS-PAGE . The fra2+ beads column followed by His-tag cleavage using TEV (20× less TEV concentration than protein concentration) in 20 mL TEV buffer  at 4 °C overnight. That protein solution was applied to a column containing 2 mL of Ni2+ beads. The stationary phase chromatography was equilibrated with TEV buffer (12 mL), and then the sample was eluted and collected completely followed by two more elutions (12 mL each) using 30 mM and 300 mM imidazole in TEV buffer, respectively. All procedures were performed at 4 °C. Those samples were analyzed by SDS-PAGE and concentrated through centrifugal filter  up to 20 mg/mL and stored at 4 °C.Protein fractions were gathered from CoTM plus  using gel filtration buffer as mobile phase. All proteins were collected in a range volume of 75–80 mL, analyzed by SDS-PAGE, concentrated up to 10 mg/mL, and then stored at −80 °C.The chromatography column Superdex 75 Increase  was equilibrated beforehand using gel filtration buffer  at flow of 1 mL/min. Samples were loaded through loop of 5 mL using that column previously which was connected to chromatograph ÄktPrimemT data for each compound was performed by qPCR machine , which had temperature raises of 3 °C per min in 71 cycles, 1 degree step, 21 s, with a starting temperature of 25 °C [All bromodomains were prepared at 2 µM of final concentration in 2 mL of DSF-assay buffer (10 mM HEPES pH 7.5 and 500 mM NaCl). Sypro Orange dye  (2 µL), used as fluorescence probe, was added into the sample protein, and then the samples were pipetted (20 µL) into 96-well microplates. All compounds were diluted beforehand (500 µM) in dimethyl sulfoxide (DMSO)  and stored into 96-well plates at −20 °C. RSV was pipetted into protein samples  and plate sealed with PCR seal. Δof 25 °C .ΔH) and dissociation constants (Kd). Thermodynamic parameters were calculated . In all experiments BRD4(1) was titrated into RSV solution (reverse titration) [Experiments were carried out on a Nano ITC microcalorimeter . The experiment was carried out at 15 °C and stirring at 350 RPM in ITC buffer . BRD4(1) was loaded using a microsyringe (250 μL) in ITC buffer. Initial titrations had injection of 4.0 μL followed by 21 injections of 8 μL spacing among them at 200 s between injections. The collected data was processed through NanoAnalyze™ software  supplied with the instrument to yield enthalpies of binding (tration) .Molecular modeling studies were performed with Maestro v9.1 Software  .The crysm) of BRDs increased at values of 2.0 °C (BRD4(1)), 2.9 °C (BRD2(1)), 1.8 °C (BRD3(1)), 1.5 °C (BRDT(1)), and 3.0 °C for BRD4(2), respectively  to interact with several BRDs of the BET family, comprising the first and second bromodomains (BD1 and BD2). In the presence of RSV, the melting temperature shifts (∆Tectively . These rKd) using ITC has demonstrated Kd value of 6.6 µM for RSV against BRD4(1) (ΔH) values, suggesting favorable polar interaction with this ligand. It was found that ΔH had values of −4.5 Kcal/mol. On the other hand, RSV appears to be revealing hydrophobic interaction not favorable due to entropic change ΔS > 0. It was found, for RSV, that ΔG values of −6.80 Kcal/mol were against BRD4(1).Orthogonal assay using ITC was performed in order to confirm the data observed for DSF studies. For this assay, BRD4(1) was used due to its well established involvement in cancer development . The obt BRD4(1) . The affΔS > 0 value. Hits optimization aiming to catch up hydrophobic interactions, such as π-stacking with Trp81 into ZA-loop region, appears to provide affinity for BRD4(1) inhibitors [The possible binding mode of RSV against BRD4(1) and its interaction with the conserved Kac binding site of this bromodomain were characterized through docking studies. The result suggests that RSV occupies the Kac binding site with interactions mimicking hydrogens bond of acetyl-lysine . In thishibitors ,19. The Modification upon chromatin structure such as methylation, acetylation, and phosphorylation have been broadly exploited as key point of understanding epigenetic mechanisms . Lysine Kd = 6.6 µM) to interact with BRD4(1). The molecular docking study allows for the proposing of the mode of interaction of RSV with BRD4(1). Our data showed that RSV has a similar binding mode into Kac of BRD4(1) compared to other BRDs-inhibitors, which mimics polar interaction of acetyl-lysine with Asn140 into Kac [ΔS > 0 value. Hydrophobic interactions such as π-stacking with Trp81 into ZA-loop region appear to provide a higher affinity of inhibitors for BRD4(1) [In this work, we demonstrated the capacity of resveratrol to interact with BET-BRDs using biophysical methods besides suggesting its binding mode into Kac of BRD4(1) through molecular docking studies. Therefore, we performed screening of RSV against BET-BRDs  by setting DSF and ITC experiments. RSV showed thermal shift value for all BET-BRDs which ranged from 1.5 to 3.0 °C. ITC experiments confirmed the ability of RSV  ,19.BRD4 has been reported as target in various cancer types as ovarian, breast, gastric, andregulation of transcriptions factor MYC . InteresHerein, we hypothesized that the anticancer effects of RSV could be related to dual activity, including SIRT-1 activation and BRDs inhibition . HoweverResveratrol was described for the first time to interact with BET-BRDs, being classified as a pan-BET inhibitor. This interesting effect can contribute to and explain the chemopreventive and anticancer effect of this polyphenol.


A/J/NaJ (B6.A10) consomic strain which carries the A/J chromosome 10 on a B6 strain background were compared. The B6.A10 mice compared to B6 were larger in body weight (p < 0.02): 27.2 ± 1.9 vs. 23.8 ± 2.7 and 23.4 ± 1.9 vs. 22.9 ± 2.3 g, for males and females, respectively, and in male soleus weight (p < 0.02): 9.7 ± 0.4 vs. 8.6 ± 0.9 mg. In the forced running test the B6.A10 mice completed only 64% of the B6 covered distance (p < 0.0001). However, there was no difference in voluntary wheel running (p = 0.6) or in fatigability of isolated soleus (p = 0.24) or extensor digitorum longus  muscles. We conclude that chromosome 10 of the A/J strain contributes to reduced endurance performance. We also discuss physiological mechanisms and methodological aspects relevant to interpretation of these findings.Phenotypic diversity between laboratory mouse strains provides a model for studying the underlying genetic mechanisms. The A/J strain performs poorly in various endurance exercise models. The aim of the study was to test if endurance capacity and contractility of the fast- and slow-twitch muscles are affected by the genes on mouse chromosome 10. The C57BL/6J (B6) strain and C57BL/6J-Chr 10 Endurance as a component of physical fitness is an important determinant of health and well-being. For instance, the risk of mortality due to cardiovascular disease in obese men is significantly reduced with increasing level of fitness  consomic strain carrying A/J Chr 10 on the B6 strain background provides a useful model for studying the genetic mechanisms contributing to impaired endurance capacity of the A/J strain. A gene encoding the key enzyme of the TCA cycle, citrate synthase (Cs), resides on mouse Chr 10. Both human  strains were purchased from Charles River and the Jackson Laboratory, respectively. Samples of the experimental animals were bred locally.All animal procedures were approved by the Lithuanian State Food and Veterinary Service (Ref. # 0230). Breeding nuclei of the C57BL/6J (B6) and C57BL/6J-Chr 10ad libitum. The forced running experimental procedure on 16 B6  and 21 B6.A10  was initiated at 12–13 week of age. Voluntary wheel running was measured in separate group of 10 B6 and 10 B6.A10 males at 15–16 week of age. The ex vivo testing was carried out on a separate group of 6 B6 and 6 B6.A10 males of 18–19 week of age.Mice were housed in standard cages, one to three same sex animals per cage, ambient temperature 20–21°C and 40–60 % humidity, with 12-h light/ 12-h dark cycle. Animals were fed standard chow diet (LabDiet 5001) and received tap water Animals were tested on a 5-lane treadmill  with an exercise area of 37 × 5 × 5 cm (L × W × H) in each lane, and with a built-in air puff stimulation at the end of the lane. All treadmill sessions were carried out during the dark cycle under red light illumination. Each exposure to the treadmill began with a 2-min habituation period on a stationary treadmill belt at a zero incline. Two habituation sessions of 15 min were carried out on 2 consecutive days prior to endurance test. During those sessions mice ran on the treadmill at a speed of 16.8 m/min at zero incline. The treadmill belt was cleaned with 70% ethanol between the groups.Two days after completion of habituation mice were subjected to an endurance test. The test is a modification of the protocol described earlier . Following 1 day habituation period running wheel activity was recorded as revolutions of the wheel over 3 consecutive days. This permitted calculation of the distance ran over that period of time. However, it was not possible to determine the actual time spent on the wheel.2, 1.8 CaCl2, 0.4 NaH2PO4, 0.1 NaEDTA, 24 NaHCO3, 5.5 glucose, pH 7.4, and bubbled with 95% O2: 5% CO2). The distal tendon was attached to a stable hook and the proximal end was tied directly to the lever of the muscle test system . Both soleus and EDL of the same hind limb were processed in an alternating order between the animals. While processing the first muscle the other was kept in a bath with Tyrode solution. Mounted muscle was left to equilibrate in the solution for 10 min. Afterwards the muscle was stimulated using a custom-made stimulator by 25 V square pulses. It has been determined in a pilot experiment that this voltage elicits maximal contraction. Muscle length was increased and twitch force measured every 30 s until no further increase in force was observed. Muscle was kept at this optimal length (L0) during the subsequent experiments. Muscles were photographed with a scale in the background in order to assess muscle length with a precision of 0.1 mm. Twitch contraction time (CT) was assessed as the time elapsed from the beginning of the contraction to its peak. Twitch half relaxation time (HRT) was measured as the time taken for the force to decline to 50% of the peak value. At L0 muscle was subjected to 300 ms (EDL) or 900 ms (soleus) trains of pulses of increasing frequencies . Maximal isometric force (P0) was estimated from plateau in force-frequency curve and usually occurred at 100 Hz for SOL and at 100–150 Hz for EDL.Contractility of extensor digitorum longus (EDL) and soleus muscles was examined. All procedures were carried out at room temperature (23–25°C). Following sacrifice, soleus and EDL muscles of randomly selected hind limb were excised for contractile measurements. Sutures were attached to the proximal and distal tendons of isolated muscles. Thereafter muscle was mounted between two platinum plate electrodes in 100 mL tissue bath  filled with Tyrode's solution  were delivered every second for EDL and every 1.1 s for SOL, respectively. Thus, duration of fatigue protocol for EDL and SOL was 180 and 198 s, respectively. The fatigue index was calculated as the ratio between the force of the final and the first contraction of the fatigue protocol multiplied by 100.f) and the density of mammalian skeletal muscle: CSA (mm2) = mass (mg)/length (mm)/1.06 (mg/mm3)  was determined by the ratio between P0 and CSA.After the measurements, the muscle was cleaned from all visible tendons, blotted, and weighed on an analytical balance . Muscle physiological cross-sectional area (CSA) was calculated by dividing wet muscle mass by the optimal fiber length (Ln = 9) and B6.A10 (n = 9) males were homogenized in ice-cold lysis buffer  with an ULTRA-TURRAX homogenizer . Following shaking for 60 min the homogenates were centrifuged at 13,000 g at 4°C for 10 min and the protein concentration was measured in the supernatant using the Bradford assay . The reaction reagent consisted of 100 mM triethanolamine-HCl, DTNB (100 μM), 0.25% Triton-X (vol/vol), 0.5 mM oxaloacetate, 0.31 mM acetyl CoA with pH adjusted to 8.0. Ten microliters of muscle homogenate was added to start the reaction in 1000 μL. The molar extinction coefficient of 13,600 M−1·cm−1 was used to assess the maximum CS activity (Vmax) at 412 nm during the first 2 min of the reaction. The assay was carried out at room temperature (~21°C), and CS from porcine heart was used as a standard  for assay calibration.CS activity was measured as previously described  software. Mean and SD is presented unless stated otherwise.A 2-way ANOVA (Strain and Sex), 2-way ANCOVA (body weight as covariate) and Mann-Whitney First we aimed at determining if consomic strain carrying Chr 10 of A/J strain would show a reduced performance in the forced running endurance test.p < 0.0001). There was no sex effect (p = 0.6) on endurance performance  compared to the B6; 27.2 ± 1.9 vs. 23.8 ± 2.7 and 23.4 ±1.9 vs. 22.9 ± 2.3 g, for males and females, respectively, the strain effect on forced running distance remained statistically significant (p < 0.0001) after inclusion of body weight as covariate.The B6 strain showed a superior endurance capacity by running significantly further compared to the B6.A10 strain, 391 ± 53 vs. 251 ± 54 m respectively  in B6.A10 compared to the B6 strain , respectively, and EDL muscles, 209 ± 21 vs. 217 ± 13 mN (p = 0.59). The isometric contraction force after adjustment for the CSA of the muscle did not differ between the strains in either soleus or EDL muscle , and 27 ± 2 and 27 ± 4% (p = 0.7) in the EDL.The peak force of isometric contraction was similar between the B6 and B6.A10 strains in both soleus, 192 ± 24 vs. 211 ± 10 mN  in B6.A10 compared to B6 strain, 487.7 ± 69.5 vs. 754.0 ± 68.6 U/g, respectively.The CS activity in gastrocnemius muscle was assessed to examine if it could have contributed to reduced forced running endurance of the B6.A10 strain. The analysis indicated that CS activity was significantly lower  has been used as a biomarker of mitochondrial content  in the B6 strain exhibit properties reminiscent of the “athlete's heart” in comparison to A/J  determining endurance capacity.AL, AR, and TV designed the study; MK carried out forced running and voluntary wheel running experiments with supervision of TV; PM performed physiological analyses of isolated muscles; AF measured enzyme activity; AL and MK wrote the manuscript with contribution of all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Elevated production of reactive oxygen species (ROS) and an altered redox state have frequently been observed in hepatocellular carcinoma (HCC); therefore, selective killing of HCC cells by chemotherapeutic agents that stimulate ROS generation or impair antioxidant systems may be a feasible approach in HCC chemotherapy. Recently, betulinic acid and its derivatives have attracted attention because they showed anti-cancer effects via a ROS- and mitochondria-related mechanism. However, the source of ROS overproduction and the role of mitochondria were poorly identified, and the weak in vivo antitumour activity of these compounds limits their development as drugs.Cytotoxicity was detected using MTT assays. In vivo anti-HCC effects were assessed using nude mice bearing HepG2 tumour xenografts. Cell cycle analysis, apoptosis rate and mitochondrial membrane potential were measured by flow cytometry. ROS production was detected using a microplate reader or a fluorescence microscope. Changes in gene and protein levels were measured by RT-PCR and western blotting, respectively. Other assays were performed using related detection kits.2 consumption rate and ATP production. Furthermore, mtDNA-depleted ρ0 HepG2 cells were less sensitive to B5G9 treatment than wt HepG2 cells, indicating the importance of mitochondria in B5G9-induced cell death.B5G9, a piperazidine derivative of 23-hydroxy betulinic acid (23-HBA), showed excellent in vivo anti-HCC effects, with a tumour growth inhibitory rate of greater than 80%, and no significant side effects. B5G9 stimulated the production of ROS, which were derived from the mitochondria, but it had no effect on various other antioxidant systems. Moreover, B5G9 induced mitochondrial dysfunction, which was characterized by morphological changes, membrane potential collapse, membrane permeabilization, and decreases in the OWe discovered a piperazidine derivative of 23-HBA, B5G9, with excellent anti-HCC effects both in vivo and in vitro and no obvious toxic effects. The underlying mechanism was associated with mitochondria-derived ROS overproduction, and mitochondria played essential roles in B5G9-induced cell death. This study identified a potential agent for anti-HCC therapy and elucidated the mitochondria-related mechanism of BA and its derivatives.The online version of this article (doi:10.1186/s13046-016-0457-1) contains supplementary material, which is available to authorized users. Hepatocellular carcinoma (HCC), the most common type of liver cancer, is the sixth most common malignancy in the world . The majReactive oxygen species (ROS), a by-product of the normal metabolism of oxygen, play an important role in cell proliferation and differentiation . HoweverIn normal physiological conditions, cellular ROS is maintained at a stable level due to the dynamic balance between ROS production and elimination. ROS overproduction or antioxidant system impairment may result in oxidative stress . MitochoBetulinic acid (BA), a naturally occurring pentacyclic triterpenoid isolated from white birch trees, has attracted widespread attentions due to its multiple biological activities, such as anti-HIV, anti-cancer, anti-malarial and anti-inflammatory effects , 19. In In this study, we found that a piperazidine derivative of 23-HBA, B5G9, exhibited excellent in vivo anti-HCC efficiency, with a tumour growth inhibitory rate of more than 80%, and no significant side effects in B5G9-treated nude mice bearing HepG2 xenografts. Moreover, the underlying mechanism of B5G9 was associated with apoptosis induction, and we further explored the relationship among the mitochondria, ROS and apoptosis.2DCFDA, MitoTracker® Red CMXRos, MitoSOX Red, a BCA protein assay kit and a Dead Cell Apoptosis Kit were purchased from Thermo Fisher Scientific . Necrostatin-1 was obtained from Selleck Chemicals . MnTBAP was purchased from Santa Cruz Biotechnology . An antibody against cytochrome c was obtained from Epitomics . Antibodies against caspase-3, caspase-9, cleaved-caspase-3, cleaved-caspase-9, PARP and β-actin were obtained from Cell Signaling . Other reagents were purchased from Sigma Aldrich .B5G9 with a purity of 98% was synthesized as described previously . Hoechst2 atmosphere.The HCC cell lines HepG2 and Hep3B were obtained from the American Type Culture Collection . Bel-7402 cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences . HepG2/ADM cells were kindly provided by Prof. Kwok-Pui Fung . The HepG2, Hep3B, HepG2/ADM and Bel-7402 cells were maintained in RPMI 1640 medium  containing 10% (v/v) foetal bovine serum  and 1% (v/v) penicillin-streptomycin  at 37 °C in a 5% CO4/well) were seeded in 96-well plates and cultured overnight. Then, the cells were treated with different concentrations of B5G9 for an additional 12 h, 24 h or 36 h. Subsequently, the cells were incubated with 30 μl of MTT (5 mg/ml) for 4 h. The formazan crystals that formed were solubilized in 100 μl DMSO, and the absorbance was measured at 595 nm using a microplate reader . Cell viability was calculated as a percentage of the vehicle control (treatment with medium containing 0.2% DMSO).HepG2, HepG2/ADM, Hep3B and Bel-7402 cells : vehicle, B5G9 (20 mg/kg and 40 mg/kg) and 23-HBA (40 mg/kg). The drugs were administered via intragastric injection every day. The vehicle group was administered 0.9% NaCl. Body weight and tumour volume were measured every other day, and tumour volume was calculated as (a × b2)/2, where a and b are the longest and the shortest diameters of the tumours, respectively [3, the mice were sacrificed and the tumours, organs and blood were collected for subsequent measurement.Six-week-old nude mice were obtained from Vital River Laboratory Animal Technology Co, Ltd. . All animals were maintained in specific pathogen free (SPF) room. The nude mice subcutaneously inoculated with 1.5 × 10ectively . After 2For histological examination, haematoxylin-eosin (H&E) staining of the viscera was performed as described previously . The imaAfter B5G9 treatment, the blood of the mice was collected and serum biochemistry and routine blood analyses were carried out as described previously , 37.HepG2 cells treated with B5G9 for 12 h were stained with Hoechst (5 μg/ml) 33342 in the dark at 37 °C for 20 min. After being washed with phosphate-buffered saline (PBS), the cells were observed using a fluorescence microscope .HepG2 cells treated with B5G9 for 12 h were fixed in 3% glutaraldehyde overnight and in 1% osmium tetroxide for an additional 1 h. The cells were then dehydrated in a gradient of 50–100% ethanol and polymerized with epoxy resin. The ultra-thin sections were stained with aqueous uranyl acetate and lead citrate. The ultrastructure of the cells was observed using a JEM-1400 Plus transmission electron microscope .HepG2 cells treated with various concentrations of B5G9 were collected and fixed in ice-cold 70% (v/v) ethanol at 4 °C overnight. Then, the cells were resuspended in PBS containing PI (10 μg/ml) and RNase (0.1 mg/ml) and incubated at 37 °C for 30 min in the dark. DNA content was measured with an Epics XL Flow Cytometer  (Ex = 488 nm and Em = 620 nm).HepG2 cells treated with various concentrations of B5G9 were collected and stained with detection buffer containing Annexin V-FITC and PI for 15 min at 37 °C in the dark. The fluorescence was detected with an Epics XL Flow Cytometer  .2DCFDA (10 μM) in the dark at 37 °C for 20 min. The fluorescence intensity was observed with a fluorescence microscope or detected by a microplate reader .HepG2 cells were treated with B5G9 for the indicated times, and the cells were stained with HA Lipid Peroxidation MDA Assay Kit  was used for the detection of cellular lipid peroxidation. Methane dicarboxylic aldehyde (MDA) was used as a characteristic product of lipid peroxidation. In brief, cell lysates from HepG2 cells treated with B5G9 or the MDA standards were mixed with thiobarbituric acid (TBA) working solution according to the manufacturer’s manual. The absorbance at 535 nm of the MDA-TBA complex was detected with a microplate reader . The concentration of cellular MDA was calculated according to the standard curve.ΔΨm detection, HepG2 cells treated with B5G9 were stained with JC-1 at 37 °C for 20 min, and the JC-1 fluorescence was detected with an Epics XL Flow Cytometer . The JC-1 fluorescence conversion from red to green demonstrates the ΔΨm collapse. For the measurement of MPT, a mitochondrial permeability transition pore detection kit  was used according to the manufacturer’s manual.For HepG2 cells were stimulated with B5G9 for the indicated times, and cells were stained with MitoTracker® Red CMXRos at 37 °C for 30 min. Mitochondrial morphology was observed with a fluorescence microscope .2 consumption rate (OCR) was measured using an Oxygen Consumption Rate Assay Kit . HepG2 cells  were seeded in a 96-well plate and cultured overnight. The cells were treated with B5G9 for the indicated times or antimycin A for 3 h. Then, MitoXpress-Xtra was added and HS Mineral Oil was overlaid in each well. Finally, the MitoXpress-Xtra fluorescence was detected with an Infinite F500 microplate reader  using time-resolved fluorescence measurement .The OATP content was determined using a luciferase-dependent method. In brief, HepG2 cells seeded in 96-well plates  were treated with B5G9 in complete medium or no-glucose medium and washed with PBS twice. Cellular ATP content was detected with a Cell Titer-Glo 2.0 Assay kit following the manufacturer’s protocol .ρ0 HepG2 cells were derived from wt HepG2 cells by maintaining the cells in EB (100 ng/ml), pyruvate (100 μg/ml) and uridine (50 μg/ml) for more than 30 generations , 39. MitFor total protein extraction, HepG2 cells treated with various concentrations of B5G9 were harvested, washed twice with ice-cold PBS and lysed in RIPA  buffer containing PMSF, phosphatase inhibitors and protease inhibitors for 30 min on ice. After centrifugation at 20,000 g at 4 °C for 10 min, the supernatants were quantified using a BCA protein assay kit. The cytoplasmic and mitochondrial proteins were extracted using a digitonin-based method as described previously . For wes2 Profiler PCR Arrays  were used. Primer sequences are shown in Additional file Total RNA from HepG2 cells was extracted using an RNeasy Mini Kit . Reverse transcription was performed using a Transcriptor First Strand cDNA Synthesis Kit  with total RNA (2 μg) as described by the manufacturer. Then, RT-PCR was carried out by adding SYBR Green I Master 10 μl) , forward primer (0.5 μM), reverse primer (0.5 μM), cDNA (2 μl) and distilled water (6 μl) per sample, and the PCR products were quantified with a LightCycler 480 PCR system . To analyse the related genes in B5G9-induced oxidative stress, RT0 μl  were upregulated after B5G9 treatment  can co-localize with a mitochondria-selective probe, indicating that elevated ROS may be produced by the mitochondria, and similar characteristics were also found in 23-HBA-treated HepG2 cells  is an abundant source of anti-HCC agents. Recently, the modification of natural products from TCM has become a promising approach for novel drug development. To enhance the antitumour efficacy of 23-HBA and reduce its side effects, we performed a series of structural modifications of 23-HBA and conducted a bioactivity screening . We founIn the current study, we evaluated the anti-HCC activity of B5G9 on four HCC cell lines, including HepG2, Bel-7402 and Hep3B cells (p53-null), and multidrug-resistant HepG2/ADM cells. B5G9 showed comparable cytotoxicity in the four hepatoma cell lines, which indicates the potential of B5G9 in the treatment of various types of HCC cells, regardless of p53 status. B5G9 also exhibited outstanding anti-proliferation activity on HepG2 tumour xenografts without significant toxicity, whereas 23-HBA had a minimal effect.2DCFDA and Mito-Tracker as well as MitoSOX Red staining. We found that 23-HBA had a similar but weaker effect, which could explain why B5G9 had greater anti-HCC activity than that of 23-HBA; however, B5G9 may have a different underlying mechanism of mitochondrial dysfunction. Moreover, we noted that B5G9 just induced a slight increase of mitochondrial ROS and apoptosis in normal liver LO2 cells compare to HepG2 cells  or bongkrekic acid , 60. In In summary, we discovered a piperazidine derivative of 23-HBA, B5G9, with excellent anti-HCC effects both in vivo and in vitro, and no obvious toxic effects. The underlying mechanism was associated with mitochondria-derived ROS production, and we further identified the importance of the mitochondria in B5G9-induced cell death. This study identifies a potential agent for anti-HCC therapy and elucidates the source of ROS induced by BA and its derivatives.


Estrogen improves cardiac recovery after ischemia/reperfusion (I/R) by yet incompletely understood mechanisms. Mitochondria play a crucial role in I/R injury through cytochrome c-dependent apoptosis activation. We tested the hypothesis that 17β-estradiol (E2) as well as a specific ERβ agonist improve cardiac recovery through estrogen receptor (ER)β-mediated mechanisms by reducing mitochondria-induced apoptosis and preserving mitochondrial integrity.We randomized ovariectomized C57BL/6N mice 24h before I/R to pre-treatment with E2 or a specific ERβ agonist (ERβA). Isolated hearts were perfused for 20min prior to 30min global ischemia followed by 40min reperfusion.Compared with controls, ERβA and E2 treated groups showed a significant improvement in cardiac recovery, i.e. an increase in left ventricular developed pressure, dP/dtmax and dP/dtmin. ERβA and E2 pre-treatment led to a significant reduction in apoptosis with decreased cytochrome c release from the mitochondria and increased mitochondrial levels of anti-apoptotic Bcl2 and ACAA2. Protein levels of mitochondrial translocase inner membrane (TIM23) and mitochondrial complex I of respiratory chain were increased by ERβA and E2 pre-treatment. Furthermore, we found a significant increase of myosin light chain 2 (MLC2) phosphorylation together with ERK1/2 activation in E2, but not in ERβA treated groups.Activation of ERβ is essential for the improvement of cardiac recovery after I/R through the inhibition of apoptosis and preservation of mitochondrial integrity and can be a achieved by a specific ERβ agonist. Furthermore, E2 modulates MLC2 activation after I/R independent of ERβ.The online version of this article (doi:10.1186/s13293-016-0104-8) contains supplementary material, which is available to authorized users. Premenopausal women have a decreased risk of ischemic heart disease compared with age-matched men, suggesting a putative cardioprotective effect related to estrogens during reproductive age. However, in randomized control trials, chronic estrogen therapy in postmenopausal women has not demonstrated readily any cardiovascular benefit , 2. To cTo study the effects of I/R in rodents, generally either myocardial infarction (MI) with subsequent reperfusion or isolated perfused heart systems with global or regional ischemia, i.e., the Langendorff model, have been used . In the 2+ sensitivity of myofilaments. Myosin light chain 2 (MLC2) plays an important role in the regulation of muscle contractile activity via its phosphorylation [I/R injury has also been shown to lead to myofibrillar remodeling and to depress Carylation , 19. Monrylation . We founrylation . TherefoExperiments were performed in accordance with the guidelines of Charité-Universitaetsmedizin, were approved by the Landesamt für Gesundheit und Soziales , and conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of health . Female C57BL/6N mice (Janvier) were bilaterally ovariectomized (ovx) at the age of 10 weeks and kept on soy-free diet  for 4 weeks. Before I/R experiments, animals were randomized into three groups [vehicle treated (untreated) or treated either with E2 (0.2 mg/kg body weight (BW))  in vivo 4, 118.0 NaCl, 4.7 KCl, 0.06 EDTA, 24.7 NaHCO3, 0.23 KH2PO4, 3.215 CaCl2, and 10.0 glucose. The aorta was quickly cannulated for retrograde perfusion at a constant pressure of 80 mmHg using a Langendorff perfusion apparatus. The apparatus was water-jacketed to maintain a temperature of the heart at 37 °C. The buffer was oxygenated with 95 % oxygen/5 % CO2 to maintain a pH of 7.4. A water-filled latex balloon that was connected to a pressure transducer was inserted into the left ventricle (LV). LV diastolic pressure (LVPdia) was set to 12 mmHg. Heart rate (HR), LV systolic (LVPsys) and LV developed pressure (LVPdp), as well as dP/dtmin and dP/dtmax, markers for relaxation and contractility, were measured and recorded continuously using ISOHEART® Isolated Heart Data Acquisition Software . The hearts were perfused for a 20-min stabilization period. Following 30 min of global ischemia, the hearts were reperfused for a total of 40 min. Recovery of cardiac function was measured at the end of 40 min of reperfusion and expressed as a percentage of the rate prior to ischemia. During the Langendorff experiment, the physiological data were monitored continuously and calculated at the end of I/R in percent of the value at the start of ischemia.Animals were anesthetized with ketamine hydrochloride (80 mg/ml)/xylazine hydrochloride (12 mg/ml) solution administered by intraperitoneal injection at a dose of 1 mg/kg BW and anticoagulated with heparin. The hearts were quickly excised and placed in ice-cold Krebs–Henseleit buffer containing in millimoles per liter: 2.1 MgSOl-lactate dehydrogenase, Roche Applied Science, Mannheim, Germany) according to the manufacturer’s instructions. Optical density was measured in duplicate for each sample and multiplied by the respective coronary flow.For determination of necrosis, lactate dehydrogenase (LDH) was used as biomarker. Effluents of each group were collected for LDH assay over 3 min directly before global ischemia and four times over 3 min immediately after the start of reperfusion. LDH was assayed with the use of a commercially available assay  method was used to evaluate apoptosis in ischemic reperfused heart tissue. Staining was analyzed by Fluorescent Microscopy . Results are expressed according to manufacturer indication in apoptotic nuclei percentage. Representative pictures are shown in Fig. Whole cell extracts were isolated using modified RIPA buffer and separated by SDS-polyacrylamide gel electrophoresis as previously described . The lefFor immunoblotting, 10 μg of protein was separated by 12 or 15 % SDS-polyacrylamide gel electrophoresis (PAGE) and subsequently transferred to a polyvinylidene difluoride (PVDF) membrane. Adequate transfer of protein was confirmed by Ponceau Red staining of the membranes. Antibodies against cytochrome c , Bcl2 , Acetylcoenzyme A acyltransferase 2 (ACAA2) , TIM23 (#611222 BD Biosciences), MitoProfile® total OXPHOS Rodent WB Antibody Cocktail , myosin light chain 2 (MLC2) , myosin light chain (pMLC2) , Phospho-p44/42 MAPK (Erk1/2) , and p44/42 MAPK (Erk1/2) (137F5)  had been used.In particular, MitoProfile® total OXPHOS Rodent WB Antibody Cocktail contains five mAbs, one each against CI subunit NDUFB8 (ab110242), CII-30 kDa (ab14714), CIII-Core protein 2 (ab14745) CIV subunit I (ab14705), and CV alpha subunit (ab14748) as an optimized premixed cocktail. The kit is suitable for western blotting analysis of the relative levels of the five OXPHOS complexes in mitochondrial preparations from mouse.Equal protein loading and fractions purity were confirmed by probing for α-tubulin for whole lysate or cytosolic fraction and mitochondrial OXPHOS complex II protein for mitochondrial fraction.Immunoreactive proteins were detected using ECL Plus  and quantified by the ImageJ 1.41 version software. At least, all samples were evaluated as duplicates. Representative western blots of all measured proteins are shown in Additional file T test for independent samples. p values ≤0.05 were considered statistically significant.Data are shown as mean ± standard error of the mean (SEM). We only included animals with cannulation times less than 2 min and coronary flow between 1 and 4 ml/min at the end of the stabilization phase. Parameters were tested by one-way ANOVA followed by post hoc Dunnett comparing all treated groups with the untreated. For RPP, additionally we performed two-sided Developed left ventricular pressure (LVPdp) and rate pressure product (RPP) showed a significant better recovery in ERβA- and E2-treated groups compared with controls Fig. . MarkersLDH in the effluent of isolated hearts, a marker of necrosis, was measured immediately before ischemia and 3, 6, 9, and 12 min immediately after start of reperfusion. All groups showed a peak in LDH levels between 3 and 6 min post-ischemia, which were significantly decreased only in the E2 group  were significantly higher than those in untreated controls  , 34. ACCMitochondria in the ERβA and E2 pre-treated groups had higher levels of TIM23, a marker of integrity of the mitochondrial inner membrane . This isThe respiratory chain in the inner mitochondrial membrane is responsible for generating ATP and consists of five major membrane protein complexes. We found here that the pre-treatment with ERβA and E2 increases the protein levels of mitochondrial complex I, supporting the notion that E2 reduces I/R damage through an ERβ-dependent mechanism.Other mechanisms could contribute to the better contractile recovery after I/R in ERβA and E2 pre-treated hearts. Our group previously reported that E2 treatment can modulate myocardial contractility via MLC2 function in a sex-specific manner . We obseThis study has several limitations. Most importantly, we did not confirm the specificity of the ERβA. This has however been well established in previous investigations . We limiThe present study suggests that the activation of ERβ by E2 or a specific agonist essentially contributes to a reduction of cardiac stress by reducing apoptosis and preservation of mitochondrial integrity, and improved functional recovery after I/R. The identification of new ERβ-mediated E2 effects opens new opportunities to therapeutically targeting these pathways in different clinical settings.


Whereas the structures of FLRT LRR domains from various species have been determined, the expression and purification of recombinant FLRT FnIII domains, important steps for further structural and functional characterizations of the proteins, have not yet been described. Here we present a protocol for expressing recombinant FLRT-FnIII domains in inclusion bodies in Escherichia coli. His-tags permitted affinity purification of the domains, which subsequently were refolded on a Ni-NTA agarose column by reducing the concentration of urea. The refolding was confirmed by circular dichroism (CD) and 1H-NMR. By thermal unfolding experiments we show that a strand-strand cystine bridge has significant effect on the stability of the FLRT FnIII fold. We further show by Surface Plasmon Resonance that all three FnIII domains bind to FGFR1, and roughly estimate a Kd for each domain, all Kds being in the µM range.The fibronectin leucine rich transmembrane (FLRT) protein family consists in humans of 3 proteins, FLRT1, -2, and -3. The FLRT proteins contain two extracellular domains separated by an unstructured linker. The most membrane distal part is a leucine rich repeat (LRR) domain responsible for both  The fibronectin leucine rich transmembrane (FLRT) protein family was first described in 1999. In mammals it includes three proteins, FLRT1, -2, and -3  each comA number of extracellular binding partners to FLRT proteins have been reported, including Myelin-associated glycoprotein, Brother of CDO, netrin receptors, and the synaptic, G-protein-coupled receptors, latrophilins (reviewed by Xenopus FLRT3 (XFLRT3) is reported to bind directly to FGFR1 . Using polymerase chain reaction (PCR) with oligonucleotides primer  cDNA for all three FLRT-FnIII domains was amplified and cloned into pET-DEST42 Gateway (Invitrogen) destination vector containing ampicillin resistance.E. coli BL21 (DE3) (Novagen) or Rosetta (DE3) (Novagen) and selected with appropriated antibiotics. A total of 10 ml starter culture of Luria Bertani (LB) broth containing 100 µg/ml ampicillin or 50 µg/ml kanamycin was inoculated with a single colony, and grown at 37 °C overnight with 250 rpm shaking.All the constructs were transformed into 600 of 0.6 was reached. Overexpression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, and the cultures were allowed to overexpress for 20 h at 15 or 25 °C. The cells were harvested by centrifugation at 6,000 × g for 10 min. Cell pellets were frozen for minimum 15 min at −20 °C. Thawed cell pellets were resuspended in denaturing buffer , pH 7.4, 1 mM Dithiothretol (DTT)) at 5 ml/g wet weight, and sonicated for 7 min with 10 s pulse and 10 s pause with an amplitude set to 50%. Following sonication the cell paste was stirred for 60 min at room temperature to solubilize inclusion bodies. The lysate containing soluble inclusion bodies was centrifuged at 20,000 × g for 30 min at +4 °C to pellet insoluble material. The supernatant was collected and filtered with 0.45 µm sterile filter before purification. 20% SDS-PAGE was used to follow purification and verify protein purity with typical sample loading of 0.5–1 mg/ml protein. SDS-PAGE gels were stained overnight with InstantBlue™ (Expedeon).Afterwards, 1 L LB containing 100 µg/ml ampicillin or 50 µg/ml kanamycin was inoculated with the overnight starter culture. The culture grew at 37 °C with 250 rpm shaking until an OD280 nm and A260 nm were stabilized. Subsequently, the washed proteins were refolded on the column by applying PBS (pH 7.4), thus generating a decreasing gradient of urea and DTT (from 8 M urea and 1 mM DTT to 0 M in 30 min). A second wash step was included with wash buffer (5 × PBS and 20 mM imidazole pH 7.4). Before eluting the protein, PBS was run through the column to decrease the salt concentration. The protein was eluted with elution buffer (PBS pH 7.4 containing 500 mM imidazole) for 5 column volumes. The purification was monitored using ultraviolet absorption at 280 nm and 260 nm. The refolded proteins were further purified by size-exclusion chromatography (SEC) using a 320 ml or 120 ml Superdex 75 16/60 GL column equilibrated with regular PBS. The collection was monitored at A280 nm and A260 nm. All refolding and purification procedures were carried out at 4 °C and the purified proteins were stored at −80 °C until use.FnIII domains from filtrates were immobilized on a 1 ml HP HisTrap Ni-NTA column (1 for each protein)  coupled to an Äkta purify system. The column was equilibrated with 10–15 column volumes of denaturing buffer  with flow rate 1 ml/min. Following equilibration the filtrate was loaded onto the column, and washed with wash buffer  for at least 30 column volumes until the AThe masses of the FLRT domains were determined by MALDI-TOF-MS. 1 µl sample and 1 µl a-Cyano-4-hydroxycinnamic acid (HCCA) matrix were mixed and dispensed onto a MALDI target and allowed to dry out at room temperature. Protein masses were acquired in linear mid-mass positive mode, averaging 2,000 laser shots per MALDI-TOF spectrum. Calibration mixtures  were used to calibrate the spectrum to a mass tolerance within 2 Da. The mass range was 5,000–14,000 Da.The Far-UV CD measurements were performed using a Jasco J-815 spectropolarimeter at 20 °C in a 1 mm path length quartz cuvette from Hellma over the wavelength range 190–260 nm. A resolution of 1 nm and a scan speed of 20 nm/min were employed. At least three consecutive scans were obtained, the resulting data was smoothed and the contribution of the buffer blank was subtracted. The monitored scans were averaged. Spectra were analyzed for secondary structural distribution using the Beta Structure Selection, BeStSel, server . Variabl2O (Invitrogen) and 500 µl of the concentrated sample of protein were transferred to an NMR tube. Following the preparation of NMR samples a 1D-spectrum was recorded at 298 K on a Varian 750 MHz spectrometer using a cold probe.For 1D-NMR measurements, 50 µl D405GDGAKTLAIHVKALTADSIRITWKA TLPASSFRLSWLRLGHSPAVGSITETLVQGDKTEYLLTALEPKSTYIICMVTMETSNAYVA DETPVCAKAETADSYGPT508, FLRT2; 420GPISERIQLSIHFVNDTSIQVSWLSLFTVMAYK LTWVKMGHSLVGGIVQERIVSGEKQHLSLVNLEPRSTYRICLVPLDAFNYRAVEDTICSE ATTHASYL519 and FLRT3; 404GSPSRKTITITVKSVTSDTIHISWKLALPMTALRLSWLKL GHSPAFGSITETIVTGERSEYLVTALEPDSPYKVCMVPMETSNLYLFDETPVCIETETAPL RM506) were input to the I-TASSER server . The FGFR1 and IgG1 proteins were immobilized by an amine coupling kit (BIAcore) with flow rate 5 µl/min. FLRT1, -2 and -3 FnIII domains in PBS (pH 7.4) were injected at 20–30 µl/min. The binding curves were analyzed by BIAevaluation version 4.1 software. A steady state affinity analysis was used to estimate Kd.Binding studies were performed using a BIAcore 2000 instrument (BIAcore) at 25 °C using 10-mM PBS (pH 7.4) as running buffer. A CM4 sensor chip was immobilized with approximately 1300 RU of FGFR1β (IIIc)/Fc chimera  and 750 RU recombinant human IgGFLRT FnIII gene were transformed into E.coli strain BL21 (DE3) or the rare codon harboring Rosetta (DE3), which were then used as expression hosts of recombinant FnIII domains for all three FLRT domains. As standard conditions for protein expression proteins, the strains harboring the plasmids were cultured at 37 °C until an OD600 of 0.6 was reached. At this density the cultures were induced with IPTG, and temperature was adjusted for expression. However, no obvious overexpression could be visualized for any of the constructs. Although several expression parameters  were tested, the non-codon optimized genes cloned in to the pET-DEST42 vector gave no (FLRT2 FnIII) or little (FLRT1 FnIII and FRLT3 FnIII) protein expression. Furthermore, the various changes in expression conditions did not result in soluble protein, and the FnIII domains were always expressed as inclusion bodies.Purified pET-DEST42 destination vectors containing each relevant non-codon optimized Since attempts to express the recombinant proteins from non-codon optimized constructs had limited success, codon optimized genes were designed and purchased from GenScript . The genes were synthesized and cloned into the pET-30-a(+), a vector that like pET-DEST42 contains the bacteriophage T7 polymerase promoter . However600 = 0.6 where 1 mM IPTG was added to induce recombinant protein expression. The culture was allowed to express overnight.In contrast to the former expression experiments with the non-codon optimized constructs, the codon optimized constructs were expressed with higher yields. The proteins were still expressed as inclusion bodies as seen from small-scale test expressions. Thus, refolding was still required. The final protocol used for expression of inclusion bodies was to grow the BL21 (DE3) culture at 37 °C to an ODFrom the non-optimized gene constructs it was only possible to express the FnIII domains from FLRT1 and FLRT3, but despite very low yields, a purification procedure could be established. Since the recombinant proteins were localized in inclusion bodies, the bacteria pellets were solubilized in denaturation buffer containing 8 M urea followed by immobilization and refolding on-column using a Ni-NTA column, and by decreasing the concentration of urea to 0 M. After washing, proteins were eluted and further purified using SEC see . From a For all three FnIII domains, MALDI-TOF-MS analysis was performed to ensure correct recombinant protein expression as well as pure sample see . MethionFor FLRT3-FnIII MALDI-TOF we observed 1 peak (12232.2 Da) corresponding to the loss of the N-terminal Met. It has previously been shown that, when a small amino acid like glycine is following the N-terminal Met, Met is likely to be cleaved off . For FLR1H-NMR, of FLRT1-FnIII . Of this fraction 30.2% is estimated to be anti3 (right-twisted) β-sheet, which is 6–7% more than for the FLRT1 and FLRT3 FnIII domains  where a strong CD signal was observed (see main see . The domains see . A sequeains see . In the C-score). C-scores should be in the range between −5 and 2, with more positive values being the better and C-scores over −1.5 indicating usually a correct fold. The best models all had C-scores better than −0.5 see With few exceptions , FnIII din vivo.Several structures of FnIII domains have been solved within the last decades, and despite high tertiary structure similarities they tend to share low sequence identity. This is also evident from the I-TASSER structure prediction where sequence identities with the used templates range from 12.6 to 23.2%. All top solutions had sequence coverage from 91.3 to 94.1%. The tertiary structure of the FnIII domains consist of a β-sheet built up by B, C, E and F β-strands, and a β-sheet composed of A, C’ and G β-strands. A hydrophobic core stabilizes the domain. The topology of the FnIII domain has previously been shown to have conserved Trp residues as well as a hydrophobic pattern building up the hydrophobic core. When inspecting the sequences of the FLRTs FnIII domains it can be seen the also contain this hydrophobic pattern see . Taken tXenopus FLRT3 FnIII domain has been shown in vivo by truncation experiments to interact with FGFR1 (in vitro. Surface plasmon resonance (SPR) analysis with FGFR1β immobilized on the chip, and FLRT FnIII domains in solution demonstrated the expected binding between FGFR1β and FLRT FnIII domains (see Kd’s for the interaction between FGFR1β and the FnIII domains. The Kds are all in the µM range (see Kd values obtained for other FGFR-FnIII domains interactions (Kds should only be taken as indicative as the measurements were only performed twice. Interestingly, although the FLRT2 FnIII domain shows lower affinity than the other two, the Kd is in a similar range suggesting that the domain is at least partly correctly folded.Since the th FGFR1 , we trieains see . On the ains see . By steaange see , which cractions . The Kdsin vitro bind FGFR1b with Kd values in the µM range. Binding of FLRT proteins to FGFRs have been reported to include both extracellular and intracellular interactions (in vitro. We speculate that differences in the primary, secondary or tertiary structures of FLRT FnIII domains might contribute to different binding affinities between different members of FLRT and FGFR families, hence ensuring variability in the biological responses to FLRT-FGFR interactions.Here we present a protocol for expression and on-column refolding of FLRT FnIII domains. It can be concluded that codon optimization had significant effects on the expression levels, when compared to non-codon optimized constructs. Moreover, it can be concluded that on-column refolding of FLRTs-FnIII domains can be performed successfully as seen from CD experiments and NMR studies, but also that protease inhibitors should be included in future studies, since the domains are prone to cleavage. Also, we concluded from a combination of modelling and stability studies that a Cys–Cys bridge between the F and G strands might be necessary to stabilize the FnIII domains. Finally, we show that all three recombinant human FLRT FnIII domains ractions , but our10.7717/peerj.3550/supp-1Supplemental Information 1Click here for additional data file.


C60 is an excellent nanofiller for PPS, and 2 wt% PPS/C60 composite displayed the optimal conductivity which achieved 1.67 × 10−2 S/cm. However, when C60 concentration reached 2 wt%, the breaking strength and tensile modulus of PPS/C60 fiber achieved maximum 290 MPa and 605 MPa, and those values were 7.72 and 11.2 times as that of pure PPS. The excellent conductive and mechanical properties of PPS/C60 were attributed to the heterogeneous nucleation of C60 during PPS crystallization, formation of a large number of covalent bond by main C60-thiol adducts and minor C60-ArCl alkylation between C60 outer surface and PPS matrix. At same time, PPS/C60 thermal properties were also investigated.Electrical conductive poly(phenylene sulfide) (PPS)/fullerene (C It has been shown that the incorporating fullerene or its derivatives into a polymer matrix could significantly improve its photovoltaic, electromechanical and thermomechanical properties5. The literature already identified several techniques to blend fullerene with polymers3, and the properties of fullerene/polymer composites are closely related to the morphologies of the polymers formed during melt or solution processing4. Chemical functionalization of fullerenes is often performed to increase the miscibility of fullerenes with host polymers6, but the chemical functionalization method makes fullerenes lost certain original precious properties, which resulted in chemical attachment of fullerenes to a polymer is not always the most effective method for getting high performance composite materials, despite a number of fullerene-attached polymers have been successfully synthesized in the past7.Fullerene/polymer composites have recently attracted considerable attention from the materials research community10. To improve the performance of PPS, the nanofiller-reinforced composites require homogenous filler dispersion and good interfacial adhesion with the host matrix. Attaching functional groups onto the filler surface has been proven to be an effective approach to prepare polymer composites, and another effective method is wrapping fillers by organic molecules to provide the π-π stacking interactions between PPS matrix and nanofiller sidewalls. The conventional fillers included single-walled carbon nanotubes (SWCNTs)12, inorganic fullerene-like tungsten disulfide (IF-WS2)13, SWCNT-IF-WS216, functionalized SWCNTs and MWCNTs20, nano particles of TiO2, ZnO, CuO and SiC21, nanoscale alumina particles22, graphite23, glass fiber24, metal inorganic salt25, nano-SiOx26, carbon and fiber27et al. However, it has been recently shown that the properties of PPS based composites hardly increase at low nanofiller loadings (up to 1 wt%)29. Because of van der Waals attraction between C60 and their large surface area, C60 tend to form agglomerates during mixing with PPS by melt blending. Therefore, it is difficult to use the conventional method to disperse C60 in the PPS matrix.PPS is widely used in practice due to its excellent properties, such as chemical resistance, low coefficient of friction, mechanical behaviors, dimensional stability and electrical property60 was selected as fillers to improve PPS electrical conductivity and mechanical property, and the influence of the 1-chloronaphthalene solution mixing method and subsequent melt process on filler dispersion, C60 interfacial adhesion with PPS matrix and composite properties were investigated to correlate the microscopic structure with macroscopic properties.Herein, C60 powder (>99.9 wt/wt purity) was purchased from Puyang Yongxin Fullerene Co., Ltd, and used as received. PPS  was synthesized from Na2S and 1,4-dichlorobenzene in N-methylpyrrole, and the end groups of synthesized PPS contained –SH and -PhCl. 1-chlornaphthalene  was purchased from J& K Chemical Co., Ltd and purified by distillation under reduced pressure before use.C60 ranging from 0.5 to 10 wt% were loaded into PPS/1-chloronaphthalene solution at 205 °C under mechanical agitation. PPS/C60 composites with 0.5 to 10 wt% nanofiller were obtained after removing 1-chloronaphthalene under vacuum condition. And then PPS and PPS/C60 composite fibers with diameters of 45~85 µm were prepared by melt spinning technology at 315 °C.PPS (5 g) was dissolved in 100 ml of 1-chloronaphthalene at 205 °C under nitrogen atmosphere. After that, a certain amount of CTm), heat of melting (ΔHm), peak crystallization temperature (Hc) were obtained. The degree of crystallinity  spectra were obtained using Bruker IFS66 at room temperature. Thermo gravimetric analyzer (TGA) analysis was carried out on a NETZSCH STA 409 TG analyzer, and the rate of temperature increase was at 10 °C/min. X-ray diffraction (XRD) data were obtained using an Elmer PHI-5600 instrument using a Mg Kα line as a radiation source and a D8 discover. The morphology of composites was characterized by a field emission scanning electron microscopy  and transmission electron micrographs . Differential scanning calorimetry (DSC) analysis was performed on a Perkin Elemer DSC-7 under nitrogen condition, and samples placed in aluminium pans were melted at 320 °C and kept at this temperature for 5 min to erase their thermal history. Subsequently, they were cooled from the melt to room temperature and then heated again up to 320 °C at a scan rate of 10 °C/min. From the DSC heating and cooling traces, peak melting temperature  was determined by the following equation:Fiber diameter data were obtained using a KEYENCE VHX-1000 microscope at room temperature. The breaking strength  and a digital microvoltmeter (Model DMV-001). The powder sample was filled into a test slot, then applying a pressure about 18 MPa. The electrical resistivity was calculated from the following equation:60 and PPS were dissolved in 1-chloronaphthalene, 1-chlornaphthalene evaporated under vacuum condition to obtain PPS/C60 composites, and the dispersion and alignment of C60 within the PPS matrix were characterized by TEM , C60 formed agglomerates inside the PPS matrix. TEM observation showed that C60 were wrapped in PPS or covered by PPS layer and the heterogeneous dispersed bright dots with dimensions from 150–350 nm in 5 wt% and 10 wt% PPS/C60 composites were detected, indicating good adhesion between C60 and PPS.After C60 agglomerates were detected in 0.5 wt%, 1 wt% and 2 wt% PPS/C60 composites, due to C60 nanofillers further disaggregation within the polymer matrix during PPS/C60 melt-process. C60 nanofillers were randomly dispersed in the PPS matrix, occurring irregular crystal with dimensions from 20–50 nm when the content of C60 is less than 2 wt%. C60 nanofillers are difficult to disperse uniformly in PPS melt, therefore PPS can not well reinforced by C60 nanofillers in melt blending technology. However, C60 aggregates can be well dispersed in PPS matrix by solution blending method, in which the solvent plays a dual role in dispersing C60 and preventing C60 agglomeration. The heterogeneous dispersed bright dots with dimensions from 80–350 nm in 5 wt% and 10 wt% PPS/C60 composites were detected, which was attributed to C60 agglomerates. Similar results can also be found through the cross-sectional SEM images of the composites in Fig. 60, 2 wt% C60 particles can be uniformly dispersed in PPS matrix, but further addition of 5 wt% C60 would cause the agglomeration of C60, as can be seen in Fig. Figure 2 A shows the SEM micrograph of PPS as a blank example, and the SEM images of Fig. 60 composites were displayed in Fig. 30. And C60 shows characteristic peaks at 2θ = 10.9°, 17.2°, 20.8°, 21.8°, 28.2°, 30.8° and 32.7° arising from the (110), (220), (310), (220), (330), (420) and (330) crystalline planes of the orthorhombic unit cell, respectively31. The C60 agglomerate peaks were hardly visible in the diffractograms of 0.5–2wt% composites, which suggested that C60 was well dispersed in PPS matrix. After 5 wt% C60 was introduced into PPS, the serious C60 agglomerate was clearly observed, and the crystal characteristic peaks of C60 shifted to lower 2θ values bacause C60 aggregates result in PPS lattice distortion32. Moreover, composite peaks become broader with reduced intensity, indicating the structural order decline which induced by the incorporation of C60. These observations are consistent with the behaviors of SWCNTs33 and MWCNTs34, where the local order of the polymer decreased after the grafting reaction.X-ray diffraction patterns of PPS/C−1 are characteristic absorption peak of C60 35. The phenyl groups of PPS exhibited the absorption peaks at 1628 and 1405 cm−1, and the two bands at 1100 and 623 cm−1 were attributed to aromatic C-S stretching vibrations. After PPS was reinforced by C60, C60 characteristic absorption peaks at 565 and 1475 cm−1 band were still observed, which demonstrated the C60 successful incorporation into PPS matrix. However, C60 band at 1218 cm−1 disappeared and a new peak at 1010 cm−1 appeared, which implied the well C60 dispersion and C60-S formation  increased from 2.22 to 2.56. Only some PPS chains could interact with C60 to lead to the longer polymer chains. However, when C60 content reached 5 wt%, 5 wt% PPS/C60 composite molecular weight declined to 3.71 × 104 g·mol−1. The deviation could be attributed to much more the low molecular weight C60 loading. A similar result was previously reported by Peng, K. J. & Liu, Y. L.41. HTGPC characterized results suggested some covalent bond formation between PPS and C60 through the C60-thiol adducts and C60-ArCl alkylation.In order to testify the existence of chain-extension reaction through the end-group reaction between PPS and CPC Table . As C60 60 had less influence on PPS crystallization temperature (Tc) increase  at 502 °C and exhibited the maximum weight loss (Tmax) rate at 541.3 °C. At 800 °C, the residual mass was about 52.2% of the initial weight. Clearly, the addition of 0.5–2 wt% C60 filler led to an improvement in the thermal stability of PPS matrix, and a maximum Ti increase (about 7 °C) was obtained at 2.0 wt% filler loading, and Tmax increment for 2.0 wt% PPS/C60 was 5.5 °C. However, the significantly decline of Ti and Tmax was detected at 10 wt% C60 loading, but the residual mass at 800 °C increased to 60.39% of the initial weight. Such results should be attributed to different factors. Firstly, C60 fillers are better dispersed within PPS matrix, which restricts chain mobility or diffusion to slow down the decomposition process11. Secondly, the covalent anchoring of PPS to C60 leads to a strong enhancement in the thermal conductivity that facilitates heat dissipation within the composite43. The reason for the thermal stability decline at 5 and 10 wt% C60 loading might be attributed to the appearance of C60 agglomerates.The TG curves for the pure PPS matrix and composites under inert atmospheres were shown in Fig. 60. The mechanical behavior of PPS/C60 fibers was investigated by single fiber strength tester technique which provides additional information about filler-matrix and filler-filler interactions. Figure 60 fiber. The results of mechanical property study indicated that the concentration of C60 had a greater influence on the mechanical performance of composites. As C60 nanofiller content increase, the breaking strength and tensile modulus of composites firstly increased and then decreased. When C60 concentration reached 2 wt%, the breaking strength and tensile modulus of composites achieved maximum 290 MPa and 605 MPa, and those value were 7.72 and 11.2 times as that of pure PPS, respectively. The breaking elongation of PPS/C60 composites always decreased with increasing C60 content , which limits its use in self-health monitoring, electro-actuation, etc32. Herein, C60 was used as conductive fillers to improve PPS electrical conductivity and the electrical performance of PPS/C60 composites were compared (Fig. 60 nanofiller content increase, the electrical conductivity of PPS/C60 composites firstly increased and then decreased. When C60 concentration reached 2 wt%, the electrical conductivity of composites achieved maximum 1.67 × 10−2S/cm, much higher than the value of pure PPS. The excellent electrical conductivity of 2 wt% PPS/C60 composites was attributed to the well dispersed C60 fillers, and the maximum conductive networks might be formed in the composite at appropriate C60 content because of the conductive network formation44. C60 fillers are well dispersed in the PPS matrix owing to the covalent bond formation by main C60-thiol adducts and mino C60-ArCl alkylation between C60 surface and PPS. However, the excessive addition of C60 nanofillers caused a significant reduction of electrical conductivity, e.g., the electrical conductivity of 10 wt% C60 composite declined to 3.79 × 10−3S/cm, due to the phenomenon of C60 aggregation.PPS is an insulating material (σ 10red Fig. . As C60 60 had been prepared by solution co-blending method because solvent promotes the tangled PPS molecular chains creeping and stretching. The electrical conductivity value of 2 wt% achieved the maximum, and the excellent electrical conductivity of 2 wt% PPS/C60 composites was mainly attributed to the covalent bond formation by main C60-thiol adducts and minor C60-ArCl alkylation between C60 surface and PPS, but C60 aggregation reduced composite electrical conductivity. Furthermore, 2 wt% C60 could effectively increase PPS crystalli zation temperature, thermal stability and mechanical performance. However, the excessive C60 loading reduced the PPS crystallization degree and caused C60 re-aggregation, which led to poorer mechanical performance of PPS/C60 composite.PPS composites with well-dispersed C


The assessment of the indoor air quality was performed taking into consideration the pollution of the atmospheric air (outdoor). I/O ratios (indoor/outdoor concentration) for each investigated parameter were also calculated. Twenty-four-hour samples of PM2.5, SO2, and NO2 were collected during spring in two sites in southern Poland (Silesia), representing urban and rural areas. Indoor samples were taken in naturally ventilated kindergartens. At the same time, in the vicinity of the kindergarten buildings, the collection of outdoor samples of PM2.5, SO2, and NO2 was carried out. The content of BaP and the sum of 15 studied PAHs was determined in each 24-h sample of PM2.5 (indoor and outdoor). In the urban site, statistically lower concentrations of SO2 and NO2 were detected indoors compared to outdoors, whereas in the rural site, such a relationship was observed only for NO2. No statistically significant differences in the concentrations of PM2.5, PM2.5-bound BaP, and Σ15 PAHs in kindergartens (indoor) versus atmospheric (outdoor) air in the two studied areas were identified. Mutagenic effect of indoor PM2.5 samples was twice as low as in outdoor samples. The I/O ratios indicated that all studied air pollutants in the urban kindergarten originated from the ambient air. In the rural site concentrations of SO2, PM2.5 and BaP in the kindergarten were influenced by internal sources .More than 80% of people living in urban areas who monitor air pollution are exposed to air quality levels that exceed limits defined by the World Health Organization (WHO). Although all regions of the world are affected, populations in low-income cities are the most impacted. According to average annual levels of fine particulate matter  presented in the urban air quality database issued by WHO in 2016, as many as 33 Polish cities are among the 50 most polluted cities in the European Union (EU), with Silesian cities topping the list. The aim of this study was to characterize the indoor air quality in Silesian kindergartens based on the concentrations of gaseous compounds (SOThe online version of this article (doi:10.1007/s11869-017-0505-9) contains supplementary material, which is available to authorized users. According to the World Health Organization (WHO) atmospheric air (outdoor) pollution contributed to 3 million premature deaths worldwide per year in 2012 due to exposure to particulate matter (PM)10 (fraction of atmospheric particles with aerodynamic diameter not greater than 10), which causes cardiovascular and respiratory disease, and cancers WHO . Many epSalmonella/microsome assay was applied, which allowed detecting a broad range of chemical substances causing genetic damage (Mortelmans and Zeiger A commonly identified congener of PAHs in the atmospheric air is benzo(a)pyrene (BaP), which in 2012 was classified to the highly genotoxic compounds. According to the International Agency for Research on Cancer (IARC), it belongs to group 1—carcinogenic to humans IARC . It is kPeople spend most of their time in indoor environments (88.9%), with limited time spent outdoors (5.8%) or in vehicles (5.36%) , nitrogen oxides (NOx), ozone (O3), carbon mon- and dioxide (CO and CO2), volatile organic compounds (VOCs), bioaerosols, PM, and PAHs, and selected heavy metals, including mercury  and rural (Złoty Potok) areas of Silesia, Poland, taking into consideration the air pollution by PM2.5, 15 PM2.5-bound PAHs, SO2, NO2) and PM2.5 were collected in two sites during the spring season (17 March–09 April 2010 in Złoty Potok and 10 April–3 May 2010 in Dąbrowa Górnicza). The first site represents an urban-industrial area in Dąbrowa Górnicza, and the second one, a rural region in Złoty Potok. The sampling site in Dąbrowa Górnicza  may be considered as an urban background site according to Directive 2008/50/EC. The sampling site in Złoty Potok  represented a regional background site (Directive 2008/50/EC). The location of the described sampling points is presented in the Supplementary Material (SMThe studied area was located in the southern part of Poland (Silesia). Indoor and outdoor samples of gaseous pollutants  in Katowice(http://www.katowice.pios.gov.pl/). Sequential Dichotomous Partisol-Plus (Model 2025) was used for outdoor 24-h PM2.5 sample collection (air volume 1 m3/h). The 24-h PM2.5 samples of indoor particles were taken with a PNS-15 sampler . All samples of PM2.5 were collected on quartz fiber filters (QMA: 47 mm in diameters). Concentrations of PM2.5 samples were determined according to the reference method for gravimetric measurements (EN 12341:2014). More detailed descriptions of the localities and sampling procedures for PM2.5 as well as atmospheric conditions are published in Błaszczyk et al.  and 0.5 m3 per 24 h for the glass frit (NO2). Volume of air was measured with a marked gas meter. The operation of the aspirator was based on a weekly exchange of sintered glass and solutions in sorbent scrubbers. The values of outdoor 24-h average concentrations of SO2 and NO2 in the two studied sites were taken from the urban and RIEP (http://www.katowice.pios.gov.pl/). In this study, in total forty 24-h samples for each compound  were collected . For each selected pollutant, in each location, ten air samples in kindergartens (indoor samples) and ten samples of atmospheric air (outdoor samples) were collected.Indoor samples for determination of SO2 takes place in 0.3% H2O2 solution. Sulfur dioxide was oxidized to sulfate ion, which was determined by ion chromatography using DX100 ion chromatograph  with AO4S column. Daily concentrations of SO2 absorbed in the glass scrubber were calculated  at a wavelength of 540 nm. Then, the daily concentration of NOThe sum of 15 PM2.5-bound PAHs was determined based on the concentrations of the following compounds: naphthalene (NP), acenaphthene (ACE), fluorene (FL), phenanthrene (PHE), anthracene (ANT), fluoranthene (FLA), pyrene (PYR), benz(a)anthracene (BaA), chrysene (CHR), benzo(b)fluoranthene (BbF), benzo(k)fluoranthene (BkF), benzo(a)pyrene (BaP), dibenzanthracene (DahA), benzoperylene (BghiP), and indenopyrene (IcdP), which were identified according to the standard methodology (EN 15549:2008). Each sample of PM2.5 deposited on the filter was extracted in an accelerated solvent extractor  with dichloromethane (DCM) as an extraction solvent. Analysis of the extract was carried out using high-performance liquid chromatography with a fluorescence detector . PAH resolution was performed using RP C18 column  and gradient elution: water-methanol. Qualitative and quantitative assessment of PAHs was made using a fluorescence detector (FLD), with time-programmable excitation and emission wavelength changes: for NP, ACE, and FL—220/330 nm; for PHE, ANT, FLA, PYR, BaA, and CHR—260/420 nm; for BbF, BkF, BaP, DahA, and BghiP—290/450 nm; and for IcdP—248/500 nm. A detailed description of the PAH analysis containing extraction and concentration procedure as well as calibration parameters and quality control description was presented in Błaszczyk et al. .Salmonella assay. Dry aerosol extracts of PM2.5 were dissolved in dimethyl sulfoxide (DMSO) in a volume corresponding to the highest volume of the air passing through the filter.Dichloromethane extracts of PM2.5 samples, remaining after the determination of PAHs, were used to investigate the mutagenic activity. In addition, dichloromethane extracts from clean filters were prepared and used as a blank sample. They were stored at −20 °C unless all extracts were tested using the Salmonella/microsome assay  and its derivative strain YG1024 (O-acetyltransferase overproducing), obtained from Prof. Takehiko Nohmi, the National Institute of Health in Tokyo, were used, with and without metabolic activation (± S9 fraction). The choice of the TA98 and YG1024 strains was based on the sensitivity of these strains to certain chemical compounds responsible for mutagenic effects caused by particulate matter. PAHs belong to chemical pollutants which can induce frameshift mutations rather than base-pair substitutions in bacterial strains of Salmonella. Results of the previously carried out studies indicate that in Poland during winter season, the most suitable strain used for testing PM mutagenicity is TA98 and its YG derivatives   0.6 ml of 0.2 M phosphate buffer (pH 7.4) containing 0.4 M MgCl3 of air. In the case of indoor samples, doses corresponding to 2, 4, and 8 m3 were used. The tested doses were added to 2.5 ml of sterilized top agar (0.6% agar and 0.5% NaCl containing 0.5 mM of histidine and 0.5 mM of biotin) and poured onto minimal glucose agar plates . The plates were then incubated at + 37 °C for 48 h (TA98) and 72 h (YG1024), and then histidine-independent revertant colonies were counted using a colony counter. In parallel, the number of spontaneous revertants and the number of revertants after adding the blank samples with and without S9 fraction were analyzed. Apart from that, levels of revertants induced by direct and indirect mutagen were tested, which for the TA98 strain were nitrocholino-4-N-oxide (NQNO) and BaP, and for the strain YG1024: 1-nitropirene (1-NP) and 2-aminofluorene (2-AF).The mutagenic effect of PM2.5 collected outdoor was tested at doses corresponding to 1, 2, and 4 mThe obtained results were analyzed using the package Statistica for Windows, version 10.3 of the sampled air was computed (rev/m3). The sample was considered to be mutagenic when the number of revertants was twice as high as that of the corresponding negative control sample and when a significantly positive dose-response relationship was observed.The mutagenic effect of PM2.5 organic extracts was calculated using the number of revertants as a dependent variable and dose of extract as an independent variable. To select the doses with linear dose-response relationship, a point-rejection method was applied. The assessment of data fitting to the model was made by the least squares method involving a regression analysis and analysis of variance instead of a maximum likelihood method proposed by Bernstein et al. , with thU test was used to compare indoor-outdoor differences, and indoor-outdoor correlations were described using Spearman’s correlation coefficients (rs). The I/O ratio of pollutant concentration was used to justify the presence of indoor sources (I/O > 1) or infiltration of the ambient air (I/O ≤ 1). Statistical significance was assumed at p ≤ 0.05.Summary statistics  was determined for each of the pollutants. Since the concentrations of the measured pollutants were not normally distributed, a nonparametric Mann-Whitney 2 and NO2 in Dąbrowa Górnicza and Złoty Potok.Guidelines of the European Parliament for atmospheric air quality  than its indoor concentrations.The indoor 24-h concentrations of SO2 are established only for atmospheric air and are 125 μg/m3 for 24-h exposure  and 20 μg/m3 for annual exposure  and from 2.8 to 10.2 μg/m3 (mean 5.9 μg/m3), respectively, whereas in the suburban area, from 1.4 to 3.5 μg/m3 (mean 2.4 μg/m3) and from 4.2 to 15.4 μg/m3 (mean 7.7 μg/m3), respectively  and 6.7 μg/m3 (active sampling), whereas the outdoor concentration of this pollutant was definitely higher (39.6 μg/m3) than in our study  were higher than in Belgium. On the other hand, in Turkey, the mineral industry and the oil and brown coal energy grid are much better developed than in Poland. Therefore, the outdoor 24-h concentrations of SO2 in the urbanized region of Turkey were much higher than in the Silesian region of Poland.Most of sulfur in fossil fuel is converted into SO2 were significantly higher than the indoor ones. In Dąbrowa Górnicza, 24-h I/O ratios varied from 0.39 to 0.98 with the median of 0.67. The I/O ratios of 24-h indoor and outdoor concentrations of SO2 in Złoty Potok varied from 0.39 to 2.52 with the median value of 1.28 2SO2) into the rooms. SO2 is chemically short-lived in virtually all types of atmospheres  correlations between the indoor and outdoor 24-h concentrations of SO2 were confirmed only in the kindergarten in Dąbrowa Górnicza .The indoor 24-h concentrations of NO2 as in Złoty Potok were recorded in the suburban areas of Stockholm  and Złoty Potok (p = 0.016)—Table 2 to 24-h outdoor concentrations in Dąbrowa Górnicza varied from 0.14 to 0.49 with the median value of 0.26. The 24-h I/O ratio of NO2 in Złoty Potok varied from 0.47 to 1.28 with the median value of 0.62  between the indoor and outdoor 24-h concentrations of NO2 was also detected  in Dąbrowa Górnicza and from 16.0 to 69.5 μg/m3 (median 30.6 μg/m3) in Złoty Potok . In the same period, the lowest outdoor concentration of PM2.5 (21.88 μg/m3) was detected in the air of the nursery school playgrounds located in the urban traffic area, whereas the highest level (88.30 μg/m3) was observed at the playground located in the rural site , indicating that the indoor PM2.5 in the kindergarten in Dąbrowa Górnicza was impacted mainly by the outdoor PM2.5, whereas in the kindergarten in Złoty Potok, such a relationship between the indoor and outdoor 24-h concentrations of PM2.5 was not observed , and the 24-h values of the I/O ratio varied from 0.58 to 3.76 with the median value of 1.66  in Dąbrowa Górnicza, which suggested that PM2.5-bound BaP and Σ15PAHs in the urban kindergarten came mainly from the outdoor environment. In the kindergarten in Złoty Potok, the lack of statistically significant correlations between the 24-h indoor and outdoor concentrations of both BaP and Σ15PAHs confirmed the earlier assumption of the existence of internal sources of air pollutants  in comparison to the urbanized city (Dąbrowa Górnicza). A different situation was observed in the case of Σ15PAHs, which are influenced not only by emissions from fossil fuel combustion, but mainly from transport sources. Therefore, during spring season in Dąbrowa Górnicza, higher concentrations of Σ15PAHs were detected in comparison to Złoty Potok. In both areas, the differences between the indoor and outdoor 24-h concentrations of BaP and Σ15PAHs were not statistically significant, and no differences between the examined areas were observed, both in the case of the indoor and outdoor air. Incidentally, high 24-h concentration of BaP was detected in samples collected in the kindergarten in Złoty Potok. The 24-h I/O ratios of indoor concentrations of BaP in Dąbrowa Górnicza varied from 0.67 to 1.20 with the median of 0.89. Indoor 24-h concentrations of BaP in Złoty Potok were higher than the outdoor ones . Cytotoxic effects were not detected for any of the analyzed samples.Table 3 with the median of 21.2 rev/m3 in Dąbrowa Górnicza and from 18.6 to 23.1 rev/m3 with the median of 19.6 rev/m3 in Złoty Potok. In the variant with metabolic activation, the 24-h mutagenic effect ranged from 27.0 to 28.4 rev/m3 with the median of 28.3 rev/m3 in Dąbrowa Górnicza and from 27.8 to 30.4 rev/m3 with the median of 27.8 rev/m3 in Złoty Potok (Table The mutagenic effect of 24-h indoor samples of PM2.5 tested using the TA98 strain without metabolic activation ranged from 21.0 to 21.9 rev/m3) , the mutagenic effect of 24-h PM2.5 samples ranged from 33.1 to 66.6 rev/m3 with the median of 56.8 rev/m3 for Dąbrowa Górnicza and from 33.5 to 62.7 rev/m3 with the median of 45.4 rev/m3 for Złoty Potok  ranged from 36.6 to 51.8 rev/m3) was similar to the value obtained in our study, whereas the indirect effect appeared to be twice as high (99.0 rev/m3). Then, the average concentration of PM10-bound BaP was much higher (28.8 ng/m3) than in the present study—Table 3, respectively) and after metabolic activation   at 21 measuring stations of the Regional Sanitary-Epidemiological Station in Katowice tested using the TA98 strain (49.0 rev/m000 at 213) and with the addition of S9 (35.8 rev/m3), was observed in winter in Belgium  and TA98 + S9 (< 25.0 rev/m3) was found in the study conducted during winter (2000/2001) in Teplice and Prague , the mutagenic effect of 24-h PM2.5 samples ranged from 114.0 to 246.4 rev/m3 with the median of 192.6 rev/m3 in Dąbrowa Górnicza and from 105.2 to 270.9 rev/m3 with the median of 150.6 rev/m3 in Złoty Potok  ranged from 93.1 to 175.5 rev/m3; + S9: 32.9 rev/m3) , as well as after its application (2.1 rev/m3) , in both variants  and the variant of activation (± S9). The mutagenic effect of 24-h indoor PM2.5 samples tested using both TA98 and YG1024 strains was twice as low as that of the 24-h outdoor PM2.5 samples.Salmonella assay in the presence of S9. In the previously conducted Polish studies, the increase of mutagenic activity tested by TA98, YG1021, and YG1024 strains after addiction of S9 fraction to PM10 samples collected during winter season was observed. In summer, indirect mutagenicity was similar or lower in comparison to the variant without metabolic activation  and rural (Złoty Potok) sites, was observed during the spring period (March to May). The results showed that the mutagenicity tested using the YG1024 strain was much higher than in the case of TA98 in almost all samples, both indoors and outdoors. The YG1024 strain is extremely sensitive to aromatic amines, hydroxylamines, and nitro-PAHs, as was previously described. Furthermore, in almost all samples, mutagenicity with S9 fraction was not significantly higher than without S9 fraction. These results indicate that indirect-acting mutagenic compounds such as aromatic amines may be responsible for this effect  were characterized in naturally ventilated kindergartens in urban (Dąbrowa Górnicza) and rural (Złoty Potok) areas in Silesia, Poland during spring season. The assessment of the indoor air quality was made taking into consideration the outdoor concentrations of the studied pollutants.The indoor air quality parameters  of indoor PM2.5 samples were lower than in the case of outdoor PM2.5 samples. This fact indicates that the total mutagenicity of PM2.5 fraction does not correspond to the results obtained in the chemical analysis.In the kindergarten located in Dąbrowa Górnicza, gaseous pollutants and PM2.5, BaP, and the sum of 15 PM2.5-bound PAHs mainly came from the atmospheric air. In the kindergarten in Złoty Potok, an internal source of SOChildren spend an important part of their time in enclosed space ; they are also more sensitive to air pollution than adults. We believe that the results of the current study will point out to the need for implementing a strategy to control air quality in places where children reside. Undoubtedly, no kitchen coal stoves should be used in kindergartens and schools, and the existing ones should be replaced by electrical or gas stoves. In the newly built kindergartens or schools, mechanical ventilation with an air purification system should be installed. In old buildings, frequent measurements of the efficiency of the ventilation systems should be carried out, and more attention should be paid to the appropriate and sufficient exchange of air in rooms where children are staying, e.g., frequent airing of rooms. Development and implementation of guidelines for harmful indoor air pollutants would also be recommended.ESM 1(DOC 9395 kb)