Apiaceous and Rutaceae families of plant (ARFP) products are popular foods and medicines in the world. We previously reported significant amounts of furanocoumarin bioactive such as 8-methoxypsoralen, 5-methoxypsoralen, and isopimpinellin in ARFP products. As both caffeine and furanocoumarin bioactive are metabolized by the same hepatic CYP1A1/2 isozyme in humans, caffeine/ARFP product interactions may occur after co-administration. The objectives of the present study were to study in vivo loss of caffeine metabolizing activity by comparing the pharmacokinetics of caffeine in volunteers before and after pre-treatment with an ARFP extract, study the correlation between the decrease in hepatic CYP1A2 activity and the content of furanocoumarin bioactive in ARFP extracts, characterize CYP1A2 inactivation using in vitro incubations containing 14C-caffeine, a furanocoumarin bioactive, and human liver microsomes (HLMs), and provide a mechanistic explanation for both in vivo and in vitro data using the irreversible inhibition mechanism. The study results showed pre-treatment of volunteers with four ARFP extracts increased the area-under-the-concentration-time-curve (AUC0-inf) ratio of caffeine in the plasma ranging from 1.3 to 4.3-fold compared to the untreated volunteers indicating significant caffeine metabolism inhibition. The increases in AUC0-inf ratio also were linearly related to the effect-based doses of the furanocoumarins in the ARFP extracts, a finding which indicated caffeine metabolism inhibition was related to the content of furanocoumarin bioactive in an ARFP product. In vitro incubation studies also showed individual furanocoumarin bioactive were potent inhibitors of caffeine-N-demethylation; the IC50 for 8-methoxypsoralen 5-methoxypsoralen, and isopimpinellin were 0.09, 0.13, and 0.29 µM, respectively. In addition, CYP1A2 inactivation by individual furanocoumarin bioactive was concentration- and time-dependent involving the irreversible inhibition mechanism. The proposed irreversible inhibition mechanism was investigated further using 14C-labeled 8-methoxypsoralen and HLMs. The formation of 14C-adducts due to 14C-8-MOP-derived radioactivity bound to HLMs confirmed the irreversible inhibition of CYP1A2 activity. Thus, furanocoumarin bioactive metabolism in humans would result in reactive metabolite(s) formation inactivating CYP1A2 isozyme and inhibiting caffeine metabolism. Once the CYP1A2 isozyme was deactivated, the enzymic activity could only be regained by isozyme re-synthesis which took a long time. As a result, a single oral dose of ARFP extract administered to the human volunteers 3.0 h before still was able to inhibit caffeine metabolism.Daily consumption of caffeinated beverages is considered safe but serious health consequences do happen in some individuals. The  A single oral dose of an ARFP extract administered to humans 3 h before is still able to inhibit caffeine metabolizing activity.The degree of caffeine metabolism inhibition is linearly related to an increasing effect-based dose of the furanocoumarin mixture in the ARFP extracts.Individual furanocoumarin chemicals inactivate CYP 1A2 enzyme in a time- and concentration-dependent manner which indicates involvement of the irreversible inhibition mechanism.The long inhibitory effects of ARFP on caffeine metabolism are explainable by CYP 1A2 inactivation.Caffeine  is a popinter- and intra-individual differences (The pharmacokinetics of caffeine in humans has been studied in great details . Caffeinferences .Apiaceous and Rutaceae families of plant (ARFP) products on human CYP enzymes. The ARFP products are popular foods and traditional medicines in the Middle East and Asia. In an earlier study, we screened over 29 products from the ARFP for linear furanocoumarins using high-performance liquid chromatography equipped with an ultra-violet detector (HPLC-UV), and gas chromatography with mass spectrometry , and to propose an inhibition mechanism to explain the in vivo and in vitro data.The objectives of the present study were: to investigate nine selected ARFP products for the potential to inhibit caffeine metabolism by comparing the pharmacokinetics of caffeine in humans before and after pre-treatment with an ARFP extract, to determine the relationship between the area-under-the-concentration-time-curve (AUC) ratio of caffeine and the content of furanocoumarin bioactive in an ARFP extract, to study It is important to study CYP 1A2 inactivation by ARFP products in humans. Although the CYP 1A2 isozyme accounts for just 13% of the total hepatic CYP pool , it playAmmi majus L. (A. majus) purchased from EverWilde , Angelica archangelica L. seeds (A. archangelica), A. graveolens seeds (A. graveolens S), Pimpinella anisum L. seeds (P. anisum), and Ruta graveolens L. leaves (R. graveolens) were purchased from Mountain Rose . Apium graveolens L. flakes (A. graveolens F) and Petroselinum crispum (Mill.) Fuss leaves (P. crispum) were purchased from A1SpiceWorld . Angelica pubescens Maxim. roots (A. pubescens) purchased from Spring Wind , Cnidium monnieri (L.) Cusson (C. monnieri) were purchased from Health and Wellness House .We selected nine ARFP products for the present study based on the results of an earlier study . The selThese nine ARFP products were authenticated by the suppliers and certified to be free of pesticides and preservatives. Further authentication was performed by measurement of furanocoumarin levels chromatographically in the aforementioned ARFP as seen in 2HPO4) (≥60.0), and monopotassium phosphate (KH2PO4) (≥60.0) were obtained from Anachemia . ISOP was obtained from ChromaDex  (98.3%) and Sigma-Aldrich (≥95.0%). Spectral grade dimethyl sulfoxide (DMSO) was obtained from Caledon . High-purity nitrogen gas (N2), oxygen gas (O2), and carbon monoxide (CO) gas was obtained from Praxair .Benzotriazole (99.0%), caffeine (≥99.0%), ethyl acetate (≥99.7%), 8-MOP (≥98.0%), 5-MOP (99.0%), and β-nicotinamide adenine dinucleotide phosphate reduced form (NADPH) (≥97.0%), were obtained from Sigma-Aldrich . Methanol (≥99.9%) and acetonitrile (≥99.9%) were obtained from Thermo Fisher Scientific  and Sigma-Aldrich. Acetic acid (≥99.7%), trichloroacetic acid (TCA) (≥99.0), dipotassium phosphate (KN-14C-methyl]-Caffeine (specific activity 50–60 mCi/mmol) was obtained from American Radiolabeled Chemicals . The methyl-14C labeled 8-MOP, specific activity of 40–60 mCi/mmol, was purchased from Vitrax Radiochemicals . Scintillation cocktail fluids were obtained from Perkin Elmer Life Sciences  and Amersham Biosciences . Ultrapure water was produced using a Millipore system  with a minimum resistivity of 16.0 MΩ cm at 25°C.[3′-Pooled HLMs were purchased from BD Gentest  and GIBCO .Male human volunteers, between 21 and 30 years old and living in the Metro Vancouver area, were recruited for the study. The volunteers had to meet specific eligibility criteria: not smoking, use medication, consume alcohol heavily or have any health concerns which may affect the test results. The volunteers were asked to avoid consuming caffeinated drinks and caffeine-containing foods for 12.0 h prior to and during the study period. The participants were also asked to refrain from consuming solid foods 3.0 h prior to the study. The study protocol was approved by the Simon Fraser University Office of Research Ethics with approval number 2012s0565 and registration number ISRCTN83028296.The ARFP products were extracted as per our previous study . An ARFPith furanocoumarin component in µM unit. The RPi (relative potency) is the IC50 of the ith furanocoumarin component relative to the IC50 of 8-MOP. The relative potency of a furanocoumarin bioactive represents an equally effective 8-MOP concentration for CYP 1A2 activity inhibition. The dose measure of the furanocoumarin mixture in an ARFP extract was predicted using the concentration addition model :Cmix=∑i=This study adopted a non-randomized, single-blinded, and crossover design in which each subject acted as his own control . Each vog. Exactly 200.0 µl aliquot of the saliva sample was removed and spiked with 100.0 µl of the internal standard (ISTD) benzotriazole (50.0 μg/ml). The saliva samples were extracted once with 4.0 ml ethyl acetate by vortexing for 2.0 min and then centrifuged at 4,000×g for 5.0 min. The organic layer was removed and evaporated down to dryness, using a gentle stream of N2 gas. The residues were reconstituted in 150.0 µl mobile-phase of the HPLC. Exactly 100.0 µl aliquot of the solution was injected into the HPLC-UV system.The saliva samples were thawed at room temperature, mixed by vortexing for 30 s, and centrifuged for 10 min at 4,000×Caffeine concentrations in saliva samples were determined using a modified HPLC procedure of max (time to reach peak plasma level) and Cmax (concentration of peak plasma level) were determined by visual inspection. The AUC0-inf (area under plasma concentration-time curve from zero to infinity) was determined by non-compartmental analysis using the PK Solver software , a furanocoumarin inhibitor (see concentrations in IC50Determination and Pre-incubation Time- and Furanocoumarin Concentration-dependent Inhibition of Caffeine Metabolism in vitro), NADPH (1.34 mm), and potassium phosphate buffer  in a final volume of 200.0 µl. DMSO was used to dissolve caffeine and the furanocoumarin inhibitor; less than 1% incubation volume of DMSO was found to have no significant effect on caffeine 3-N-demethyation activity. Incubation was conducted at 37°C in a metabolic incubator with a 60 cycles/min shaking rate. At the conclusion of 10 min incubation, the reaction was terminated by the addition of 10% TCA solution (50.0 µl). The incubation mixture was centrifuged at 4,000×g. An aliquot (300.0 µl) of the supernatant was applied to a 3.0 ml Sigma-Aldrich Superclean ENVI-Carbon solid phase extraction (SPE) tube . Demethylated metabolites of caffeine  were eluted from the SPE tube with 2.0 ml of water. The eluant was collected into a scintillation vial. After the addition of 15.0 ml scintillation cocktail, the radioactivity in the vial was counted in a liquid scintillation counter. The results were expressed as residual counts of the control incubation.The caffeine-In vitro caffeine-N-demethylase activity was determined with various concentrations of a furanocoumarin inhibitor: 0.01–11.12 µm for 8-MOP; 0.06–7.10 µm for 5-MOP; and 0.01–38.45 µm for ISOP. CYP 1A2 activity inhibition was plotted against log furanocoumarin concentration after normalizing the inhibition data from 0–100%. The resulting inhibition-concentration curves were fitted separately to a four-parameter logistic function to determine the IC50 values. Curve fitting was performed using the GraphPad Prism version 5.04 . The IC50 represented the concentration of the furanocoumarin chemical to evoke a half maximal inhibition of the CYP 1A2 activity.14C-caffeine (0.2 µCi). The final incubation volume was 320.0 µl. At the conclusion of a 10 min incubation, the reaction was stopped by the addition of a 10% TCA solution. The incubation mixture was extracted by a SPE tube, and the radioactivity in the eluant was counted in a liquid scintillation counter as described earlier.These studies were conducted in two steps: pre-incubation with a furanocoumarin inactivator followed by dilution and incubation to measure the caffeine metabolizing activity. In the pre-incubation step, HLMs (1.2 mg/ml) were pre-incubated with different furanocoumarin concentrations in the presence of NADPH for various time periods at 37°C. The pre-incubation times of the studies were: 0.5, 1.0, 1.5, and 2.0 min for 8-MOP and 5-MOP, and 0.5, 1.0, 2.0, and 3.0 min for ISOP. The furanocoumarin concentration ranges of the studies were: 1.07–17.14 µm for 8-MOP. 1.13–18.07 µM for 5-MOP, and 0.47–15.02 for ISOP. In the final incubation step, 32.0 µl of the pre-incubation mixture was transferred to an incubation vial containing fresh NADPH (1.0 mm), non-labeled caffeine (6.4 µm), and in vitro rate constant (kobs) was calculated using the slopes of linear regression analyses from natural logarithm (Ln) percentage remaining activity against pre-incubation time plots. The maximum inactivation rate constant (kinact) was calculated using the non-linear regression analyses of plots of the inactivator concentrations against kobs values. The inactivator concentration (KI) was extrapolated at 50% kinact value. The plotting was performed using the GraphPad Prism version 5.04 .The observed 14C-8-MOP (0.2 µCi), potassium phosphate buffer , and NADPH (1.0 mm) in a 1.34 ml final incubation volume. The incubation mixture was pre-warmed at 37°C for 5.0 min and the reaction was started with the addition of HLMs. After a 10 min incubation, the reaction was terminated by the addition of ice-cold 10% TCA solution. Unchanged 14C-8-MOP and water-soluble metabolites in the incubation were washed off repeatedly with three cycles each of methanol, acetonitrile, and acetone. Our preliminary studies indicated that the three-cycle solvent washes were sufficient to remove all unbound radioactivity from the precipitates of HLMs. The 14C bound to the protein precipitates was determined using a liquid scintillation counter after the addition of 15.0 ml scintillation cocktail. The effects of different incubation conditions on 14C-adducts formation were studied by omitting NADPH cofactor, depleting O2, adding CO or using heated HLMs in the complete incubation mixture.These studies were conducted as per t-test, two-tailed and 95% confidence interval using Microsoft Excel. The difference between two comparable data sets were considered significant when p ≤ 0.05.The data were analyzed using the Student’s paired A. majus seeds, A. archangelica roots, C. monnieri fruits or R. graveolens leaves. Caffeine concentrations in the plasma were found to increase significantly after each ARFP extract pre-treatment as seen in A. majus seeds extract significantly increased the AUC0-inf of caffeine by 432%, the Tmax from 0.8 to 2.4 h, and the Cmax by 16.6%. A. archangelica roots extract pre-treatment increased the AUC0-inf of caffeine by 232%, the Tmax from 0.8 to 1.3 h, and no change for Cmax value. If 2x weights of A. majus seeds and A. archangelica roots were used in the extraction  of caffeine was decreased significantly after pre-treating the volunteers with these plant extracts. Thus, pre-treatment of humans with one of these 4 ARFP extracts resulted in an increase in caffeine AUC0-inf with a concomitant decrease in CL, a finding which clearly showed these ARFP extracts were potent inhibitors of caffeine metabolism.A. pubescens roots, A. graveolens seeds, A. graveolens flakes, p. aniseum seeds, or p. crispum leaves. Pre-treatment of human volunteers with the remaining 5 ARFP extracts did not change the concentration-time profiles of caffeine significantly. A. graveolens seeds extract increased the AUC0-inf of caffeine by 12%, the Tmax from 0.7 to 1.2 h, but the Cmax was reduced by 8.2% but the increase in AUC0-inf was statistically insignificant compared to the untreated volunteers. The pre-treatment with A. graveolens flakes extract increased the mean Tmax of caffeine from 0.8 to 1.5 h, but had no effect on the Cmax and AUC0-inf. The pre-treatment with P. aniseum seeds extract increased the Tmax of caffeine from 0.6 to 1.1 h, reduced the Cmax by 16.9%, and had no effect on the AUC0-inf. The pre-treatment with P. crispum leaves increased the AUC0-inf of caffeine by 27.5% but the increase was statistically insignificant. P. crispum leaves pre-treatment also increased the Cmax of caffeine by 5.0% but had no effect on the Tmax. Interestingly, treatment with A. pubescens roots decreased AUC0-inf by 30.0% and Cmax by 40% but both were found not significant (p > 0.05). However, treatment with A. pubescens roots increased Tmax from 2.4 to 3.1 h. In contrast, the CL of caffeine in these studies was decreased after ARFP pre-treatment. Thus, none of the pharmacokinetic parameters, with the exception of CL for A. pubescens roots treatment , was changed significantly after the volunteers were pre-treated with these 5 ARFP extracts.R2 = 0.94). Thus, A. majus seeds, A. archangelica roots, C. monnieri fruits, and R. graveolens leaves, which had large effect-based dose measures and were potent caffeine metabolism inhibitors, were located at the upper part of the straight line. The remaining ARFP products which were less potent inhibitors and had small effect-based dose measures, were located at the lower part of the straight line. In other words, the extent of CYP 1A2 inhibition is determined by the additive combination effects of just 3 furanocoumarin bioactive  in the ARFP extract.N-demethylase activity was significantly inhibited when 8-MOP, 5-MOP, or ISOP was added to the incubation. Also, caffeine-N-demethylase activity appeared to decrease with an increasing furanocoumarin concentration in the incubation. 10 concentration curves of 8-MOP, 5-MOP, and ISOP on caffeine metabolism. The inhibition-concentration curves were parallel to one another, indicating the linear furanocoumarins and caffeine were bound to similar metabolic sites on the CYP 1A2 isozyme. The IC50 of 8-MOP, 5-MOP, and ISOP on caffeine metabolism were determined to be 0.09 ± 0.05, 0.13 ± 0.11, and 0.29 ± 0.22 µm (mean ± standard deviation (SD)), respectively. Thus, the inhibition potency of the furanocoumarin bioactive decreased in the order of 8-MOP > 5-MOP > ISOP. The IC50 values were used to calculate a relative potency  for the furanocoumarin bioactive in the ARFP extracts. The inhibitory equivalence factor for 8-MOP, 5-MOP, and ISOP were 1.00, 0.69, and 0.31, respectively, assuming the 8-MOP value was equal to 1.00. The effect-based dose for the furanocoumarin mixture in an ARFP extract was predicted using the concentration addition model. Little or no caffeine was metabolized by the HLMs if NADPH were omitted from the incubation (data not shown). Caffeine-obs and the concentration of individual furanocoumarin in these studies. The KI values for 8-MOP, 5-MOP, and ISOP were estimated at 0.78 ± 0.32, 3.73 ± 3.66, and 4.48 ± 0.56 µm, respectively. The estimated kinact values were 0.17 ± 0.01, 0.35 ± 0.12, and 0.65 ± 0.03 min−1, respectively.14C-8-MOP metabolism studies. 14C-8-MOP biotransformation in NADPH-fortified HLMs resulted in the formation of 14C-adducts which were studied using different incubation conditions. 14C-adducts were formed when boiled HLMs were used or NADPH and O2 were omitted in the incubation. An increase of the CO concentration in the incubation decreased the amounts of 14C-adducts formed. These results indicated that 14C-8-MOP reactive metabolite(s) was formed and bound irreversibly to the HLMs during 14C-8-MOP biotransformation.The proposed irreversible inhibition mechanism was studied further with A. majus, A. archangelica, C. monnieri, and R. graveolens, are the most potent inhibitors among the tested products since they can alter the pharmacokinetics of caffeine in humans by increasing the AUC ratio of caffeine with a concomitant decrease in CL  is observed in the untreated or control volunteers in our study , 4. The kinetics . As suchkinetics . This iskinetics , 4). In kinetics . This isP. crispum . The long inhibitory effects of ARFP products are consistent with the following studies: Firstly  patients is reported to range from 0.6 ± 0.2 h to 2.3 ± 0.9 h  and C. monnieri herbal products are shown to be 1.3 ± 0.3 h pyrene hydroxylase activities are about 25 µm in HLMs  the ectively . Also, 8ectively , 3) the ectively , and 4) ectively  have pro2 level in the incubation results in decreased formation of 14C-adducts (14C-8-MOP biotransformation is mediated by an oxidation reaction of HLMs and 14C-adducts are the by-products of 14C-8-MOP metabolism. We hypothesize the formation of 14C-adducts to involve the following steps: the furan ring of 14C-8-MOP is first oxidized by CYP 1A2 to an electrophilic epoxide intermediate(s) which then binds irreversibly to the CYP 1A2-substrate complex and inactivates the CYP 1A2 isozyme itself. Our hypothesis is supported by the reports that 8-MOP is metabolized by epoxidation of the furan ring in dogs (The removal of NADPH or the reduction of O-adducts . These r in dogs , and fur in dogs . In othe in dogs . Once thPre- or co-administration of caffeine and ARFP products may result in CYP 1A2 inactivation, caffeine overdose, and serious health consequences. As such, care must be exercised when furanocoumarin-rich plant products are co-administered with drugs that are substrates of the CYP 1A2 isozyme. Our studies also support the development of regulations for proper labeling of natural health products and functional foods that contain inhibitors of CYP enzymes.


They are able not only to deposit and transport •NO, but are also to act as antioxidant and antiradical agents. However, the mechanics of hemoglobin-bound DNICs (Hb-DNICs) protecting Hb against peroxynitrite-caused, mediated oxidative modification have not yet been scrutinized. Through EPR spectroscopy we show that Hb-DNICs are destroyed under the peroxynitrite action in a dose-dependent manner. At the same time, DNICs inhibit the oxidation of tryptophan and tyrosine residues and formation of carbonyl derivatives. They also prevent the formation of covalent crosslinks between Hb subunits and degradation of a heme group. These effects can arise from the oxoferryl heme form being reduced, and they can be connected with the ability of DNICs to directly intercept peroxynitrite and free radicals, which emerge due to its homolysis. These data show that DNICs may ensure protection from myocardial ischemia. Dinitrosyl iron complexes (DNICs) are a physiological form of nitric oxide ( Square brackets are used to point out the total charge of the entire complex, and to show that the total charges of both sides of this equation are equal. In the reaction Equations (9) and (10) this formula is used without square brackets.During reaction 7, unstable intermediates, nitrosyl-peroxynitrite-associated complexes, are formed . The forThe resistance of DNICs to peroxynitrite depends on the availability of the nitrosyl DNICs group. The glutathione DNICs complex is the most resistant to oxidation: The cysteine DNICs complex is oxidized more efficiently: nd DNICs : or chelatable iron pool (CIP) . LIP usu•NO . It is ath DNICs . It is pormation .− and its free radical derivatives, e.g., •NO2 and •CO3  complex, and, consequently, the appearance of nitroxyl (NO−) and nitrosonium (NO+) ions, whose reactivity is higher than that of a neutral •NO molecule pyran-2-carboxylic acid—methyl ester (ThioGlo1)—“Calbiochem” ; 4-hydroxy-oxyl (4-hydroxy-TEMPO)—“Oxis” .The following reagents were used in the present work: bovine methemoglobin (oxidized Hb), pyridine, sodium dithionite, glycerol, glycine, Coomassie brilliant blue R-250, 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), dimethyl sulfoxide (DMSO), 2,4-dinitrophenylhydrazine (DNPH), 4 solution (5.5 mM) in 100 mM K,Na-phosphate buffer (pH 6.8). Hb-DNICs were obtained by adding 1 ml of 1 mM Hb solution in 0.1 M K,Na-phosphate buffer (pH 6.8) to 400 μL of 5 mM phosphate DNICs. Hb-DNICs (~1.3 mM) emerged after 5 min of incubation in the amount of ~1.8 complexes per a Hb tetramer. The DNICs concentration was determined based on the overall intensity of the electron paramagnetic resonance (EPR) signal of the complexes, with the 4-hydroxy-TEMPO spin label as an external standard. The DNICs preparations were stored at −70 °C.DNICs was synthesized as described in ,15. DNIC2 solution and 0.6 M H2O2 solution in 0.7 M HCl. It was stabilized with 0.9 M NaOH solution. Unreacted H2O2 was removed by adding MnO2 powder, which was then separated by filtration. Peroxynitrite concentration was determined using a characteristic absorption band at 302 nm (ε = 1.67 mm−1cm−1).Peroxynitrite was synthesized following , rapidlyEPR spectra were recorded using the E-109E spectrometer  at room temperature (~25 °C). Samples (80 µL) were placed in gas-permeable Teflon capillaries PTFE 22  before measurements. Conditions for recording EPR spectra: microwave power—10 mW, microwave field frequency—9.15 GHz, RF modulation amplitude—0.4 mT (for DNICs). The EPR signal of the 4-hydroxy-TEMPO spin tag was recorded under the following conditions: 10 mW microwave power, 9.14 GHz microwave field frequency, and 0.05 mT RF modulation amplitude. Carbonyl Hb derivatives were quantified using the method  with min2 = (E363 + E370) × 3.5 + (E370 + E428) × 29 + (E430 + E434) × 2 + (E520 + E535) × 7.5.S1—aldehyde-dinitrophenylhydrazones,S2—neutral ketone-dinitrophenylhydrazones.SThe samples were prepared in the following way. Peroxynitrite was added to 0.1 mL of 0.15 mM metHb solution in 20 mM K,Na-phosphate buffer (pH 6.8) to the final concentrations of 0.38; 0.6; 1.0; 1.6; 2.55; and 4.2 mM, and incubated at room temperature for 15 min. Then 0.5 mL of DNPH solution in 2 M HCl was added to the mixture and incubated for 1 h. The protein precipitate formed after adding 0.5 mL of 20% TCA solution, and after 10 min samples were centrifugated at 3000 g for 15 min. The precipitate was thrice washed with a mixture of ethanol and ethyl acetate (1:1) (0.4 mL) to remove unbound 2,4-DNFG. The final precipitate was air-dried and dissolved in 1 mL of 8 M urea before measurement. A similar procedure was performed with the iron complex DTPA, which was added to the final 2.5 mM concentration. The samples were diluted five times prior to measurements.Optical absorption spectra were recorded on the Cary 300 UV-VIS spectrophotometer  at room temperature in a 1 cm optical cuvette at a scanning speed of 600 nm/min. 556-A539, using the difference A556-A539, given (Ε556-539 = 4.3 mM−1cm−1).The heme concentration in metHb solution was measured using the pyridine hemochrome method ; 135 µL Reduced sulfhydryl groups were quantified using the ThioGlo1 thiol-specific fluorescent label. The adding of the ThioGlo1 protein to the solution resulted in the formation of a thiol adduct with maximum emission of fluorescence emission at 500 nm at an excitation wavelength of 379 nm . Samples were prepared as follows: 5 μL of 2.5 mM ThioGlo1 in DMSO was added to 5 μL of a reaction mixture containing 0.03 mM metHb, and incubated for 4 min. The resulting solution (10 μL) was added to a quartz spectrofluorimetric cuvette containing 490 μL of a 20 mM K-phosphate buffer (pH 6.8). The fluorescence was registered on RF-5301 PC spectrofluorimeter  with high sensitivity and at a medium scanning speed (in accordance with the instrument description). The slit width was 3 nm and 5 nm for excitation and emitted light respectively. The state of tryptophan and tyrosine residues in the metHb molecule was studied via fluorescence spectroscopy. The tryptophan autofluorescence was selectively excited at a λ = 295 nm and recorded at 330 nm. The slit width was 5 nm for the excitation light and 10 nm for the emitted one. Dityrosine formation was detected at excitation λ = 325 nm and emission λ = 400 nm , the speThe protein samples were prepared as described above in Electrophoresis was performed in 12% PAAG blocks with a size of 15 cm × 15 cm × 1 mm according to the Laemmli method  with a VElectrophoresis was carried out at 4 °C, I = 50 mA, and U = 150 V. Elf-4  was used as a power source for electrophoresis. After completing protein separation, the gel was fixed and stained with Coomassie brilliant blue R-250 solution.p value was less than 0.05.The measurements were performed in at least three replicates for each sample. The statistical data were processed based on 3–4 analytical repetitions. The data are presented as a mean ± standard deviation. Statistical differences were determined by means of a one-way ANOVA analysis followed by post hoc Tukey’s multiple comparison test. The differences were considered to be statistically significant when the


Flos populi extract (FPE) on the growth, antioxidation capability, innate immune response, and disease resistance in gibel carp. A total of 480 fish were fed with five different diets containing 0, 0.5, 1.0, 1.5, or 2.0 g kg−1 FPE  for 45 days. The fish were challenged with A. hydrophila after the feeding trial. Compared with the control, the feed efficiency (FE), weight gain (WG), final body weight (FBW), and specific growth rate (SGR) were significantly improved in groups D1.0 and D1.5. Dietary FPE significantly increased serum superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities, as well as glutathione (GSH) content. The contents of protein carbonyl (PCC) and malondialdehyde (MDA) in serum decreased significantly. Additionally, FPE supplementation in diets resulted in significant improvement in serum lysozyme (LZM) and myeloperoxidase (MPO) activities, as well as immunoglobulin M (IgM) and complement 3 (C3) concentrations. The hepatic antioxidant enzymes (CAT and SOD) activities increased, whereas content of MDA decreased in fish treated with dietary FPE than those of control both pre- and post-challenged. After 12 h-challenge, an obvious downregulation of hepatic Kelch-like-ECH-associated protein 1 (Keap1), splenic tumor necrosis factor-α (TNF-α), interleukin (IL)-8, IL-1β, and toll-like receptor 2 (TLR2) mRNA levels was observed in fish treated with dietary FPE, whereas hepatic Nrf2 transcription level was upregulated compared to the control. Furthermore, compared to group D0.5, higher relative percent survival (RPS) was observed in gibel carp fed dietary 1.0–2.0 g/kg FPE. Our results reveal that FPE supplemented diet has a stimulatory effect on antioxidant capacity and nonspecific immune response, along with improved growth performance and enhanced resistance against A. hydrophila infection in juvenile gibel carp.This study investigated the effects of dietary  Carassius auratus gibelio) is considered one of the most important cultivated freshwater fish species in China. It is easy to culture and breeds rapidly   is enriched with a blend of flavonoids and their glucopyranosides , phenolics and cardiac glycosides  and were reared in an indoor recirculating system. Before feeding experiments, 850 fish were all acclimated in fiber glass cylinders (200 L) under the experimental conditions for 2 weeks by feeding the control diet. During the acclimation, fish were fed up to apparent satiation thrice daily.Two weeks later, fish had accustomed to the experimental conditions. After 24 h of fasting, the selected 600 healthy fish of uniform size : 19.96 ± 0.06 g, initial protein content of fish: (12.60 ± 0.08)%) were distributed randomly into 20 tanks (200 L each) at a density of 30 fish per tank. Quadruplicate tanks were assigned to each dietary group in a random manner. Throughout the entire experimental period, fish were fed their respective diets per day at 07:30, 12:30, and 17:30. The trial lasted for 45 days. A lower pressure blower was used to supply sufficient oxygen.During the trial, the water flowing rate (0.2 L/min), the water dissolved oxygen (7.38 ± 0.05 mg/l), pH (6.8–7.0), temperature (26.7 ± 1 °C), and ammonia (≤ 0.04 mg/l) of the tanks were recorded daily, along with a natural photoperiod. A portable analyzer  was used to monitor the water physicochemical parameters inside the tanks daily. The feeding amount was adjusted according to BW measurement every two weeks. After 30 min of feeding, excess feed and fish feces were removed by siphon and about 33% of the tank water was renewed once a day to maintain water quality. The experimental program was approved by the Ethics and Animal Welfare of Nanjing Forestry University  (permit number: NJFU (Su) 2016–0024).Before sampling, all the fish were fasted for 24 h. The fish numbers in each tank were recorded, and the total weight of fish per tank was measured. Then, each fish was weighed to calculate SGR , WGR , FE , FI , PRE , and SR  according to our previous publications  were recorded 12–96 h post bacterial infection. The survival rate (%) was calculated as  × 100. The relative percentage survival (RPS) (%) was calculated through the formula of Amend  under the same management conditions  for challenge with bacterial septicemia pathogen d Amend, : RPS (%)Sample collection.When the feeding trial finished, after weighing the fish, 3 fish/tank were randomly selected and anesthetized on ice with diluted MS-222  at the concentration of 100 mg/L. Blood sample was then rapidly drawn from the caudal vein using 2 ml heparinized plastic syringes. The collected blood samples were centrifuged , and the plasma samples were obtained and stored at − 80 °C for subsequent analysis. After the fish were dissected, individual liver tissue was isolated and immediately put into liquid nitrogen. In the challenge test, 12-h post-injection, 12 alive fish per group were sampled for liver and splenic tissues. All the samples were stored at − 80 °C until further processing.The activities of serum alanine aminotransferase (ALT), aminotransferase (AST), alkaline phosphatase (AKP), lysozyme (LZM), and myeloperoxidase (MPO) were determined by the colorimetric method, and the contents of immunoglobulin M (IgM) and complement 3 (C3) were determined by the immunoturbidimetric method. The analyses were carried out using commercial kits (Nanjing Jiancheng Bioengineering Institute of China) with a Synergy2 multifunctional microplate reader .After sampling, hepatic sample was placed in a centrifuge tube; then, ice-cold phosphate-buffered saline  was added. The hepatic samples were homogenized by a hand-held homogenizer in an ice bath. 5 min later, the homogenate was then centrifuged at 5000 rpm at 4 °C for 10 min, then the supernatant was collected for the following analysis. The protein concentration in the supernatant was measured by the method of Bradford .The contents of glutathione (GSH), protein carbonyl content (PCC), and malonaldehyde (MDA), and the activities of catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) were determined by the method of spectrophotometric, colorimetry, TBA, ammonium molybdate, DTNB, and hydroxylamine, respectively, using a commercial kit according to the manufacturer’s instructions . Indicators of the hepatic antioxidant capacity were expressed as U/mg protein.CT−ΔΔ method. The mRNA expressions of the target genes were normalized to β-actin with the quantity of the control group scaled to 1.Based on previous work  between the values obtained from pre and 12-h post-challenge tests were marked by asterisks above histogram bars using independent t test. The data were represented as mean ± standard error of mean (SEM). The significant difference level was set at P < 0.05.All data were statistically analyzed using Statistical Package SPSS version 22.0 software and subjected to one-way ANOVA analysis. Duncan’s multiple range test was used to detect the significance of the difference in mean values among different treatments. Significant differences (P < 0.05). The D1.0 and D1.5 groups presented higher (P < 0.05) FBW, compared with the other two groups. The WGR in group D1.0 was higher (P < 0.05) compared with that of D0.5 and D2.0 groups, respectively. SR of the four FPE groups was significantly increased (P < 0.05), in comparison with the control. No significant difference (P > 0.05) of FI and PRE in gibel carp was found among the five groups.In comparison with the control, groups D1.0 and D1.5 presented the highest FBW, WGR, SGR, and FE of fish after 45 days of the feeding trial Table  (P < 0.0P < 0.05) than those of in the control group. In contrast, AKP activities of groups D1.0, D1.5, and D2.0 were higher (P < 0.05) by comparison with the control.ALT activities of fish in groups D1.0, D1.5, and D2.0 were considerably lower than that of the control group Table . AST actFigure P < 0.05) increase. In contrast, the serum MDA and PCC contents showed the opposite trend to SOD and GPx contents (P < 0.05 or P < 0.01).Activities of GPx and SOD in serum increased in the treatment groups Fig. . compareP < 0.05) in D1.0 and D1.5 groups. Fish fed FPE supplemented diets displayed increased serum MPO activity (P < 0.05) and IgM concentrations (P < 0.01). In comparison with the control group, the serum C3 levels were higher (P < 0.05) in D0.5, D1.0, and D1.5 groups.As presented in Fig. P < 0.01). CAT activities of groups D0.5, D1.0, and D1.5 were significantly higher (P < 0.05) compared with the control. MDA content is the opposite of CAT activity.Figure P < 0.05) relative to pre-infection levels. The hepatic MDA content in D1.0, D1.5, and D2.0 groups showed a significant decrease (P < 0.05), compared with the control. There was a remarkable increment (P < 0.05) in the activity of SOD in the groups D1.0, D1.5, and D2.0, as well as in the activity of CAT in the groups D1.5 and D2.0.At 12-h post-bacterial challenge, hepatic CAT activity decreased while the content of MDA increased (Nrf2 and Keap1) in liver and immune-related genes  in the spleen of gibel carp before challenge and 12-h post-challenge. Before the challenge, no difference (P > 0.05) was observed on the expression of Nrf2, Keap1, TLR2, TNF-α, IL-8, and IL-1β.Figure Keap1 and splenic TLR2, IL-1β, TNF-α, or IL-8 were remarkably upregulated in the injected fish than that of the pre-challenged one (P < 0.05). The hepatic Nrf2 expression was inhibited by the 12-h post-challenge relative to pre-infection levels (P < 0.05). However, post the bacterial challenge, fish in FPE groups had lower expressional levels of Keap1, TNF-α, and IL-1β than those of fish in the control group (P < 0.05). The opposite was true for the expression of hepatic Nrf2 (P < 0.01). Furthermore, groups D1.0, D1.5, and D2.0 also exerted a preventive effect on the increased levels of TLR2 and IL-8 in the spleen of post-challenged fish (P < 0.01).At 12-h post-challenge, gene expressions of hepatic A. hydrophila infection. The survival rates of the fish post-challenge were 62.00% (control group), 73.00% (D0.5 group), 78.40% (D1.0 group), 79.60% (D1.5 group), and 76.50% (D2.0 group), respectively. The RPS of the four treatment groups vs. the control group was 28.95%, 43.16%, 46.32%, and 38.16%, respectively. Furthermore, groups D1.0, D1.5, and D2.0 showed higher RPS (P < 0.05) compared with group D0.5. Typical symptoms of hemorrhagic septicemia were observed in dying or dead fish. Aeromonas hydrophila colonies were also isolated from dead fish.As shown in Fig. The proposed hypothesis of the present study is that dietary FPE is expected to improve the growth and health of gibel carp. Medicinal herb feed additives can improve feed conversion and digestibility by stimulating appetite, increasing digestive enzyme activity  fed with dandelion (Taraxacum spp.) extract  is one of the three major isotypes of immunoglobulin, which responds to pathogens both in local and systemic pathogens -[α-rhamnopyranoside(1–4)]-β-glucoside (Xu et al. Fish immunity is closely associated with inflammatory response and antioxidant status (Zhao et al. A. hydrophila (Mu et al. Moringa oleifera Lam leaves rich in flavonoids and polyphenols can normalize the transcriptional levels of pro-inflammatory cytokines via regulating TLR2 signaling (Zhang et al. Radix Bupleuri extract treatment reduced inflammatory response and IL-1β, TNF-α, and IL-8 mRNA levels by inhibiting TLRs-MyD88-NF-κB signaling pathway (Jia et al. TLR signaling pathway in fish immune tissue is activated significantly after the invasion of A. hydrophila of gibel carp after 96-h challenge with A. hydrophila. Antibacterial activity of FPE was shown previously that against Salmonella typhi, Shigella flexneri, and Escherichia coli in vitro (Xu et al. Pathogen infection is usually accompanied by an increase in free radical production (Liu et al. A. hydrophila, as well as mitigate the excessive inflammatory response of gibel carp. Therefore, FPE at 1.0 and 1.5 g kg−1 levels is recommended as a functional feed additive for gibel carp.Taken together, our results indicated that dietary FPE could notably improve antioxidant capability, feed utilization, nonspecific immune, and disease resistance of gibel carp against


A second form of familial systemic amyloidosis caused by the β2m variant, D76N, results in amyloid deposits in the viscera, without renal dysfunction. Although the folding and misfolding mechanisms of β2 microglobulin have been widely studied in vitro and in vivo, we lack a comparable understanding of the molecular mechanisms underlying toxicity in a cellular and organismal environment. Here, we established transgenic C. elegans lines expressing wild-type (WT) human β2m, or the two highly amyloidogenic naturally occurring variants, D76N β2m and ΔN6 β2m, in the C. elegans bodywall muscle. Nematodes expressing the D76N β2m and ΔN6 β2m variants exhibit increased age-dependent and cell nonautonomous proteotoxicity associated with reduced motility, delayed development and shortened lifespan. Both β2m variants cause widespread endogenous protein aggregation contributing to the increased toxicity in aged animals. We show that expression of β2m reduces the capacity of C. elegans to cope with heat and endoplasmic reticulum (ER) stress, correlating with a deficiency to upregulate BiP/hsp-4 transcripts in response to ER stress in young adult animals. Interestingly, protein secretion in all β2m variants is reduced, despite the presence of the natural signal sequence, suggesting a possible link between organismal β2m toxicity and a disrupted ER secretory metabolism. Aggregation of β The 1500 bp myo-3p body-wall muscle promoter  was ligated into the pPD95_75 vector using HindII and SalI sites. The signal sequence for human WT β2m was generated by oligonucleotide annealing of SalI-signalpeptideB2m-for and BamHI-signalpeptideB2m-rev. Oligos were mixed in a 1:1 ratio in 10 μL, then placed at 94 °C and allowed to cool until 30 °C. Serial dilutions of the oligos were made . 5 μL of the undiluted oligos and the serial dilutions were ligated into the successfully sequenced myo-3p-pPD95_75 vector. Ligations were transformed into DH5α cells. Colony restriction digestion was performed using BamHI and SalI restriction enzymes and analysed for a 100 bp band, successful constructs were sent for sequencing. Finally, human WT, D76N β2m and ΔN6 β2m cDNA (Radford lab) were PCR amplified using primers BamHI-B2m-for and MscI-B2m-rev to contain BamHI and MscI restriction sites and ligated into the myo-3p-ss-pPD95_75 vector. Successful colonies containing a 367 bp β2m band were sent for sequencing.BamHI-B2m-for5′ AAAGGATCCATTCAAAGAACTCCAAAAATTC 3′MscI-B2m-rev5′ AAATGGCCATTACATGTCTCGATCCCACTTA 3′SalI-signalpeptide B2m-for5′ TCGACAAAAATGTCTCGCTCCGTGGCCTTAGCTGTGGCCTTAGCTGTGCTC GCGCTACTCTCTCTTTCTGGCCTGGAGGCTG 3′BamHI-signalpeptide B2m-rev5′ GATCCAGCCTCCAGGCCAGAAAGAGAGAGTAGCGCGAGCACAGCTAAG GCCACGGAGCGAGACATTTTTG 3′C. elegans strain, using a DNA solution containing either the co-injection marker only ), or in combination with 25 ng/µL of the β2m construct. Multiple extrachromosomal lines were generated, based on the red fluorescent pharyngeal co-injection marker. The established transmitting lines used in this study have ~70% meiotic stability.The transgenes were microinjected into the gonads of the N2 Bristol v/v) NP-40). Extracts were centrifuged at 13,000 rpm for 1 min to pellet the carcasses and the supernatant collected. The protein extract was run on a 10% (w/v) Tris-Tricine gel and proteins were electro-transferred to 0.22 μM nitrocellulose membranes. The blots were blocked using 5% (w/v) milk overnight, washed four times in PBS/Tween (0.1%) for 5 min, then probed with primary anti-human β2m rabbit polyclonal antibody  at a 1:1000 (v/v) dilution in PBS/Tween (0.1%) and 1% (v/v) milk for 2 h. Blots were washed then probed with 1:10,000 (v/v) dilution HRP-conjugated goat anti-rabbit secondary antibody for 1 h. Blots were developed using ECL .For Western blot analysis, nematodes were collected and lysed on ice using a motorised pestle in lysis buffer  SDS-PAGE gel . The gel was fixed in solution A (50% (v/v) ethanol, 10% (v/v) acetic acid) for 30 min, then incubated in solution B (5% (v/v) ethanol, 1% (v/v) acetic acid) for 15 min on a gentle rocker. The gel was washed in dH2O three times for 5 min, and then incubated in solution D (0.02 g sodium thiosulphate in 100 mL dH2O) for 2 min while gently rocking. The gel was washed again in dH2O three times for 30 s, then incubated in solution E  for 20 min. After three washes in dH2O for 20 s, the gel was developed in solution F  until protein bands could be visualised and the reaction was stopped using solution G (5% (v/v) acetic acid). The gel was then stored in dH2O and imaged.For silver staining of SDS-PAGE, nematode extracts were loaded on an 4–20% (v/v) Triton X-100, complete protease inhibitor ). Extracts were shock frozen in liquid nitrogen, and the frozen nematode pellet was ground using a motorized pestle, then lysed on ice in the presence of 0.0025 U/mL benzonase . Lysates were centrifuged at 1000 rpm for 1 min in a tabletop centrifuge and the supernatant was extracted. 2% (v/v) N-Lauroylsarcosine was added to the supernatant and was ultracentrifuged at 100,000 g for 1 h at 4 °C. The supernatant was removed, and the resulting pellet was resuspended in the same volume of dH2O as the supernatant.Animals were aged to day 1 or day 8 adults using NGM containing 50 µg/uL FUdR plates. Nematodes were collected using M9 buffer, and resuspended in lysis buffer .Thirty L4-stage nematodes were transferred onto separate individual NGM plates smented with OP50-1. At time-points of 24-, 48-, 72- and 96- hours, individual animals were transferred onto a new NGM plate using a sterilised platinum wire, and the number of eggs laid over the previous 24 h was counted and recorded. Three independent experiments were performed for each strain .Animals were synchronised using egg-laying methods, and 100 L4-stage nematodes were transferred onto five NGM plates with OP50-1 . Every 24 h for 8 days, each nematode was assessed by the touch-nose response for paralysis and the numbers of mobile, paralysed or censored animals were recorded. Mobile nematodes were determined if they were able to move freely, either when touched by a platinum wire, or when unprompted. Paralysed animals were determined if little movement was seen when touched by a platinum wire, but still displayed minimal movement or pharyngeal pumping to indicate they were still alive. Censored worms included worms that had died, displayed the ‘bag of worms’ phenotype, crawled up the side of the plate, or could not be detected. Mobile worms were moved onto a new plate each day to ensure any progeny did not influence the results. Three independent experiments were performed for each strain . At least three videos of each strain were recorded. Videos were analysed in ImageJ using the WrmTrck plugin  to quantn = 50 worms) and error bars indicate S.E.M.Progeny were synchronised by egg-laying and 50 L4-stage animals were selected and placed onto an NGM plate. The next day (day 1 adults), plates were incubated at 35 °C for 6 h then moved immediately into a 20 °C incubator. Animals were left to recover for 16 h at 20 °C, then survival of animals was scored. Animals were scored as alive if movement or pharyngeal pumping was observed. The percentage of alive and dead animals were scored and mean survival rates were determined using Student’s t test. Three independent experiments were performed for each strain (n = 50 worms per experiment); error bars indicate S.E.M.Nematodes were synchronised by egg-laying and L4-stage animals were selected and placed onto new NGM plates. Nematodes were left overnight to grow and 50 day-1 adult worms were transferred using a sterilised platinum wire into M9 buffer containing 50 μg/mL tunicamycin. Animals were incubated in tunicamycin for 6 h at 20 °C on a rotator. After incubation, nematodes were washed three times in M9 buffer and plated onto NGM plates. After 16 h of recovery, animals were scored for survival. Alive animals were determined by their ability to move spontaneously or when stimulated by touch, or pharyngeal pumping was observed. Dead animals were determined through lack of movement or pharyngeal pumping. Percentages of alive and dead animals were calculated and plotted. Three independent experiments were performed for each strain , and qRT-PCR was performed as described previously ,53.C. elegans, age-synchronised nematodes at indicated time points were immobilised in 10 mM sodium azide on 2% agarose (w/v) pads on a microscope slide. Images were captured on a LSM880 upright laser scanning confocal microscope  with a 40 × oil objective, using the Zen software. tagRFP was detected using a 561 nm excitation laser and an emission range from 565–650 nm. Representative confocal images are displayed as maximum z-stack projections.To quantify LBP-2::tagRFP aggregation in the tail region of myo-2p::mCherry co-injection marker in the head region of β2m and ctrl animals. Aggregates were quantified in a semi-automated process. ImageJ was used to subtract the background and apply a threshold to a maximum intensity projection images. Aggregates were defined as fluorescent puncta when larger than 1 µm2. Two independent experiments were performed for each strain (n = 10–21 worms per condition per experiment) and error bars indicate S.E.M. For statistical significance, a two-way ANOVA followed by a post-hoc Tukey’s test was performed.For aggregation quantification, the tail region was defined as the region from the tip of the tail to the anal sphincter. We chose the tail region because of interference of the


N,N-dimethylthiocarbamate was applied as the selective recognition site for HOCl. Before adding HOCl, the fluorescent probe exhibited weak fluorescence. Upon adding HOCl, the fluorescent probe displayed remarkable fluorescence enhancement. The fluorescence intensity at 502 nm showed a linear response to the concentration of HOCl from 3.0 × 10−7 to 1.0 × 10−5 mol·L−1. The detection limit was estimated to be 1.5 × 10−7 mol·L−1 for HOCl. The fluorescent probe showed fast response and outstanding selectivity toward HOCl. It owned good biocompatibility and had also been successfully applied in the confocal imaging of exogenous and endogenous HOCl in living cells.Hypochlorous acid (HOCl) was crucial for maintaining the homeostasis in cells and plays vital roles in many physiological and pathological processes. In this work, a highly selective fluorescent probe for hypochlorous acid in living cells was constructed and prepared based on a naphthalene derivative. A naphthalene derivative was utilized as the fluorescent group, and  Amonse (MPO) , 4, was se (MPO) . Despitese (MPO) , atherosse (MPO) , kidney se (MPO) , and neuse (MPO) . Thus, mp-methoxyphenol [p-aminophenyl ether [Many analytical methods had been utilized to detect HOCl, such as electrochemical analysis , mass spxyphenol , p-aminoyl ether , 21, thiyl ether –24, thioyl ether , 26, hydyl ether –29, rhodyl ether , selenidyl ether , 32, unsyl ether –35, oximyl ether , 37, thiyl ether , 39, andyl ether . These syl ether , 40 and yl ether , 36. Thuπ-acceptor (D-π-A) structure had been extensively applied to construct fluorescent probes due to their many advantages such as superior light stability, better chemical stability, and high fluorescence quantum yield. In the past few years, the naphthalene-based fluorescent probes for various substances had been reported including thiophenols [2O2 [− [− [N, N-Dimethylthiocarbamate was applied as a sensing unit for HOCl. Studied results demonstrated the probe possessed excellent sensitivity, a wide pH range, superior selectivity, and fast response time. Furthermore, the probe displayed almost no cell cytotoxicity and had been effectively utilized in the confocal imaging of exogenous and endogenous HOCl in living cells.Naphthalene derivatives owing a donor-ophenols , HNO [42ols [2O2 , cysteinols [2O2 , hydrogeols [2O2 , ONOO− [ [2O2 [− , and F− O2 [− [− . In thisN, N-diisopropylethylamine were bought from Tianjin Heowns Biochemical Technology Company. p-Toluenesulfonic acid monohydrate was purchased from China National Pharmaceutical Group Chemical Reagent Company. 6-Hydroxy-2-naphthaldehyde and 2-aminothiophenol were obtained from Saen Chemical Technology (Shanghai) Company. Sodium hypochlorite was purchased from Tianjin Fuyu Fine Chemical Company. Hydrogen peroxide (H2O2) and tertbutyl hydroperoxide (TBHP) were obtained from 30% to 70% aqueous solutions, respectively. Hydroxyl radical (·OH) and tert-butoxy radical (OtBu) were produced by reaction of 1 mM Fe2+ with 200 µM H2O2 or 200 µM TBHP, respectively. Superoxide (O2−) was generated from potassium superoxide (KO2) solid diluted in dimethyl sulfoxide (DMSO). Singlet oxygen (1O2) was yielded by the reaction of 1 mM ClO− with 200 μM H2O2. Peroxynitrite (ONOO−) solution was prepared by the reaction of H2O2 and NaNO2, and its concentration was assessed from absorption at λ = 302 nm (ɛ = 1670 L·mol−1·cm−1) [ɛ292 nm = 350 L·mol−1·cm−1) [Dimethylaminothioformyl chloride and −1·cm−1) . Unless −1·cm−1) .−1 PBS buffer . Except the fluorescence data recorded by time scanning, all other fluorescence and absorption data were measured at 3 min after adding HOCl at room temperature.The NMR spectrum was measured with a Bruker DRX-500 NMR spectrometer using tetramethylsilane (TMS) as the internal standard. The mass spectrum was obtained on an Agilent 6420 triple quadrupole LC/MS high resolution mass spectrometer. Fluorescence tests were performed on a Hitachi F-7000 fluorescence spectrophotometer equipped with a 1 cm quartz absorption cell . The UV-visible absorption spectrum was measured by an Evolution 260 Bio UV-Vis spectrophotometer with a 1 cm quartz absorption cell. The pH values were measured by the Mettler Toledo pH meter. Fluorescence imaging of living cells was recorded by an Olympus FV-1200 single photon laser confocal microscope. Data processing was mainly obtained in SigmaPlot software. The data obtained by fluorescence spectrophotometry and UV-visible spectrophotometry were measured in 0.01 mol·LThe synthetic route for fluorescence probe 1 is illustrated in p-toluenesulfonic acid monohydrate  at room temperature. Then, the reaction mixture was heated to reflux for 12 hours, and the solvent was evaporated under reduced pressure to obtain a crude product. The crude product was subjected to column chromatography with petroleum ether/ethyl acetate  as the eluent to obtain compound 2  as a yellow solid. 1H NMR , δ (ppm): 10.11 , 8.54 , 8.14 , 8.08 , 8.05 , 8.00 , 7.84 , 7.55–7.52 , 7.46–7.43 , 7.20–7.16 . 13C NMR , δ (ppm):168.19, 157.63, 154.18, 136.63, 134.82, 131.20, 127.83, 127.78, 127.74, 127.57, 127.06, 125.75, 124.65, 123.08, 122.73, 120.26, 109.40. MS (ESI) m/z: 278.0638 (M + H)+.2-Aminothiophenol  was dissolved in 5 mL ethanol and then added dropwisely to a 35 mL ethanol solution containing 6-hydroxy-2-naphthaldehyde  and 2Cl2: anhydrous DMF = 4:1, V/V). Then, 0.45 mL N, N-diisopropylethylamine (DIPEA) was added to the above solution. Next, N, N-dimethylaminothioformyl chloride  was slowly added to the above mixed solution in batches. The reaction mixture was stirred at room temperature. Then, the solvent was evaporated under reduced pressure, and the crude product was purified by column chromatography using dichloromethane/methanol  as the eluent to obtain probe 1 . 1H NMR , δ (ppm): 8.58 , 8.21 , 8.11 , 7.97 , 7.93–7.89 , 7.53–7.49 , 7.41–7.38 , 7.31 , 3.48 , 3.40 . 13C NMR , δ (ppm): 187.52, 168.05, 153.76, 152.89, 135.03, 134.86, 131.24, 130.64, 129.91, 128.66, 127.58, 126.54, 125.40, 125.07, 123.72, 123.12, 121.66, 119.68, 43.32, 38.85. MS (ESI) m/z: 365.0790 (M + H)+, 387.0610 (M + Na)+.Under nitrogen flow, compound 2  was dissolved in 20 mL mixture solvent  for three times. Next, the fresh media containing probe 1  and the purified product of compound 1 with HOCl  were introduced into the wells, respectively, and incubated for 24 h. Subsequently, 10 μL MTT (5 mg·mL−1) was added to per well, and the PC-12 cells were fostered for 4 h allowing the formation of formazan. Finally, the above loading media was deserted, and 150 μL DMSO was added to the wells. The 96-well plates were shaken for 10 min, and the absorbance at 490 nm was measured utilizing a microplate reader . The survival rate was assessed referring to A/A0 × 100% . Similarly, MTT assay of the probe for RAW 264.7 cells was performed.To assess the cytotoxicity of the probe, 3--2, 5-diphenyltetrazolium bromide (MTT) assay was carried out. First, PC-12 cells were hatched in 1640 medium supplemented with 10% fetal bovine serum, 100 units mLμM fluorescent probe 1 at 37°C for 30 min and then washed with DPBS for three times and imaged. In the control experiment imaging exogenous HOCl, PC-12 cells were cultured in the medium containing 5.0 μM probe for 30 min at 37°C, and the supernatant was sucked out. After cleaning with DPBS, the complete culture medium containing 10 μM HOCl was added, incubated for another 30 min, and imaged. The PC-12 cells were washed with DPBS for three times for imaging. In the experiment of imaging endogenous HOCl, RAW 264.7 cells were cultured in a medium containing 5.0 μM fluorescent probe 1 at 37°C for 30 min and then washed with DPBS for three times and imaged. In the control experiment imaging endogenous HOCl, RAW 264.7 cells were stimulated with PMA (1.5 μg/mL) for 30 min and then incubated with 5.0 µM probe 1 for 30 min and imaged. Next, RAW 264.7 cells were stimulated with 1.5 μg/mL PMA for 30 min, cultured with 5 μg/mL taurine, incubated with 5.0 µM probe 1 for 30 min, and imaged. The fluorescence imaging of cells was acquired on the Olympus FV1200-MPE single photon confocal inverted microscope with a 40× objective lens.The PC-12 cells and RAW 264.7 cells were incubated in laser confocal culture dishes at 37°C for 24 h to allow good cell growth and then cleaned three times with DPBS. In the experiment of imaging exogenous HOCl, PC-12 cells were cultured in a medium containing 5.0 −1 PBS buffer   . As show of HOCl . As show−7 mol·L−1 to 1.0 × 10−5 mol·L−1, the fluorescence intensity of probe had a linear relationship with the concentration of HOCl (F = 63.5503 + 223.0080 × 106 × C (r = 0.9988), where F denotes the measured fluorescence intensity, C represents the concentration of HOCl, and r is the linear correlation coefficient. The detection limit was calculated by three times standard deviation of blank solution [−7 mol·L−1 for the fluorescent probe. Compared with the detection limit of 5.86 × 10−7 mol·L−1 in reference [−7 mol·L−1 in reference [In order to study the linear relationship between the fluorescence intensity of probe 1 and the concentration of HOCl, different concentrations of HOCl were added to the probe. The excitation wavelength of probe was 350 nm. When the concentration of HOCl was in the range of 3.0 × 10 of HOCl . The linsolution , 49. Theeference  and the eference , the fluµM HOCl (We studied the time response of probe for HOCl by recording the fluorescence intensity of the probe changes with time before and after adding 10 µM HOCl . As showµM) without and with HOCl (10 µM) at different pH values were recorded . The above investigated results confirmed the proposed response mechanism of the probe toward HOCl in Referring to previous literature , 50–52, drolysis . In ordedrolysis . As showµM)  . As showµM probe for fluorescence imaging, it was found that the green channel had almost no fluorescence (µM probe for 30 min and then incubated with 10 µM HOCl for another 30 min and imaged. As shown in µM probe, and weak fluorescence was observed in green channel (μg/mL PMA for 30 min and then hatched with 5.0 µM probe for 30 min, a remarkable fluorescence increase in RAW 264.7 cells was found (µM probe for 30 min, much weaker fluorescence was obtained (In order to prove the practicality of probe in biological detection, we used fluorescent probe to perform laser confocal fluorescence imaging of HOCl in PC-12 cells and RAW 264.7 cells. PC-12 cells were seeded in a 35 mm laser confocal culture dish and cultured for 24 hours. After incubating for 30 min in a medium containing 5.0 rescence . At the  channel . When thas found . Howeverobtained . MeanwhiN, N-Dimethylthiocarbamate was chosen as the selective sensing unit for HOCl. When HOCl was absent, the reported probe displayed weak fluorescence. In the presence of HOCl, the probe exhibited green fluorescence. The probe had a short response time, a wide working pH range, and the high selectivity and specificity for hypochlorous acid. At the same time, the probe had good biocompatibility and is suitable for the detection of hypochlorous acid. In addition, confocal fluorescence imaging of living cells showed that the probe could be utilized to image exogenous and endogenous HOCl in living cells.On the whole, we constructed and prepared a novel naphthalene-based fluorescent probe for the determination of hypochlorous acid.


CENPW expression affects biological processes in liver cancer cells remains unknown. In this article, we found that CENPW was overexpressed in liver cancer tissues. Low CENPW expression was correlated with a better prognosis in hepatocellular carcinoma (HCC) patients, compared to high CENPW expression. The results of qRT-PCR and western blot assay showed that CENPW was effectively knocked down in HCC cells using siRNA transfection. Cell proliferation, migration, and invasion were inhibited. Cell apoptosis rates were increased. The cells were arrested in the G2/M phase of the cell cycle. Subsequently, 127 differentially expressed genes (DEGs) were identified based on RNA-seq data. GO and KEGG enrichment and PPI network analysis were performed. The novel DEGs were found and mainly enriched in nucleosome assembly and the complement system. In summary, our study indicated that overexpression of CENPW implied unfavorable prognosis and CENPW might be the potential predictive biomarker in liver cancer. Downregulation of CENPW might inhibit the HCC developmentby regulating the expression of the molecules in nucleosomes and the complement system.Centromere protein W (CENP-W), identified as a centromeric component, plays an important role in the cell life cycle. However, how   Worldwide, liver cancers are the fourth leading cause of cancer mortality and rank sixth in the incidence of various cancers . ApproxiCENPW gene, CENP-W is identified as a centromeric component [2 structure [Centromere protein W (CENP-W), which used to be called cancer-upregulated gene 2 (CUG2) protein, is overexpressed in various human cancers. Encoded by the omponent ,13. CENPomponent . Furtheromponent . Later, tructure . Such 10tructure . Howevertructure  and enhatructure  and facitructure . We wereCENPW was reported to be upregulated in many human cancer tissues, and exogenous CENPW overexpression was shown to induce tumorigenesis. Recent cellular experiments have mostly focused on the overexpression of exogenous CENPW [CENPW has scarcely been reported. In addition, there are no data about the function of CENPW in HCC cells. Therefore, we explored the downregulation of the CENPW gene, seeking the potential diagnostic biomarker and therapeutic target for HCC. In our study, we firstly confirmed the overexpression of the CENPW gene in various cancer tissues including HCC via analysis of the Oncomine database. Survival analysis was then carried out by using the Kaplan-Meier plotter database, showing the correlation between CENPW expression and the prognosis of liver cancer. Next, endogenous CENPW expression of human HCC cells was knocked down by siRNA transfection. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blot were carried out to verify the downregulation of CENPW expression. Cell cycle, apoptosis, viability, migration, and invasion assays were performed, indicating the relationship between CENPW expression and tumor cell behavior. Further, RNA-seq was used to discover the downstream regulation of CENPW to explore the potential molecular mechanism. Overall, our study provided a clue for the biological behavior of HCC cells after knocking down CENPW expression, implying CENPW as a potential diagnostic biomarker and the target for HCC gene therapy.us CENPW . In cont2.2.1.CENPW gene in different cancers was analyzed in the Oncomine 4.5 database (https://www.oncomine.org). The threshold was set as follows: P-value of 0.0001, fold change of 2, and gene rank of top 10%. Further, CENPW expression in HCC tissues was compared to that in normal liver tissues, and the differential expression with P < 0.01 was considered statistically significant. The analysis was based on the HCC studies of Chen Liver [CENPW expression was evaluatedin subgroups of HCC patients, based on gender, age, cancer stages, and tumor grade, using UALCAN .The mRNA differential expression level of the en Liver  and Wurmen Liver . Moreove2.2.CENPW expression in liver cancer was analyzed by using the Kaplan-Meier plotter database . According to the median values of RNA sequencing data, liver cancer patients were split into the low and high CENPW expression groups. The overall survival (OS) and relapse-free survival (RFS) analyses were performed on 364 and 313 patients, respectively. Kaplan-Meier plots were obtained. Differences with P < 0.01 were considered significant. Log-rank P-values and hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated.The correlation between survival and 2.3.2 at 37°C.The human hepatocellular cell line Hep3B was purchased from American Type Culture Collection , and Huh7 was kindly provided by Prof. Hong Shan . Hep3B and Huh7 cells were maintained in high-glucose Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  and 1% penicillin streptomycin (Gibco) in a humidified incubator  with 5% CO2.4.CENPW and negative control siRNA-NC were synthesized  and purified by high-performance liquid chromatography (HPLC). The siRNAs were transfected by using LipofectamineTM 3000 Transfection Reagent . After transfection for 24 to 48 h, cells were used for subsequent experiments.Four pairs of siRNAs  targeting 2.5.CENPW gene. The primers were as follows: for the β-actin gene:5ʹ-AGGTGGACAGCGAGGCCAGGAT-3ʹ (forward), 5ʹ-TTGCCGACAGGATGCAGAAGGA-3ʹ (reverse); and for the CENPW gene: 5ʹ-TAGCAGAAGAGTCCAGGACAAAC-3ʹ (forward), 5ʹ-ACCTCTGCTCTTCTTTAGAATTACC-3ʹ (reverse). CENPW expression was normalized to β-actin expression, and relative expression was calculated using the 2−ΔΔCt method.Using the FastPure Cell/Tissue Total RNA Isolation Kit , total RNA was extracted from Hep3B and Huh7 cells. The purity and concentration of the total RNA were detected using a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific). HiScriptIII RT SuperMix for qPCR (+gDNA wiper) (Vazyme Biotech) was applied for reverse transcription to synthesize cDNA. qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech) and CFX96 Touch Real-Time PCR Detection System . The β-actin gene was selected as the internal control against the 2.6.HCC cells were harvested after 48 h of transfection and lysed by RIPA Lysis Buffer . After 14,000 × g for 10 min at 4°C, total protein concentration was measured by the Enhanced BCA Protein Assay Kit (Beyotime). The protein was separated by 18% SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked by 5% skim milk and incubated with the primary antibodies against CENP-W  and GAPDH . After incubation with the secondary antibody, the bands were detected with enhanced chemiluminescence. The density of the blots was measured by ImageJ version 1.51. CENP-W expression was normalized to GAPDH expression, and relative protein expression was calculated.2.7.After transfection for 48 h, Hep3B cells and Huh7 cells were inoculated into 96-well plates with 100 μl complete medium . Then, 10 μl of CCK-8  solution was added into each well at 0, 24, 48 and 72 h. After 2 h incubation, optical density (OD) was measured at a 450 nm wavelength by a microplate reader . Cell proliferation curves were calculated and plotted.2.8.4 cells were inoculated into the upper chamber in 200 μl of serum-free medium, and 600 μl of complete medium with 10% FBS was placed into the lower chamber. Transwell chambers were coated with Matrigel (Corning) only for the invasion assay. After 24 h incubation at 37°C, cells were fixed with 4% paraformaldehyde for 30 min at room temperature and stained with 0.1% crystal violet overnight. The nonmigrating cells were removed before the cells on the lower surfaces of the inserts were counted under a microscope .Transwell migration and invasion assays were performed using 24-well chambers with 8 μm pore-size membranes . After transfection for 48 h, serum-free medium was used to starve the cells for another 12 h. Then, 6 × 102.9.5 cells were resuspended in 100 μl 1 × binding buffer and then stained with 5 μl annexin V-fluorescein isothiocyanate (FITC) and 5 μl propidium iodide (PI) for 10 min in the dark at room temperature. Another 400 μl 1 × binding buffer was added after incubation. Cells were detected on a flow cytometer  within 1 h. Data were managed in FlowJo version 10.4.After 48 h of transfection, Hep3B and Huh7 cells were harvested and washed with cold phosphate buffered saline  twice. Using the Annexin V-FITC/PI Apoptosis Detection Kit (Vazyme Biotech), 5 × 102.10.5 cells were harvested for cell cycle analysis. Cells were washed with cold PBS twice and fixed with 70% ethanol at −20°C overnight. Then, the cells were washed in PBS to remove ethanol and stained with 500 μl PI/RNase staining buffer . After incubation for 15 min in the dark at room temperature, the cells were detected on a Beckman Coulter flow cytometer. Cells were analyzed on a flow cytometer within 1 h. Data were analyzed using FlowJo version 10.4.After siRNA transfection for 48 h, 5 × 102.11.https://report.bgi.com,BGI). DEGs were identified with |Log2 fold-change (FC)| http://geneontology.org)biological process and Kyoto Encyclopedia of Genes and Genomes  pathway enrichment analysis were performed. The PPI network was established using theSearch Tool for the Retrieval of Interacting Genes/Proteins  and Cytoscape version 3.7.2. As previously described, the DEGs were randomly selected for qRT-PCR to validate the RNA-seq data.Total RNA was extracted from the siRNA-NC group and the siRNA-758 group of Huh7 cells after siRNA transfection for 48 h and sent to the Beijing Genomics Institute . Following quality control, total RNA was performed to mRNA library construction and subjected to the BGIseq500 platform (BGI) for RNA-seq detection. The raw sequencing data were filtered and the clean reads were mapped to the human genome. After that, a database was built and analyzed by Dr. Tom online system  from at least three replicates and were analyzed by one-way analysis of variance (ANOVA) or independent sample t-test with SPSS version 20.0. A value of P < 0.05 was considered statistically significant.The results of 3.3.1.CENPW by using the Oncomine database. Compared to those in normal tissues, CENPW expression levels were higher in various types of cancers, including brain and central nervous system (CNS) cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, gastric cancer, head and neck cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer and other cancers . The fold change was within 2.916, and the overexpression gene rank was within the top 2% in Chen Liver , implying that CENP-W might play roles other than its role as a component in a nucleosome-like structure. Further, using UALCAN in subgroup analyses, we confirmed that the transcription level of CENPW was significantly higher in HCC patients than that in normal people, including the early stage of HCC . Notably, in the subgroup of cancer stages, when compared to the stage 1, the CENPW expression of stage 2 (P = 0.0064 < 0.01) and stage 3 (P = 0.0002 < 0.001) were also significantly increased . These results implied the expression of CENPW might isolate well the early stage of HCC and be a potential biomarker in HCC early diagnosis.We assessed the differential expression of  cancers ). Considen Liver ), while en Liver ). We als3.2.CENPW expression was correlated with prognosis in liver cancer patients, we used the Kaplan-Meier plotter database to evaluate the prognostic potential of CENPW based on the RNA sequencing data. Notably, poor prognosis in liver cancer  was correlated with high CENPW expression , European Genome-phenome Archive (EGA) and The Cancer Genome Atlas (TCGA) databases. To investigate whether pression . The res3.3.CENPW mRNA expression in Hep3B and Huh7 cells was calculated after transfection for 24 h with the siRNAs . On the whole, siRNA-758 exhibited the best knockdown efficiency in both HCC cells and its data showed 14.81% ± 3.26% free CENPW mRNA in Hep3B cells and 27.14% ± 2.41% in Huh7 cells by normalizing to the siRNA-NC group as a negative control. Therefore, we chose siRNA-758 for subsequent experiments.The relative e siRNAs . CENPW m3.4.P < 0.01). The results confirmed that the siRNA-758 transfection knocked down the CENP-W protein expression level.The CENP-W expression in protein level was detected by western blot assay . Compare3.5.P < 0.01) from 24 to 72 h in the siRNA-758 group compared to the siRNA-NC group  in the siRNA-758 group, indicating the contribution of CENPW to cell motility (P < 0.01) in the siRNA-758 group, showing that CENPW confers aggressive features in HCC cells (CENPW reduced the migration and invasion abilities of HCC cells.The motility and aggressiveness of Hep3B and Huh7 cells were examined by the Transwell assay. The migrating cells were significantly reduced (motility . SimilarCC cells . Knockin3.7.P < 0.01) and Huh7 (P < 0.05) cells  cells . The res3.8.P < 0.01) and Huh7 (P < 0.05) cells  cells . Accordi3.9.HIST1H2BJ) and cAMP-specific 3',5'-cyclic phosphodiesterase 7B (PDE7B), while downregulated DEGs included histone H4(HIST4H4), histone H2A(HIST1H2AL), histone H1.1(HIST1H1A), COMMD3-BMI1 readthrough (COMMD3-BMI1), 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-2 (PLCG2) and Complement C4-B (C4B_2).Normalized to the siRNA-NC group, 127 DEGs (82 upregulated and 45 downregulated) in the siRNA-758 group of Huh7 cells were identified . The upregulated DEGs included histone H2B  also had DEGs enrichment to a certain degree.To identify the DEGs associated pathway, KEGG pathway enrichment was carried out . The DEG3.12.We established a PPI network for 127 DEGs, containing 45 nodes and 48 edges . The DEG3.13.C4B_2, FAM72C, UGT2A2, and LOC102724951. The primers were as follows: for the C4B_2 gene: 5ʹ-GTGTGCATCTGGCGGAACG-3ʹ (forward), 5ʹ-TGGGGACCGAGTCAAAATACAG-3ʹ (reverse); for the FAM72C gene: 5ʹ-TCCTGTCTTCTTTCCTGCAACA-3ʹ (forward), 5ʹ-AGTAGGACGTTTACACCTGTGG-3ʹ (reverse); for the UGT2A2 gene: 5ʹ-TGATGGCAAGACTTCAGAAAGG-3ʹ (forward), 5ʹ-TGTTACTGGGTCTGCTACCAA-3ʹ (reverse); and for the LOC102724951 gene: 5ʹ-ACCACCGTAGTGGAAGAGAGA-3ʹ (forward), 5ʹ-GCTATGATGCCTATTTCCTCAGC-3ʹ (reverse). Consistent with the RNA-seq data, the expression of C4B_2, FAM72C and LOC102724951 was confirmed to be downregulated, while that of UGT2A2 was upregulated obviously , 5ʹ-AGGGCTGTCCTGAATAAGCAG-3ʹ (reverse); for the OCT4 gene: 5ʹ-CTTGAATCCCGAATGGAAAGGG-3ʹ (forward), 5ʹ-GTGTATATCCCAGGGTGATCCTC-3ʹ (reverse); for the SOX2 gene: 5ʹ-GCCGAGTGGAAACTTTTGTCG-3ʹ (forward), 5ʹ-GGCAGCGTGTACTTATCCTTCT-3ʹ (reverse); for the MYC gene: 5ʹ-GGCTCCTGGCAAAAGGTCA−3ʹ (forward), 5ʹ-CTGCGTAGTTGTGCTGATGT−3ʹ (reverse). The expression ofNANOG, OCT4, and SOX2 were significantly increased , while that of MYC presented no statistical difference (data not shown).Further, we investigated the differential expression of the reprogramming genes by qRT-PCR, such as 4.CENPW overexpression led to tumorigenesis and cell death, suggesting CENPW as a novel oncogene [CENPW as one of the prognostic genes for HCC based on the gene co-expression network analysis, implying the prognostic value of CENPW for HCC [CENPW expression in the prognosis and development of liver cancer is still unknown. We confirmed the overexpression of CENPW in liver cancer using the Oncomine database. The results indicated that the CENPW expression level in HCC tissues was significantly higher than that in normal liver tissues (P < 0.01). In addition, the survival curves generated by the Kaplan-Meier plotter database showed that better prognosis of patients with liver cancer was correlated with lower CENPW expression. We suggest that CENPW is a potential predictive biomarker for liver cancer prognosis.Previous studies showed that oncogene . A recen for HCC . HoweverCENPW gene, has three transcript variants. They are called isoforms a, b and c, with molecular weights of 11.8, 10.1 and 7.9 kDa, respectively. Isoform b is regarded as the canonical isoform, the most prevalent isoform in cells. In this article, we transiently knocked down CENPW expression in HCC cells by siRNA transfection. The siRNA-758 was designed to target the most conserved domain, which was shared by all three isoforms. In other words, all isoforms of CENP-W were knocked down after siRNA-758 transfection. In this way, the cellular behavior changes in the siRNA-758 group reflected all of the three CENP-W isoforms. Then, qRT-PCR and western blot were carried out to confirm the decrease in CENPW expression level. In Hep3B cells, 14.81% ± 3.26% of CENPW mRNA was left, while 27.14% ± 2.41% was left in Huh7 cells. The efficiency of siRNA-758 reached approximately 70% to 90%. A series of functional experiments in HCC cells implied that CENPW was an oncogene. Knocking down CENPW caused subsequent changes in HCC cells. The proliferation was obviously decreased from 24 to 72 h, and the cell apoptosis rate increased. The abilities of cell migration and invasion were also decreased significantly. In the cell cycle assay, the cells were arrested in G2/M phase, indicating that the cells had difficulty in mitosis after DNA synthesis. These results were reasonable, as CENPWis a centromeric component that plays a key role in cell division.Centromere protein W (CENP-W), encoded by the CENPW downregulation mainly caused changes in nucleosome assembly, nucleosome positioning, and DNA and chromatin regulation, relating to histones H4, H2B, H2A, H1.1, H1.4, and H1.5  was enhanced by CENPW silencing in HCC cells , implying the possibility of reprogramming into cancer stem cells which characterized by inhibition of cell proliferation and metabolic changes [CENPW might be a potential predictive biomarker and a therapeutic target for liver cancer.The results of RNA-seq and bioinformatic analysis showed that the and H1.5 . Interesand H1.5 , implyin network . By furt changes ,25. Base5.CENPW gene is a highly expressed oncogene in liver cancer, and is related to the prognosis of HCC patients, being a potential diagnostic biomarker in HCC. Based on our cellular experiments, the downregulation of CENPW expression might inhibit HCC development. Some important DEGs associated with CENPW knockdown were identified by RNA-seq, mainly correlating with nucleosomes and the complement system. These DEGs might be the CENPW downstream targets and potential therapeutic targets for HCC.Click here for additional data file.


The clinical significance of carbon-ion radiotherapy (CIRT) for octogenarians with locally advanced non-small-cell lung cancer (LA-NSCLC) remains unclear. We aimed to evaluate the clinical outcomes of CIRT alone for octogenarians with LA-NSCLC.We evaluated 32 patients who underwent CIRT alone between 1997 and 2015. The median age was 82.0 years . In terms of clinical stage (UICC 7th edition), 7 (21.9%), 10 (31.3%), 11 (34.4%), and 4 (12.5%) patients had stage IIA, IIB, IIIA, and ΙΙΙB disease, respectively. The median CIRT dose was 72.0 Gy , and the median follow-up period was 33.1 months.All patients successfully completed CIRT. Regarding grade ≥ 2 toxicities, 1 (3.1%), 3 (9.4%), and 4 (0.7%) patients developed grade 3 radiation pneumonitis, grade 2 radiation pneumonitis, and grade 2 dermatitis, respectively. No grade ≥ 4 toxicities were observed. The 2 year LC, PFS, and OS rates were 83.5%, 46.7%, and 68.0%, respectively.CIRT alone is safe and effective for octogenarians with LA-NSCLC. Primary lung cancer is one of the most common cancers worldwide . Japan h. conducted a multicenter study on CIRT alone in 64 patients with LA-NSCLC and reported similar 2 year LC and OS rates of 81.8% and 62.2%, respectively -fluorodeoxyglucose positron emission tomography combined with CT (18F-FDG PET/CT) were also performed. Histological or cytological diagnosis was confirmed via bronchoscopic biopsy, CT-guided biopsy, or sputum cytology in 28 (87.5%) patients and via CT, radiography, and/or 18F-FDG PET/CT in the remaining patients.We collected information on grade ≥ 2 toxicities. Treatment-related toxicities were graded according to the National Cancer Institute’s Common Terminology Criteria for Adverse Events (version 4.0) .Patients were fixed using an individually tailored immobilization device , and CT images were obtained in the supine or prone position using respiratory sensors to monitor the respiratory phase , 8. Targn = 21, 65.6%). The total dose was applied to the isocenter, and it enclosed the PTV conformably, with a 95% isodose line. The patients with lymph node metastasis underwent metastatic and prophylactic lymph node irradiation at a median dose of 49.5 Gy (RBE) [The prescribed dose ranged from 68.0 to 76.0 Gy [relative biological effectiveness (RBE)] in 12–16 fractions, 4 days per week. Their prescribed doses were converted to 96.9 to 115.0 Gy (RBE) as the biologically effective dose using α/β = 10. A dose escalation study conducted during our investigation showed the potential of short-course CIRT alone at 72 Gy (RBE)  and 77.1% (95% CI 54.3–90.5%), respectively ] with stage ΙΙΙ LA-NSCLC [Only one study has focused on treatment options for octogenarians with LA-NSCLC . In the LA-NSCLC . The mos. reported a median OS of 5 months when patients older than 70 years with stage ΙΙΙ LA-NSCLC selected non-curative treatment [Several studies on photon radiotherapy alone for patients older than 70 years with stage ΙΙΙ LA-NSCLC reported that the median PFS and OS of radiotherapy alone were 6.8 months and 11–18.1 months, respectively . Driessereatment . MeanwhiThe side effects of definitive treatment are generally more severe in elderly patients than in younger patients. Therefore, for elderly patients, the clinical goals must be decided by considering not only survival, but also quality of life and treatment tolerance. Administration of EGFR-TKI is limited to patients with NSCLC with activating EGFR mutation, although it is effective and relatively safe . In addiOur study had several limitations. First, the present study was a single-center retrospective analysis. Second, our results were derived from a small sample size of only 32 patients. Third, only octogenarians with Eastern Cooperative Oncology Group performance status of 0–2 were treated with CIRT alone, and thus there might have been patient bias. Finally, the total doses and fractionation varied (68–76 Gy (RBE) in 12–16 fractions) among the patients. Further large-scale multicenter prospective trials are warranted.In conclusion, CIRT alone is a relatively safe and effective treatment modality for octogenarians with LA-NSCLC.