Bacillus thuringiensis strain SG4. Strain SG4 effectively degraded cypermethrin under different conditions. The maximum degradation was observed at 32 °C, pH 7.0, and a shaking speed of 110 rpm, and about 80% of the initial dose of cypermethrin (50 mg·L−1) was degraded in minimal salt medium within 15 days. SG4 cells immobilized with sodium alginate provided a higher degradation rate (85.0%) and lower half-life (t1/2) of 5.3 days compared to the 52.9 days of the control. Bioaugmentation of cypermethrin-contaminated soil slurry with strain SG4 significantly enhanced its biodegradation (83.3%). Analysis of the degradation products led to identification of nine metabolites of cypermethrin, which revealed that cypermethrin could be degraded first by cleavage of its ester bond, followed by degradation of the benzene ring, and subsequent metabolism. A new degradation pathway for cypermethrin was proposed based on analysis of the metabolites. We investigated the active role of B. thuringiensis strain SG4 in cypermethrin degradation under various conditions that could be applied in large-scale pollutant treatment.Cypermethrin is popularly used as an insecticide in households and agricultural fields, resulting in serious environmental contamination. Rapid and effective techniques that minimize or remove insecticidal residues from the environment are urgently required. However, the currently available cypermethrin-degrading bacterial strains are suboptimal. We aimed to characterize the kinetics and metabolic pathway of highly efficient cypermethrin-degrading  Cypermethrin ((±)-α-cyano-3-phenoxybenzyl (±)-cis-trans3--2,2-dimethylcyclopropanecarboxylate) is a synthetic pyrethroid that is commonly used against agricultural and household pests. Pyrethroids are applied worldwide to protect crop fields against insects and enhance agricultural production ,2. PyretBacillus, Pseudomonas, Acinetobacter, Roultella, Aspergillus, Candida, Trichoderma, and Cunninghamella have been used for the degradation of cypermethrin and other pyrethroids [Bacillus thuringiensis strain SG4 in cypermethrin biodegradation under conditions similar to the natural environment. Cypermethrin persistence in the environment varies according to the physiochemical conditions of the soil ,11. Micrethroids ,22,23,24ethroids ,22,23,24ethroids . Howeverethroids . In this−1, which was filter sterilized and kept in the refrigerator. Minimal salt medium 2SO4 2.0 g·L−1, Na2HPO4·12H2O 1.5 g·L−1, KH2PO4 1.5 g·L−1, MgSO4·7H2O 0.2 g·L−1, CaCl2·2H2O 0.01 g·L−1, FeSO4·7H2O 0.001 g·L−1; pH 7.0) and nutrient broth  were used for the cultivation of pesticide-degrading soil bacterial strains. Analytical grade chemicals and solvents were used in this study.Technical-grade cypermethrin was obtained from the Department of Chemistry, G.B Pant University of Agriculture and Technology, Pantnagar, India. It was dissolved in acetonitrile to prepare a stock solution of 1 mg·mL−1), and residue concentrations at various incubation intervals were quantified using high-performance liquid chromatography (HPLC) . The SG4 bacterial strain was isolated from the soil samples and selected for further studies based on its cypermethrin-degradation ability. The strain was identified based on a polyphasic approach using its morphological, biochemical, and molecular characteristics [Cypermethrin-contaminated soil samples were collected from the agricultural fields in Pantnagar, Uttarakhand, India. Enrichment was carried out in MSM to isolate bacteria from collected soil samples. We used 1 g soil for bacterial isolation using the pour plate method. Bacterial colonies were transferred to 50 mL MSM containing cypermethrin , pH , and shaking speed 90, 110, 130 rpm) conditions. Strain SG4 was incubated in MSM (pH 7.0) containing 50 mg·L0 rpm con by HPLC ,30,31.2 (0.4 M) through a needleless syringe to prepare fine beads (2 mm) inside the laminar airflow. Beads were stored overnight at 4 °C [−1) was added to the flasks along with 20 sodium alginate beads. We removed 1 mL aliquots of the broth from each flask on 5th, 10th, and 15th days and cypermethrin was extracted from the samples. Non-inoculated flasks spiked with cypermethrin served as the control and samples were quantified by HPLC. Cypermethrin biodegradation was tested with immobilized bacterial culture in sodium alginate beads at 32 °C for 15 days. Sodium alginate beads were prepared by mixing 4% sodium alginate with homogenized bacterial culture(s). The mixture was poured drop by drop into pre-chilled autoclaved CaCl−1) was added along with 20 agar discs. We removed a 1 mL aliquot of the broth from each flask on the 5th, 10th, and 15th days and cypermethrin was extracted. Non-inoculated flasks spiked with cypermethrin served as the control and all the extracted samples were quantified by HPLC.Agar discs were also used to analyze the cypermethrin biodegradation with immobilized bacterial isolates at 32 °C for 15 days. Agar solution (2.5%) was sterilized and cooled (44 °C) to prepare agar discs. Homogenized bacterial cultures were mixed with molten agar and poured into Petri dishes aseptically inside a laminar airflow ,34. Agar−1) in MSM (50 mL) inoculated with an active culture of strain SG4. Non-inoculated flasks with 50 mg·L−1 cypermethrin served as the control, and the experiment was repeated three times. For analysis, a 10 mL mixture of samples and acetone was prepared, and 10 mL ethyl acetate was added. After vortexing (1 min), the upper layer was collected in new tubes and sodium sulfate (5 g) was added. Samples were transferred to round-bottomed flasks and evaporated. Cypermethrin was collected in a dried round-bottomed flask with acetonitrile and analyzed using FTIR. Cypermethrin metabolites were extracted with acetonitrile after 15 days of inoculation. Bond stretching was analyzed in the F-IR system at a wavelength of 400–4000 cm−1, at the Department of Biophysics, G. B Pant University of Agriculture and Technology, Pantnagar, India [FTIR analysis was performed to estimate bond stretching during cypermethrin degradation . The soil slurry method was used to investigate cypermethrin degradation by strain SG4. We placed 50 g autoclaved soil in 250 mL flasks, and 20 mL minimal salt broth was added ,38. We t18 reverse-phase column and an ECD-3000RS electrochemical detector. A 10 μL mixture of acetonitrile and water  was used as the mobile phase at a flow rate of 1.0 mL·min−1.The cypermethrin residues of each experiment were analyzed in triplicate. Cypermethrin was extracted by adding 5 mL bacterial culture into 20 mL acetone in a flask. After shaking for 1 h, the mixture was carefully washed with 10 mL acetone and filtered through a Buchner funnel . The filC0 is the initial concentration of cypermethrin in the medium, Ct is the concentration of cypermethrin at time t, k is the degradation rate constant (day−1), and t is the reaction time. The first-order kinetic equation was followed to fit experimental data of cypermethrin degradation kinetics as follows :(1)Ct=C0t1/2) of cypermethrin was calculated asThe biodegradation half-life  i.e., 1 h after the application (100%). The k of each concentration was computed from the slope of the line [To calculate the line .p < 0.05 to examine specific differences between treatments. Experiments were arranged as completely randomized designs with three replicates. Data were analyzed by one-way analysis of variance (ANOVA), and means were compared according to Bonferroni’s multiple comparisons test using IBM-SPSS software . Statistical significance was determined by lowest significance differences (LSD) test at Bacillus thuringiensis strain SG4 (accession number KT186610). Cypermethrin-degrading bacterial isolate SG4 was enriched and isolated from contaminated agricultural fields and characterized as Gram-positive rod-shaped bacilli. The morphological and biochemical characteristics of strain SG4 are provided in k value of 0.1035 day−1 and a t1/2 of 6.7 days. In the non-inoculated control, the k and t1/2 values were 0.990 day−1 and 165.0 days, respectively −O−, and C–H bonds of cypermethrin. Conversely, peaks were absent in the strain-SG4-treated sample, revealing effective cypermethrin degradation . Strain SG4 efficiently degraded cypermethrin in soil slurry and immobilized the culture with sodium alginate/agar discs. B. thuringiensis (Bt) is a bacterium well-known for its broad capabilities and it is widely used in biological control [Pseudomonas, Micrococcus, Acinetobacter, Aspergillus, Trichoderma, and Candida [Since the late 1980s, extensive cypermethrin applications in the agriculture and household sectors have led to toxic effects on organisms, necessitating its removal from contaminated environments. In this study, a cypermethrin-degrading bacterium was isolated from a pyrethroid-contaminated agricultural field. The bacterial isolate was characterized as  control . Bt has  control ,42,43. H control . Other c Candida ,45,46. T Candida . All the Candida .k value of 0.1035 day−1). Therefore, these conditions were used for all the experiments. These results were consistent with findings reported by Chen et al. [Temperature, pH, and shaking speed are considered important factors that influence the xenobiotic degradation ability of microbes ,28,30. Un et al.  who foun2–CN, and (C=O)−O bond [Bacillus sp. SG2 after 15 days of cypermethrin degradation. Biodegraded cypermethrin with Bacillus sp. SG2 revealed major changes in the range of 100 to 1650 cm−1 and 2259 to 3431 cm−1 [Cypermethrin and its intermediate metabolites were effectively detected and characterized by FTIR analysis . The mai)−O bond . Ether c431 cm−1 . Photoly431 cm−1 . The FTI431 cm−1 .Stenotrophomonas, Acinetobacter, Bacillus, Raoultella, Pseudomonas, and Brevibacterium, and fungal strains Aspergillus, Candidia, and Trichoderma [The study of an organism’s metabolic pathways is crucial for analyzing the biodegradation potential of a microorganism. Intermediate metabolites are sometimes more toxic than the parent compound ,53,54. Wchoderma ,59,60,61B. thuringiensis strain SG4 enhanced cypermethrin degradation compared with freely suspended cells. Tallur et al. suggested that agar disc and sodium alginate stabilize membrane permeability and offer protection against toxic intermediates [t1/2 was reduced by 177 days in soil compared with soil without the SG4 strain. Unlike most bacterial isolates, strain SG4 has an exceptional ability to degrade pollutants in soil, highlighting its potential as a potent strain for the bioremediation of pyrethroid-contaminated environments.The addition of sodium alginate and agar discs with mediates . During mediates ,62,63. Wmediates ,64,65. IB. thuringiensis SG4 uses cypermethrin as a carbon and nitrogen source, enabling its colonization of a niche area in a natural ecosystem where cypermethrin is present under limited nutrient conditions. Strain SG4 degraded cypermethrin in minimal medium, soil slurry, and in an immobilized culture assay with sodium alginate and agar disc beads. Our findings suggest that strain SG4 can efficiently degrade cypermethrin and similar ester-bond-containing pesticides. Our study of metabolites revealed that strain SG4 follows a complete degradation pathway to degrade cypermethrin. The degradation kinetics of cypermethrin under various treatments indicated that the strain could be used in large-scale applications in contaminated soil and water areas. Analysis with high-throughput techniques suggested that strain SG4 should be further explored to increase the understanding of the molecular machinery that regulates its degradation ability of toxic insecticides.


Purpose:N6-methyladenosine (m6A) mRNA methylation is affected by dietary factors and associated with lipid metabolism; however, whether the regulatory role of resveratrol in lipid metabolism is involved in m6A mRNA methylation remains unknown. Here, the objective of this study was to investigate the effect of resveratrol on hepatic lipid metabolism and m6A RNA methylation in the liver of mice.Methods: A total of 24 male mice were randomly allocated to LFD (low-fat diet), LFDR (low-fat diet + resveratrol), HFD (high-fat diet), and HFDR (high-fat diet + resveratrol) groups for 12 weeks (n = 6/group). Final body weight of mice was measured before sacrificing. Perirhemtric fat, abdominal and epididymal fat, liver tissues, and serum were collected at sacrifice and analyzed. Briefly, mice phenotype, lipid metabolic index, and m6A modification in the liver were assessed.Results: Compared to the HFD group, dietary resveratrol supplementation reduced the body weight and relative abdominal, epididymal, and perirhemtric fat weight in high-fat-exposed mice; however, resveratrol significantly increased average daily feed intake in mice given HFD. The amounts of serum low-density lipoprotein cholesterol (LDL), liver total cholesterol (TC), and triacylglycerol (TAG) were significantly decreased by resveratrol supplementation. In addition, resveratrol significantly enhanced the levels of peroxisome proliferator-activated receptor alpha (PPARα), peroxisome proliferator-activated receptor beta/delta (PPARβ/δ), cytochrome P450, family 4, subfamily a, polypeptide 10/14 (CYP4A10/14), acyl-CoA oxidase 1 (ACOX1), and fatty acid-binding protein 4 (FABP4) mRNA and inhibited acyl-CoA carboxylase (ACC) mRNA levels in the liver. Furthermore, the resveratrol in HFD increased the transcript levels of methyltransferase like 3 (METTL3), alkB homolog 5 (ALKBH5), fat mass and obesity associated protein (FTO), and YTH domain family 2 (YTHDF2), whereas it decreased the level of YTH domain family 3 (YTHDF3) and m6A abundance in mice liver.Conclusion: The beneficial effect of resveratrol on lipid metabolism disorder under HFD may be due to decrease of m6A RNA methylation and increase of PPARα mRNA, providing mechanistic insights into the function of resveratrol in alleviating the disturbance of lipid metabolism in mice. Lipids are critical nutrients and energy substances in both human and animals, whereas long-term high-fat diet (HFD) could result in defective nutritional metabolism, particularly in hepatic lipid metabolism . HepaticResveratrol  is a natural polyphenolic compound found in plants. It is well known that resveratrol has antioxidative , anti-inN6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotes, which accounts for over 60% of all RNA chemical modifications    , demethynd WTAP) , and m6A YTHDF3) . m6A RNAficiency . In additabolism . Recentl betaine  and curc betaine . Li et a6A levels. Thus, the aim of this study was to investigate the effect of resveratrol on lipid metabolism and m6A RNA methylation in the liver of mice.We speculated that resveratrol in a HFD alleviated liver lipid metabolism disorders maybe due to the changes of mAll experimental procedures were conducted in conformity with the Chinese Guidelines for Animal Welfare and were approved by the Animal Care Advisory Committee of Nanjing Agricultural University, China (NJAU-CAST-2015-095). Twenty-four C57BL/6J male mice (5 weeks of age) were from Yangzhou Institute of Experimental Animals [SCXK (Su) 2012-0004]. After 3 weeks of acclimation, mice were randomly distributed into four groups of six mice each as follows: 10% LFD and dietary supplemented with 276 mg/kg of resveratrol (LFDR), 60% HFD and dietary supplemented with 400 mg/kg of resveratrol (HFDR) . There iResveratrol  used in the experiment was bought from Sigma-Aldrich. We used high performance liquid chromatography (HPLC) analysis to confirm the concentration of resveratrol. All diets were manufactured by Trophic Animal Feed Co., Ltd. . Composition and nutritional levels of mice diet were based on AIN93 . The LFDMice body weight in the HFD group was higher up to 4 g (>4 g) than in the LFD group at the end of 12 weeks, suggesting that we successfully built a model of obesity . Final bg for 1 min in ice-bath using homogenizer . The homogenate was spun at 3,000 g for 15 min at a temperature of 4°C, and the supernatant was collected and analyzed immediately.The liver sample (0.2 g) from −80°C was suspended in ice-cold physiological saline  and then homogenized at 13,500 The levels of total cholesterol , triacylglycerol , and low-density lipoprotein cholesterol  were measured using commercial kits  by a microplate reader  with a detection wavelength of 510, 510, and 546 nm, respectively. All experimental procedures were performed according to the manufacturer’s protocols. All hepatic measurements were normalized to concentrations of total protein for intersample comparisons.The liver sections fixed in 10% paraformaldehyde were dehydrated with graded dilutions of ethanol and embedded in paraffin. Then tissues (5 µm) were deparaffinized with xylene and rehydrated with graded dilutions of ethanol. The slides were stained with hematoxylin and eosin (H&E). A light microscope  was used to photograph and evaluate the pathological changes.For oil-red staining, fresh livers frozen at −80°C were sectioned (5 µm thick), fixed in a slide, and dissolved in propylene glycol (2 min). Slides were transferred to oil-red O solution  for 1 h, then immersed in 85% propylene glycol (1 min), and washed two times in water. Finally, slides were counterstained in hematoxylin solution (10 s) and mounted using glycerin.−ΔΔCT method after normalization to housekeeping gene GAPDH. Samples in the LFD group were used as calibrator.Total RNA of snap-frozen liver was extracted using TRIZol reagent . The RNA integrity was examined on 1% of agarose gel using GelRed staining. The RNA contents were quantified by Thermo NanoDrop 2000 Ultra Trace Visible Spectrophotometer . After that, 1,000 ng total RNA was reverse-transcribed into cDNA in a 20 μl reaction volume using the PrimerScript RT Reagent kit . Real-time PCR was performed on the QuantStudioTM Design & Analysis Software . Primers were synthesized by Invitrogen Biotech Co., Ltd.  and listed in 6A RNA Methylation Quantification Kit was used to detect total RNA m6A levels  according to previous studies . The m6A level was calculated by OD intensity.A total of 200 ng aliquots of mRNA was extracted from liver. EpiQuikTM m studies . Brieflyg and 4°C for 30 min. The protein concentrations were measured using a commercial kit . Samples (40 μg of protein) were mixed with 5× sample buffer and boiled at 100°C for 10 min. Separation of the protein samples was performed on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and electrotransferred onto the polyvinylidene fluoride (PVDF) membranes  with transfer buffer. The PVDF membranes were incubated overnight with primary antibody at a temperature of 4°C after being blocked in tris-buffered saline (TBS) containing 5% nonfat milk and 0.1% Tween-20 for 1 h at RT. After three times of washing, horseradish peroxidase-conjugated secondary antibodies  were applied to incubation of the membranes for 90 min at RT. The bands were visualized using enhanced chemiluminescence (ECLPlus) detection kit . The images were analyzed by a luminescence image analyzer LAS-4000 system  and were quantified by Gel-Pro Analyzer 4.0 software . Some information about primary antibodies is as follows: methyltransferase like 3 , YTH domain family 2 , alkB homolog 5 , FTO , and β-actin .Proteins from each 20 mg liver were extracted using tissue lysis buffer  at a temperature of 4°C. Then, the homogenate was centrifuged at 12,000 p values < 0.05 were considered as statistically significant level. p values < 0.01 were regarded as very significant.Data were analyzed by the two-way analysis of variance (ANOVA) and were presented as means ± SD (standard deviations) after confirming normally distributed patterns. The classification variables were dietary resveratrol supplementation (LFD + HFD × LFDR + HFDR), HFD (LFD + LFDR × HFD + HFDR), and their interaction (LFD × LFDR × HFD × HFDR). The significant difference among groups was examined by Duncan’s multiple range tests when significant difference of resveratrol × HFD interaction was examined. The SPSS 21.0 statistical software  was used to analyze the present results. p < 0.05, p < 0.05, p < 0.05, p < 0.05, During the entire 12-week period, mice body weight in the HFD group was higher than that of the HFDR group . The finp < 0.05, p < 0.05, Mice given HFD (HFD and HFDR) exhibited significant increase of the liver, abdominal, epididymal, and perirhemtric fat weight  in serum of HFDR mice (p < 0.05) than HFD mice. However, the differences in TAG and TC in serum were not found between the HFD and HFDR groups. In addition, there was a significant enhancement (p < 0.05) in the concentrations of TAG, TC, and LDL in the liver of HFD mice compared with mice fed LFD alone. Notably, resveratrol could reverse the increase of TAG induced by HFD (p < 0.05).The contents of TC and LDL in the serum of HFD mice were significantly higher than those of LFD mice . However, the levels of ACC and PPARγ mRNA in HFDR mice were reduced by resveratrol compared to untreated HFD group (p < 0.05). We also noted that dietary resveratrol in the LFD increased (p < 0.05) the expression of PPARα, PPARβ/δ, CPT1α, CYP4A10, CYP4A14, ACOX1, and FATP4 mRNA relative to LFD.We next measured the expression of lipid metabolism regulatory genes. Compared with LFD mice, HFD downregulated the expression of hepatic 6A methylation, we tested m6A and m6A-related genes and proteins. Compared with LFD mice, HFD significantly downregulated the gene expression of ALKBH5 and FTO while it obviously increased the level of YTHDF3  the levels of METTL3, YTHDF2, FTO, ALKBH5, and mRNA and decreased the mRNA expression of YTHDF3 in HFDR mice , a late adipocyte differentiation marker. Fellous et al. recently confirmed that activation of PPAR receptors could promote the expression of FABP4 and other downstream genes  as a methyl donor .JW, YL, and XZ designed the research and wrote the paper, and JW was a major contributor in writing the manuscript. JW, YL, and ZG searched and read the literature. JW, YL, WW, and JY performed experiments. CW, LZ, and TW provided essential suggestion and revision. XZ had primary responsibility for final content. All authors read and approved the final manuscript.This study was supported by grants from the National Natural Science Foundation of China (No. 31872391).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.


Embryos  of Japanese medaka were exposed to 0.001 ppb, 0.1 ppb, or 10 ppb DEHP for 21 days and returned to control water (without DEHP) for maturation (4 months old). At day 9 of the exposure study, mortality was significantly increased in medaka embryos (before hatching) treated with 0.001 ppb and 10 ppb DEHP. Continual exposure of young hatchlings for an additional 12 days  resulted in a significant increase in mortality in fish exposed to 0.001 ppb, 0.1 and 10 ppb DEHP. Significant reduction in egg production was observed in adult female medaka (4 months old) with prior exposure to 0.1 ppb and 10 ppb DEHP for 21 days during early development. Fertilization and hatching success were also significantly reduced in breeding pairs with prior exposure to 0.001 ppb, 0.1 ppb and 10 ppb DEHP during early life stage. Histological analysis of adult male gonads revealed a significant decline in mature sperm count accompanied by an increase in interstitial space in fish exposed to 0.1 ppb and 10 ppb DEHP during early development. Likewise, the amount of vitellogenic (mature) oocytes observed in the ovaries of adult female with transient exposure to 0.1 ppb and 10 ppb DEHP was significantly reduced when compared with the solvent control group. Our data suggest that transient exposure to ultra low concentrations of DEHP during sensitive time windows of development results in irreversible reproductive impairment which may impact fish populations negatively.Di-(2-ethylhexyl) phthalate (DEHP) is a well-known endocrine disruptor and it is ubiquitously distributed in the environment. However, very few studies have investigated the effects of short-term exposure to environmentally realistic concentrations of DEHP during early developmental stages and its chronic effects. This study monitored the long-term effects of transient exposure to DEHP in early life stages (F These chemicals are commonly found in wire and cables, self-adhesives, medical devices, cosmetics, food packages and many househould and personal care products . PhthalaAlthough DEHP has been shown to impair reproduction, reduce sperm quality and lower testosterone levels in several fish species, such as zebrafish, goldfish and fathead minnow, majority of these studies only looked at the effects of DEHP on the adults ,22; GolsOryzias latipes, were exposed to three concentrations of DEHP  for 21 days since 4 h post fertilization (4 hpf). The objectives of the present study were to investigate (i) if exposure to environmentally realistic concentrations of DEHP affect survival of embryos and young hatchlings, and the time fertilized eggs requires to hatch (hatching time), (ii) if transient exposure to environmentally realistic concentrations of DEHP during early developmental stages permanently affects reproduction success in adult fish in terms of egg production, fertilization and hatching success; and (iii) if transient exposure to environmentally realistic concentrations of DEHP during early life stage permanently alters oogenesis and spermatogenesis. Japanese medaka was chosen as the model-organism due to (i) the transparency of the medaka’s eggs allows embryonic development to be observed without sacrificing the embyros; (ii) extensive information is available of the physiology, embryology and genetics of Japanese medaka, and (iii), the ease of culture of this species in the laboratory, such as daily spawning, external fertilization and relatively short generation time (approximately 3 months) [In this study, embryos of Japanese medaka,  months) ,25.2All animal procedures were conducted in accordance and approved by the UIC Animal Care and Use Committee.2.1DEHP was obtained from Dr. Ehrenstorfer GmbH . Stock solutions of DEHP  were prepared by dissolving DEHP in acetone , and the final acetone concentration in the exposure water was 0.02 % (v/v), i.e., 200 μl of each stock solution  was added into 1 L embryo rearing medium (ERM) to prepare 0.001 ppb, 01 ppb and 10 ppb DEHP working grade solutions. Solvent control contained acetone only.2.2Oryzias latipes, were cultured in flow-through tanks under a constant 14 h/10 h light/dark cycle at 24 ± 2 °C, with pH maintained at 7.4 ± 2. Adult fish were fed twice daily with Otohime Fish Diet  and fresh Artemia nauplii. During breeding, adult medaka, i.e., breeding pairs , were raised in 1X Embryo Rearing Medium (ERM) prepared by diluting 25 mg of neomycin sulfate, 50 μl stock solution (35 % w/v) of sodium thiosulfate, 125 μl Amquel® and 200 μl of stock solution (0.1 % w/v) of methylene blue in 1 L of pure water.Adult Japanese medaka, 2.2.1Eggs from 10 breeding pairs were pooled and sorted using stereomicroscope . Transparent eggs with doubled membrane (approximately 4 hpf) were assigned to petri dishes each containing 30 fertilized eggs for exposure study. Preliminary range finding test on DEHP on embryonic toxicity were conducted by exposing 4 hpf embryos to 0.0001 ppb, 0.001 ppb, 0.01 ppb, 0.1 ppb, 1 ppb and 10 ppb DEHP, with control and solvent control groups for 9 days . Nominal concentration was used in all cases. Solutions were renewed daily throughout the 9-day exposure period and a total of 90 fertilized eggs  were used for each control and treatment groups. Based on the results obtained from the range finding test, subsequent long-term monitoring (5 months) on the effects of transient exposure to DEHP during early developmental stages were conducted in survived hatchlings with prior exposure to 0.001 ppb, 0.1 ppb and 10 ppb DEHP.2.2.2Fertilized eggs (4 hpf) were exposed to control group, solvent control group , 0.001 ppb DEHP, 0.1 ppb DEHP and 10 ppb DEHP treatment groups for 21 days. Nominal concentration was used in all cases. Solutions were renewed daily throughout the 21-day exposure period and a total of 90 fertilized eggs  were used for each control and treatment groups. After 21-day exposure, surviving larvae/young hatchlings were returned to ERM (minus DEHP and solvent) and flow-through tanks for maturation (to reach 4 months of age). Temperature, light/dark cycle, pH and feeding regimes were identical as those used for brood stocks.2.3Artemia nauplii. Stereomicroscope was used to record the number of eggs produced and fertilization success  per breeding pair. Fertilized eggs were placed in petri dishes containing 1X ERM and observed for hatching success (9-day observation period). ERM was renewed daily and dead embryos were discarded.At 4 months old, adult medaka which have been transiently exposed to DEHP during early development were arranged in breeding pairs for the reproduction study. To breed, one male and one female adult medaka were placed in 1 L of 1X ERM and were allowed to breed freely for 40 days. Five pairs of adult fish (n = 5) were used for each control and treatment groups with ERM renewed daily. Eggs were collected twice daily 1 h after the mating pairs were fed with fresh 2.4At the end of the experiment, five male (n = 5) and five female (n = 5) adult medaka (approximately 5 months old) for each control and treatment groups were dissected. Their gonads were fixed in 4 % paraformaldehyde, dehydrated in graded ethanol series, cleared in xylene and embedded in paraffin. Tissues were sectioned at 5 μm thickness and stained by Hematoxylin and Eosin for morphological analysis using a Leica light microscope . Images of male and female gonads were captured by SPOT Insight Color Digital Camera with SPOT Software . Image Pro Plus (IPP) 6.0  was used to manually point count different cell types. Five images (20X magnification for ovaries and 40X magnification for testes) were captured randomly per fish and used for quantitative analyses. A digital grid mask (24*18 = 432) intersection points from IPP was superimposed on each image. For testes, spermatocyte/spermatogonia, spermatids/spermatozoa and interstitial space were counted in red, blue and green dots, respectively. For ovaries, stage I oocytes (perinucleolar oocyte), stage II oocytes  and stage III oocytes (vitellogenic oocytes) were counted in red, green and blue dots, respectively.Percentage of each cell type was calculated using the formula2.5p ≤  0.05) between the controls and DEHP treatment groups was identified, pairwise comparisons between each individual treatment and control groups were carried out using the Holm-Sidak method. Likewise, whenever significant difference (p ≤  0.05) was detected within the same control and/or treatment group, pairwise comparisons on the amount of eggs produced, amount of eggs fertilized and successful hatch outs were carried out using the Holm-Sidak method. In cases where data failed to follow Gaussian distribution, log10 or arcsin squart root transformation would be performed prior to analysis. Statistics were performed using the statistical software SigmaPlot, Version 12 . Graphs were plotted using Graphpad Prism, Version 7.00 .One-way analysis of variance was used to test the null hypotheses that exposure to different waterborne concentrations of DEHP did not cause significant changes in (i) mortality and hatching success of embryos and hatchlings during the 9-day and 21-day exposure periods; (ii) spermatogenesis in adult male medaka transiently exposed to DEHP during early development; and (iii) oogenesis in adult female medaka transiently exposed to DEHP during early development. Two-way analysis of variance was used to test the null hypothesis that transient exposure to different concentrations of DEHP during early developmental stages do not cause significant changes in the amount of (i) eggs produced, (ii) eggs fertilized and (iii) eggs hatched by these adult fish. If a significant difference (33.1p ≤  0.05)  . No sign≤  0.05) . Due to p > 0.5)  .Fig. 2Av3.2Significant increase in mortality was observed in hatchlings exposed to 0.001 ppb, 0.1 ppb and 10 ppb DEHP for 21 days since 4 hpf, with mortalities recorded at 55.0 ± 6.0 %, 56.0 ± 5.6 % and 59.7 ± 9.4 %, respectively, when compared with the solvent control (26.7 ± 3.3 %) .Fig. 3Mo3.3After exposure to 0.001 ppb, 0.1 ppb or 10 ppb DEHP for 21 days, survived hatchlings were returned to 1X ERM (minus DEHP and acetone) for maturation. At approximately 4 months old, a 40-day breeding study was performed on the survived adult medaka to determine if prior DEHP exposure affected reproduction success of the adult fish. Number of eggs successfully fertilized by breeding pairs with prior exposure to 0.001 ppb. 0.1 ppb and 10 ppb DEHP during early development were significantly lower than those of the control and solvent control groups . MoreoveHatching success, in terms of number of successful hatch outs, was significantly reduced in breeding pairs with prior exposure to 0.001 ppb, 0.1 ppb and 10 ppb DEHP when compared with those of the control and solvent control groups . Hatch rAlthough within each treatment group , the number of successful hatch outs was significantly lower than the number of eggs produced by the breeding pair , no sign3.4Histological analysis revealed that pre-exposure to 0.1 ppb and 10 ppb DEHP during early development significantly affected oogenesis in female medaka b and 7 .3.5A histo-morphological inspection of testes revealed that transient exposure to DEHP during early development significantly reduced mature sperm (spermatozoa and spermatids) count from 27 ± 4 % in the control and 24 ± 6 % in the solvent control to 14 ± 4 % and 13 ± 6 % in the 0.1 ppb and 10 ppb DEHP treatment group, respectively  . The lu4In the past two decades, majority of studies focused on the acute effects of DEHP in murine species ,9,27;. U4.1In contrast to most toxicological studies which exhibit monotonic dose-response relationship , non-monotonic dose response was observed in 9-day old medaka embryos exposed to 0–10 ppb DEHP. At the high end of the exposure spectrum, i.e., 10 ppb DEHP, mortality was significantly higher than that of the control groups. As the dose diminished, i.e., 0.01 ppb–1 ppb DEHP, mortality of 9-day old medaka embryos decreased to that of the control level. Further reduction in DEHP concentration, i.e., at 0.001 ppb, however, resulted in significantly higher mortality than those of the control groups . Non-monSignificantly higher mortality was observed in medaka hatchings exposed to 0.001 ppb, 0.1 ppb and 10 ppb DEHP 21 days since 4 hpf , which cAlthough a few studies have reported significant delays in hatch out time in medaka embryos exposed to 0.1–1 ppb DEHP ,35, no s4.2Throughout the study period (approximately 5 months in length), no significant difference was found between the control and solvent control group in terms of mortality, reproduction success  and morphological analyses of the gonads of male and female medaka, indicating that 0.02 % v/v acetone has neglectable effect on Japanese medaka. Transient exposure to 0.001 ppb, 0.1 ppb and 10 ppb DEHP during early development significantly reduced egg production in 4 month-old female medaka . Similarboule gene [cyp19a in testes, and plasma 17β-estradiol and testosterone levels [Transient exposure to 0.001 ppb, 0.1 ppb and 10 ppb DEHP during early development not only resulted in permanent reduction in egg production in adult female fish, fertilization success of eggs produced by these breeding pairs  was also significantly reduced . The redule gene , peroxisule gene , cyp19a e levels ,42,48. EAlthough exposure to DEHP has been shown to increase reactive oxygen species (ROS) production by reducing sperm motility and inhibiting follicle growth in mice and rats , such prAlthough the reduction in the number of eggs laid by female fish and fertilization success could be explained by the adverse effects of DEHP on the actions of sex steroid hormones resulting in the inhibition of oogenesis  and sper0 adults. Foran et al. [0 generation than by exposure to EE2 or young hatchlings alone. It is worth noting that in this study, although significant reductions in egg production and fertilization success were observed in adult fish transiently exposed to DEHP during early development, hatch rates  were not significantly affected (0), development of embryos (F1 generation) did not appear to be affected, as almost all fertilized eggs managed to hatch out successfully (1, F2 and F3 generations remain virtually unknown. Recently, exposure to DEHP has been found to significantly alter the expression of certain genes in the male gonads via DNA methylation on their promoter regions, thereby reducing sperm quantity and quality [In this study, significant reductions in egg production and fertilization success were observed in adult fish transiently exposed to DEHP during early development . Our resn et al.  exposed n et al. , in earlaffected . Resultsessfully . Althoug quality ,60. Furt5To better protect the environment and human health, the Canadian Council of Ministers of the Environment recommends an upper limit of 16 ppb DEHP in surface water  and the The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.B.B.H.Y., and A.B.Q designed the experiment. A.B.Q. performed the experiments. B.B.H.Y. and A.B.Q. analyzed dat. All authors prepared and reviewed the manuscript.Bonny Bun Ho Yuen: Conceptualization, Writing - review & editing, Funding acquisition. Anna Boya Qiu: Visualization, Validation, Investigation, Methodology, Formal analysis. Bruce Hao Chen: .


Gallbladder cancer (GBC) is the most common tumor of the biliary tract. The incidence of GBC shows a large geographic variability, being particularly frequent in Native American populations. In Chile, GBC represents the second cause of cancer-related death among women. We describe here the establishment of three novel cell lines derived from the ascitic fluid of a Chilean GBC patient, who presented 46% European, 36% Mapuche, 12% Aymara and 6% African ancestry.TP53 and ERBB2 with some differences among the three cells lines, and revealed a novel exonic mutation in NF1. The PUC-GBC3 clone was the most aggressive according to histopathological features and the tumorigenic capacity in NSG mice.After immunocytochemical staining of the primary cell culture, we isolated and comprehensively characterized three independent clones  by short tandem repeat DNA profiling and RNA sequencing as well as karyotype, doubling time, chemosensitivity, in vitro migration capability and in vivo tumorigenicity assay. Primary culture cells showed high expression of CK7, CK19, CA 19-9, MUC1 and MUC16, and negative expression of mesothelial markers. The three isolated clones displayed an epithelial phenotype and an abnormal structure and number of chromosomes. RNA sequencing confirmed the increased expression of cytokeratin and mucin genes, and also of The first cell lines established from a Chilean GBC patient represent a new model for studying GBC in patients of Native American descent. Gallbladder cancer  accounts for 80–95% of biliary tract cancers and is the sixth most common gastrointestinal cancer worldwide . The regOne of the major characteristics of GBC is its late diagnosis and ineffective treatment . The lacTo improve the clinical prognosis of GBC patients, more efficient methods for early diagnosis and more effective therapeutic approaches must be developed. Research on GBC is challenging not only because it is a rare tumor worldwide, but also due to the difficulty of accessing human tissue samples and the lack of experimental models. Obtaining fresh frozen tissue samples is extremely demanding because most of the early lesions are thin, flat, incidental and masked with gross acute inflammatory changes, and chemotherapy and palliative care is frequently offered to patients with advanced tumor after diagnosis confirmation by fine needle aspiration biopsy . The proA large proportion of the current knowledge on GBC biology comes from studies performed using GBC cell lines, which have played a critical role in identifying therapeutic targets and facilitating the rapid screening of new anticancer compounds. Most GBC cell lines derive from primary tumors and metastatic sites and have been isolated from Japanese –16, KoreTo our knowledge, this is the first report on the development and characterization of cell lines derived from a GBC patient of Native American descent. We describe the isolation of three cell lines from the ascites of a single patient, which conserved the genomic features and malignant behavior of the primary GBC tumor. Our finding suggest that the three established cell lines reflect the heterogeneity of GBC tumors and provide a useful model to investigate the mechanisms underlying late stage GBC progression and metastasis, gain new insight into drug resistance mechanisms and test new therapeutic strategies.We successfully established a primary culture of tumor cells derived from the ascitic fluid of a 60-year-old man diagnosed with metastatic GBC, who showed the following percentages of genetic ancestry: 46% European, 36% Mapuche Native American, 12% Aymara Native American and 6% African. Moderate to strong expression (2+ to 3+) of the epithelial markers CK7 (cytokeratin 7) and CK19 (cytokeratin 19) was observed in 100% of the ascites-derived cells, mesothelin was not expressed. Almost 90% of the cells showed high expression levels (3+) of the tumor carbohydrate antigen 19-9 (CA 19-9) and moderate expression (2+) of the mucins MUC1 (mucin 1) and MUC16 (mucin 16)  gene and poor prognosis of GBC [The primary culture consisted of epithelial cells with different morphologies, which grew as heterogeneous cell populations. In order to generate monoclonal cell lines, we used a limiting dilution procedure to isolate and spread individual cells. We established three cell lines as separate cultures, namely PUC-GBC1, PUC-GBC2 and PUC-GBC3. In consistency with the primary culture, the newly established cell lines showed high expression levels of the epithelial markers CK7 and CK19  and no mesothelin expression  for the three cell lines. The karyotype for PUC-GBC1 was: XY,-X, +3, +4, +5, +5, −7, +10, +11, +12, +12, +12, +13, add(13)(q33), +14, +15, +16, +16, −17,+ 19, −21, −21, −22, −22. For PUC-GBC2: XY, +1, +2, +3, +4, +5, +9, +10, +10, +13, +14, +14, add(q31), +19, −20, −22 [KRT7 and KRT19) and mucin (MUC1 and MUC16) genes, as well as TP53 with some differences in expression among the cell lines. For example, TP53 was weakly expressed in PUC-GBC3. The principal component analysis (PCA) of the three cell lines and the 20 genes with the largest variability in gene expression (highest coefficient of variation) revealed that the expression profiles of PUC-GBC2 and PUC-GBC3 were more similar to each other than to PUC-GBC1  and cell metabolism (CYP2B6 and SLC44A4) were overrepresented in PUC-GBC1. Additional file The analysis of RNA sequencing data confirmed the increased expression of cytokeratin . According to SIFT (Sorting Intolerance from Tolerance) and Polyphen-2, this mutation has a damaging amino acid impact (p.S1100L). Additional file CDKN1A (rs1801270), MDM4 (rs4252716), PIK3CA (rs2230461), ERBB2 (rs1058808) and ERBB3 (rs55699040), a frameshift deletion in CDKN2A (NA), among others , along with ICP = 0.0073)  and PUC-GBC1 (P = 0.031) at day 38.In vivo tumorigenicity assays revealed palpable tumors in all mice within 15 days. As shown in Fig. The hematoxylin- and eosin-stained tissue sections of the mice tumor xenografts showed histological features of adenocarcinoma, with high mitotic index, anisokaryosis and anisocytosis. Particularly, PUC-GBC1 and PUC-GBC2 xenografts were histologically similar and characterized by the presence of tubular structures constituted by cells with enlarged, hyperchromatic nuclei, frequent mitotic activity, abundant apoptotic bodies, and murine stroma dominating the tumor mass. In contrast, PUC-GBC3 tumors had a more aggressive appearance, with a more solid growth and less gland formation; also, isolated signet ring cells and scarce infiltration of the host stroma were noted Fig. .Fig. 9XeERBB2 expression for all GBC cell lines, and particularly PUC-GBC1 and PUC-GBC2  showed a strong positivity of the epithelial markers CK7, CK19 and E-cadherin , which were also expressed in the primary tumor  protein (collagen α‑1(III)) associated with tumor progression [POSTN, also overrepresented in PUC-GBC2, might explain the higher migratory ability of these cells. POSTN encodes for periostin, a multifunctional ECM protein that contributes to the remodeling of the tumor microenvironment during tumor progression [HNF1A, a transcription factor recently reported to be enriched in pancreatic cancer stem cells [TP53 and ERBB2 expression for PUC-GBC1 and PUC-GBC2, and revealed a novel exonic mutation in NF1.We report in this study on the phenotypic, genomic and functional characteristics of three new GBC cell lines derived from the metastatic ascites of a Chilean male patient; these were named PUC-GBC1, PUC-GBC2 and PUC-GBC3. Mapuche Native American ancestry has been strongly associated with the risk of GBC, and we found that the percentage of Mapuche Native American ancestry of the male donor of the primary tumor cells was 36%. For comparison, the first (third) quartiles of Mapuche ancestry reported in a recent study that included 1805 Chileans were 28% (43%) [gression , 30 and gression –33. At tgression , 35. Simgression , 38. On em cells  and a keem cells . RNA-SeqWe confirmed the epithelial origin of the primary culture and the derived cell lines by evaluation of the immunocytochemical expression of CK7 and CK19  in all cell lines, which has not been reported as an oncogenic mutation but likely possesses a pathogenic role according to the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/). Additionally, we found a nsSNV in MDM4 and a frameshift deletion in CDKN2A, both genes encoding proteins related with regulation of p53  and submitted to immunocytochemistry analysis to confirm the epithelial origin and evaluate the expression of tumor markers. This analysis was repeated at passage 17. Primary culture cells were subcultured until they reached more than 20 passages. Then, three individual clones were obtained by serial dilution of the primary cell culture in a 96-well plate. Once established, all cell lines were cultured in DMEM supplemented with 5% FBS and 1% P/S. All cultures were tested for mycoplasma contamination with a commercial PCR kit .The primary cell culture was established from the ascites of a 60-year-old male patient with GBC. Histological examination classified this tumor as moderately differentiated tubular adenocarcinoma with a T3NxM1 stage. There was evidence of vascular and lymphatic system invasion and peritoneal carcinomatosis. Ascitic fluid (50 mL) was obtained from the patient, delivered to the laboratory and centrifuged at 1000 rpm for 10 min, and the pellet was rinsed twice with sterile phosphate-buffered saline  containing antibiotics . The supernatant was removed and saved as ascitic fluid supplement at -20 °C, and the remaining cells were resuspended in DMEM/F12 medium  supplemented with 5% FBS, 50% of ascitic fluid  and 1% P/S, seeded into 6-well culture plates, and incubated at 37 °C in a humidified atmosphere of 5% COPrimary antibodies and working dilutions used for immunostaining analysis were mouse monoclonal anti-cytokeratin 7  and mouse monoclonal anti-p53 (Cat. No IS616: Clone DO-7) from Dako ; mouse monoclonal anti-mesothelin  from Biocare Medical, LLC ; mouse monoclonal anti-CA 15-3  from Biogenex ; mouse monoclonal anti-cytokeratin 19 , mouse monoclonal anti-CA 19-9 , mouse monoclonal anti-CA 125 , rabbit monoclonal anti-calretinin , mouse monoclonal anti-vimentin , mouse monoclonal anti-E-cadherin  and rabbit monoclonal anti-HER2  from Roche Tissue Diagnostics .5 per well in 24-well plates and allowed to attach for 24 h. Following fixation in 4% paraformaldehyde for 15 min, PBS wash and permeabilization with 0.1% Triton X-100, cells were incubated with primary antibodies at 1:100 dilution for 1 h. Then, cells were washed four times with 0.2% BSA  in PBS and incubated for 30 min with the SuperPicture™ Polymer Detection Kit . The bound antibody was visualized with a red chromogen . Antibody for HER2/neu was immunostained on the Benchmark XT automated stainer  according to the manufacturer’s instructions. Images were acquired using the EVOS XL Core Imaging System .The epithelial and tumor origin of the primary culture from the ascites and the derived cell lines was evaluated by immunocytochemistry. Cells were seeded at 2 × 10The primary tumor and xenografts were analyzed immunohistochemically on formalin-fixed paraffin-embedded (FFPE) sections (2 μm thickness) using the automated immunostainer BenchMark ULTRA and the ultraVIEW universal DAB detection kit . Digital images were captured using the Aperio AT2 Digital Pathology Scanner . The expression was evaluated by the pathologist (J.C.R.) considering the intensity of the staining  and the percentage of labeled cells. Immunohistochemical scoring of HER-2 expression was based on the system proposed for gastric and gastroesophageal junction cancer .® Genomic DNA mini kit .The genetic profiling of the cell lines derived from the primary cell culture was established using the polymorphic STR loci detection service offered by the Department of Molecular Medicine at Aarhus University Hospital (IdentiCell service for human cell line authentication). DNA of each cell line was purified using the PureLink® Solution, Irvine Scientific, Santa Ana, CA, USA) for 45 min at 37 °C. Then, cells were harvested to arrest metaphases and treated with the hypotonic solution (0.075 M KCl) for 10 min at 37 °C. After two changes in the fixative , the cell suspension was incubated overnight at -20 °C. The next day, the fixative was changed another three times and two drops of the cell suspension were dropped from a distance of about 50 cm onto clean dry slides tilted at an angle of about 45°, allowing the cells to roll across the slide. After air drying, one slide was stained with Giemsa (3:1 ratio of Gurr Buffer and Giemsa Stain) and analyzed under a light microscope at 10× and 100× magnification. If the metaphase cells were abundant and well spread, the remaining slides were used for chromosome analysis using trypsin G-banding. To determine ploidy, chromosomes were counted from a minimum of 50 metaphase spreads using GenASIs Karyotyping .The cytogenetic karyotyping analysis was performed using a standard air-dried method. Briefly, cells in an exponential growth phase were treated with a final concentration of 0.1 µg/mL colcemid . The doubling times were determined from the growth curves using the data generated from 3 independent experiments, each with three technical replicates.3 cells/well in 100 μL cell culture medium. After an overnight attachment period, cells were treated with either gemcitabine (starting at 300 µM), cisplatin (starting at 350 µM) or fluorouracil (starting at 2 mM) with a serial dilution of 1/3. Following 72 h of drug incubation, cell viability was assessed by incubating the cells for 2 h at 37 °C with a MTS-PMS colorimetric solution , and absorbance of each well was read at a wavelength of 490 nm using a multiwell plate reader . The half maximal inhibitory concentration was calculated from the dose response curves . Three independent experiments were performed, each with three technical replicates.The cytotoxic drugs used for in vitro chemosensitivity assays were gemcitabine , cisplatin  and Fluorouracil . These drugs are the main chemotherapy agents used to treat GBC. Cells were seeded into 96-well plates at a density of 5 × 10® 24-well plates containing polycarbonate filters with an 8-micron pore size . Complete medium was placed in the lower chamber to act as a chemoattractant, and 5 × 104 cells were seeded with serum-free medium into the upper chamber. After 24 h, cells were fixed in methanol for 15 min and stained with 0.05% crystal violet in 25% methanol/PBS for 15 min. Cells on top of the membrane were removed using a cotton swab, and filters were washed with PBS. Cells on the underside of filters were viewed and counted under a microscope in 10 randomly selected fields. Three independent experiments were performed, each with three technical replicates.Migration assays were performed using Transwellscid gamma (NSG™) mice obtained from The Jackson Laboratory . The animals (n = 9) were randomized into three groups, anesthetized with isoflurane (3% for induction of anesthesia and 1.5% for maintenance) and 3 x 106 cells suspended in 150 μL PBS/matrigel  were injected bilaterally and subcutaneously on the back of the anesthetized mice. Animals were examined every week for the development of tumors. Tumor volumes were estimated twice a week from caliper measurements (volume = 0.52 × (width)2 × length). When tumors had grown to 1.5–2.0 cm3 after 4 weeks, mice were euthanized by carbon dioxide (CO2) and the tumor xenografts were removed, weighted and photographed. Then, tumors were fixed in 10% neutral buffered formalin, and processed for routine histopathological examination.The in vivo tumorigenicity assay was performed using 7 to 8-week-old male NOD http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc). PRINSEQ (0.20.3) [http://broadinstitute.github.io/picard/) and variant calling with GATK 3.5 [Total RNA was extracted and sequenced on an Illumina Hi-Seq 2000. The quality of the sequencing reads was assessed using FastQC (0.11.2) ((0.20.3)  and cuta(0.20.3)  were use(0.20.3) , towardsGATK 3.5 . ThreshoGATK 3.5 . Gene exGATK 3.5  and normThe identified variants with an allele frequency > 5% according to the 1000 Genome Project  were used to infer the genetic ancestry proportions of the donor of the primary GBC tumor cells . SurrogaStatistical analyses were performed using R programming environment in Rstudio© version 1.0 . A Kruskal–Wallis test was used to compare multiple groups, followed by Dunn’s post hoc multiple comparisons test. Probability values smaller than 0.05 were considered statistically significant.Additional file 1. Representative micrographs of immunocytochemical staining for CK7, CK19, Mesothelin, Vimentin and Calretinin in the three clones isolated from the ascites-derived primary culture .Additional file 2. Gene expression of normalized transcripts per million in Chilean GBC cell lines.Additional file 3. Exonic mutations in Chilean GBC cell lines.Additional file 4. Venn diagrams depicting unique and shared (overlapping circles) variants in the three cell lines. (a) non-synonymous; (b) synonymous; and (c) frameshift. The green circle depicts the variants identified in PUC-GBC1, the orange circle depicts the variants identified in PUC-GBC2, and the blue circle depicts the variants identified in PUC-GBC3.Additional file 5. Sequencing statistics.


In recent years, CD109 was also associated with different tumor entities and identified as a possible future diagnostic marker linked to reduced patient survival. Also, different cell signaling pathways were proposed as targets for CD109 interference including the TGFβ, JAK-STAT3, YAP/TAZ, and EGFR/AKT/mTOR pathways. Here, we identify the metalloproteinase meprin β to cleave CD109 at the cell surface and thereby induce the release of cleavage fragments of different size. Major cleavage was identified within the bait region of CD109 residing in the middle of the protein. To identify the structural localization of the bait region, homology modeling and single-particle analysis were applied, resulting in a molecular model of membrane-associated CD109, which allows for the localization of the newly identified cleavage sites for meprin β and the previously published cleavage sites for the metalloproteinase bone morphogenetic protein-1 (BMP-1). Full-length CD109 localized on extracellular vesicles (EVs) was also identified as a release mechanism, and we can show that proteolytic cleavage of CD109 at the cell surface reduces the amount of CD109 sorted to EVs. In summary, we identified meprin β as the first membrane-bound protease to cleave CD109 within the bait region, provide a first structural model for CD109, and show that cell surface proteolysis correlates negatively with CD109 released on EVs.Cluster of differentiation 109 (CD109) is a glycosylphosphatidylinositol (GPI)-anchored protein expressed on primitive hematopoietic stem cells, activated platelets, CD4 CD109 iinocytes , lung cainocytes , pancreainocytes , breast inocytes , glioma inocytes , and maninocytes . It has inocytes . Many siinocytes  and TGFβinocytes , impact inocytes , or inflinocytes , and morinocytes  as well inocytes . CD109 iinocytes . Solomoninocytes  and a GPinocytes . The CD1inocytes . Proteolinocytes . Furtherinocytes  with a tinocytes , which hinocytes   3D reconstruction of CD109, which, together with molecular modeling, results in an experimentally derived structural model of CD109, indicating its position at the cell surface.Unless described otherwise, chemicals and detergents were purchased from Carl Roth GmbH & Co. KG, Karlsruhe, Germany, and disposable material were obtained from SARSTEDT AG & Co. KG, Nümbrecht, Germany.TM, Carlsbad, CA, United States) with 10% fetal bovine serum  and 1% Penicillin/Streptomycin  at 37°C, with 5% CO2, and 95% humidity. Transient transfection was performed as described before  and cultivated in Dulbecco’s Modified Eagle’s Medium high-glucose medium  and Cellometer® Auto T4 Plus . The cells were seeded at a density of 1 × 106 cells/ml in fresh media and incubated for 30 min. Subsequently, they were transfected again with three parts PEI to one part DNA diluted in Opti-MEM I Reduced Serum Media . For increased overexpression, cells were supplemented with 3.5 mM valproic acid  12 h post transfection. It has been determined that media harvest after 96 h produces the highest yield.HEK293F cells  were cultured in serum- and protein-free FreeStyle 293 expression medium without additional supplements  at 37°C, with 8% COBoth expression constructs pCMV3-SP-CD109-N-His  and pCMV3-SP-CD109-N-HA  contain an antibiotic resistance in mammalian cell systems against hygromycin B. To determine the minimum antibiotic concentration needed to kill all the non-transfected cells in 1 week, the amount of hygromycin B was determined by performing a kill curve. Afterwards, the concentration of 0.15 mg/ml and 0.2 mg/ml hygromycin B was chosen. HEK293T cells (passage no. 6) were transfected as described above and changed after 16 h onto selective (hygromycin B-containing) medium with 10% FBS. Every other day, medium was discarded and cells were washed with Dulbecco’s phosphate-buffered saline  and given fresh selective medium over 9 days. Polyclonal colonies were resuspended in selective medium and seeded statistically 1 cell/well in a 96-well plate, again for 9 days. Monoclonal cell colonies were then transferred to grow in 6-well plates, checked for protein expression via Western blot of supernatant and cell lysates, and used for experiments. Stable cell lines HEK293T CD109 N-term HA-tag and N-term His-tag between passages 8 and 12 (after transfection) have been used for studies.g, 4°C, lysis buffer  was added and the suspension was incubated for 30 min on ice. Afterward, cell lysates were centrifuged at 13,000 × g for 15 min at 4°C to remove cell debris. Protein amount was determined using PierceTM BCA Protein Assay Kit . Supernatant was then mixed with sodium dodecyl sulfate (SDS)-containing loading buffer and proteins were denatured for 10 min at 98°C. After separation by SDS-PAGE, proteins were either stained with Coomassie Brilliant Blue R-250 or transferred to a nitrocellulose 0.2-μm  membrane, blocked with 3% milk or BSA  in Tris-buffered saline  for 1.5–2 h and then incubated with the primary antibody overnight at 4°C (see TM TyphoonTM Biomolecular Imager infrared imaging system (for Coomassie gel detection). Concanavalin A  (ConA) precipitation from cell culture supernatants was performed as described previously   followed by 1-h incubation at 37°C. The PCR product was analyzed by agarose gel electrophoresis and sequencing  to confirm the right mutation.The expression construct pCMV3-SP-CD109-N-His  was used as a template to generate a mutant construct of the soluble protein. The individual mutagenesis primer was designed as shown: 5′-TTGTGAGGATTGAGCTTCAGGC-3′ and 5′-GCCTGAAGCTCAATCCTCACAA-3′, synthesized by Thermo Fisher Scientific Inc.  and Merck Millipore . The mutagenesis reaction contained 0.5 μl of Phusion High-Fidelity DNA Polymerase (2 U/μl) , 10 μl of 5 × Phusion HF-Puffer , 10 mM of each primer, 10 mM dNTPs , 14 μl of DMSO , 100 ng of plasmid DNA, and double-distilled water to a final volume of 50 μl. The probe was initially denatured at 98°C for 1 min, followed by 30 cycles, each containing denaturation at 98°C for 20 s, annealing at 60.6°C for 5 min, and elongation at 72°C for 8 min. The last step after finishing the PCR cycles was to hold 72°C for 10 min. Subsequently the parental template DNA of the generated PCR product was digested by adding 1 μl of TM Ni-NTA-Agarose  in batch procedure. The purification was done according to the manufacturer’s protocol with minor changes. Briefly, the harvested cell culture medium was cleared from the remaining cells by centrifugation at 2,000 × g, 4°C for 5 min, and afterward cell debris was eliminated by centrifugation at 16,000 × g, 4°C for 45 min. The supernatant was added to the equilibrated Ni-NTA-Agarose beads, with NPI-10 buffer , and incubated for 1 h at 4°C. Protein-bound beads were washed in three washing steps, twice with NPI-30  and the third time with an NPI-50 buffer . The recombinant protein was eluted in four steps, thrice with NPI-150 buffer 50  and the last time with NPI-250 buffer . The collected eluent fractions were concentrated and finally the buffer was exchanged using Amicon Ultra-4, 50 K columns .The purification of recombinant His-tagged protein from the cell culture media was performed by Ni-NTA affinity chromatography using Protinog, 4°C to eliminate remaining cells, and again at 10,000 × g, 4°C for 60 min to remove cell debris; 500 μl of isolation reagent was given to 1,000 μl of supernatant and incubated overnight at 4°C. The next day, the samples are centrifuged again for 60 min at 10,000 × g, 4°C, and the supernatant was discarded. EVs were resuspended in 50 μl of ice-cold DPBS and used for further analysis, such as Western blot.To determine whether meprin β affects the amount and fragmentation of CD109, EVs were isolated for quantification purposes by using total exosome isolation reagent . The purification was performed according to the manufacturer’s protocol. The transfection was done as described above, medium was saved 40 h post transfection, centrifuged for 30 min at 2,000 × For extracellular measurements in negative staining transmission electron microscopy (TEM) or for dynamic light scattering analysis (DLS), samples must be free from any matrix as it would be found in the abovementioned isolation kit. Therefore, EVs were isolated using ultracentrifugation as described previously .Sciences, Hatfield, United States). The glow discharge was performed by using the Mini Sputter Coater System , with a strong current of 25 mA for 30 s. The purified and previously buffer exchanged  protein sample as well as the EV samples were added into the glow-discharged carbon-coated EM grid for 30 s, and access volume was removed using filter paper. The EM grid was stained twice with 1% aqueous uranyl acetate solution , blotted again with filter paper, and air dried. The micrographs were collected by JEOL 1400 Plus TEM  operating at 100 kV with a nominal magnification of 50,000×.Negative staining TEM was performed as described before . The negFor single-particle analysis, images were taken utilizing SerialEM  and were®  and measured with the Spectroscatter 201 . Each sample was prepared in three technical replicates and each was measured 10 times with a 90° scattering angle at 20°C.For DLS analysis, EV samples, generated using ultracentrifugation, were adjusted to the same protein amount (measured as described above) and stored in DPBS. Samples were filled into precision cells made of Quartz SUPRASILTerminal Isotopic Labeling of Substrates (TAILS) analysis was performed as described by 4HCO3 (ABC)] for 15 min. The supernatant was removed, and the gel pieces were dehydrated by adding pure acetonitrile for 10 min with the tubes rotating in a rotator. The acetonitrile was removed first by pipetting and the samples were afterward dried to completion using a vacuum evaporator . The dried samples were rehydrated, and disulfide bonds were reduced using reduction buffer  for 1 h at 56°C. Buffer was removed by pipetting and cysteine residues of proteins were subsequently alkylated with 50 mM iodoacetamide (IAA) dissolved in 50 mM NH4HCO3, pH 8.0, for 45 min at room temperature (RT) in the dark. Samples were dehydrated again by adding pure acetonitrile and dried with vacuum evaporation. The vacuum-dried gel slices were incubated with 1 μg of trypsin per tube in 50 mM triethylammonium bicarbonate (TEAB) buffer, pH 8.0, at 37°C overnight. Digested peptides were extracted by adding twice 150 μl with 30% acetonitrile and once 150 μl of pure acetonitrile for 15 min at 25°C agitating at 1,400 rpm in a thermo shaker . As the next step, we performed reductive dimethylation as described previously , 7% acetic acid, and 45% ethanol. The gel was afterward washed several times with deionized water to remove excess dye. In-gel digestion was performed in principle as described previously  and detaeviously  for eacheviously . The elu6; max. IT = 20 ms; scan range 300–1,650 m/z with a top 10 MS/MS DDA method. Normalized collision energy was set to 25 and MS/MS scan mode was operated at a resolution of 17,000; AGC target 1 × 105 and max IT of 120 ms. Database search was performed using MaxQuant , Oxidation (M); fixed modifications: Carbamidomethyl (C), FDR of 1% on peptide and protein level was applied.Peptides were separated on an EasyLC1000 HPLC  with the following reversed-phase chromatography gradient: 0–67 min 0–22% mixture B, 67–88 min 22–40% mixture B, and 89–92 min 40–95% mixture B  and directly sprayed into a Q Exactive Plus mass spectrometer . The mass spectrometer was operating in positive scan mode with a full scan resolution of 70,000; AGC target 3 × 10MaxQuant . VersionFor quantification, light label and heavy label were assigned on either lysine residues or N-terminal amino group with a maximum allowance for three labeled amino acids per peptide. Proteins with at least two peptides, one of them unique, were considered as identified. Proteins matching the reverse database or a common contamination list, as well as proteins containing only peptides with a modification, were removed. The quantification ratios from MaxQuant of the dimethyl labels were log2 transformed.2O) and 20 mM sodium cyanoborohydride (NaBH3CN) for 3 h at 25°C for 3 h in HEPES buffer (pH 7.5). Excessive reagents were quenched in three washing steps: once with 50 mM ABC and two times with 30% ACN/50 mM ABC. Gel bands were dehydrated with ACN and vacuum centrifugation. Chymotrypsin (50 ng) was added, and the samples were digested overnight at 37°C in the presence of ABC. Peptides were extracted from the gel band using 1% formic acid (FA), 50% acetonitrile (ACN), 1% FA, and 100% ACN with the aid of sonication. Pooled supernatants were dried down by vacuum centrifugation and stored at −20°C and resuspended in 3% ACN and 0.1% trifluoroacetic acid (TFA) prior to analysis.The gel bands were washed and destained, reduced with DTT (10 mM), and alkylated with IAA (55 mM) using standard techniques, but in the presence of 50 mM HEPES (pH 7.5) rather than ABC. Protein N-terminus and lysine residues were reductively dimethylated using 40 mM formaldehyde  and heavy  formaldehyde in the presence of NaCNBH3, as described previously  and inactive meprin β (E153A) was made up to 30 μl in a denaturing buffer 100 mM TEAB and 1% SDS and inactivated by the addition of Tris(carboxyehtyl)phosphine  at 95°C for 10 min. Samples were reduced for another 30 min and then alkylated with IAA (12.5 mM) for 30 min at RT and then quenched with DTT. Samples were precipitated onto a mixture of SpeedBeads, Magnetic Carboxylate particles , and then labeled with light . The samples were washed on a trap column  for 4 min with 3% ACN/0.1% TFA at a flow rate of 30 μl/min prior to peptide separation using an Acclaim PepMap 100 C-18 analytical column . A flow rate of 300 nl/min using eluent A (0.05% FA) and eluent B (80% ACN/0.04% FA) was used for gradient separation (5–40% B). Spray voltage applied on a metal-coated PicoTip emitter  was 1.6 kV, with a source temperature of 250°C. For Q Exactive Plus, measurements were acquired at a resolution of 70,000 at m/z 200. The 10 most intense precursors with charge states ≥ 2 + were subjected to fragmentation with HCD with NCE of 27%; isolation width of 3 m/z; resolution, 17,500 at m/z 200. For measurements on the Q Exactive HF Full scan, MS spectra were acquired between 350 and 1,400 m/z at a resolution of 120,000 at m/z 200. The 10 most intense precursors with charge states greater than 2 + were selected with an isolation window of 1.4 m/z and fragmented by HCD with normalized collision energies of 27 at a resolution of 30,000. Lock mass (445.120025) and dynamic exclusion (30 s) were enabled on both machines.The MS raw files were processed by Proteome Discoverer 2.2  and MS/MS spectra were searched using the Sequest HT algorithm against common contaminants and the canonical and reviewed human database. An MS1 tolerance of 10 ppm and an MS2 tolerance of 0.02 Da was implemented. Carbamidomethylation (57.02146 Da) on cysteine residues and dimethylation on lysine residues (28.031 Da) were set as fixed modifications while oxidation of methionine (15.995 Da) residues and dimethylation on the peptide N-terminus were set as dynamic modifications. For in-gel N-terminomics, samples were searched with semi-chymotrypsin specificity with three missed cleavages allowed, while for quantitative reductive dimethylation, the sample was searched with semi Arg-C specificity with two missed cleavages allowed. For quantitative reductive dimethylation, two sequest nodes were used to search the data: one node with “light” dimethylation (+28.031 Da) set as dynamic modification on peptide N-termini, and as a fixed modification on lysine residues, while the other implemented “heavy” dimethylation (+32.056 Da) on the aforementioned lysine residues and peptide N-termini. High-confidence peptides were set to a 1% FDR, while medium-confidence peptides were allowed a 5% FDR. For in-gel dimethylation, N-terminal peptides were only considered if they were identified with at least two PSMs.1 via the PRIDE partner repository (The mass spectrometry (MS) proteomics data have been deposited to the ProteomeXchange Consortiumpository  with the2 .If not differently specified, the results are given as mean ± standard error of the mean (SEM). To determine if two groups of data were significantly different, unpaired non-parametric Mann–Whitney in vitro, recombinant CD109  was incubated with recombinant meprin β, the closely related meprin α (2O) and heavy (CD2O) labeling for meprin β and the E153A variant, respectively, and identified a cleavage site between 676H and 677D that was unique for CD109 transfected with meprin β . Cleavage of CD109 by meprin β at this position seems not to be a shedding event, where ectodomains of membrane-bound proteins are released by meprin β from the cell surface . To struo CD109, ] was ideo CD109,  display o CD109, , red is o CD109,  showed yo CD109,  revealedo CD109, , none ofo CD109, . After to CD109,  through o CD109, , it was o CD109, . Purity o CD109, . For supo CD109,  and transelected . The resselected .To determine the molecular model that best fits the calculated 3D reconstruction, we docked all calculated homology models into the 3D density and calculated the average map value (UCSF Chimera) . This vaIt was shown before that CD109 can be sorted to exosomes  and incrDrosophila CD109 ortholog TEP1 and not to α2-macroglobulin, as suggested from sequence alignments  regulates the Drosophila Yki pathway (ortholog to Yap/Taz)  . This fuYap/Taz) , and herYap/Taz)  and the Yap/Taz)  that proYap/Taz) . As no oYap/Taz) . HoweverTaken together, we provide a comprehensive analysis of CD109 fates at the cell surface. We demonstrate how proteolytic processing at the cell surface reduces the amount of full-length CD109 sorted to EVs. Proteolysis results in the release of fragmented CD109 into the supernatant, and using single-particle analysis and homology modeling, we could also provide the first experimentally solved structure of CD109, allowing us to deduce its orientation on the cell membrane and the identified cleavage sites.https://www.ebi.ac.uk/pride/, PXD023727.The datasets presented in this study can be found in online repositories. The name of the repository and accession number can be found below: PRoteomics IDEntification Database (PRIDE), WL and PA: conceptualization. WL, SB, TK, EP, FP, PS, and PA: formal analysis and investigation. WL and PA: writing—original draft preparation. All authors: writing—review and editing. CB-P, FZ, and PA: funding acquisition. CB-P, RL, and FZ: resources. PA: supervision.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.


The singlet oxygen quantum yield value (ΦΔ) for (1–7) was measured using 1,3-diphenylisobenzofuran (DPBF) as a singlet oxygen trapping agent and 5,10,15,20-(tetraphenyl)-porphyrin (H2TPP) as a reference standard; besides, parasite viability was estimated by the MTT assay. After metal insertion into the porphyrin core, the ΦΔ increased from 0.76–0.90 and cell viability changed considerably. The ΦΔ and metal type changed the cytotoxic activity. Finally, (2) showed both the highest ΦΔ (0.90) and the best photodynamic activity against the parasites studied (IC50 of 1.2 μM).In this study, a family of porphyrins based on 5,10,15,20-Tetrakis(4-ethylphenyl)porphyrin ( Leishmania spp are extra and intracellular protozoan parasites that infect a variety of animals . However, this zoonosis also affects human beings when they invade the habitat of both natural reservoirs and transmitting vectors thereof +; Anal. Elem. Calc. for C52H46N4 (%): C (85.91), H (6.39), N (7.71), Anal. Elem. Found. (%) C52H46N4, C (85.95), H (6.34), N (7.71).2–7) were synthesized by mixing (1) with the metal chloride salt for each metal in DMF. The mixture was stirred for 6 h at room temperature. Then, the reaction mixture was cooled in ice-water bath; the formed precipitate was filtered and dried at room temperature; (2–7) were purified through column chromatography with silica gel (2.5 × 24 cm), petroleum ether:ethyl acetate (PE:EA) was used as mobile phase. Details of the spectroscopic characterization are listed in Compound  were determined in air using the relative method with 1,3- diphenylisobenzofuran (DPBF) as a singlet oxygen trapping agent and 5,10,15,20-(tetraphenyl)-porphyrin (H2TPP) as a reference standard. The tests consisted of preparing a 1 × 10−9 M solution of each compound in Dimethylformamide (DMF) by triplicate, and calculations were determined according to Equation (1) [∆st is the singlet oxygen quantum yield of standard H2TPP in DMF (0.64), W y Wst are the DPBF photobleaching rates in the presence of complex (1 and 2) and standard porphyrin, respectively. Data for Singlet Oxygen Quantum Yield calculation are provided in The Φtion (1) ,57,58,59x and Fest correspond to the area under the curve in the fluorescence emission spectrum for compounds (1), (2) and standard. Ax and Aest correspond to absorbance at excitation wavelength for compounds (1), (2) and standard; ɳx and ɳest correspond to the refraction index for solvents (ɳethyl acetate = 1.3724 and ɳwater = 1.33336). Data for Singlet Oxygen Quantum Yield calculation are provided in The comparative method was used to determine fluorescence quantum yield. Fluorescein dissolved in water was standard, and sensitizers were dissolved in ethyl acetate. The fluorescence quantum yield values were determined by taking the maximum of the Soret band as the excitation wavelength . Quantum fluorescence yield was calculated with the following equation ,42,60,61Leishmania panamensis (M2903) and Leishmania braziliensis (UA140) were used in the in vitro study. The parasites were cultured in RPMI-1640 supplemented with 10% fetal bovine serum, 1% glutamine and 4% antibiotics (200 U penicillin/200 μg Amikacin) under incubation conditions of 5% CO2. The metacyclic promastigotes in the infectious stage were isolated from stationary cultures of 5 days using a uniform procedure based on a modified density gradient purification.emission lamp = 420 nm–450 nm for using light intensity 80 J·cm−2. All the measurements of the optical densities were taken in microplates of 96 U-bottom wells, using the Multiskan Sky ThermoScientific equipment. Standard deviation was obtained from 12 independent experiments—these were correlated with a percentage variation coefficient <5%. We applied an ANOVA test to determine the differences or similarities between treatments and the positive control. In addition, a post hoc analysis was performed using Tukey statistics. Finally, differences were considered to be significant when p < 0.05.Parasite viability was estimated by the MTT assay, converting a yellow tetrazolium salt, 3--2,5-diphenyltetrazolium bromide into an insoluble product (formazan); the amount of formed formazan depends on the number of viable parasites present ,59,62. T1–7) showed suitable singlet oxygen quantum yields, which induced inhibition of the L. braziliensis and L. panamensis growth when the compounds were irradiated with a visible light source. The non-irradiated treatments generated little or no inhibitory response of the parasites. All the results indicate that (1–7) have suitable properties to be used in photodynamic therapy. All the compounds showed better cytotoxic against L. panamensis than against L. braziliensis. Compound (2) was the best photosensitizer of all the compounds included in this study, as it showed a larger ΦΔ value (0.90) and a better IC50 value compared to that of the positive control. Therefore, compound (2) is the best candidate to be tested in photodynamic application against L. braziliensis.Porphyrins (


TC toxicity also positively correlated with the ion bonds ofN4-Glu252, but negatively correlated with the ion bonds of N4-Asp379. Hydrogen bonds and ion bonds for above key sites were closely related to the inhibition effect of TCs on CAT.As potential inhibitors target to biological enzymes, antibiotics may have certain impacts on the biochemical treatment process. With micrococcus catalase (CAT) served as the target molecule, the impact and inhibition mechanism for typical tetracyclines (TCs) were evaluated. Toxicity experiments showed that TCs had significant inhibition on CAT in the sequence of tetracycline>chlortetracycline>oxytetracycline>doxycycline. To clarify the inhibition mechanism between TCs and CAT which was explored with the assistance of fluorescence spectroscopy and MOE molecule simulation. According to fluorescence analysis, TCs quenched the fluorescence signal of CAT by the mode of static quenching. Combined with toxicity data, it could be presumed that TCs combined with the catalytic active center and thus inhibited CAT. Above presumption was further verified by the molecular simulation data. When TCs combined with the catalytic center of CAT, the compounds have increased combination areas and prominent energy change . IBM SPSS statistics showed that TC toxicity positively correlated with the hydrogen bonds such as O TCs have been widely used in animal husbandry and aquaculture as the advantages of low-cost and broad antibacterial spectrum –4. BecauIn view of TC biotoxicity, controls on their levels become of great importance. At present, the main treatment methods are the chemical method and biochemical method –14. Comp2O2 into molecular oxygen and water, removes hydrogen peroxide from the body, and protects cells from H2O2 toxicity  = 1 + Kqτ0[Q] , qK, and τ0 were the quenching constant, the concentration of the quencher, the fluorescence quenching rate constant, and the average lifetime of the fluorescent molecules in the absence of the quencher. For biomacromolecules, τ0 = 10−8 s.Fluorescence spectra were measured by a three-dimensional fluorescence spectrometer . Firstly, 2 mL of 0.2 mol/L phosphate buffer (pH 7.8), 1 mL of 400 mg/L CAT solution, and 1 ml different amounts (0 mg/L-1500 mg/L) of TC solution were sequentially added to 50 mL colorimetric tubes. In addition, 2 mL of 0.2 mol/L phosphate buffer (pH 7.8) and 1 mL of 1500 mg/L TC solution were added to another 50 mL colorimetric tube, then make the final volume to 50 ml with ultra-pure water, and mixed and kept for 30 min. The fluorescence spectra of samples were measured at an excitation wavelength of 276 nm and an emission wavelength range of 287-380 nm. Fluorescence quenching type was calculated by the dynamic quenching formula of Stern-Volmer:  Kqτ0[Q] . F0 and http://www.rcsb.org/pdb/home/home.do)  = 1 + Kqτ0[Q]  . The colBased on the toxicity experiment and fluorescence spectroscopy experiment, the interaction for TCs to CAT could be preliminary explained, but the specific molecular mechanism was not clarified. According to molecular docking simulation, the interaction models between TCs and CAT could be obtained, and the discrepant inhibition mechanism could be clarified.http://www.rcsb.org/pdb/home/home.do) [molecular hole method of MOE software, the interaction models between TCs and CAT in the iron porphyrin catalytic active center and no-catalytic active center were obtained  . Based oThe interaction between TCs and CAT usually involved in changed combination area and energy. To further clarify the combination site, relevant parameters were obtained by MOE, and the rate of combination areas to small molecule areas and energy changes for TC-CAT complexes at different combinatiolizan sites were calculated see . CombinaBased on data in the catalytic activity center, the combination area ratio for different TC-CAT complexes was determined as follows: chlortetracycline>tetracycline=oxytetracycline>doxycycline. Besides, the energy change in the catalytic active center for different TCs combined with CAT had different decreased in the sequence of chlortetracycline>oxytetracycline>tetracycline>doxycycline. Combined with toxicity data, combination area ratio and energy change were not well correlated with TC toxicity. The combination area and energy change of chlortetracycline were the largest, which could be related to its special chlorine atom structure. Furthermore, the correlation between TC toxicity and combination area ratio/energy change was evaluated by IBM SPSS statistics see . The resThe specific interaction sites between TCs and CAT can be seen through the ligand interaction diagram provided by the MOE software see . AccordiSimulation information of the hydrogen bonds for TC-CAT complexes was shown in 13→Glu252, O1←Arg195, O6→Asp249, O10→Glu252, O1←Lys256, 6-ring-Arg195, N21→His368, O11←Asn127, C41→Asp379, C51→Asp379, 6-ring-Gln357, O6→Asn369, O6←His368, O11←Asn369, O13←His368, C5→Asp379, N4→Glu125, O13→Ala366, O13→His354, 6-ring-Asn369, and 6-ring-Gly126, but negatively correlated with O10→Pro363, O10→Lys455, O12→Asn127, O13→Asp379, O13→Thr324, O13←Val367, O21←Arg384, C41→Val367, C42→Asp379, N21→Asp379, and N21→Glu125. By statistical analysis, 379 residues in CAT, including six hydrogen bonds of C41→Asp379, C51→Asp379, O13→Asp379, C5→Asp379, C42→Asp379, abnd N21 → Asp379. Besides, the correlation sequence of residues including 2-3 interaction sites was verified as follows >Asn127 >His368 >Glu125 >Asn369 >Arg195 >Glu252 . 6→Asp249, O1←Lys256, 6-ring-Gln357, O10→Pro363, O10→Lys455, O13→Thr324, O21←Arg384) were also important parameters to evaluate inhibition mechanism of TCs on CAT. However, Gly126, Arg334, Val351, Ser316, Tyr200, Glu416, So4505, Asn361, Asn323, and Leu348 residues were less correlated with TC toxicity >N21 >6-ring >O6 >O1 >O11 >O12 . 21←Arg384, C4A→Val367, C42→Asp379, O1C→Glu252, C41→Asp379, C51→Asp379, C5→Asp379, N4→Glu125), which also cannot be ignored. Combining MOE simulation information and related IBM SPSS statistics, these closely related hydrogen bond changes can be used as a key indicator to evaluate the interaction between TCs and CAT and are also very important for the regulation of TC toxicity.According to statistical analysis, hydrogen bonds related to the key sites of TCs also were ranked see . The int Figures : O10  on CAT was evaluated. The inhibition sequence was verified as follows: tetracycline>chlortetracycline>oxytetracycline>doxycycline. With the assistance of fluorescence spectroscopy and MOE molecule simulation, the interactions between TCs and CAT were further evaluated. Fluorescence spectroscopy showed that the reduced fluorescence intensity of CAT should be attributed to static quenching between TCs and CAT in the catalytic active center. Molecular simulation showed that combination sites for TC-CAT complexes were in the catalytic active center by calculating the combination area ratio and energy change in the catalytic active center/noncatalytic active center. Combined with toxicity data, the combination area ratio and energy change in the catalytic active center had basically positive correlation with TC toxicity. In addition, combined with the simulation information and related IBM SPSS statistics, TC toxicity was positively correlated with the hydrogen bonds of O