Pif1 is a multifunctional helicase and DNA processing enzyme that has roles in genome stability. The enzyme is conserved in eukaryotes and also found in some prokaryotes. The functions of human PIF1 (hPIF1) are also critical for survival of certain tumour cell lines during replication stress, making it an important target for cancer therapy. Crystal structures of hPIF1 presented here explore structural events along the chemical reaction coordinate of ATP hydrolysis at an unprecedented level of detail. The structures for the apo as well as the ground and transition states reveal conformational adjustments in defined protein segments that can trigger larger domain movements required for helicase action. Comparisons with the structures of yeast and bacterial Pif1 reveal a conserved ssDNA binding channel in hPIF1 that we show is critical for single-stranded DNA binding during unwinding, but not the binding of G quadruplex DNA. Mutational analysis suggests that while the ssDNA-binding channel is important for helicase activity, it is not used in DNA annealing. Structural differences, in particular in the DNA strand separation wedge region, highlight significant evolutionary divergence of the human PIF1 protein from bacterial and yeast orthologues. Accurate DNA replication requires a suite of enzymes including helicases that translocate on DNA. Helicases can catalyse protein displacement from DNA  but theySaccharomyces cerevisiae as a gene involved in mitochondrial DNA recombination and stability , while the 5′–3′ polarity of unwinding place them in the SF1B subgroup. Seven conserved amino acid motifs  have been identified in SF1 helicases that are located in two structurally related RecA-like domains, 1A and 2A. The RecA domains and two additional domains, 1B and 2B, form the helicase core  are conserved relative to ScPif1  its cellrevisiae . Howeverrevisiae . Importarevisiae ,34. hPIFBacteroides spp. Pif1 (BsPif1) protein. Interestingly, the full-length 433 amino acid BsPif1 is structurally similar to hPIF1HD  residing in residues 206–620 . The fun hPIF1HD  although hPIF1HD .4− bound, mimicking a transition state intermediate. These structures allowed us to model the structural events along the chemical reaction coordinates of ATP hydrolysis and how they may be linked to helicase activity. This and the crystal structures of BsPif1  was expressed with an N-terminal hexa-histidine-maltose binding protein (His-MBP) tag, followed by a thrombin cleavage site. All purification steps were at 4°C. For both constructs, cells were lysed by sonication in 50 mM Tris–Cl pH 8.0 (4°C), 550 mM NaCl, 5 mM DTT, 10% (v/v) glycerol, 1 mM PMSF (3 ml/g of cells) and centrifuged at 25 000 × g for 30 min at 4°C. The NaCl concentration of the cleared lysate was adjusted to ∼1 M and nucleic acids removed by polyethylenimine P (5% w/v) precipitation. 0.226 grams of ammonium sulphate was added per ml of cleared solution and protein precipitated by centrifugation at 25 000 × g for 20 min at 4°C. The pellet was re-suspended in 50 mM Tris–Cl pH 8.0 (4°C), 500 mM NaCl, 2.5 mM DTT, 20 mM imidazole, 10% (v/v) glycerol and applied to a HisTrap Ni-Sepharose column  and eluted in a gradient developed to 200 mM imidazole over 15 column volumes (CV). The His or His-MBP tags were removed by thrombin cleavage, leaving four residues of the tag, GSRM.Human PIF1 helicase domain proteins were expressed codon-optimized using pET system plasmids (pET15b) in ArcticExpress™ BL21(DE3) cells (Agilent Technologies) at 6°C for 3 days. hPIF1HD (residues 206–621) was expressed with an N-terminal hexa-histidine and thrombin cleavage site tag (His-tag) and hPIF1HDhPIF1HD was further purified with a second round of Ni-sepharose chromatography, size exclusion chromatography , 300 mM NaCl, 2.5 mM DTT, 5% (v/v) glycerol, 1 mM EDTA, 1 mM PMSF) and cation exchange chromatography  glycerol, 2.5 mM DTT, 0.1 mM PMSF, gradient from 100–350 mM NaCl, 15 CV) and finally dialysed against 10 mM Tris–Cl pH 8.0, 200 mM NaCl, 2% (v/v) glycerol, 5 mM DTT, 0.1 mM PMSF, 0.1 mM EDTA.206–641 was further purified by SEC (as above), followed by a second round of Ni-sepharose chromatography and cation exchange chromatography  and finally dialysed against 10 mM Tris–Cl pH 8 (4°C), 300 mM NaCl, 2% (v/v) glycerol, 5 mM DTT, 0.1 mM PMSF, 0.1 mM EDTA.hPIF1HD−1 for storage (−80°C). Concentrations were determined from absorbance at 280 nm readings determined in 7M guanidinium hydrochloride using the calculated molar extinction coefficients.Proteins were concentrated to ∼30 mg ml−1). Crystals of the hPIF1HD-AMP-PNP complex were grown in hanging drops (4°C) by mixing the protein at a final concentration of 11 mg ml−1 in 10 mM Tris–Cl pH 8.0, 2% (v/v) glycerol, 5 mM MgCl2, 5 mM AMP-PNP, 2.5 mM TCEP, 0.3 M NaCl with a reservoir solution in a 1:1 ratio. The well solution contained 0.3 M Na-acetate, 17–27% (w/v) PEG 2K MME in 0.1 M Tris–Cl pH 7.5. For phasing, crystals were soaked briefly in mother liquid supplemented with 0.8 M KBr. All diffraction data were collected at cryo-conditions using synchrotron radiation at Diamond Light Source  and processed with XDS  glycerol, 5 mM MgCl2, 5 mM AMP-PNP, 2.5 mM TCEP, 0.3 M NaCl, 0.3 M Na-acetate, 10% (w/v) PEG 8K and 10% (w/v) PEG 1K. The apo structure of hPIF1HD206–641 was obtained from crystals belonging to the P212121 space group with one protein molecule per asymmetric unit grown using 15 mg ml−1 protein solution in 0.1 M MES pH, 6.0, 0.5% (v/v) glycerol, 5 mM MgCl2, 2.5 mM TCEP, 150 mM NaCl 0.2 M Li2SO4 and 25% (w/v) PEG 2KMME at 4°C. The hPIF1HD-ADP•AlF4− co-crystals in the P3121 space group with two molecules per asymmetric unit were grown using 13 mg ml−1 protein solution in 0.1 M MES pH 6, 1% (v/v) glycerol, 5 mM MgCl2, 5 mM ADP, 6 mM AlCl3, 50 mM NaF, 150 mM NaCl, 2.5 mM TCEP, 0.2 M Ca-acetate, 8% (w/v) PEG 20K and 8% (w/v) PEG 550MME. The coordinates of the refined Br derivative structure were used as a molecular replacement search model using the data obtained from the hPIF1HD-AMP-PNP, apo hPIFHD206–641 and ADP•AlF4−-hPIF1HD crystals. An unambiguous solution was found using MOLREP , and using the ssDNA coordinates from 5FHD. The human PIFHD-ssDNA model was then energy minimized in Maestro  substrate (0.4 nM), end-labelled with 32P using polynucleotide kinase (pnk) and [γ-32P]ATP (6000 Ci/mmol) and purified from poly-acrylamide gels as described previously (−1 BSA. Reactions were incubated for 20 min at 20°C before resolving complexes on 5% poly-acrylamide gels (29:1) using 0.25× TBE running buffer. The radiolabelled tetramolecular G4 DNA substrate used was formed from the single-stranded precursor 5′-TTTTTTTTTTGGGGTTTTGGGG as described previously ((35) competitor ssDNA. Reaction products were visualized and quantified following exposure of the dried gels to phosphorimaging plates. Data analysis was performed using the program PRISM (GraphPad), as were all other biochemical data described below.Single stranded DNA binding reactions were performed with a poly-Teviously . The bineviously . The rea55-CGAATTCGAGCTCGGTACCC and 5′-GGGTACCGAGCTCGAATTCG, as described previously  NP-40. Reactions (0.1 nM substrate) were incubated at 20°C for 30 min and terminated by the addition of 0.2 volumes of 120 mM EDTA, 0.6% (w/v) SDS, 60% (v/v) glycerol and 0.1% (w/v) bromophenol blue before polyacrylamide gel electrophoresis (8% (19:1), 0.25× TBE/0.05% (w/v) SDS) and exposure of dried gels to phosphorimaging plates.A radiolabelled partially single- and double-stranded test substrate was generated by annealing the following oligonucleotides: 5′-(T)eviously . The rea(30) as described previously , 1 mM DTT, 0.1 mg ml−1 BSA and 0.1% (v/v) NP-40. Phosphate release was determined after 10 min (20°C) when ∼3% of the ATP was hydrolysed.A charcoal binding assay  was usedeviously . The rea55-CGAATTCGAGCTCGGTACCC and 5′-GGGTACCGAGCTCGAATTCG-(C)30. The reaction buffer was 20 mM HEPES–NaOH pH 7.5, 135 mM NaCl, 1 mM DTT and 0.1% (v/v) NP-40. The annealed substrate was gel-purified and heat denatured before addition to the reaction . Reactions were incubated and processed as described for the helicase assays.Strand annealing assays employed two radiolabelled oligonucleotides with a complementary sequence of 20 bases and non-complementary 55 nucleotide 5′ or 30 nucleotide 3′ overhangs-5′-(T)Variants of hPIF1HD (residues 206–620) were made by site directed mutagenesis by primer extension and purified as described above. Protein concentrations of mutant and wild-type proteins for biochemical assays were determined using the Bradford assay (BioRad) from triplicate measurements performed in parallel using BSA as a standard.4−, Figure 4− structure  mirrors a previously published low resolution (3.6 Å) crystal structure of the hPIF1 helicase domain . Notably, average temperature factors  or 206–620 . In the 1.44 Å apo-hPIF1 crystal structure residues after 620 are not seen in the electron density suggesting they are disordered. Our two constructs  are active in helicase/DNA binding assays and demonstrate characteristic DNA dependent ATPase activity.High resolution X-ray structures were determined for the helicase domain of hPIF1 Figure , an apo = 35.5%, ), it is  factors  indicateBacteroides Pif1 structures  induces changes at the inter-domain 2A–2B contact area, allowing the segment β18-η7-β19 comprising helicase motif V see , to reacy Figure . Accordi4− structure . It is also possible that movement of the Walker A motif could cause the main-chain N–H of G231 . In thetructure  shows th1 Figure , left to1 Figure . Interesd Figure , right, d Figure –C. A fuld Figure  for ATP ure 5 in ).ATP binding results in a 4 Å movement of the N-terminus of helix α9  and C (residues 494–499) motifs within domain 2B  that are(35) substrate was assayed, Figure Kd of 2.3 ± 0.3 nM was determined from the quantified data shown in Figure (35) ssDNA with similar affinity to wild-type (1.9 ± 0.6 nM), while the binding affinity of R294 was reduced ∼2–3-fold (apparent Kd 5.7 ± 1.6). Binding of variant R290A was reduced ∼10–20-fold (apparent Kd 40.4 ± 14 nM), although the failure to reach near saturation binding precludes the determination of a more accurate apparent Kd value. Variants E307Q  and K414A also bound ssDNA at close to wild-type levels . All amino acid substitutions in the ssDNA binding channel including F573 and those from the B and C motifs  result in large decreases in ssDNA binding affinity, with variant K556A showing the greatest defect. As above, insufficient binding extents  with polydeoxynucleotide substrates greater than 30 bases . Here, ss Figure  precludeWild-type and variant hPIF1 proteins were tested in an ATP-dependent helicase assays for their ability to displace a 20 base radiolabelled oligonucleotide from a partially single- and double-stranded test substrate. Unwinding reactions were performed at low salt concentration (20 mM NaCl) as the strand displacement activity of hPIF1 is significantly inhibited at higher salt concentrations and the protein inactive in unwinding assays performed at ∼50 mM NaCl. The inclusion of 0.1% NP40 was also required for optimal activity. Reaction products were separated by polyacrylamide gel electrophoresis and quantified by phosphorimaging. The protein concentration range chosen was at the upper-end of the range where maximum unwinding is observed for wild-type protein Figure  and wherase , with excess T(35) ssDNA and at a reduced salt concentration (75 mM NaCl) relative to direct ssDNA binding assays  only for the variant H269A, H555A and K556A .Overall, the results of hPIF1 binding to G4 DNA were significantly different to the results observed for ssDNA binding Figure . Only smA Figure . Curious4− bound hPIF1 is clearly indicated in the overall atomic temperature factors of the refined models  and Walker B (motif II), are clearly defined in the chemo-mechanical chain of events. Second, events at the ATP binding site and in particular the ATP γ-phosphate are relayed to positional and conformational adjustments in defined protein segments. Third, such structural changes result in an increased flexibility of the protein structure that could affect protein–DNA interactions. The increased flexibility of the ADP•AlFd models .4− bound. Indeed, the higher affinity of BsPif1 towards ssDNA observed with the ADP•AlF4− transition-state mimic may have facilitated determination of the protein-DNA complex X-ray structures  or ADP (product) ssDNA binding affinities are lower than those observed in the absence of cofactor  and N296  interact with DNA (E. coli and D. radiodurans. Mutation and deletion of the short linker region between the BsPif1 B and C motifs (ase activity. The BsPif1 substitution N296A (C motif) retained ∼60% ssDNA binding activity while for the corresponding substitution in hPIF1 (N495A) binding affinity is reduced ∼50 fold. Together, the mutational data indicate that the mode of ssDNA binding is substantially conserved in Pif1 proteins. However, they also indicate that the residues in the B and C motifs may make a greater contribution to ssDNA binding in the mammalian compared to bacterial Pif1 proteins.Our hPIF1 structures allowed the generation and testing of a model for hPIF1 ssDNA binding. Although the model does not allow a precise structural and mechanistic interpretation of the data, significant conclusions can be made. All substitutions of conserved residues in the putative hPIF1 ssDNA binding channel resulted in a substantial ∼50-fold decrease in ssDNA binding affinity in the absence of nucleotide cofactors. With the exception of K485E, which retained ∼10% activity, all ssDNA binding channel mutants were inactive in helicase assays and had decreased DNA-dependent ATPwith DNA ,27, formwith DNA . The reswith DNA ,27,35. L87KFSEYK92 in BsPif1, 289QRPGVR294 in hPIF1 and 323RRSRKH328 in ScPif1. Although several residues of the BsPif1 segment interact with ssDNA, which in the crystal structure (pdb code 5FHD) is observed to bend sharply as it enters the ssDNA binding channel, alanine substitution of the residues results in only modest reductions in ssDNA binding and DNA unwinding activity , is on the outer surface of the DNA-free hPIF1 structures, and based on our ssDNA-ADP•AlFl Figure . ScPif1 l Figure , suggestl Figure ,52 and hl Figure  engage bAlthough it is unknown how Pif1 proteins bind G4 DNA some parallels could be drawn from the structure of the SF2 helicase DHX36  bound to a 3′ ssDNA tailed G4 DNA substrate , where tIn conclusion, the structural analysis indicates how ATP binding and hydrolysis events are coupled to DNA binding. The mutational data indicate that the ssDNA binding channel is substantially conserved in all Pif1 proteins. However, sequence and structural variability in functional motifs (e.g. the wedge region), the presence of additional domains (e.g. the 2C domain insertion in ScPif1) and the analysis of variant hPIF1 forms presented here indicate that caution is required in extrapolating directly from the analysis of microbial Pif1 proteins to human. Further studies of hPIF1, in particular structural studies on complexes with DNA substrates and helicase activity inhibitors, are required to fully understand its mechanism and to establish possible routes for therapeutic intervention.4− structures have been deposited with the Protein Data Bank under accession codes 6HPT, 6HPH and 6HPQ and 6HPU respectively.The coordinates and structure factors of the apo hPIF1, hPIF1-AMP-PNP, hPIF1-AMP-PNP(Br) and hPIF1-ADP•AlFSupplementary DataClick here for additional data file.


Cryptococcus neoformans, employs multiple mechanisms to compete for iron during cryptococcosis, a disease primarily of immunocompromised hosts. In this study, we examined the role of endocytic trafficking in iron uptake by characterizing a mutant defective in the Sec1/Munc18 (SM) protein Vps45. This protein is known to regulate the machinery for vesicle trafficking and fusion via interactions with SNARE proteins. As expected, a vps45 deletion mutant was impaired in endocytosis and showed sensitivity to trafficking inhibitors. The mutant also showed poor growth on iron-limited media and a defect in transporting the Cfo1 ferroxidase of the high-affinity iron uptake system from the plasma membrane to the vacuole. Remarkably, we made the novel observation that Vps45 also contributes to mitochondrial function in that a Vps45-Gfp fusion protein associated with mitotracker, and a vps45 mutant showed enhanced sensitivity to inhibitors of electron transport complexes as well as changes in mitochondrial membrane potential. Consistent with mitochondrial function, the vps45 mutant was impaired in calcium homeostasis. To assess the relevance of these defects for virulence, we examined cell surface properties of the vps45 mutant and found increased sensitivity to agents that challenge cell wall integrity and to antifungal drugs. A change in cell wall properties was consistent with our observation of altered capsule polysaccharide attachment, and with attenuated virulence in a mouse model of cryptococcosis. Overall, our studies reveal a novel role for Vps45-mediated trafficking for iron uptake, mitochondrial function and virulence.The battle for iron between invading microorganisms and mammalian hosts is a pivotal determinant of the outcome of infection. The pathogenic fungus,  Cryptococcus neoformans is a causative agent of cryptococcal meningitis, a disease that is estimated to cause ~ 15% of AIDS-related deaths. In this context, cryptococosis is one of the most common causes of mortality in people with HIV/AIDS, closely behind tuberculosis. Unfortunately, very few antifungal drugs are available to treat this disease. However, understanding mechanisms involved in the pathogenesis of C. neoformans can lead to new therapeutic avenues. In this study, we discovered a new role for a regulatory protein involved in vesicle transport. Specifically, we found that the Vps45 protein, which regulates vesicle fusion, participates in the trafficking of iron into fungal cells, supports mitochondria function, mediates antifungal resistance and is required for virulence. These discoveries shed light on the molecular mechanisms underlying the uptake and use of iron as an essential nutrient for the virulence of C. neoformans. Further investigations could lead to the development of drugs that target Vps45-mediated processes. Cryptococcus neoformans attacks immunocompromised people to cause cryptococcosis, a particularly devastating disease in HIV/AIDS sufferers   was performed to observe internalized vesicle trafficking and vacuolar membrane. The cells were stained with 5 μM for FM4-64 in PBS. The cells were incubated for an additional 90 min at 30°C and 37°C and pictures were taken every 15 min using an Olympus Fluoview FV1000 laser scanning confocal system. Colocalization analyses were performed on images of whole cells using the ImageJ coloc2 test. Positive correlations between Vps45-GFP and subcellular region were determined by Pearson’s R value (0.10–0.65) and Costes P-value (0.95–1.00) . A total7 mL-1 for staining as follows . For Eosin Y (Sigma), cells were resuspended in McIlvaines buffer, pH 6.0, and stained with 250μg mL-1 of the dye. For Pontamine , cells were stained in PBS with 100μg mL-1. For Concanavalin A-FITC (Sigma-Aldrich), cells were stained with 30μg mL-1 in Hepes-buffered saline, pH 7.0, containing 10mM each MgCl2 and CaCl2. Cells were stained for 15 min at room temperature, and then washed three times in appropriate buffers. Cells were then resuspended in PBS with 10mM NaN3 and analyzed by flow cytometry on a BD FACSCalibur. The acidic pH sensor carboxy-DCFDA (5-(and-6-)-Carboxy-2’7’-dichlorofluorescein diacetate) (ThermoFisher Scientific) was used to assess intracellular acidification. Overnight cultures were stained with 50μM carboxy-DCFDA and incubated for 30 min at 30°C and 37°C. Cells were spun down and resuspended in 10mM NaN3 in PBS. 50, 000 cells were analyzed by flow cytometry on a BD LSR II-561. JC-1 . Overnight cultures were stained with 5μM JC-1 for 30 min at 30°C and 37°C, washed 3 times, resuspended in media, and analyzed by flow cytometry on a BD LSR II-561. Flow analysis was performed using FlowJo software.Cells were grown overnight at 30°C and 37°C in YPD, washed in PBS, and resuspended at 10Capsule formation was examined by differential interference contrast microscopy on an Axio imager M.2 microscope (Zeiss) with magnification X1000 after incubation for 48 h at 30°C and 37°C in defined LIM and staining with India ink. Capsule measurements were performed using ImageJ. Capsule shedding from cells was examined with a blot assay using the anti-GXM 18B7 antibody as described .3 at 30°C or 37°C, washed 3 times with metal-chelated PBS, and lyophilized. The lyophilized cells were digested with 5 mL of HNO3 and 3 mL of H2O2 using a microwave digestion system (START D). Total iron and calcium contents were analyzed using an OPTIMA 5300 DV (PerkinElmer) system.Cellular iron and calcium contents were measured by inductively coupled plasma mass spectrometry (ICP-MS). Briefly, cells were pre-grown iron starved cells inoculated for 48h in YNB + 150 μM BPS ± 100μM FeCl°C with 5% CO2. Macrophages were stimulated 2 h prior to infection with 150ng mL-1 phorbol myristate acetate (PMA). Fungal cells were grown in YPD overnight and PBS-washed cells were opsonized in DMEM with 0.5 μg mL-1 of the monoclonal antibody 18B7 for 30 min at 37°C. Stimulated macrophages were infected with 2x105 opsonized fungal cells (MOI 1:1) for 2 and 24 h at 37°C with 5% CO2. Macrophages containing internalized cryptococci cells were washed thoroughly 3 times with PBS and then lysed with sterile water for 30 min. Lysate dilutions were plated on YPD agar and incubated at 30°C for 48 hrs, at which time the resulting CFUs were counted. Statistical significance of intracellular survival was determined by two-tailed unpaired t-tests .Macrophage infections were performed as described previously . Brieflyvps45Δ-34 mutant and the complemented strain vps45Δ::VPS45 [-1) and xylazine (5.5 mg kg-1) and suspended on a silk thread by the superior incisors. A suspension of 2X105 cells in 50μL was slowly inoculated into the nares of the mice. The health status of the mice was monitored daily post-inoculation and mice reaching the humane endpoint were euthanized by CO2 anoxia. Fungal burden of organs  and cardiac blood was assessed. The organs and blood were aseptically removed. Blood was retrieved from the heart using sterile syringes pre-rinsed with 500 units of heparin. Organs were weighed, and homogenized using a Retsch MM301 mixer mill. The samples were serial diluted, plated on YPD containing chloramphenicol (30μg mL-1) and incubated at 30°C for 2 days; CFUs were then counted. Statistical analyses of survival differences in mice were performed with the log rank test and a two-tailed unpaired Mann-Whitney test was used to assess the fungal load .The virulence of the WT strain H99, the Δ::VPS45  was asseThis study was carried out in strict accordance with the guidelines of the Canadian Council on Animal Care. The protocol for the virulence assays employing mice (protocol A17-0117) was approved by the University of British Columbia Committee on Animal Care.S1 Table(PDF)Click here for additional data file.S1 FigVPS45 locus yielded fragments of 4329 bp in the WT strain, 1582 bp in the deletion mutants, and 4478 bp and 1582 bp in complemented strains.Extracted DNA was digested with BglI and genomic hybridization to detect the (TIF)Click here for additional data file.S2 Figvps45 mutant, complemented (A), and GFP-tagged (B) strains were grown for 24h in YNB-BPS at 37°C. 1X106 cells/mL were inoculated in YNB-BPS + 100μM FeCl3, stained with 5 μM FM4-64 and transferred in a chamber slide where the cells were maintained at 37°C. Confocal microscopy images were taken every 15 min for 90 minutes. Colocalization analyses using ImageJ coloc2 test revealed a positive correlation between VPS45-GFP and endocytic membranes as determined by Pearson’s R value (0.15–0.45) and Costes P-value (0.99–1.00).WT, (TIF)Click here for additional data file.S3 FigVPS45 or VPS45-GFP) were grown on YPD in the presence of 10μg mL-1 fluconazole, 25μM aminodarone and 6mM chloroquine. Cells were pre-cultured in YPD overnight at 30°C, serial diluted, and 5μL were spotted onto YPD plates containing the indicated drugs. Plates were incubated at 30°C or 37°C for 2 days.The WT, mutants and complemented strains ((TIF)Click here for additional data file.S4 FigVPS45 at 37°C.A) Localization of Cfo1-GFP in the absence of  Strains  containing a CFO1-GFP construct were cultured overnight in YPD, washed 3 times and counted. 1X106 cells/mL were inoculated in YNB-BPS and incubated at 37°C for 1h. Then cells were stained with 5 μM FM4-64, transferred in a chamber slide and maintained at 37°C. Confocal images were taken every 15 minutes for 2h. B) Localization of Cfo1-GFP after 24h incubation in YNB, YNB + 150μM BPS, and YNB-BPS + 100μM FeCl3 at 37°C. The GFP label indicates evaluation of the green fluorescent protein and DIC indicates differential interference contrast microscopy. C) Visualization of full-length Cfo1-GFP by Western Blot in the WT strain (1), the vps45 mutant (2) and the complemented strain (3) cells incubated for 3h in YNB+150μM BPS at 30°C or 37°C. Western Blot on Cfo1-GFP using the mouse monoclonal GFP antibody (B-2) (Santa Cruz Biotechnology). Cfo1 is expected to be glycosylated like its homologue Fet3p in S. cerevisiae and this would explain the observed slower migration of the band (~140 kDa) versus the predicted size of the fusion  (59). D) Blue silver staining of the SDS-PAGE gel with ~10 ug of total cell protein extracted by bead beating and sonication from cells incubated for 3h in YNB + 150μM BPS at 30°C or 37°C.(TIF)Click here for additional data file.S5 FigVPS45 did not influence mitochondria morphology in iron-chelated conditions. Confocal images of mitochondria stained with 500nM mitotracker were taken of the WT, mutant and complemented strains inoculated for 24h in YNB ± 150μM BPS at 30°C (B) and 37°C (C). D). The WT, mutants and complemented strains were grown in the presence of inhibitors of the mitochondrial electron transport chain and ROS. Cells were pre-cultured in YPD overnight at 30°C, serial diluted, and 5μL were spotted onto YPD plates containing 75μg mL-1 rotenone, 1mM malonic acid, 2μg mL-1 oligomycin A, 500μM paraquat, 50μM plumbagin, 50μM diphenyleneiodonium chloride (dpi) or 5μg mL-1 menadione. Plates were incubated at 30°C and 37°C for 2 days. E) Example of flow cytometry assessment of mitochondrial membrane potential with the JC-1 dye. The percentages of cells with polarized, mixed and depolarized mitochondria membrane potential were determined by gating the fluorescence of unstained single cells population to stained single cells population.A) Mean R values of positive correlation between VPS45-GFP and mitochondria of cells grown under different conditions. Colocalization analyses were performed on images of whole cells (15–45 cells) using the ImageJ coloc2 test. Positive correlations were determined by Costes P-value (0.95–1.00). Disruption of (TIF)Click here for additional data file.S6 Fig5 cells/mL were inoculated into LIM (A and C), LIM + 100 μM FeCl3 (B and D) and incubated at 30°C (A and B) or 37°C (C and D) for 48h.Cells were pre-grown in defined LIM for 48h at 30°C, washed and counted. 5X10(TIF)Click here for additional data file.


However, currently, the tolerance to cadmium is very low in cyanobacteria. To further engineer cyanobacteria for the environmental application, it is thus necessary to determine the mechanism that they respond to high concentration of cadmium.Cadmium has been a significant threat to environment and human health due to its high toxicity and wide application in fossil-fuel burning and battery industry. Cyanobacteria are one of the most dominant prokaryotes, and the previous studies suggested that they could be valuable in removing CdSynechocystis PCC 6803 (named ALE-9.0) tolerant to CdSO4 with a concentration up to 9.0 µM was successfully isolated via adaptive laboratory evolution over 802-day continuous passages under cadmium stress. Whole-genome re-sequencing was then performed and nine mutations were identified for the evolved strain compared to the wild-type strain. Among these mutations, a large fragment deletion in slr0454 encoding a cation or drug efflux system protein was found to contribute directly to the resistance to Cd2+ stress. In addition, five other mutations were also demonstrated related to the improved Cd2+ tolerance in ALE-9.0. Moreover, the evolved ALE-9.0 strain was found to obtain cross tolerance to some other heavy metals like zinc and cobalt as well as higher resistance to high light.In this study, a robust strain of 2+ tolerance in Synechocystis PCC 6803, and demonstrated the feasibility of adaptive laboratory evolution in tolerance modifications. This work also provided valuable information regarding the cadmium tolerance mechanism in Synechocystis PCC 6803, and useful insights for cyanobacterial robustness and tolerance engineering.The work here identified six genes and their mutations related to CdThe online version of this article (10.1186/s13068-018-1205-x) contains supplementary material, which is available to authorized users. In addition, removal of toxic metal ions such as Cd2+ from water by cyanobacteria has been widely evaluated in recent years and is considered as a promising alternative treatment in wastewater purification [2+ by Gloeothece magna suggested that they would probably be cultivated in water bodies contaminated by Cd2+ to ameliorate its toxicity effectively [2+ in cyanobacteria.In recent years, environmental pollution caused by heavy metals has caused serious problems, including contaminating water, entering food chain and posing threats to growth of living organisms in nature , 2. Amony metals . Cadmiumy metals . Nowadayy metals . On the y metals , while wy metals , which wfication . For insectively . Therefo2+ have been investigated in the past decades. In a previous study, the direct influence of Cd2+ to photosynthetic machinery was found to be multiphase effects in model cyanobacteria Synechocystis sp. PCC 6803 (here after Synechocystis). The results showed that Cd2+ first limited photosystem I acceptor side, and 7 h later, it disturbed photosystem II under the existence of light [2+ toxicity caused the generation of reactive oxidative species (ROS) and the consumption of glutathione as well as the thiol-group containing protein [2+ penetrated rapidly into the cells and replaced other heavy metals like Ca2+ and Zn2+ by competing for enzymes and disturbing membrane potential [2+ triggered the integrated reprogramming of the whole metabolism in Synechocystis, which was controlled by the Slr1738 regulator [2+, like smtA in Synechococcus PCC 7942 [sll0649 in Synechocystis [2+ toxicity involved a variety of complex reaction mechanisms. However, up to now, the detailed mechanism of Cd2+ toxicity to cyanobacteria has still not been fully understood. Although some algae like Phormidium ambiguum and Scenedesmus quadricauda var. quadrispina show good tolerance to Cd2+ at the concentration of up to 0.35 mM [Synechocystis sp. PCC 6803 is a model organism with known genomic information [2+ stress [2+ tolerance of Synechocystis and understand how the Cd2+ tolerance is regulated, so that to guide the tolerance engineering in other algal or cyanobacterial species.Cellular responses of cyanobacteria to high concentration of Cdof light . In addi protein . Furtherotential . In anotegulator . MeanwhiPCC 7942  and sll0 0.35 mM , they arormation  and relaSaccharomyces cerevisiae, four strategies to isolate cobalt-tolerant cells were performed by ALE, yielding the most resistant mutant to cobalt stress from 2.5 to 8 mM, which indicated the efficiency of ALE to improve strains [Synechocystis to improve 1-butanol tolerance from a concentration of 0.2–0.5% (v/v) and a further metabolomic basis for rational tolerance engineering was determined [Adaptive laboratory evolution (ALE) is a strategy to improve strains via constant batch transfer under specific growth conditions , and has strains . In additermined .Synechocystis to Cd2+ stress. As a result, after 128 continuous passages of approximately 802 days, tolerance of the evolved strain was improved from 4.6- to 9.0-µM Cd2+. With the aid of high-throughput re-sequencing technology, the mutations in the genome of the evolved strain compared with the wild-type strain were identified and further functionally characterized. This study demonstrated the feasibility of ALE in tolerance modifications and provided useful insights for cyanobacterial robustness and tolerance engineering.In this study, the ALE strategy was employed to improve the tolerance of Synechocystis strain was evolved by serial passaging for 128 passages (802 days) in BG11 medium supplemented with CdSO4, as a selective pressure to enrich population with Cd2+ tolerance. The starting Cd2+ concentration for WT was set as 4.6 µM as our previous study showed that WT showed a slight growth deficiency at this Cd2+ concentration level [4, Synechocystis could achieve late exponential phase (OD750 nm = 1.5) from an initial inoculum (OD750 nm = 0.1) within 96 h. When CdSO4 was added, cell growth rate decreased obviously. In this ALE process, a simple rule was established that once the evolutionary strain could reach OD750 nm of 0.5  within 96 h, the Cd2+ concentration was increased. Thus, the Synechocystis strain was cultivated with increasing Cd2+ concentration from 4.6 µM gradually to 9.0 µM  (Table Wild-type (WT) on level . Under n2+, ALE-9.0 grew slightly, but not significantly, slower than WT, while under 9.0-µM CdSO4 condition, the growth of ALE-9.0 was dramatically better than WT, as WT can hardly survive under 9.0-µM Cd2+, demonstrating the improved Cd2+ tolerance in ALE-9.0. To show the effect of ALE process better, tenfold serial dilutions of WT and ALE-9.0 liquid cultures were spotted onto BG11 agar plates with different concentrations of CdSO4 . In all, one deletion, nine single-nucleotide polymorphisms (SNPs) and four structural variations (SVs) were detected in ALE-9.0 compared to WT after the evolution process, and then, all these mutations were individually verified by Sanger sequencing. As a result, seven SNPs and one SV were identified, and the deletion turned out to be one insertion . It is speculative that these mutations might lead to an increased tolerance of Synechocystis to Cd2+ in the strain ALE-9.0.Notably, WT could only endure 4.6-µM CdSOon Table . Among t2+  occurred in the later stage of the whole ALE process  evolved in the middle term of this evolution process and tolerant to 7.0-µM Cd2+ Fig. b was als4 stress conditions. To evaluate this hypothesis, five samples  were selected for qRT-PCR analysis. The expression change was presented by the ratio of the relative expression level of the genes under stress condition to that in normal BG11 medium, respectively, and a fold change > 2.0 was used as a cutoff. As shown in Table 4 stress compared with WT cultured in normal BG11 medium, probably because that these genes did not respond to Cd2+ under this concentration. However, transcriptional levels of most genes were increased significantly in ALE-9.0 when CdSO4 was added. Particularly, the most significant changes were found in slr0721 and slr0798. Although the relative expression level of slr0721 had no significant change in ALE-9.0 under 4.6-µM CdSO4, it was up-regulated almost ninefold under 9.0-µM CdSO4. Meanwhile, slr0798 was up-regulated 17.5-fold under 4.6-µM CdSO4 condition and 36.5-fold in ALE-9.0 under 9.0-µM CdSO4, indicating its important roles in the tolerance to Cd2+. Another gene, slr1753, was also up-regulated 2.8-fold under 4.6-µM CdSO4, while 4.0-fold under 9.0-µM CdSO4. Transcriptional level of ssr1480 was increased by about 2.3-fold and sll1586 increased by about 3.5-fold in ALE-9.0 under both 4.6- and 9.0-µM CdSO4 stress conditions. Meanwhile, slr0623 was found up-regulated 2.3-fold under 4.6-µM CdSO4 and 1.8-fold under 9.0-µM CdSO4. On the other hand, the expression of slr0454, slr0774, and slr1302 was only slightly changed in ALE-9.0 under both Cd2+ conditions (fold changes less than 2.0).Besides the genetic differences between WT and ALE-9.0, the expression level of the mutated genes could possibly also change under CdSOslr0721, slr0798, slr1753, sr1480, sll1586, and slr0623, were significantly induced upon the exposure to Cd2+ after this ALE process, indicating their relevant roles in Cd2+ tolerance of ALE-9.0.According to the above results, the expression levels of six genes, 2+ tolerance. Relevant genes were replaced with chloramphenicol-resistance cassettes in WT and ALE-9.0 by homologous recombination, respectively (the related names of mutants were shown as ∆XWT and ∆XALE-9.0), and knock of neutral site slr0168 in WT (WT-C) and ALE-9.0 (ALE-9.0-C) was selected as controls  family. Consistent with our results, this kind of protein was reported previously to help Gram-negative bacteria to keep the intracellular homeostasis under high metal concentrations [Escherichia coli (E. coli), which has been found mediated resistance to ions including nickel, cadmium, and cobalt [Among these nine genes, overexpression of four genes, trations . In addid cobalt .Fig. 5Gr2+ tolerance under Cd2+ stress condition when the WT or mutated genes were overexpressed  even had negative effects on the tolerance to Cd2+, probably due to that the expression of these genes was already saturated, or their overexpression has brought extra metabolic burden to WT or ALE-9.0.For the remained five genes, none of them showed positive effect on Cd4, CoCl2, CuSO4, high light, ethanol, and H2O2. The results showed that compared to WT, ALE-9.0 grew better under stress conditions of ZnSO4 and CoCl2  were observed between WT and ALE-9.0  were also obtained.Possible cross tolerance of ALE-9.0 to other stresses was also investigated, including ZnSOCl2 Fig. a, while  WT Fig. b. Althouays Fig. c. H2O2 ad-lactic acid produced by the evolved strain has been increased 2.0-fold than the original strain in Leuconostoc mesenteroides [Corynebacterium glutamicum [2+ tolerance of Synechocystis and an evolved strain tolerant to 9.0-µM CdSO4 was isolated after 802 day series passages. The slightly slower growth of ALE-9.0 compared with WT under normal BG11 condition  TrxA, which is a class of small redox proteins known to be present in most microorganisms. Consistent with our results, research in E. coli showed Trx was inhibited by Cd2+ and posed a positive role in protection from Cd2+ [2+ directly bound to Trx by forming a chelator and decreased thiol-disulfide transferase activity, and this kind of Cd2+ sink might help against Cd2+ stress. slr0721 showed similar results as slr0623 both in gene knockout and overexpression analysis. slr0721 encodes the decarboxylating NADP-dependent malic enzyme, participating in tricarboxylic acid cycle. A study on malic acid and Cd2+ stress in Miscanthus sacchariflorus proved exogenous addition of malic acid could alleviate Cd2+ toxicity through enhancing photosynthetic capacity and restraining ROS accumulation [slr0721ALE-9.0 in ALE-9.0 did not show significant change under 4.6 µM, but was increased significantly under 9.0-µM CdSO4  pathway was considered as a major translocation process of protein from cytosol across the cytoplasmic membrane in bacteria [slr0774 could lead to different efficiency of SecD or help SecD interact with other membrane protein better, leading to high Cd2+ tolerance. In addition, slr1753 was found as an outer membrane fraction for its homology to a cell-surface glycoprotein in Clostridium thermocellum [2+ resistance [slr1753 possibly was able to help optimize the constitution and content of saccharide on cell-surface, leading to different Cd2+ tolerances. Since the ALE-9.0 could tolerate 9.0-µM Cd2+, except for the function of each single gene, the combinations of these genes and mutations to work together most probably also exist [On the other hand, five mutated genes, rom Cd2+ . Accordimulation . Even thmulation , 43, we olerance . Consistbacteria  and SecDbacteria . It is tmocellum . Besidessistance , it is tso exist  which is2+ tolerance, some genes including slr0649, slr0946, slr1738, and sll1598 were also demonstrated to be related to Cd2+ tolerance in the previous studies [Mutations occur randomly during the course of ALE and are selected naturally when a particular mutation enhances the activity of a protein and/or thereby the better tolerant or survival . For Cd22+, Co2+, and high illumination intensity  under a light intensity of approximately 50-μmol photons/m2/s in an illuminating or shaking incubator of 130 rpm at 30 °C  [E. coli strain DH 5α was used for constructing and collecting plasmids. E. coli was grown on LB agar plate or in LB liquid medium in incubator at 37 °C or shaking incubator at 200 rpm supplemented with appropriate antibiotic .The wild-type , China) . Proper 2+ tolerance was carried out in 20 mL liquid BG11 medium in a 100 mL shake flask. The CdSO4 stock solution was prepared with CdSO4·8/3H2O of analytical pure, purchased from Aladdin . The initial WT stain was cultured with 4.6-µM CdSO4 from an inoculum of OD750 nm 0.1. Cd2+ concentration in BG11 medium was increased by 0.3–0.4 µM when the culture reached OD750 nm of 0.5 within 96 h. The simplified process is shown in Fig. 2+ in the last passage, the culture was centrifuged and transformed into fresh BG11 medium during evolution. Serial adaptation passages were conducted until the final tolerance to CdSO4 achieved 9.0 µM. After confirming that the strain can survive under 9.0-µM CdSO4, the evolved strain was screened on BG11 agar plate with 9.0-µM CdSO4, and four clones were isolated and re-cultured in BG11 liquid medium. After re-confirmation of the tolerance, one clone showing greatest growth state was selected for further analysis . Final results involved SNP (single-nucleotide polymorphism), InDel , and SV .Isolation of genomic DNA was carried out as described previously . Total DTo validate the SNP, InDel, and SV revealed by re-sequencing, Sanger sequencing was performed. Primers used to amplify gene fragments are listed as “primers for Sanger sequencing” in Additional file E. coli DH5α was used for vector construction and amplification. For knockout of relative original and mutated genes, the plasmids’ framework was obtained from above plasmid used for sequencing by PCR with primers for gene knockout  were set out for different genes.To monitor growth profile under Cd4, 1.8 µM for CuSO4, 17 µM for CoCl2, 1 mM for H2O2, and 1 mM for ethanol. High light was set as 200-μmol photons/m2/s.Growth under other examined stress conditions was also measured in the same way as above. Concentrations of chemicals used were as follows: 20 µM for ZnSOAdditional file 1: Fig. S1. Growth patterns of OE-C and overexpression of other genes in normal BG11 medium and under corresponding CdSO4 concentration. (a) slr0774, (b) slr1302, (c) ssr1480, (d) sll1586, (e) slr1753. The error bars represented the calculated standard deviation of the measurements of three biological replicates. Fig. S2. Cross tolerance of WT and ALE-9.0 against other stresses. Cell growth at 48 and 84 h in normal BG11 media, 2% ethanol, 1.8 μM CuSO4 or 1 mM H2O2. ALE: adaptive laboratory evolution.Additional file 2: Table S1. All the primers used in this study.


MEIS1 gene is associated with an increased risk in developing RLS, but the effect of MEIS1 dysfunction on sensorimotor function remain unknown. Mouse models for a dysfunctional D3 receptor (D3KO) and Meis1 (Meis1KO) were developed independently, and each animal expresses some features associated with RLS in the clinic, but they have not been compared in their responsiveness to treatment options used in the clinic. We here confirm that D3KO and Meis1KO animals show increased locomotor activities, but that only D3KO show an increased sensory excitability to thermal stimuli. Next we compared the effects of dopaminergics and opioids in both animal models, and we assessed D1 and D3 dopamine receptor expression in the spinal cord, the gateway for sensorimotor processing. We found that Meis1KO share most of the tested behavioral properties with their wild type (WT) controls, including the modulation of the thermal pain withdrawal reflex by morphine, L-DOPA and D3 receptor (D3R) agonists and antagonists. However, Meis1KO and D3KO were behaviorally more similar to each other than to WT when tested with D1 receptor (D1R) agonists and antagonists. Subsequent Western blot analyses of D1R and D3R protein expression in the spinal cord revealed a significant increase in D1R but not D3R expression in Meis1KO and D3KO over WT controls. As the D3R is mostly present in the dorsal spinal cord where it has been shown to modulate sensory pathways, while activation of the D1Rs can activate motoneurons in the ventral spinal cord, we speculate that D3KO and Meis1KO represent two complementary animal models for RLS, in which the mechanisms of sensory (D3R-mediated) and motor (D1R-mediated) dysfunctions can be differentially explored.Restless Legs Syndrome (RLS) is often and successfully treated with dopamine receptor agonists that target the inhibitory D3 receptor subtype, however there is no clinical evidence of a D3 receptor dysfunction in RLS patients. In contrast, genome-wide association studies in RLS patients have established that a mutation of the  MEIS1 and BTBD9 , suggesting a possible interaction between MEIS1 and dopamine (DA) function.Restless Legs Syndrome (RLS) is a highly prevalent , and the D3R subtype has a very high affinity to DA  and Meis1 (Meis1KO) were developed independently  and their appropriate wild-type (WT) controls , dopamine D3 receptor knockout mice , n = 13) and their appropriate WT controls  . All efforts were made to minimize the number of animals used, and a total of 55 male mice (age range ~9–12 months) were tested in this study. Behavioral testing was performed on Behavioral testing procedures have been described in detail recently , and for D3KO and their controls with a TSE LabMaster System . Infrared sensors were used to record ambulatory activity in the X-Y plane). Counts across all these axes were summed to give total ambulatory activity. Meis1KO and their WT controls were monitored for 7 days. Only the data from the last 4 days were pooled and analyzed. For D3KO and their controls, after 2 days of acclimation, data were collected for 2 consecutive days to calculate locomotor activity.We tested the effects of levodopa , the D3 receptor agonist, pramipexole , the D3 receptor antagonist, SB277011-A , the D1 agonist, SKF 38393 , the D1 receptor antagonist, SCH 39166 , and morphine . Drug concentrations were chosen based on previous publications by others and us , and stored at −20°C until use. Spinal cords were homogenized in 1 ml of RIPA buffer with protease and phosphatase inhibitors . The homogenized spinal cords were centrifuged , and supernatants were aliquoted and stored individually at −80°C. Following homogenization, standard protein concentrations were established with a Bradford protein assay , and plates were read on an Epoch Microplate Spectrophotometer  at 595 nm using the Gen5.1 software package .For Western blots, 30 μg of each lumbar spinal cord protein samples were denatured using 2x Laemmli buffer containing 5% β-mercaptoethanol and 1% SDS at 95°C for 10 min and loaded onto a 12% Criterion™ TGX Stain-Free™ Protein Gel  and run ~45 min at 200 V. The proteins were transferred to a low fluorescent PVDF membrane using Trans-Blot® Turbo™ RTA Midi LF PVDF Transfer Kit . Membranes were probed with the primary antibodies and with secondary antibodies  by the iBind™ Flex Western Device  based on sequential Lateral Flow (SLF) technology using iBind™ Flex Fluorescent Detection (FD) Solution Kit . Membranes were imaged with an Odyssey imaging system .The primary antibodies used for Western blot to detect receptor-specific protein expression were: anti-dopamine receptor D1  and anti-dopamine receptor D3 . The secondary antibodies used were goat anti-rabbit 680RD  and goat anti-mouse 680RD .post-hoc comparisons ; t-tests or paired t-tests were used for comparison between two sets of data (treatment against respective control vehicle treatment only). Significance levels were set at p < 0.05. For Western blot analysis, images were analyzed with ImageStudio and ImageJ  and statistical analyses were performed with SigmaPlot 11.0.Following the experiments, behavioral data were transferred and stored in Excel format, then analyzed and plotted offline with SigmaPlot . For statistical comparisons, we employed parametric or non-parametric comparisons as appropriate when comparing multiple groups  with appropriate p = 0.97, n = 7 each, power: 0.05). In cohort 2 (tested about 1 year later) the average WT latency was 6.6 ± 0.4 s, while Meis1KO latencies were 6.7 ± 0.9 s . Thus despite the difference between the two Meis1KO cohorts and their WT controls, there was no effect within each cohort between WT and knockout. In contrast, individual withdrawal latencies in D3KO ranged per trial from 4.8 to 11.1 s,  while the latencies of their respective WTs (WTD3KO) ranged from 5.9 to 13.7 s . A t-test comparison revealed that the difference between WTD3KO and D3KO was significant .We first tested and compared pain withdrawal latency responses in Meis1KO and D3KO lines under baseline conditions , and compared them to their respective controls. Meis1KO and their respective controls were tested as two independent cohorts. In cohort 1 the average WT latency was 10.2 ± 1.1 s, while Meis1KO latencies were 10.2 ± 0.5 s  were not significantly different from each other , while those of D3KO were significantly reduced over their respective WTs .To illustrate the effect of the genotype alone on thermal pain withdrawal reflex latencies, we normalized the WTn = 6), while responses of Meis1KO increased from 6.3 ± 1.2 s to 10.1 ± 1.1 s (n = 7). Similarly, in cohort 2, WT withdrawal latencies increased from 6.5 ± 1.2 s to 9.3 ± 1.9 s (n = 5), while responses of Meis1KO increased from 6.3 ± 0.6 s to 7.9 ± 1.9 s (n = 7). After normalization to each WTmeis1 control, the data are as follows: WTmeis1; Ctrl: 99.3 ± 7.1%; after treatment: 132.5 ± 14.8%, p = 0.08, paired t-test, n = 11, power: 0.83, Figure p = 0.024, paired t-test, n = 14, power: 0.59, Figure D3KO the raw data were as follows; Ctrl: 8.3 ± 0.3 s; after treatment: 11.4 ± 0.6 s (n = 7), while D3KO were not responsive to 2 mg/kg morphine . After normalization to the pre-treatment control, the data for WTD3KO are: 99.8 ± 2.8%; after treatment: 137.1 ± 7%, p = 0.002, paired t-test, power: 0.99; Figure p = 0.62, paired t-test, power: 0.05, Figure meis1, WTD3KO, and Meis1KO animals respond similar to morphine, while D3KO are not affected.While opioids are commonly used for treating chronic neuropathic pain, they have also become a treatment of choice in dopamine agonist-refractory RLS .meis1, treatment with PPX (0.5 mg/kg) increased withdrawal latencies from 100.2 ± 6.6% to 144.4 ± 13.7%, . In Meis1KO, the PPX treatment also led to a significant increase in withdrawal latencies from 100.1 ± 8.9% to 131.3 ± 12.3% . The effect of PPX was similar in the WT of the D3KO mice (WTD3KO) to the effect observed in WTmeis1 and Meis1KO, but it did not alter the responses in the D3KO. In WTD3KO, withdrawal latencies rose significantly from 100 ± 6.3% (Ctrl) to 204.8 ± 9% . Taken together, the results of these dopaminergic modulators suggest that D3 receptor-mediated actions affect WT and Meis1KO similarly, while activation of D1 receptor signaling pathways exert similar effects in Meis1KO and D3KO but not WTs.In WTMeis1, Meis1KO, and D3KO , and that D3KO showed a similar significant increase over their WT controls .As the pharmacological and Western blot data suggested a role of the D1 receptor system in the control of spinal cord function, and as D1 receptor activation can recruit spinal cord networks to generate locomotor-like activities, we next tested if Meis1KO and D3KO show altered locomotor activities over their respective WT controls Figure . We founin vivo and in vitro studies  controls under different drug treatment conditions. We found that, under baseline conditions (sham), withdrawal latencies of WT and Meis1KO were similar, while those of D3KO were decreased, suggesting heightened excitability in these animals to a thermal stimulus and confirming earlier Opioids are powerful modulators of nociceptive pathways, and we have shown previously that low morphine exposure  was unsuccessful in modulating thermal pain withdrawal reflexes in D3KO and that this effect could be mimicked by acutely blocking D3 receptors in the isolated WT spinal cord preparation  as the immediate precursor of DA, to test the effects of raised DA levels on sensorimotor function in the three animal lines but observed no clear effect in either animal line. While L-DOPA plays an important modulatory role in controlling, at least temporarily, RLS symptoms Akpinar, , it can in vitro while they do not alter SRAs in D3KO  may be the component that drives this behavioral outcome. We here confirmed that both Meis1KO and D3KO animals express increased locomotor activities (Accili et al., All DA receptor-mRNAs are expressed in the neurons of the rodent spinal cord (Zhu et al., The presence of a D3 receptor protein in the D3KO may appear unexpected, but the D3KO mouse used in our studies is not a genetic knockout of the D3 receptor; rather it is a targeted mutation of the D3 receptor gene in the second intracellular loop of the predicted protein sequence that prevents the incorporation of the D3R into the membrane (Accili et al., In contrast to the D3 receptor data, the results of the D1 receptor protein expression point to a similarity of Meis1KO and D3KO, but not with either of them and the WT. We have shown previously that D3KO express not only decreased thermal pain withdrawal latencies but also an increased spinal D1 receptor protein expression (Brewer et al., Descending DA projections to and the presence of DA receptors in the spinal cord are well established (Holstege et al., Figure SM, M-LD, SL, YunL, C-TL, and MK performed the experiments. SM, M-LD, MK, C-TL, and SC analyzed the data. SC and YuqL designed the experiments. SM, KB, YuqL, and SC wrote the manuscript. SM, M-LD, MK, SL, YunL, YuqL, C-TL, KB, YL, and SC approved of the manuscript.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.


Morinda citrifolia has always been sought out for its remarkable remedies. Damnacanthal, an anthraquinone that can be extracted from the roots of Morinda citrifolia is a promising compound that possesses a variety of biological properties. This study aimed to study the therapeutic effects of damnacanthal in combination with doxorubicin in breast cancer cells. Collectively, the combination of both these molecules enhanced the efficacy of induced cell death in MCF-7 as evidenced by the MTT assay, cell cycle, annexin V and expression of apoptosis-related genes and proteins. The effectiveness of doxorubicin as an anti-cancer drug was increased upon addition of damnacanthal. These results could provide a promising approach to treat breast cancer patients.Despite progressive research being done on drug therapy to treat breast cancer, the number of patients succumbing to the disease is still a major issue. Combinatorial treatment using different drugs and herbs to treat cancer patients is of major interest in scientists nowadays. Doxorubicin is one of the most used drugs to treat breast cancer patients. The combination of doxorubicin to other drugs such as tamoxifen has been reported. Nevertheless, the combination of doxorubicin with a natural product-derived agent has not been studied yet.  Breast cancer is a complex disease that results from the interaction of multiple environmental, hormonal, and lifestyle risk factors with the individual’s genome . AlthougMorinda citrifolia (Noni or Yor). Morinda citrifolia L. (Rubiaceae) is a small tree, known commercially as noni that grows widely throughout the Pacific and is one of the most significant sources of traditional medicines among Pacific Islander societies in Hawaii, Fiji, Vanuatu, New Guinea, New Caledonia, and the Solomon Islands. All parts of the plant, including the roots, barks, stems, leaves, and fruits have been used traditionally as folk medicines for the treatment of many diseases, including diabetes, hypertension, and cancer [Morinda citrifolia are the source of important compounds, i.e., anthraquinones, which have been proven to have anti-viral, anti-bacterial, and anti-cancer activities. The most medicinally valuable anthraquinone in the roots of this plant is damnacanthal, which has been used for the treatment of chronic diseases such as cancer and heart disease [For centuries, scientists and medical professionals have been investigating chemical constituents in all parts of d cancer . Accordid cancer  noni frud cancer  reported disease .50 value (the concentration that inhibited 50% viability of the MCF-7 cell population by the treatment) were used to treat MCF-7 cells with or without the presence of doxorubicin.It would be interesting if damnacanthal were used in combination with other drugs that are used in treating patients diagnosed with estrogen receptor positive breast cancer, since tamoxifen and doxorubicin shows antagonistic effects when used in combination on MCF-7 cells . Althoug50 value of doxorubicin on MCF-7 was estimated at around 0.55 ± 0.02 µg/mL. In 50 value dropped to less than 0.55 ± 0.02 µg/mL when MCF-7 cells were treated with a combination of doxorubicin and damnacanthal. The CD50 value dropped to 0.50 ± 0.03 µg/mL, 0.20 ± 0.02 µg/mL and 0.12 ± 0.05 µg/mL when MCF-7 cells were exposed to 2.5 ± 0.12 µg/mL, 8.2 ± 0.07 µg/mL and 10.3 ± 0.15 µg/mL of damnacanthal, respectively. The pattern is also the same in 50 value was reduced to less than 0.55 ± 0.02 µg/mL when MCF-7 cells were treated with a combination of doxorubicin and damnacanthal. The CD50 value was significantly reduced to 0.30 ± 0.12 µg/mL, 0.11 ± 0.01 µg/mL and 0.10 ± 0.03 µg/mL when MCF-7 cells were exposed to 2.5 µg/mL, 8.2 µg/mL and 10.3 µg/mL of damnacanthal, respectively.50 value also dropped to less than 0.55 ± 0.02 µg/mL when MCF-7 cells were treated a combination of doxorubicin and damnacanthal. The CD50 value significantly decreased to 0.45 ± 0.13 µg/mL, 0.12 ± 0.11 µg/mL and 0.10 ± 0.15 µg/mL when MCF-7 cells were exposed to 2.5 µg/mL, 8.2 µg/mL and 10.3 µg/mL of damnacanthal, respectively. From the results, the percentages of viability of MCF-7 cells after treatment with combination of doxorubicin and damnacanthal were decreased compared to the percentages of viability of MCF-7 cells when treated with doxorubicin alone. This indicates that damnacanthal can act in combination with doxorubicin.Lastly in AO/PI staining is considered the appropriate method for evaluating the changes of nuclear morphology and is used to quantify the cellular profile of viable, apoptotic and necrotic cells. Viable (green intact cells), apoptotic (green shrinking cells with condensed or fragmented nucleus) and late apoptotic and necrotic cells (red cells) were quantified from a population of 200 cells for the data to be statistically significant. The same procedure was carried out on cells treated for 72 h. Results were expressed as a proportion of the total number of cells examined. The experiment was repeated at least three times with triplicate samples for each experiment. The results of AO/PI staining is shown in p < 0.05) amount of cells in sub-G0/G1 phase was detected in MCF-7 cells treated with G2 (doxorubicin at 0.55 µg/mL), G5  and G6 . This sub-G0/G1 population is higher than the MCF-7 cells treated with G3 and G4 which are damnacanthal alone. The sub-G0/G1 population increased further from 2% for the control group to 8.7%, 18.7% and 8% after exposure to G2, G5 and G6, respectively, after 72 h. The percentage of sub-G0/G1 population are higher in G5 and G6 of MCF-7 cells which is the combination of doxorubicin and damnacanthal compared to the MCF-7 cells treated with either doxorubicin or damnacanthal alone. Apart from that, as shown in 1 phase and G2/M phase. The percentage of G1 phase was increased after MCF-7 cells were treated with G3 and G4, which is damnacanthal alone. Meanwhile the percentage of G2/M phase was increased in treatment G2 which is doxorubicin alone. After 72 h of incubation, the early apoptosis population of MCF-7 cells treated G2, G5 and G6 were decreased compared to 24 h of incubation time. However, the early apoptosis population of MCF-7 cells treated with G4 treatment is slightly increased.In comparison with the spontaneous cell death as observed in G1 (untreated control cells), substantial apoptotic cells were detected in MCF-7 cells treated with G2 (doxorubicin at 0.55 µg/ml), G3 , G4 , G5  and G6 . Changes in the distribution of cell populations in various cell cycle phases after 72 h of MCF-7 cell treatment with G2 (doxorubicin at 0.55 µg/mL), G3 , G4 , G5  and G6  is displayed in +/PI+) late apoptosis population for MCF-7 cells was detected. The percentage of dead cell population also increased in G2, G5 and G6, as shown in While the early apoptosis population for MCF-7 cells treated with G2, G5 and G6 decreased, a pronounced increment in the double-positive  as compared to control (G1). Meanwhile, the expression level of caspase-7 was found to increase significantly (p < 0.05) in G5 and G6 treatments as compared to control. p < 0.05) as compared to control (G1). Meanwhile, the expression level of caspase-3 was found significantly increased (p < 0.05) in the G2, G4, G5 and G5 treatments. Expression level of caspase-7 increased significantly (p < 0.05) when the cells were treated with G4, G5 and G6.From In this study, expression levels of protein that are involved in apoptosis; Bcl-2, p53, ER-alpha and XIAP were evaluated using BD FACS Calibur multicolor flow cytometer . p < 0.05) with treatments G2, G3, G4, G5 and G6 as compared to control G1. Meanwhile, percentage expression level of Bcl-2, XIAP and ER-alpha protein decreased significantly (p < 0.05) with treatments G2, G3, G4, G5 and G6 as compared to control.Percentage expression level of p53 protein increased significantly , CD50 (8.2 µg/mL) and CD60 (10.3 µg/mL) values were determined based on the reported results. From the results, the population of MCF-7 cells decreased more when it was treated with the combination of doxorubicin and damnacanthal compared to the use of doxorubicin alone. The combination of damnacanthal and doxorubicin thus led to an enhancement of the cytotoxicity of doxorubicin. In principle, this result is similar to the cytotoxic effect of mistletoe  extract combined with doxorubicin on Jurkat cells [Doxorubicin is an anthracycline antibiotic, formed from natural products produced by species of the soil fungus toxicity . Magrathtoxicity  suggesteat cells . It was Staining of apoptotic cells with fluorescent dyes such as AO and PI is considered the appropriate method for evaluating the changes in nuclear morphology . This AO0/G1 peak will observed before the G1 peak in the fluorescence histogram [0/G1 population, indicating the occurrence of apoptosis in MCF-7 cells treated with the combination of damnacanthal and doxorubicin. Cell cycle analysis revealed that MCF-7 cells treated with damnacanthal have induced apoptosis and cell cycle is arrested at the G1 phase while MCF-7 cells treated with doxorubicin have induced apoptosis and cell cycle is arrested at the G2/M phase. This finding was similar to that of Ling et al. [0/G1). DNA histograms for MCF-7 cells treated with the combination of damnacanthal and doxorubicin exhibited a prominent decrease of the G1 phase cell population at 72 h of incubation.Cell cycle analysis is important for evaluating cell cycle parameters of surviving cells upon treatment or cell damage. When cells are damaged the cell cycle is interrupted at certain checkpoints and this prevents the normal cells from being transformed into cancer cells . It is aistogram . Therefog et al. . They reg et al.  reported−/PI−), early apoptosis (annexin V-FITC+/PI−) and late apoptotic or necrotic cells (annexin V-FITC+/PI+) [The early apoptosis process involves the alteration of cell membranes. In normal living cells, phosphatidylserine (PS) is located on the inner cytoplasmic surface of cell membrane. While apoptotic cells will expose PS on their intact cell membrane’s surface ,22 the PTC+/PI+) . HoweverTC+/PI+) .1 use consistent nomenclature  [The expression of p21 gene is controlled by p53 protein. p21 is a potent cyclin-dependent kinase inhibitor (CKI) where it binds to and inhibits the activity of cyclin-CDK2 or -CDK1 complexes, and thus functions as a regulator of cell cycle progression at G groups) . Besides groups)  and also groups) . In the  groups) . Like p2 groups) . This ex groups) . Differe groups) . Initiat groups) . After u groups) . Cytochr groups) . Apaf-1  groups) . The rel groups) .3--2,5-diphenyl tetrazolium bromide (MTT), RPMI-1640, phosphate buffer saline (PBS), bovine serum albumin (BSA) and ethylenediaminetetraacetic acid (EDTA) were all purchased from Sigma . Foetal bovine serum (FBS) was purchased from PAA  and was heat-activated for 30 min in a 56 °C water bath prior to use. Tryple E from Gibco Invitrogen , DMSO . 96-well and 6-well plates, FITC-conjugated Annexin V kit were purchased from .5 cell/wells incubated for 24 h, 48 h and 72 h . One hundred µL of diluted doxorubicin at 10 µg/mL was added into row A and row B. A series of two-fold dilution of extract was carried out down from row B until row G. The row H was left untouched and the excess solution (100 µL) was discarded and 100 µL of diluted compound  was added into wells in the 96-well plate. Finally, the plate was read at 570 nm and 630 nm as reference wavelength by using a µ Quant ELISA Reader . The experiment was repeated for at least three times with triplicate samples for each experiment. The percentage of proliferation was calculated using the following formula:The effect of the combination of damnacanthal and doxorubicin treatment on cell viability of MCF-7 cells was determined using a colorimetric technique, which is an MTT assay on a 96-well plate (BD) in RPMI 1640 media with 10% of FBS per well at cell density 1 × 105 cells) were seeded in 6-well plates and incubated at 37 °C in a 5% CO2 atmosphere. Twenty four h later, the medium in each well was removed and replaced with the various treatments . The concentration of doxorubicin at 0.55 µg/mL was obtained from the CD50 value of doxorubicin toward MCF-7 cells and the concentration of doxorubicin at 0.2 µg/mL was obtained from the CD25 value of doxorubicin towards MCF-7 cells. Meanwhile, the concentrations of damnacanthal used were same as in the cell viability assay described in 2 and 90% humidity for 24 and 72 h. After 24-h incubation, detached and anchored cells in the medium were collected. Anchored cells were detached with the use of tryple E . The cell suspension was washed with PBS. Ten µL of the cells were then put on a glass slide and mixed with 10 µL of acridine orange (50 µg/mL) and propidium iodide (50 µg/mL) at a ratio of 1:1 in 1 mL of cells. Within 30 min, the slide was analyzed using fluorescent microscope  using a combination of excitation and barrier filters at 450–490 nm and long pass filter.MCF-7 cells . The plate was incubated at 37 °C in an incubator with 5% CO2 and 90% humidity for 24 and 72 h. After the corresponding period, the samples were washed and transferred to a centrifuge tube (BD Biosciences). The cells were pelleted and fixed with 80% ethanol and incubated at 4 °C for 2 h. Then, the cells were re-pelleted and washed twice with PBS-sodium azide-EDTA buffer. The cell pellet was finally dissolved and stained in PBS buffer consisting of 0.1% Triton X-100, 10 mM EDTA, 50 µg/mL RNase and 2 µg/mL propidium iodide (PI) in the dark. The cell pellet was then incubated for half an hour in 4 °C prior to analysis by FACS-Calibur flow cytometer . The analysis of stained cells by flow cytometer was conducted within 24 h. The experiment was repeated for three times with triplicate samples for each experiment.The perturbation in the distribution of cells in the different phases of the cell cycle was determined by flow cytometry. 5 × 105 of MCF-7 cells were seeded into each well of a 6-well plate. After incubation for 24 h the cells were treated with various treatments  were added. Group treatments used were same as in the AO/PI staining assay  with propidium iodide (PI). The procedures were carried out according to the instructions provided by the manufacturer. Briefly, the culture was washed with PBS and re-suspended in 500 µL total volume that contain 100 µL of cells (5 × 105 cells/mL), 10 µL PI (Sigma), 5 µL AnnexinV-FITC (BD Bioscience) and 400 µL binding buffer (BD Bioscience). After 15-minute incubation in dark, the cells were analyzed with FACS-Calibur flow cytometer (BD Biosciences). The experiment was repeated for three times with triplicate samples for each experiment.5 × 10ng assay . The plaThe MCF-7 cells were treated with with various treatments . Meanwhile untreated (G1) was used as controls. The expression profiles were analyzed at two time points, 12 and 24 h. After the treatment period, cells were trypsinized and washed twice with PBS in order to prepare for RNA extraction. Total RNA was extracted from the treated and untreated cell lines for both time points using the RNeasy® Mini Kits . The eluted RNA was kept in −80 °C for future use. After RNA extraction and quantification, RNA from each sample was reverse transcribed to cDNA using the GenomeLab™ GeXP Start Kit . The RT reaction then was run in a thermal-cycler  with the following program: 48 °C for 1 min; 37 °C for 5 min; 42 °C for 60 min; 95 °C for 5 min; hold at 4 °C.2, the gene-specific forward chimeric primer plex, fluorescently-labeled universal forward primer, unlabeled universal reverse primer and Thermo-Start® DNA polymerase   . The 96-5 of MCF-7 cells were seeded into each well of a 6-well plate and after incubation for 24 h the cells were treated with various treatments . The plate was incubated at 37 °C in an incubator with 5% CO2 and 90% humidity for 72 h. After the corresponding incubation, cells were harvested and washed. The fixation and permeabilisation was done by using BD Cytofix/CytopermTM Fixation/Permeabilisation (BD Bioscience). Briefly, the cells were resuspended thoroughly and 250 µL of Fixation/Permeabilisation solution were added into each tube and incubated for 20 min at 4 °C. After 20 min, the cells were washed twice by 1 × BD Perm/Wash™ buffer. For each treatment, the cells were divided into 4 centrifuge tube (BD Bioscience). Then, the cells were stained with fluorochrome-conjugated monoclonal anti-mouse p53 and Bcl-2 , anti-mouse XIAP (BD Biosciences) and anti-mouse estrogen receptor alpha (ER α) . The cells were washed twice with PBS to remove non-specific binding materials. The cells were stained again with secondary fluorochrome-conjugated monoclonal antibody mouse anti-p53, anti-Bcl2, anti-XIAP and anti-ER α . After that, the cells were washed twice to remove non-specific binding. Finally, the cells were analyzed by BD FACS Calibur multicolor flow cytometer (BD Biosciences).5 × 10p < 0.05) was taken as statistically significant.Results were expressed as Mean ± Standard Error (S.E.M). Differences between means were evaluated using ANOVA test (one way) followed by Tukey’s test and (25 and IC50 levels was able to reduce the IC50 value of doxorubicin to MCF-7 cells from 5.5 to 4.0 and 2.0 µg/mL, respectively. Based on the results from a cell viability assay, DNA cell cycle analysis and annexin V-FITC/PI staining, this natural product and drug combination targets MCF-7 cells via induction of apoptosis. Moreover, gene expression and protein detection show that this natural compound-drug combination, particularly at 0.2 µg/mL of doxorubicin and 8.2 µg/mL of damnacanthal, activated the apoptosis-related genes the most comparing to the other treatment groups. Additional in vivo studies on the combination of damnacanthal and doxorubicin are however needed to clarify the efficacy and safety of damnacanthal-doxorubicin in cancer treatment.In conclusion, the present results indicate that damnacanthal is effective in sensitising MCF-7 cells to doxorubicin treatment. Damnacanthal at IC


Dbp and Hnf4α as potential regulatory mediators. Cell-based experiments confirm that Dbp and Hnf4α activate Cyp3a11 transcription by their binding to a D-box and a DR1 element in the Cyp3a11 promoter, respectively. Bmal1 binds to the P1 distal promoter to regulate Hnf4α transcriptionally. Cellular regulation of Cyp3a11 by Bmal1 is Dbp- and Hnf4α-dependent. Bmal1 deficiency sensitizes mice to toxicities of drugs such as aconitine and triptolide (and blunts circadian toxicity rhythmicities) due to elevated drug exposure. In summary, Bmal1 connects circadian clock and Cyp3a11 metabolism, thereby impacting drug detoxification as a function of daily time.Metabolism is a major defense mechanism of the body against xenobiotic threats. Here we unravel a critical role of Bmal1 for circadian clock-controlled Cyp3a11 expression and xenobiotic metabolism. Bmal1 deficiency decreases the mRNA, protein and microsomal activity of Cyp3a11, and blunts their circadian rhythms in mice. A screen for Cyp3a11 regulators identifies two circadian genes  Lin, Wang et al. show that a core clock transcription factor Bmal1 regulates the expression of a major drug detoxification enzyme Cyp3a11 and the daily rhythm of drug metabolism through Dbp and Hnf4α. This study suggests that Bmal1 connects circadian clock and Cyp3a11 metabolism to regulate drug detoxification as a function of daily time. Circadian rhythms are produced and regulated by circadian clocks that are organized in a hierarchical and interactive manner2. The master clock located in the suprachiasmatic nucleus of hypothalamus coordinates all peripheral clocks through neuronal connections and hormonal signals4. At the molecular level, all circadian clocks are composed of a network of genes/proteins that form several feedback circuits5. The main circuit consists of BMAL1  and CLOCK (circadian locomotor output cycles kaput) ) as the positive elements, as well as CRY (cryptochrome) and PER (period) as the negative elements6. The BMAL1/CLOCK heterodimer activates the transcription of clock-controlled genes (CCGs) including CRY/PER6. Once reaching a critical concentration, CRY/PER proteins inhibit the transcriptional activity of BMAL1/CLOCK, thereby downregulating their own expressions7. When CRY/PER are unable to repress BMAL1/CLOCK, a new cycle of CRY/PER (and other CCGs) transcription can start. Additional transcriptional factors such as RORs/REV-ERBs and DBP /E4BP4 (E4 promoter-binding protein) contribute to circadian rhythms via regulation of BMAL1 and PER8. In addition to transcriptional regulation, post-transcriptional modifications  and GSK3 (glycogen synthase kinase-3)) play a role in maintaining the robustness of circadian circuits9.Circadian rhythms are found in virtually all living things including animals, plants and microbes. In mammals, many aspects of physiology and behaviors  are subjected to circadian rhythms, allowing adaptation of physiological needs to the time of day in an anticipatory way. Disruption of circadian rhythms has been linked to various health problems such as sleep disorders, obesity, diabetes, and cancers10. Cytochromes P450 (CYPs) are a major class of enzymes responsible for xenobiotic metabolism11. Of all CYP enzymes, CYP3A4 (Cyp3a11 in mice) is perhaps the most important one as it contributes to the metabolism of about 50% of drugs12. Many drug-processing genes including CYPs present temporal variations in tissue expression14. Circadian expression of these genes underlies the chronopharmacokinetics, contributing to circadian time-dependent drug efficacy and/or toxicity15. For instance, the temporal variations in the hypnotic effects of hexobarbital are attributed to the circadian oxidase activities in rats16. Mitoxantrone toxicity closely depends on dosing-time with the least toxicity and the highest drug clearance at 16 h after light onset in mice17.Metabolism (biotransformation) is a main mechanism for xenobiotic/drug detoxification in the body19. A previous study shows that the clock output genes  family of transcription factors, Dbp, Hlf (hepatocyte leukemia factor) and Tef (thyrotroph embryonic factor)) control diurnal expression of Cyp2b10 via rhythmic regulation of the CAR receptor, a well-known activator of the enzyme20. This original study suggests molecular clock-controlled metabolism and detoxification. However, whether and how the clock machinery  controls drug-metabolizing enzymes remain unknown. One of more important questions awaits investigations: whether and how Cyp3a11/CYP3A4 (the most important enzyme for drug metabolism/detoxification) is controlled by the clock system.Circadian rhythms in xenobiotic metabolism have been recognizedPER and CRY) via binding to the E-box elements present in promoter regions21. Although in most cases both BMAL1 and CLOCK are required in transactivation of clock-controlled genes, BMAL1 can independently regulate the expression of certain genes such as Dio2 (type II iodothyronine deiodinase) and p21Waf1/CIP123. In addition to controlling circadian rhythms, BMAL1 has regulatory roles in the development of various diseases such as hypertension, diabetes, dyslipidemia and obesity25.The BMAL1/CLOCK heterodimer essentially is a transcriptional activator that stimulates the transcription of target genes , and to determine the molecular mechanisms involved. We further clarified the impact of Bmal1 on time-dependent xenobiotic metabolism and detoxification. Overall, to our knowledge, our results defined a previously unreported paradigm for circadian clock-controlled xenobiotic detoxification.Cyp3a11/CYP3A4 in various hepatoma and colon carcinoma cell lines   Fig. . Additio-2) Fig. . Bmal1 iays Fig. . Taken tCyp3a11 based on in silico sequence analysis (Genomatix program). Therefore, an indirect rather than direct mechanism was involved in Bmal1 regulation of Cyp3a11. Interestingly, of six known Cyp3a11 regulators, Dbp and Hnf4α mRNAs were decreased in mouse liver and small intestine as a result of Bmal1 deletion  was found in the promoter region of mouse ion Fig. . In lineion Fig. . In addills Fig. . These dCyp3a11 mRNA in Hepa-1c1c7  cells  activity  revealed that Bmal1 bound to the distal region (from −6.1 to −6.0 kb) of the P1 promoter of Hnf4α  activity  component of tes Fig. 27. A higyme Fig. . Interesyme Fig. . It was ity Fig.  of Cyp3aity Fig.  consisteity Fig.  and 5f. ity Fig. . This waity Fig. .Fig. 5BmTripterygium wilfordii Hook F, an herbal medicine for treatment of leukemia and inflammation. Cyp3a11/CYP3A4 is the major enzyme contributing to triptolide metabolism and detoxification  component of ion Fig. 29. We cotes Fig. . Similartes Fig.  was corrtes Fig. . Bmal1 ates Fig. . This wates Fig. . Taken t30, Bmal1 regulated Cyp3a11 using an indirect mechanism. This indirect mechanism involved two circadian genes Dbp and Hnf4α (both are positive regulators of Cyp3a11). Bmal1 transcriptionally activated Dbp and Hnf4α to induce Cyp3a11 expression ion Fig. . Dbp andHnf4α gene is known to have two promoters (named P1 and P2) that generate two different mRNA transcripts. Hnf4α P1 appears to be more important due to a superior activity in the recruitment of coactivators. Hnf4α P1 rather than P2 controls pancreatic β-cell function and has a tight association with metabolic disorders such as diabetes32. It was noteworthy that Bmal1 mainly acted on the P1 promoter   in Cyp3a11 promoter for Hnf4α binding herein differed from that (−1581/−1524 bp) in the study of Inoue et al.34. Hnf4α was not responsive to −1581 bp DR1 element in our reporter assays  were not in a full agreement with circadian Cyp3a11 protein levels. This was probably because neither testosterone nor midazolam is a pure selective substrate of Cyp3a11. In addition to Cyp3a11, other Cyp enzymes such as Cyp1a2, Cyp3a13, Cyp7a1, Cyp7b1 and Cyp2b9 can metabolize testosterone albeit at lower activitiesHnf4α was a clock-controlled gene and that Bmal1 participated in circadian regulation of Hnf4α. Bmal1 ablation blunted the expression rhythmicity of Hnf4α in mice  display sex-specific expressions in the liverBmal1−/− mice  rhythms may contribute to the loss of Cyp3a11 rhythms. This is because food-derived chemicals may activate xenobiotic receptors such as PXR and CAR to induce Cyp3a11 expression. It is acknowledged that disrupted feeding rhythms might make a contribution to the loss of Cyp3a11 expression rhythms. We also observed down-regulated P450 oxidoreductase  in CYP3A4 is the orthologous gene of Cyp3a11 in humans46. We provided in vitro evidence that human BMAL1 positively regulates CYP3A4 expression  may be also applicable to humans despite of the lack of sufficient human-related studies.ion Fig.  consisteBmal1 controls the circadian rhythmicity of Cyp3a11 and drug chronotoxicity through regulation of Dbp/Hnf4α. Our findings contribute extensively to a deep understanding of clock-controlled drug metabolism, and will facilitate the practice of chronotherapeutics.In summary, the core clock gene Dbp (2 kb)-Luc, pcDNA 3.1, pcDNA-Bmal1, pcDNA-Dbp, pcDNA-Hnf4α P1, and pcDNA-BMAL1 were obtained from Biowit Technologies . The antibodies used in Western blotting were as follows: anti-Bmal1 , anti-Cyp3a11 , anti-Dbp , anti-Hnf4α , and anti-Gapdh . All the primary antibodies were diluted with 5% BSA at the ratio of 1:1000. The final concentration of antibodies was 1 μg/ml. The secondary antibody was purchased from Huaan Biotechnology and diluted with 5% skim milk at the ratio with 1:5000 . For ChIP assay, anti-Bmal1 antibody was purchased from Abcam  and normal rabbit IgG from Cell Signaling Technology . siRNA and negative control (siNC) were synthesized by Transheep Technologies .Hepa1-6 and HepG2 cells were purchased from American Type Culture Collection . Hepa1c1c7, CT-26, and Caco-2 cells were purchased from the cell bank of Chinese Academy of Sciences . The biochemical assay kits for CK, MDA, ALT and AST were purchased from Jiancheng Bioengineering Institute . BCA assay kit, cytoplasmic/nuclear protein extraction kit, and EMSA kit were purchased from Beyotime . Midazolam and NADPH were purchased from Sigma-Aldrich . JetPrime transfection kit was purchased from POLYPLUS Transfection . ChIP kit was purchased from Cell Signaling Technology . RNAiso Plus reagent and PrimeScript RT Master Mix were purchased from Takara . ChamQ Universal SYBR qPCR Master Mix was purchased from Vazyme . Dual-Luciferase® Reporter Assay System was purchased from Promega . Aconitine was purchased from MCE . Quercetin, ketoconazole, vitamin K3, quinidine, 4-methylimidazole, testosterone and triptolide were purchased from Aladdin . pGL4.10 -Luc, Cyp3a11 (1.34 kb)-Luc, Cyp3a11 (0.85 kb)-Luc, Cyp3a11 (0.22 kb)-Luc, Hnf4α-P1 (6.1 kb)-Luc, Hnf4α-P1 (5.9 kb)-Luc, Hnf4α-P1 (2.0 kb)-Luc, Hnf4α-P2 (2.0 kb)-Luc, Cyp3a11 (2 kb)-Luc containing mutated E-box, D-box or DR1 site, and Cyp3a11 (2 kb)-Luc containing double mutated D-box and DR1 sites were obtained from Transheep Technologies . The gene promoters were amplified by PCR reactions using specific primers  according to the manufacturer’s instructions.Bmal1−/− mice (on a C57BL/6J background) were maintained on a12 h L:12 h D cycle (light on 7:00 a.m. to 7:00 p.m.), with free access to food and water. Bmal1−/− mice were generated by deleting a portion of exon 5  were sacrificed every 4 h , and livers and small intestines were harvested.All wild-type (C57BL/6J) and Bmal1−/− mice as previously described50. The doses for aconitine and triptolide were 0.3 and 0.6 mg/kg, respectively. Plasma MDA , CK (creatine kinase), ALT , and AST (aspartate aminotransferase) levels were measured using the biochemical assay kits . Electrocardiogram (ECG) recordings of C57BL/6 mice treated with aconitine (i.p. at 0.3 mg/kg) at ZT2, ZT8, ZT14 or ZT20 were monitored using Dual Bio Amp/Stimulator .Toxicological studies for aconitine and triptolide were performed with wild-type and Bmal1−/− mice  by intraperitoneal injection at ZT2 or ZT14. At each time point, five mice were rendered unconscious with isoflurane for blood sampling. After centrifugation, the plasma samples were collected and subjected to LC/MS/MS analysis.For pharmacokinetic studies, the drug (acontine (0.3 mg/kg) and triotplide (0.6 mg/kg)) was administered to wild-type or Hepa1-6, Hepa-1c1c7, CT-26, HepG2 and/or Caco-2 cells were used in the gene overexpression and knockdown experiments. Luciferase reporter, electrophoretic mobility shift and chromatin immunoprecipitation assays were based on Hepa1-6 cells. Hepa-1c1c7, CT26 and Caco-2 cells were cultured in DMEM medium supplemented with 10%FBS, Hepa1-6 cells in RPMI 1640 supplemented with 10%FBS, and HepG2 cells in the α-MEM medium supplemented with 10%FBS. Once reaching a confluence of 60–70%, the cells were transfected with indicated plasmids using a JetPrime transfection kit according to the manufacturer’s protocol. Six hours later, the culture medium was replaced with fresh medium. On the next day, the cells were collected for further experiments.DNA was extracted from mouse tail 1–2 mm). PCR was performed with 400 ng DNA template. The products were subjected to the 2% agarose gel electrophoresis, and the bands were imaged using the Omega LumTM G imaging system (Aplegen). The primers are listed in Supplementary Table  mm. PCR 13. In brief, total RNA was extracted from mouse tissues and cells using RNAiso Plus reagent according to the manufacturer’s instructions. 500 ng RNA was used as a template for cDNA synthesis using the PrimeScript RT Master Mix. The sequences of all primers applied to qPCR are listed in Supplementary Table −ΔΔCT method. The primers (listed in Supplementary Table http://www.ncbi.nlm.nih.gov/BLAST/) and by performing a melting curve analysis. All qPCR assays were performed according to the MIQE (Minimum Information for Publication of qPCR experiments) guideline51.qPCR assays were performed as previously describedTM G imaging system (Aplegen). The densities of protein bands were analyzed by Fluorchem 5500 software. Uncropped scans of representative blots are provided in Supplementary Fig. Mouse tissues or cells were lysed in RIPA buffer containing 1 mM PMSF. Protein concentrations were detected using a BCA assay kit. The samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% acrylamide gels), and transferred onto PVDF membranes. After blocking with 5% skimmed milk for 1 h, the membranes were sequentially probed with the primary antibodies and horseradish peroxidase-conjugated secondary antibody. Protein bands were detected by enhanced chemiluminescence and imaged by the Omega Lum52. In vitro metabolic activity was determined using the published incubation procedures53. After 2-h-incubation at 37 °C, the reactions were terminated by adding ice-cold acetonitrile. The enzymatic activity was determined by LC/MS/MS analysis of the generated metabolites from substrates. Incubation conditions are shown in Supplementary Table Mouse liver/intestine microsomes and HEK3a11 cell lysate were prepared as previously described50.Drugs and their metabolites were quantified using an LC/MS/MS system as previously described14. In brief, the cells were transfected with 250 ng of (Hnf4α or Cyp3a11) luciferase reporter plasmid, 50 ng of pRL-TK vector, and/or 250 ng  overexpression plasmid. After 6 h, the medium was changed to fresh medium. On the next day, the medium was removed and the cells were collected. The cells were lysed with 45 μl passive lysis buffer (Promega) and the luciferase activities were detected using the Dual-Luciferase® Reporter Assay system. Relative luciferase activity was expressed as the ratio of firefly over renilla luciferase activity.Luciferase reporter assays were performed as previously described13. In brief, mouse livers were fixed in 1% formaldehyde for 20 min at room temperature. The reactions were terminated by the addition of glycine. After 5 min centrifugation, the livers were collected and homogenized by using a Dounce homogenizer. Cells were incubated with ChIP buffer containing micrococcal nuclease. After addition of 0.5 M EDTA, the precipitate was suspended in ChIP buffer and subjected to sonication. An aliquot of sheared chromatin was used as Input sample, whereas the remaining was immunoprecipitated with anti-Bmal1, anti-Hnf4α or anti-Dbp overnight at 4 °C. On the next day, the protein-DNA complex was incubated with protein G-coupled Dynabeads (30 μl) at 4 °C for 2 h. The immune complex was separated from beads and eluted in ChIP buffer. The immunoprecipitated chromatin was decross-linked at 65 °C for 4 h and protein was digest with proteinase K. The purified DNA was amplified by real-time PCR with specific primers . The mixture was incubated at room temperature for 10 min. Then, the biotin-labeled probes (0.5 nmol) were added into the mixture and incubated at room temperature for 20 min. The DNA−protein complexes were loaded onto 4% nondenaturing polyacrylamide gels. After 30-min electrophoresis, the products were transferred onto Hybond-N + membranes for 45 min. The membranes were visualized by using enhanced chemiluminescence and Omega Lum G imaging system . The primers are listed in Supplementary Table Nuclear proteins from the cells were first extracted using a cytoplasmic/nuclear protein extraction kit. EMSA assays were performed with a chemiluminescent EMSA kit as previously describedt test. Circadian parameters were obtained through cosine curve fitting using the cosinor periodogram program . The level of significance was set at P < 0.05 (*).Data are presented as mean ± SD (standard deviation). Statistical analyses on time-dependent data were performed using a two-way analysis of variance (ANOVA), followed by a post-hoc Bonferroni test (a post-hoc test was run only when a significant omnibus F-test was obtained) using GraphPad Prism software version 7 . The homogeneity of variance assumption for ANOVA was verified by Levene’s test. Statistical differences between two groups were analyzed by Student’s Further information on research design is available in the Supplementary informationDescription of additional supplementary itemsSupplementary Data 1Reporting SummaryPeer Review File