The fractalkine (CX3CR1) ligand is expressed in astrocytes and reported to be neuroprotective. When cleaved from the membrane, soluble fractalkine (sCX3CL1) activates the receptor CX3CR1. Although somewhat controversial, CX3CR1 is reported to be expressed in neurons and microglia. The membrane-bound form of CX3CL1 additionally acts as an adhesion molecule for microglia and infiltrating white blood cells. Much research has been done on the role of fractalkine in neuronal cells; however, little is known about the regulation of the CX3CL1 ligand in astrocytes.The mechanisms involved in the up-regulation and cleavage of CX3CL1 from human astrocytes were investigated using immunocytochemistry, Q-PCR and ELISA. All statistical analysis was performed using GraphPad Prism 5.A combination of ADAM17 (TACE) and ADAM10 protease inhibitors was found to attenuate IL-1β-, TNF-α- and IFN-γ-induced sCX3CL1 levels in astrocytes. A specific ADAM10 (but not ADAM17) inhibitor also attenuated these effects, suggesting ADAM10 proteases induce release of sCX3CL1 from stimulated human astrocytes. A p38 MAPK inhibitor also attenuated the levels of sCX3CL1 upon treatment with IL-1β, TNF-α or IFN-γ. In addition, an IKKβ inhibitor significantly reduced the levels of sCX3CL1 induced by IL-1β or TNF-α in a concentration-dependent manner, suggesting a role for the NF-kB pathway.In conclusion, this study shows that the release of soluble astrocytic fractalkine is regulated by ADAM10 proteases with p38 MAPK also playing a role in the fractalkine shedding event. These findings are important for understanding the role of CX3CL1 in healthy and stimulated astrocytes and may benefit our understanding of this pathway in neuro-inflammatory and neurodegenerative diseases.The online version of this article (doi:10.1186/s12974-016-0659-7) contains supplementary material, which is available to authorized users. The chemokine fractalkine CX3CL1) is expressed in the central nervous system (CNS) at relatively high levels in comparison to most other cytokines  is expre. AlthougTransmembrane proteins, such as CX3CL1, can be proteolytically cleaved at the juxtamembrane region, which results in the detachment of their extracellular region (ectodomain) in a process known as ectodomain shedding . This shWidespread ADAM expression has been demonstrated in the CNS, where a number of previous studies have suggested the expression of ADAM10 and ADAM17 in astrocytes . For exaMany studies performed on peripheral cells implicate ADAM10 in the constitutive shedding of the CX3CL1 ligand from various cell types, while ADAM17 is thought to be the main protease regulating inducible cleavage of this ligand . While aInhibitors used ADAM10/ADAM17 inhibitor (BMS-561392), specific ADAM17 inhibitor (BMS-566394), specific ADAM10 inhibitor , MMP inhibitor , IKKβ inhibitor  and the p38 MAPK inhibitor VX-702  were prepared as 10 mM stock solutions dissolved in 90 % dimethyl sulfoxide . The cytokines used were interleukin-1β , tumour necrosis factor α  and interferon-γ .2 in a humidified incubator and grown in human astrocyte speciality media  or standard DMEM/F12 media  supplemented with 1 % astrocyte growth supplement , 10 % fetal bovine serum  and 1 % penicillin/streptomycin  in T75 culture flasks (Corning) unless otherwise indicated in the figure legends. Given human astrocyte speciality media were approximately twice the cost of the standard cell culture media with supplements, we assessed the levels of sCX3CL1 using both media, with the only difference being the base media. Variations in the levels of sCX3CL1 released from the astrocytes between experiments can be primarily attributed to this reason and are highlighted accordingly in the figure legends. The cells were grown for 14 days until 90 % confluent and then re-plated in six- or 24-well plates and used when 80 % confluent.Human astrocytes from fetal brains were purchased from ScienCell Research Laboratory, USA , as per the ethics outlined by the supplier and as we have described previously , 46–48. For mRNA expression of CX3CL1, human astrocytes were grown in six-well plates until 80 % confluent. Prior to stimulation, cells were starved in serum-free media for 3 h. The cells were then pre-treated with either VX-702 (p38 inhibitor) or an IKKβ inhibitor in serum-free media for 30 min, after which IL-1β, TNF-α or IFN-γ was added and incubated for 3 or 18 h. After treatment, the media were removed and cells were washed twice with phosphate-buffered saline (PBS) in preparation for Q-PCR analysis. RNA extraction was performed using either Qiagen or Macherey-Nagel methods as described by the manufacturers. Using Qiagen kits, the cell pellets were suspended in RLT lysis buffer  and then frozen at −80 °C. RNA was isolated using RNeasy mini kit . To eliminate genomic DNA, an on-column DNase digestion was carried out with the RNase-free DNase set (Qiagen 79254). After reverse transcription of mRNA  using the High-Capacity cDNA Reverse Transcription Kit , RT-PCR was performed with the 7900HT Fast Real-Time PCR System  according to the Standard Thermal Cycler Protocol . The threshold was set manually for all samples. The analysis was performed with the SDS 2.3 software. For the Macherey-Nagel method , cells were suspended in RA1 buffer with 1 % beta-mercaptoethanol and frozen at −80 °C. To eliminate genomic DNA, an on-column DNase digestion was carried out with the RNase-free DNase. After reverse transcription of mRNA  using the High-Capacity cDNA Reverse Transcription Kit , RT-PCR was performed according to the Standard Thermal Cycler Protocol . TaqMan Gene Expression Assays using TaqMan probes of human CX3CL1  and HPRT   were used. The relative expression of CX3CL1 to the reference gene HPRT was determined. Each condition was run in triplicates, and the experiments were repeated independently three times unless otherwise indicated in the figure legends.2SO4, and absorbance was read immediately using a plate reader at 450 nm (Labsystem Multiskan). The standard curve was calculated by plotting the standards against the absorbance values, and the cytokine levels were measured in pg/ml.All cells were starved in serum-free media before treatments. Human astrocytes were pre-treated with inhibitors for 30 min followed by 18-h treatment with cytokines as indicated in the figure legends. Media were then collected, and cells were lysed in PTxE lysis buffer (1 mM EDTA and 1 % Triton-X in PBS) and frozen at −20 °C. Soluble fractalkine (sCX3CL1) levels in the supernatant and total cellular content were measured with human CX3CL1 ELISA kit according to the manufacturer’s instructions . This DuoSet detects the N-terminal chemokine domain of human fractalkine. The level of detection for fractalkine indicated by the manufacturers is 0.63–20 ng/ml, although we noted significant and detectable changes in colour at lower levels. Briefly, 96-well ELISA plates  were coated overnight at room temperature with capture antibodies diluted in PBS. The plates were washed three times with wash buffer , PBS, pH 7.4) and then blocked for 1 h at room temperature with the appropriate reagent diluent. The plates were then washed three times with wash buffer, and any remaining buffer was removed from the wells by aspiration. A standard curve was prepared using serial dilutions of the recombinant protein diluted in the appropriate reagent diluents. The samples and standards were then incubated in the antibody-coated ELISA plate for 2 h at room temperature. The plate was then washed three times with wash buffer, and detection antibody (diluted in reagent diluent) was added to each well for 2 h. Following three more washes, streptavidin-HRP diluted in reagent diluents was added to each well and incubated for 20 min at room temperature, protected from light. After an additional three washes, the wells were incubated with substrate solution  for 15 min at room temperature protected from light. The colour reaction was stopped with the addition of 1 M HHuman astrocytes were plated on glass coverslips in six-well plates (VWR) and cultured for at least 24 h until 80 % confluent. The cells were then washed with PBS and fixed with 4 % paraformaldehyde (PFA) for 10 min at room temperature. Permeabilization and nonspecific binding was reduced by incubating cells for 1 h at room temperature in blocking buffer  and 0.1 % Triton-X 100). For all antibody incubation and wash steps, diluted blocking buffer (PBS supplemented with 0.5 % BSA and 0.01 % Triton-X 100) was used. The cells were incubated overnight at 4 °C with the rabbit anti-CX3CL1, which binds the N-terminal chemokine domain ; washed three times; and incubated with anti-rabbit secondary antibody conjugated to Alex488  for 1 h. After washing three times in PBS supplemented with 0.01 % Triton-X 100, the cells were then incubated with Hoechst nuclear stain  for 10 min and washed twice again. The coverslips were then mounted on glass slides in Vectashield (Vector Labs) and the edges sealed with varnish. Samples were stored at 4 °C in the dark until imaged. The cells were visualized with a confocal microscope (Leica SP8).t test was used to compare the means between two groups. Detailed data analysis methods are provided in the “All statistical analysis was performed using GraphPad Prism 5. In experiments where three or more groups were compared, an ordinary one-way analysis of variance (ANOVA) was performed and was followed by post hoc tests. Tukey’s post hoc test was used for experiments where all columns were compared to each other. Student’s As a result of the low expression of CX3CL1 on astrocytes, its functional expression has not been well characterized to date. In agreement with previous observations , astrocyTo determine whether IL-1β, TNF-α and IFN-γ alter the levels of CX3CL1 by increasing protein synthesis, we examined their effect on CX3CL1 mRNA using Q-PCR Fig. . Treatmep < 0.001 n = 3; one-way ANOVA and Tukey’s post hoc test). Importantly, pre-treatment with a pan MMP inhibitor, RS-130830, attenuated the CX3CL1-induced increase by IL-1β , TNF-α  and IFN-γ , respectively, compared to the matched treated group (one-way ANOVA and Tukey’s post hoc test)  are a family of proteases that are capable collectively of degrading proteins in the extracellular matrix (ECM). MMPs are also involved in the cleavage of cell surface receptors and ligands such as cytokines and chemokines . Thus, Mst) Fig. . Notablyp < 0.001 compared to control). Importantly, pre-treatment with the ADAM10/ADAM17 dual inhibitor significantly decreased the levels of CX3CL1 induced by IL-1β . In contrast, the selective inhibitor for ADAM17 did not alter the levels of CX3CL1 induced by IL-1β . During our experiments, we noted variations in the absolute levels of CX3CL1, between experiments. These can be linked to the different batches of human astrocytes used, the age/passage number of these cells and the use of speciality or standard media. Importantly, however, while the absolute levels of CX3CL1 differed between experiments, the effects of the cytokines IL-1β, TNF-α and IFN-γ were similar as the effects of the inhibitors used. To convince ourselves further of a role for ADAM10 in the inducible increase in the levels of CX3CL1, we replicated experiments examining the effects of the ADAM10 inhibitor by co-stimulating astrocytes with TNF-α and IFN-γ (1 ng each). In this experiment, we still found that the ADAM10 inhibitor significantly attenuated TNF-α/IFN-γ-induced levels of fractalkine  . The effects of this ADAM10 inhibitor were also not associated with any significant changes in cell viability , without causing any changes in cell viability . From this data, we conclude that the pro-inflammatory cytokines IL-1β and TNF-α increase the levels of sCX3CL1 by promoting NF-kB-dependent synthesis which results in subsequent enhanced shedding of CX3CL1 via an ADAM10-dependent mechanism.The transcription factor NF-kB regulates the production of several pro-inflammatory cytokines . ReciproIn the current study, we investigated fractalkine (CX3CL1) expression and regulation in human astrocytes. We showed firstly that CX3CL1 is expressed in human astrocytes and is constitutively shed at low levels. On stimulation with pro-inflammatory cytokines such as IL-1β, TNF-α and IFN-γ, this low level of sCX3CL1 is significantly up-regulated. We noted that a broad-spectrum MMP inhibitor attenuated this increase in CX3CL1. Using a dual inhibitor of ADAM10/ADAM17 and selective ADAM10 and ADAM17 inhibitors, we showed further that ADAM10 is the main protease responsible for the cytokine-mediated inducible shedding of sCX3CL1. We also found these cytokines stimulated the mRNA levels of CX3CL1 suggesting a mechanism involving increased mRNA and protein synthesis. Using a p38 MAPK inhibitor, we demonstrated that the pro-inflammatory cytokine-mediated increase in sCX3CL1 likely involved a phosphorylation event mediated by p38 MAPK. An inhibitor of IKKβ also attenuated the increase in sCX3CL1 induced by the pro-inflammatory cytokines IL-1β and TNF-α, suggesting involvement of the NF-kB pathway. Taken together, the data indicates that pro-inflammatory cytokines regulate CX3CL1 expression on human astrocytes and that ADAM10 is the main protease involved in the liberation of sCX3CL1 from human astrocytes.Both ADAM10 and ADAM17 have been implicated in the development of neurodegenerative diseases such as Alzheimer’s disease , 58, 59.Similar to studies investigating the release of CX3CL1 from neurons, we show here that the shedding of CX3CL1 occurs both constitutively and in response to inflammatory stimuli in astrocytes , 30. We CX3CL1 signalling is crucial for proper development during brain maturation , synapti−/− mice have more synapses, a reduced number of microglia in the hippocampus and delayed maturation of functional glutamate receptors [CX3CL1 or CX3CR1 gene-disrupted animals are able to mature to adulthood. However, CX3CR1eceptors , 71. Diseceptors , 69. In eceptors . The geneceptors , 74. In eceptors . In conteceptors . Howevereceptors . Notablyeceptors . Studieseceptors . In addieceptors . TherefoHere, we find that CX3CL1 shedding from astrocytes is mediated by pro-inflammatory cytokines via an ADAM10-dependent pathway, where p38 MAPKs as well as NF-kB signalling may also play a role. This study suggests that the regulation of CX3CL1 in glial cells may be an important process during inflammatory events.


Pseudomonas fluorescens strain MFE01 secretes in abundance two Hcp proteins (haemolysin co-regulated proteins) Hcp1 and Hcp2, characteristic of a functional type 6 secretion system. Phenotypic studies have shown that MFE01 has antibacterial activity against a wide range of competitor bacteria, including rhizobacteria and clinically relevant bacteria. Mutagenesis of the hcp2 gene abolishes or reduces, depending on the target strain, MFE01 antibacterial activity. Hcp1, encoded by hcp1, may also be involved in bacterial competition. We therefore assessed the contribution of Hcp1 to competition of P. fluorescens MFE01 with other bacteria, by studying MFE01 mutants in various competitive conditions.hcp1 had pleiotropic effects on the MFE01 phenotype. It affected mucoidy of the strain and its motility and was associated with the loss of flagella, which were restored by introduction of plasmid expressing hcp1. The hcp1 mutation had no effect on bacterial competition during incubation in solid medium. MFE01 was able to sequester another P. fluorescens strain, MFN1032, under swimming conditions. The hcp2 mutant but not the hcp1 mutant conserved this ability. In competition assays on swarming medium, MFE01 impaired MFN1032 swarming and displayed killing activity. The hcp2 mutant, but not the hcp1 mutant, was able to reduce MFN1032 swarming. The hcp1 and hcp2 mutations each abolished killing activity in these conditions.Mutation of Our findings implicate type 6 secretion of Hcp1 in mucoidy and motility of MFE01. Our study is the first to establish a link between a type 6 secretion system and flagellin and mucoidy. Hcp1 also appears to contribute to limiting the motility of prey cells to facilitate killing mediated by Hcp2. Inhibition of motility associated with an Hcp protein has never been described. With this work, we illustrate the importance and versatility of type 6 secretion systems in bacterial adaptation and fitness. Proteus mirabilis uses the killing activity of T6SS for self-recognition: this T6SS seems to be activated when opposing P. mirabilis swarms meet. The result of its action is a visible boundary called the Dienes line [Environmental bacteria are in perpetual war against several competitors, and thus require weapons to conquer new territory. The type 6 secretion system (T6SS) of Gram-negative bacteria is an effector translocation apparatus resembling an inverted bacteriophage puncturing device -3. It isnes line ,8.P. aeruginosa Hcp are not only structural proteins but also play a crucial role as chaperone and receptor for T6SS effectors. Various Hcp proteins transport their own effectors and this effector selection by Hcp seems to be specific [The T6SS machinery comprises at least 13 proteins, the core components, and sometimes, additional proteins ,10. Somespecific .P. aeruginosa H1-T6SS gene cluster exhibits posttranscriptional regulation involving two sensor kinases, RetS and LadS [P. aeruginosa H1-T6SS being turned on [P. protegens Pf-5 and P. syringae pv. syringae T6SS gene clusters [The diversity of T6SS regulation reflects the vast array of its functions. The and LadS -15. Highurned on . It is lclusters ,18. HowePseudomonas sp. is believed to be a global adaptive stress response to adverse environmental conditions [P. aeruginosa infection in cystic fibrosis (CF) patients [in vitro [P. aeruginosa CF isolates acquire a mucoid phenotype through mutation of the anti-sigma factor MucA, a negative regulatory factor that sequesters AlgU, a positive regulator of alginate production [P. aeruginosa strains do not express T6SS genes [P. aeruginosa, some Vibrio cholerae strains are mucoid and have an active T6SS [P. fluorescens strains produce alginate or neutral and amino sugars which give a mucoid phenotype [P. fluorescens mucoid phenotype, like that in P. aeruginosa, was reported to be unstable [P. fluorescens isolate. It constitutively secretes two characteristic T6SS proteins at 28°C: Hcp1 and Hcp2. It also exerts antibacterial activity during contact on a solid surface with competitive bacteria; this activity is associated with Hcp2 [Environmental bacteria are often surrounded by an extracellular matrix, forming a protective capsule called the glycocalyx . This exnditions ,21. It ipatients ,23 but iin vitro . Some P.oduction ,25. A prSS genes . Unlike ive T6SS . Some enhenotype ,29. The unstable . We previth Hcp2 . The aimP. fluorescens strains were observed after growth on LB agar plates at 28°C. Environmental strains MFE01 [The phenotypes of four ns MFE01  and MFE0ns MFE01  and clinns MFE01  did not Pseudomonas species. We introduced the mucA gene of the Pseudomonas protegens strain Pf-5 (previously described as a P. fluorescens strain) [mucA did not switch-off mucoidy at 28°C, suggesting that the mucoid phenotype of MFE01 is not due to a mutation of the mucA gene or is MucA independent   into MFEhenotype .P. fluorescens strains MFE01 and MFE07 and found that these strains secreted large amounts of proteins identified as Hcp by Mass Spectroscopy (MS)  with a score of 120 for a score threshold of 84. The spot for the protein secreted by MFE07 spot matched significantly with gi:77458272  with a score of 65 for a score threshold of 54. The non-mucoid P. fluorescens strains MFP05 and MFN1032 did not secrete detectable amounts of Hcp proteins into the extracellular medium.We prepared supernatants of cultures of mucoid The optimal growth temperature of MFE01 is 28°C but it can grow at 37°C. Clearly visible at 28°C, the mucoid phenotype and Hcp secretion were both switched off at 37°C, suggesting a common regulation of these two phenotypes  V. cholera strains impaired in Hcp secretion: the expression of vasH restored the mucoid phenotype, but not the Hcp secretion. However, vasH can activate transcription of genes other than T6SS genes via sigma-54 [V. cholerae may be due to a common regulation system.Unterweger and colleagues observed a correlation between the mucoid phenotype and Hcp secretion in  strains . Sigma-5sigma-54 ,36 suggehcp mutants, and found that the hcp1 mutation (MFE01∆hcp1 strain) lead to the loss of mucoidy at 28°C whereas the hcp2 mutation (MFE01∆hcp2 strain) did not affect the mucoid phenotype  restored the wild-type phenotype, suggesting a direct link between hcp1 expression and mucoidy. We analysed and compared the extracellular matrices (ECM) of MFE01 and MFE01∆hcp1. The mucoid phenotype is a consequence of exopolysaccharide (EPS) accumulation [hcp1 was observed concerning the proportion of characteristic sugars of the Pseudomonas EPS  with a score of 94 for a score threshold of 86. Thus, the mucoid phenotype appears to involve the accumulation of both EPS and flagellin in ECM, which are perturbed by hcp1 deletion. This was unexpected because the literature generally reports an inverse cross-talk between the mucoid phenotype and flagellar assembly. For example, the alternative sigma factor, σ22 (synonym AlgT or AlgU), is a positive regulator of alginate biosynthesis and a negative regulator of flagellum biosynthesis [ECM protein fractions from supernatants of MFE01, MFE01∆E Figure A. Proteihcp1, MFE01∆hcp2, MFE01Δhcp1+pPSV35 (empty vector control) and MFE01∆hcp1+hcp1 were assayed for swimming; this confirmed that MFE01∆hcp1 had lost motility that was restored by hcp1 introduction in trans. The restoration of wild-type phenotype in MFE01∆hcp1+hcp1 is inconsistent with a possible polar effect of the hcp1 deletion , and we therefore studied the behaviour of MFE01 in conditions permitting its motility [P. fluorescens MFN1032 was spotted into the centre of a ring formed by MFE01 or its mutants . This putative protein matched with the WP_016772499.1 sequence . These proteins are described as T6SS needle sheath proteins TssC and are essential components of this system. MFE01∆tssC, a mutant disrupted in this T6SS core component gene, was non motile in swimming conditions and non-mucoid at 28°C as well as MFE01∆hcp1 . During co-culture on LBG containing X-gal at 28°C with E.coli containing pUC19 (E.colipUC19), MFE01∆hcp2 and MFE01∆tssC allowed E.colipUC19 growth (blue spots) whereas MFE01∆hcp1 and MFE01 inhibited E.colipUC19 growth (white spots)  50 μg/ml (E. coli) or 200 μg/ml (P. fluorescens); tetracycline (Tc) 15 μg/ml; gentamicin (Gt) 15 μg/ml (E. coli), or 50 μg/ml or 100 μg/ml .All strains and plasmids used are listed in Table g for 10 minutes at 20°C and passing them through a Millipore membrane with 0.22 μm pores. TCA  was added to the supernatant to a final concentration of 10% and the mixture was incubated overnight at 4°C. The supernatant was removed by centrifugation at 13000 × g, for 30 minutes at 4°C. The protein pellet was washed twice with 5 ml of 20 mM Tris base (VWR) in cold acetone (Merck) and centrifuged at 13000 × g, for 30 minutes at 4°C. The dry pellet was then resuspended in distilled water. Proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, samples were mixed with an equal volume of 2 × Laemmli sample buffer (with β-mercaptoethanol), boiled for 5 min at 100°C and then cooled to room temperature before loading.Hcp secretion was studied by harvesting the supernatants by centrifuging the cultures at 5000 × Strains were plated on LB agar with 0.1 mM IPTG and incubated for 24 h at 28°C; the colonies were then examined for the mucoid phenotype. Swimming assays were performed as previously described .-gfp [The protocol described by Alteri and colleagues  was used-gfp  were spog, for 5 minutes at room temperature. The cell pellets were collected on toothpicks which was used to stab 0.3% agar LB plates, with 0.1 mM IPTG; the plates were then incubated at 28°C overnight.Overnight cultures were centrifuged at 3000 × -gfp stability (presence or absence) in our conditions, MFN1032 containing pSMC21-gfp was cultivated in LB medium containing kanamycin until OD580 nm of 1. Serial dilutions were then plated on LB plates without Kanamycin during 24 h at 28°C. Colonies were scrapped and suspended in NaCl 9 g/L and adjusted to OD5580 nm of 1. Serial dilutions were plated on selective (with kanamycin) and non-selective (without kanamycin) plates and counting. Statistics were done by pairwise strain comparisons (non-parametric Mann–Whitney-two tailed Test): no significant difference was observed between the two conditions .Antibacterial competition assays in solid media were performed as described by Decoin and coworkers . To ensuE.coli growth inhibition, Pseudomonas fluorescens MFE01 and mutants were cultivated at 28°C in LB medium with shaking at 180 rpm overnight. Escherichia coli DH5αmcr transformed with pUC19 (E.colipUC19), allowing blue detection on medium containing X-Gal, was cultivated at 37°C in LB with ampicillin (50 μg/ml) with shaking at 180 rpm overnight. The OD580 nm was adjusted to 0.5 and the strains were mixed at a 1:1 ratio. A volume of 20 μl was spotted in LB agar plates supplemented with X-Gal (40 μg/ml) and incubated overnight at 28°C.For hcp1 was generated by deletions in the 5′ (44 bp) and 3′ extremities (18 bp) of hcp1. 5′ and 3′ truncated hcp1 was generated by PCR with the Hcp3F and Hcp4R primers (see Table hcp1 construct was introduced between the EcoRI and HindIII sites (blunt-ended) of the transferable suicide plasmid pME3087 (6.9 Kb) [hcp1, was verified by sequencing and was then transferred into MFE01 by biparental mating: E. coli S17-1 [hcp1 and recipient MFE01 cells were mixed and spotted onto LB agar plates and incubated overnight at 37°C. The mating mixture was then suspended in 1 ml of sterile NaCl 9 g/L and 0.1 ml aliquots were spread on LB agar plates supplemented with tetracycline (12 μg/ml), to select for the presence of the integrated plasmid, and rifampicin (25 μg/ml), to kill the E. coli S17-1 donor bacteria and to ensure the selection of MFE01. The resulting hcp1 mutant (containing two truncated hcp1 genes) was named MFE01∆hcp1.MFE01∆(6.9 Kb) . The resli S17-1  containihcp2 mutation was introduced into MFE01∆hcp1 strain by the protocol described by Decoin and co-workers [hcp1∆hcp2. The same protocol with PCR modifications was used to introduce a markerless tssC mutation into MFE01 strain. This tssC deletion was achieved by PCR with the muta1tssC and muta2tssC primers (<650 bp product) or the muta3tssC and muta4tssC primers (<650 bp product)  sequence was amplified with the PFL_1449mucAF and PFL_1449mucAR primers . The PCR product was digested with SacI (NEB) and XbaI (NEB) and ligated into pPSV35 [E. coli was transformed with the ligation product and the resulting plasmid was checked by PCR. Fresh MFE01 colonies were washed twice in cold sterile distilled water and transformed with 5 μl of plasmid DNA by electroporation in 1 mm cuvette at 1.8 kV (Savant electroporater). LB was then added and the mix incubated for 1 h at 28°C with shaking (180 rpm). The samples were then plated on LB agar supplemented with gentamicin and with 0.1 mM IPTG.The rs Table  by standcA PFL_14 sequencehcp1 gene was amplified from the P. fluorescens MFE01 genome with the Hcp1F and Hcp2R primers  and checked by PCR and asymmetric digestion with BamHI (NEB) to verify the orientation of the insert. MFE01∆hcp1 was transformed with pPSV35-hcp1 by electroporation as described in the section “hcp1+hcp1.The g for 30 min). Clear supernatants were collected and three volumes of ice-cold ethanol were added; the samples were incubated for 24 h at 4°C, and EPS were collected by centrifugation . The EPS-containing pellets were resuspended in water and lyophilised.Strains were tested for EPS production on LB agar at 28°C. ECM was extracted as described by Dignac and colleagues with modifications . BiomassLyophilised EPS were hydrolysed by treatment with 2M trifluoroacetic acid for 2 hours at 110°C. Monosaccharides were then derivatised: methanol-1M HCl (Supelco) was added and the samples incubated at 80°C overnight; then a mix of hexamethyldisiloxan:trimethyldisiloxan:pyridine  was added and the samples incubated at 110°C for 20 minutes. The resulting derivatives were dried then dissolved in 1 ml of cyclohexane and injected into the 3800 GC system equipped with a CP-Sil5-CB column (Agilent Technologies). Elution was performed with the following gradient of temperature: 120°C to 160°C at a rate of 10°C per minute, 160°C to 220°C at a rate of 1.5°C per minute, 220°C to 280°C at a rate of 20°C per minute. Quantification was based on the internal standard and response factor determined previously for each monosaccharide.580nm = 0.1. Bacteria were allowed to attach to the glass surface  during 2 h at 28°C under static conditions. Biofilm growth was then performed under a constant flow of LB medium with antibiotics at appropriate concentration if necessary  for 48 h at 28°C.Pure culture biofilms were grown in continuous-culture three-channel flow cells . The system was assembled and prepared as follows: system sterilization during 4 h with bleach at 1.2% and rinsing with sterile NaCl 9 g/L. Before flow chamber inoculation, LB medium was injected into the system at 28°C. Overnight cultures were centrifuged at 8 000 × g during 5 min at room temperature. Pellets were recovered and washed twice with 2 mL of sterilized physiological water. Channels were inoculated with 1 mL of pure bacterial suspension at DO3) per μm2 of glass surface. The results were the mean of at least five independent experiments.Microscopic observations were performed with a LSM 710 system, Zeiss, Germany by using a 63x oil immersion objective. Biofilms were observed after a 15 min incubation with 5 μM Syto 9® green fluorescent nucleic acid stain (life technologies) . Biofilm stacks were analysed with COMSTAT software. The calculated parameter was the biovolume, which is the volume of bacteria  data included a score probability greater than a score threshold defined by MASCOT software.Mass spectroscopy (MS) analyses were performed with a MALDI-TOF AutoflexIII (Brucker) in positive ion mode as described by Barbey et al. . Statistp-value <0.05 was considered to be statistically significant.Non-parametric Mann–Whitney Tests (two tailed) with GraphPad Prism version 6.0  were used for statistical analyses. A


Generally, the oocytes have a very low maturation rate under in vitro conditions. In the current study, we found that melatonin promotes the maturation of oocytes in which mitochondria play a pivotal role. It was identified that; (1) mitochondria are the major sites for melatonin synthesis in oocytes and they synthesize large amounts of melatonin during their maturation; (2) melatonin improves mitochondrial function by increased mtDNA copy, mitochondrial membrane potential (ΔΨm) and mitochondrial distribution and ATP production in oocytes; (3) the meiotic spindle assembly is enhanced; (4) melatonin reduces ROS production and inhibits 8-oxodG formation, thereby protecting potential DNA mutation from oxidative damage. As a result, melatonin improves the quality of oocytes, significantly accelerates the developmental ability of IVF embryo. The results provide novel knowledge on the physiology of oocyte’s maturation, especially under in vitro conditions.The physiology of oocyte  This may be attributed to the relatively high levels of antioxidants existing in the follicular fluid [Female fertility is mainly determined by the quality of oocytes which are susceptible to multifaceted factors such as environment stressors, availabilities of essential nutrients, etc. ,2. Maintetc. ,4. Theseetc. , GDF9 antabolism ,7, NPPC/s arrest ,9, Mos/M control . A newlyntegrity ,12. Notantegrity ,14. The ar fluid ,17. TheyN-acetyl-5-methoxytryptamine) is a derivative of tryptophan. This molecule is a naturally occurring potent free radical scavenger and a broad-spectrum antioxidant [MT1 and MT2 [SNAT gene  expression and melatonin increases progressively in oocytes during follicular development [Melatonin   (It was observed that rescence A or RT-Prescence B. Melato < 0.01) C. Meanwh < 0.01) D. SNAT was distributed in mitochondria and cytoplasm (SNAT substrate serotonin. Results suggested that melatonin production was higher in serotonin-supplemented mitochondria than in the respective controls after 15 min (26.35 ± 11.980 versus 1.92 ± 1.985 ng/mL) and 30 min (44.96 ± 17.16 versus 1.93 ± 0.658 ng/mL) culture  showed that ytoplasm A. To fur culture B. in vitro conditions. Analysis of an additional 1650 oocytes indicated that melatonin did not affect the germinal vesicle breakdown and cleavage rate (−5 and 10−7 M) groups than that in control group  (−7 M) group were also significantly higher (p < 0.05) (65.1 ± 2.55) than that in control group (53.9 ± 2.49)  C, the ce ± 2.49) D.−5 and 10−7 M) significantly improved the mtDNA copy number of MII-stage oocyte  (−7 M) decreased the massive clustering distribution rate of mitochondria in MII-stage oocyte    (The data from 238 oocytes showed that melatonin treatments (10ctively) A. Analys < 0.05) B. The ΔΨ < 0.05) C. ATP le= 0.068) D.−7 M melatonin showed the normal rate of meiotic spindles was 76.2% ± 1.94% which was significantly higher than 67.0% ± 2.22% in control group (p < 0.05)  .p < 0.01); In contrast, the levels of GSH were significantly higher in melatonin treated oocytes than those in controls    were examined. The gene expression of Bcl-2 was up-regulated by melatonin (1.50 ± 0.150) compared with control (0.91 ± 0.122) (p < 0.05). In contrast, the gene expression of Caspase3 was down-regulated by melatonin treatment compared to the control . The expression of p53, however, was not influenced by melatonin (The results showed that the levels of ROS were significantly lower in melatonin-treated oocyte (0.19 ± 0.006) than that of control (0.23 ± 0.007) ( < 0.01) A. The 8- < 0.05) B. Finallelatonin C.MT1 and MT2 were identified in human granulosa cells [SNAT existed in oocytes during their maturation and the melatonin was also detected in the culture medium of oocytes , thus, the mechanism by which SNAT was transported across mitochondria membrane is currently unknown. To further identify the subcellular site of melatonin synthesis in oocytes, mitochondria were targeted since mitochondria were hypothesized as the original sites of melatonin production . Mitochoelatonin . Thus, win vitro oocyte maturation. Melatonin treatment improved the developmental potential of IVF embryo  of oocytes and enhanced their ATP production  and human chorionic gonadotrophin (hCG) were obtained from Ningbo Hormone Products Co., Ltd. . Melatonin, Sodium Pyruvate, BSA, Fetuin, Hyaluronidase, Penicillin and Streptomycin were purchased from Sigma-Aldrich . MEMa medium was purchased from Gibco .ad libitum. PMSG (10 I.U.) was injected into mice abdomen in advance of 44–46 h to stimulate oocyte growth to sufficient size, and then hCG (10 I.U.) was injected to trigger oocyte maturation and ovulation. All animal studies have been approved by the animal care committees of China Agricultural University.Three to five week old CD-1 mice (melatonin-proficient) were purchased from Vital River Laboratories Co., Ltd. . Mice were housed under controlled conditions with temperature (22–26 °C) and a light/dark cycle 12/12 h. Animals were allowed to access to food and water MT1, MT2 and SNAT  were added, respectively, and incubated at 4 °C for overnight. Oocytes were then washed three times with PBS containing 0.5% BSA and 0.01% Triton X-100 (15 min per wash) and incubated with donkey anti-goat IgG-FITC  goat anti-rabbit conjugated Alexa Fluor-568  at 37 °C for 1 h. After washing, the cell nucleus was counterstained with Hoechst (Vector Laboratories) and analyzed by epifluorescence microscope . Mice were superovulated with an intraperitoneal injection of PMSG followed by hCG as mentioned above. Oocytes were collected at 0, 4 and 16 h respectively after hCG injection by puncturing the ovaries or oviducts with 1 mL disposable syringes. Retrieved oocytes were denuded with incubation in 100 IU/mL of hyaluronidase and then washed with PBS containing 0.5% BSA. The zona pellucida was removed via rapidly incubation in the acidified Tyrode’s solution . Thereafter, the oocytes were fixed and permeated in 4.0% neutral-buffered paraformaldehyde containing 0.3% Triton X-100 at 37 °C for 15 min. Nonspecific binding was blocked using PBS supplemented with 0.5% BSA, 0.1% Triton X-100, and 5% fatal bovine serum (FBS) at 37 °C for 1 h and then antibodies of SNAT primary antibody at 4 °C for 12–16 h and followed by incubated with anti-rabbit IgG (whole molecule)−gold-conjugated secondary antibody  for another 2 h. After washing, these specimens were counterstained with 7% uranyl acetate, and washed again with distilled water. Images were acquired using a JEM-2200FS microscope . PMSG (10 I.U.) was injected into mice abdomen in advance of 46 h to stimulate oocyte growing to sufficient size. The ovaries were collected from the abdomen cavity and they were fixed for 48 h in 2.5% glutaraldehyde. After washing, these ovaries were further dehydrated with a graded series of ethanol, xylene and, then, were embedded in epoxy resin. The follicles with the typical GV-stage oocytes were sectioned (100 nm) and placed on nickel grids and incubated in blocking solution (0.05% glycine in PBS) for 10 min. Then, the specimens were incubated with the −4 M serotonin , PMSG (1 I.U./mL), hCG (2 I.U./mL), 0.3% BSA, Fetuin (1 mg/mL), Sodium Pyruvate (20 mM), Penicillin (75 μg/mL) and Streptomycin (50 μg/mL). The 100 µL of maturation medium was collected at 4 h, 8 h of the culture, respectively, for melatonin assay. All sample preparation and HPLC detection of melatonin were performed as previously reported [To measure the melatonin levels in ovary and serum during the course of oocyte maturation, mice were anesthetized with pentobarbital sodium by intraperitoneal injection at 4, 8 h after hCG injection. Blood was collected from caudal vein and centrifuged at 3000 r for 10 min to obtain the serum. The ovary samples of mice were weighed and homogenized immediately in 1 mL methyl alcohol and centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was collected for melatonin assay. All procedures were performed under ice bath. To detecting the melatonin content in maturation medium, GV-stage oocytes were acquired by puncturing the ovary at 44–46 h after PMSG injection and denuded with hyaluronidase. For each group, 100 oocytes were cultured in MEMa medium supplemented with 10reported . The exp−4 M serotonin (the substrate of melatonin biosynthesis) at 37 °C for 15 and 30 min to test their melatonin-biosynthetic capacity. Melatonin in PBS was extracted and measured as mentioned previously. The experiments were independently repeated six times.GV-stage oocytes were collected (See above), then divided into two groups  and cultured for 16 h in maturation medium. Then, mitochondrion were isolated using mitochondria isolation kit  following the manufacturers’ instructions. The isolated mitochondria were then cultured in PBS supplemented with 10Following mitochondria isolation, its purity was verified by Western blot. VDAC1 and GADPH were selected as mitochondrial marker and cytoplasm marker, respectively. In brief, mitochondria samples were lysed in laemmli sample buffer . Proteins were then separated by SDS-PAGE (12% acrylamide running gel) and transferred to a nitrocellulose membrane . Nonspecific binding to the membrane was blocked with 5% nonfat milk in Tris (10 mM), pH 7.5, NaCl (150 mM), and 0.1% Tween 20 at 37 °C for 1 h. Membranes were then incubated with primary antibodies  ) at 4 °C, overnight, followed by incubation with horseradish peroxidase-conjugated secondary antibody  at 37 °C for another 1 h. Analysis was performed with enhanced chemiluminescence detection reagents  and X-Omat BT film , according to the manufacturers’ instructions. −9, 10−7, 10−5 M melatonin, respectively, at an incubator  for 16 h. After germinal vesicle breakdown (GVBD), the matured oocytes were denuded with hyaluronidase . The percentage of GVBD oocytes (rate of GVBD) was recorded. Each cauda epididymidis was removed from adult male mice and the sperm were collected by gently squeezing the tissue with tweezers and placed into HTF medium  for capacitation at 37 °C for 1 h. The denuded oocytes were then incubated with sperms in HTF medium for 5 h. Pronuclei were washed to diminish the sperms and they were transferred into 60-μL KSOM medium  supplemented with 10% FBS for later development. Microscopic examination was then performed to monitor the development rates of both 2-cell and blastocyst. The cell numbers of blastocysts were assessed by Hoechst staining and the cells were mounted on a clean glass slide covering with a coverslip and examined using an epifluorescence microscope.GV-stage oocytes were collected by puncturing the ovary at 44–46 h after PMSG injection and then they were cultured in 60-μL of maturation medium containing 0, 10MT-ND5) was selected as an index to determine the mtDNA copy numbers. MT-ND5 gene was amplified by PCR, then PCR products were extracted using the Tiangen gel extraction kit , and cloned into the pEASYTM-T5 Zero Cloning vector  to obtain the recombinant plasmids. A single MII oocyte was added to a PCR tube with 5 µL of lysis buffer , incubated at 55 °C for 30 min to lyse oocyte, and 95 °C for 10 min to inactivate Proteinase K. Then, amplification was performed by LightCycler 480 SYBR Green I Master Mix  on LightCycler 480 II PCR machine . Standard curves were determined using recombinant plasmids, which were serially diluted by 101-105 times. The real-time qPCR reactions consisted of SYBR Green (10 µL), forward and reverse primers (30 µM), template (5 µL) and ddH2O was added up to a total volume of 20 µL. The procedure was as follows: 95 °C for 10 min; 35 cycles of 95 °C for 10s and 60 °C for 10 s. For each sample, the mtDNA copy numbers are calculated using the threshold cycle number (Ct) and corrected from the standard curve. MT-ND5 primer sequences are listed in The mitochondria-encoded NADH dehydrogenase subunit 5 gene . Cells were cultured in maturation medium for 16 h. Then, MII-stage oocytes were collected and denuded from the adherence of cumulus cells and they were incubated in M2  medium supplemented with 100 nM dye at 37 °C for 40 min. Oocytes were then washed and analyzed by epifluorescence microscope . Oocytes with a uniform granulated distribution of active mitochondria were scored as granulated distribution (GD), the oocytes with a massive clustering distribution of mitochondria were scored as massive distribution (MD). The images were observed and scored by three independent persons who are unaware this study. The experiment was independently repeated 3 times.MitoTracker Red CMRox (Life Technologies) was used to detect mitochondrial distribution. GV-stage oocytes were collected and divided into two groups . JC-1 fluorescence has two emission peaks, with red fluorescence indicating high mitochondrial membrane potential and green fluorescence indicating low mitochondrial membrane potential. Briefly, MII-stage oocytes in each group were exposed to 10 µg/mL of JC-1 at 37 °C for 15 min, and then washed with M2 medium to remove surface fluorescence. Fluorescence was observed using an epifluorescence microscope. The distribution of JC-1 dimers with red fluorescence and monomers with green fluorescence were detected using a red filter and green filter of the microscope, respectively. Fluorescence intensity was analyzed using ImageJ software , the ratio of red to green fluorescence was used to analyze ΔΨm. The experiments were independently repeated 3 times.The ΔΨMII-stage oocytes were washed with PBS-PVA and a group of 10 oocytes were collected for ATP measurement, ATP levels were determined using a commercially available adenosine 5′-triphosphate (ATP) bioluminescent somatic cell assay kit  according to the manufacturer’s instructions. Briefly, oocytes were transferred into a 96-well plate with 45.8 µL ATP assay buffer, then 0.2 µL ATP probe, 2 µL ATP converter and 2 µL developer Mix were added. The plate was placed at room temperature for 30 min. ATP levels were measured using a luminometer . The experiments were independently repeated 6 times.−7 M Melatonin-treated, Control) and cultured for 16 h in maturation medium. MII-stage oocytes’ collection and immunofluorescence stain processed as described above. Mouse anti-α-tubulin was selected as the primary antibody , goat anti-mouse LgG-FITC was selected as the secondary antibody . For the spindle analyses, oocytes with barrel-shaped bipolar spindles having distinct and well-organized microtubule fibers, along with tightly aligned chromosomes on the metaphase plate, were scored as normal. The normal spindle rate is the percentage of oocytes with normal spindle. All experiments were independently repeated at least 3 times.GV-stage oocytes were collected though puncturing the ovary which stimulated by PMSG, then divided into two groups  and cultured for 16 h in maturation medium. Then, MII-stage oocytes were collected and denuded from adherence of cumulus cells, then they were incubated (in the dark) for 30 min in M2 medium containing H2DCFDA (10 µM) or Cell Tracker Blue (10 µM), respectively. The MII-stage oocytes were collected and denuded from the adherence of cumulus cells, and placed in 30 µL M2 droplets, and then the fluorescence was observed using an epifluorescence microscope. The fluorescence intensity was analyzed using ImageJ software . The experiments were replicated 6 times.The 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA)  and Cell Tracker Blue CMF2HC Molecular Probes  were used to detect intracellular ROS and GSH level, respectively. GV-stage oocytes were collected through puncturing the ovary and divided into two groups  and cultured in maturation medium. Oocytes were then collected and denuded from adherence of cumulus cells at 1, 4 h, respectively, after in vitro maturation and they were fixed in pure methanol at −20 °C for 20 min. Thereafter, they were incubated in PBS with 0.3% Triton x-100 for another 15 min. They were blocked with 5% FBS, 0.5% BSA in PBS at 37 °C for 1 h. Oocytes were then incubated with Alexa 488-conjugated avidin (10 μg/mL)  in blocking solution at 37 °C for 1 h. After washing, DNA was counterstained with Hoechst and analyzed by epifluorescence microscope . The fluorescence intensity of 8-oxodG was analyzed using ImageJ software. The experiments were replicated 6 times.GV-stage oocytes were collected by puncturing the ovary, then divided into two groups  and cultured for 16 h in maturation medium. The amplification was performed by LightCycler 480 SYBR Green I Master Mix  on LightCycler 480 II PCR machine . The real-time qPCR reactions consisted of l SYBR Green (10 µL), forward and reverse primers (30 µM), template (2 µL) and ddH2O was added up to a total volume of 20 µL. The procedure was as follows: 95 °C for 10 min; 35 cycles of 95 °C for 10 s and 60–62 °C for 8–15 s; melting curve from 65 to 95 °C, increasing in an increment of 0.5 °C every 5 s. Normalization was performed using the housekeeping gene Actin as control. Primer sequences are listed in Oocytes were collected and denuded from the adherent cumulus cells at 0, 4 and 16 h, respectively, after hCG injection. The reaction procedure of RT-PCR was as follows: the initial denaturation at 95 °C for 5 min; denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s and then extension at 72 °C for 30 s. The above procedures were repeated 35 cycles with a final extension at 72 °C for 5 min. For qPCR assay, GV-stage oocytes were collected and divided into two groups  with the aid of SPSS 19.0 statistical software followed by the Student


Compared with the control, significant change in Nav1.8 current density was observed when the CCR2 inhibitor INCB3344 (10 nM) was applied. Furthermore, inhibition of PKC by AEB071 significantly eliminated CCL2-induced elevated Nav1.8 currents. In vitro PKC kinase assays and autoradiograms suggested that Nav1.8 within DRG neurons was a substrate of PKC and direct phosphorylation of the Nav1.8 channel by PKC regulates its function in these neurons. Moreover, p65 expression was significantly higher in CCL2-induced neurons (P<0.05), and was reversed by treatment with INCB3344 and AEB071. PKC–NF-κB are involved in CCL2-induced elevation of Nav1.8 current density by promoting the phosphorylation of Nav1.8 and its expression.CCL2 [chemokine (C–C motif) ligand 2] contributes to the inflammation-induced neuropathic pain through activating VGSC (voltage-gated sodium channel)-mediated nerve impulse conduction, but the underlying mechanism is currently unknown. Our study aimed to investigate whether PKC (protein kinase C)–NF-κB (nuclear factor κB) is involved in CCL2-induced regulation of voltage-gated sodium Nav1.8 currents and expression. DRG  neurons were prepared from adult male Sprague–Dawley rats and incubated with various concentration of CCL2 for 24 h. Whole-cell patch-clamps were performed to record the Nav1.8 currents in response to the induction by CCL2. After being pretreated with 5 and10 nM CCL2 for 16 h, CCR2 [chemokine (C–C motif) receptor 2] and Nav1.8 expression significantly increased and the peak currents of Na v1.8 current density and expression, which mediates nerve impulse conduction and induces inflammatory nociception. PKC phosphorylates Nav1.8 to increase its current density and PKC–NF-κB are involved in inducing the up-regulation of Nav1.8.Cytokine CCL2 is responsible for promoting voltage-gated sodium Na Peripheral nerve injury, tissue inflammation and neuropathic disorders often result in neuropathic pain, which affects approximately 7% of the general populations and is insufficiently treated with currently available drugs . The typv1.8 is a critical VGSC isoform that is preferentially expressed in primary somatosensory afferent neurons to sense stimuli, which produces a slow-inactivating, TTX-R (tetrodotoxin-resistant) current and is responsible for action potential propagation within sensory neurons of the DRGs. Compelling evidence showed that there were no change of acute nociceptive thresholds and mild deficits in inflammation-induced hypersensitivity in a Nav1.8 mutant mouse model }, where G is the voltage-dependent sodium conductance, Gmax is the calculated maximal sodium conductance, V1/2 is the potential at which activation is half-maximal, Vm is the test pulse voltage potential at which the current is evoked, and k is the slope. The resulting curves were normalized and fitted using a Boltzmann distribution equation as follows: I/Imax=1/{1+exp [(V1/2−Vm)/k]}, where Imax is the peak current elicited after the most hyperpolarized prepulse, and Vm is the preconditioning pulse potential.The peak current values at each potential were plotted to generate I–V curves. G values were determined by the following equation: G=I/ for 12 h and immunoprecipitation of Naescribed . In brie2, 100 μM ATP and 5 μCi of [γ-32P]ATP]. Activated PKC  (Invitrogen) was added to each reaction tube (30°C) and allowed to proceed for 2–3 min. The kinase reactions were performed in the presence or absence of the active PKC-specific inhibitor (AEB071), and were terminated with 5 vol (1 ml) of PBS containing 20 mM EDTA. Protein A beads were again collected by centrifugation and washed three times in PBS plus EDTA to further reduce the background. Proteins were eluted from the beads using the 2-lithium dodecyl sulfate–PAGE sample buffer for gel analysis. The presence of the sodium channel was verified by Western analysis using the pan-sodium channel antibody, and the phosphorylated channels were detected via autoradiography. The IP proteins were separated on NUPAGE Bis–Tris 4-12% gels (Invitrogen) using a 3-(N-morpholino)propanesulfonic acid-based buffer system. Gels for autoradiography were stained by Coomassie Brilliant Blue and then destained to compare loading levels tested in the kinase assays.The kinase assays were performed in 200-μl reactions with the IP samples described above [25 mM HEPES, pH 7.4, 2 mM DTT (dithiothreitol), 10 mM MgClP<0.05 was considered statistically significant.Data are expressed as mean±S.D. All statistical analyses were carried out using SPSS 13.0 (SPSS). Statistical comparisons were performed by one-way analysis of variance followed by the Student–Newman–Keuls test. v1.8 in DRG neurons, the expression of CCR2 and Nav1.8 were evaluated using RT–PCR and Western blotting after the neurons were incubated with 1, 5 and 10 nM exogenous CCL2 for 24 h. As shown in v1.8 (mRNA and protein level) increased in a dose-dependent fashion following the treatment with various concentrations of CCL2. In comparison with the control, 5 and 10 nM CCL2 significantly enhanced the expression of CCR2 and Nav1.8 (P<0.05). Thus, 10 nM CCL2 was applied for the following studies. Moreover, as shown in v1.8 and CCR2 compared with the sole CCL2 treatment (P<0.05).To investigate the effects of CCL2 on CCR2 and Nav1.8 sodium currents in DRG neurons. In our study, the family of Nav1.8 sodium currents was generated by applying a range of membrane potentials between -70 mV and +40 mV for 100 ms according to the previous reports. The current–voltage (I–V) analysis revealed that the mean peak current amplitude was significantly higher in CCL2-treated neurons than in control neurons . However, the mean peak current amplitude of Nav1.8 in DRG neurons was significantly reduced after being treated with CCL2+CCR2 inhibitor INCB3344 (10 nM) compared with the control and CCL2-treated neurons (P<0.05), whereas no significant change in Nav1.8 current density was observed when INCB3344 (10 nM) was applied alone  while no significant decrease was observed when AEB071 was applied alone compared with the control , it is still a limitation that we failed to isolate the small-sized DRG neurons for the present study. Kao et al. [v1.8 in DRG neurons. Belkouch et al. [v1.8 current densities in both small- and medium-diameter sensory neurons, which was reversed by the administration of CCR2 antagonist INCB. Furthermore, Gβγ was suggested to be involved in the crosstalk between CCL2 and Nav1.8. Our study partly confirmed the previous conclusions that the CCL2–CCR2 axis participate in the modulation of Nav1.8 expression and current density. Meanwhile, we found that PKC–NF-κB contributed to the regulation of Nav1.8 expression and current density, another underlying mechanism that was implicated in Nav1.8 modulation.As an important member of the VGSCs that are responsible for nerve impulse conduction, Na neurons . Although et al.  revealed+-channel function is affected by phosphorylation [+ channels. It was also found that all α-subunits of VGSCs share a common topology and contain several potential PKC phosphorylation sites in the first and third intracellular loops. Chen et al. [+ channels and reduces peak Na+ current in hippocampal neurons. Chang et al. [v1.8 current in a PKC-dependent pattern. Moreover, a compelling study determined that Nav1.8 was a substrate of PKC that mediates mechanical hyperalgesia [It is believed that Narylation  and PKC n et al.  proved tg et al.  found thralgesia . Therefov1.8 peak current densities, and the in vitro PKC kinase assays also confirmed that PKC contributed to the phosphorylation of Nav1.8 in response to CCL2. Furthermore, regarding the CCL2-induced up-regulation of Nav1.8, we evaluated the role of the PKC–NF-κB pathway in this phenomenon, and found that CCL2 induced the activation of NF-κB, whereas the inhibition of PKC and NF-κB reduced the Nav1.8 expression. However, more details about the effects of CCL2 on PKC as well as the interaction of PKC and Nav1.8 await clarification.In the present study, we found that the inhibition of PKC by AEB071 significantly reduced the CCL2-induced Nav1.8 in DRG neurons. PKC contributed to the CCL2-induced inflammatory hyperalgesia through promoting the phosphorylation of Nav1.8 and activating NF-κB to modulate the expression of Nav1.8.In summary, our findings demonstrate that CCL2 induced the up-regulation and an increase of peak current density of Na


The semi-solid sintering induced by eutectic transformation to form a bimodal microstructure in Ti62Nb12.2Fe13.6Co6.4Al5.8 alloy is a fundamentally different approach from other known methods. The fabricated alloy exhibits high yield strength of 1790 MPa and plastic strain of 15.5%. The novel idea provides a new insight into obtaining nano-grain or bimodal microstructure in alloy systems with high melting point by semi-solid forming and into fabricating high-performance metallic alloys in structural applications.It is well known that semi-solid forming could only obtain coarse-grained microstructure in a few alloy systems with a low melting point, such as aluminum and magnesium alloys. This work presents that semi-solid forming could also produce novel bimodal microstructure composed of nanostructured matrix and micro-sized (CoFe)Ti Currently, the liquid phase in semi-solid forming based on eutectic transformation has a relatively loose random-packed structure in aluminum alloys and magnesium alloys17According to binary alloy phase diagrams62Nb12.2Fe13.6Co6.4Al5.8 alloy engineered for both high strength and large ductility. The as-fabricated bimodal microstructure is different from those reported so far, as examples in refs In this work, based on the aforementioned assumption, a material forming method coupling sintering nanocomposite powder with subsequent semi-solid treatment induced by eutectic transformation, referred to as semi-solid sintering, was introduced to fabricate bimodal microstructure in a Ti62Nb12.2Fe13.6Co6.4Al5.8 powder, i.e. the starting powder used for semi-solid sintering. As seen from 62Nb12.2Fe13.6Co6.4Al5.8 nanocomposite powder, confirming that there exists a semi-solid interval between 1080 °C and 1200 °C for the Ti62Nb12.2Fe13.6Co6.4Al5.8 alloy.62Nb12.2Fe13.6Co6.4Al5.8 alloy at 1100 °C. Interestingly, the sample consists of a lath-shaped (CoFe)Ti2 twins with a size scale of several tens of microns dispersed in a complicated matrix Ti2 twins, is different from those in the samples fabricated by thermo-mechanical treatment, powder consolidation, recrystallization method, or rapid solidification4567891011122 and bcc TiFe phases with grain sizes of 200–400 nm embedded into equiaxed β-Ti matrix with a grain size of 400–600 nm  continues until forming liquid phase. At stage III , the punch displacement displays an instantaneous increase around 1080 °C, indicating the formation of liquid phase induced by the eutectic transformation between the β-Ti and TiFe. This is in good agreement with the onset melting temperature (~1080 °C) on the DSC curve Ti2 twins   , which i2 and bcc β-Ti, respectively, to transform into liquid phase. Two melting endothermic peaks with peak temperatures of 1125 °C and 1180 °C respectively are distinctly observed on the DSC curve Ti2 is a solid solution of Fe atoms substituting for Co positions in fcc CoTi2 compound. The solution of Fe atoms may enhance largely the eutectic transformation temperature of the fcc (CoFe)Ti2 and bcc β-Ti2 remains solid state and grows from ultrafine grains  dispersed in a nanostructured eutectic matrix with β-Ti phase and TiFe phase (in a light color) Ti2 can also be supported by the additional experiments carried out in Ti66Nb13Fe8Co6.8Al6.2 alloy with lower Fe content . As seen from 2 twins and β-Ti in sintered Ti66Nb13Fe8Co6.8Al6.2 alloy (66Nb13Fe8Co6.8Al6.2 powder further confirm that the (CoFe)Ti2 phase is in a solid state at sintering temperature of 1100 °C. As such, the formation of such a liquid phase results from the preferential eutectic transformation between the β-Ti and TiFe in the Ti62Nb12.2Fe13.6Co6.4Al5.8 alloy. From another point of view, if the formation of liquid phase originates from a ternary eutectic transformation of the bcc β-Ti, bcc TiFe and fcc (CoFe)Ti2, it is impossible to obtain the bimodal microstructure in the present case according to the formation mechanism of bimodal microstructure in rapid solidification78910111262Nb12.2Fe13.6Co6.4Al5.8 alloy is certainly attributed to the preferential eutectic transformation between the β-Ti and TiFe phases.In order to further elaborate the formation mechanism of the present novel bimodal microstructure, the Tit color) . This on.2 alloy , and abs.2 alloy  during s62Nb12.2Fe13.6Co6.4Al5.8 alloy compared with the sintered sample at 950 °C. All the mechanical properties, i.e. strength and strain in deformation, were averaged by three tests. As seen from 7891011122 twins Ti2 twins are separated, hindered and restricted by the surrounded nanostructured matrix Ti2 twins. The large plasticity is ascribed to the profuse shear bands in nano-sized β-Ti phase and associated block effects by nano-sized TiFe phase. The findings will help guide endeavors to obtain high-performance metallic materials in alloy systems with a high melting point.In summary, this work has successfully prepared a bimodal microstructure in Ti62Nb12.2Fe13.6Co6.4Al5.8 (at.%) alloy powder from respective element powder by mechanical alloying in a high-energy planetary ball mill  under a purified argon gas atmosphere. Approximately 3 g of the powder was removed from the mill-vial every 10 h for X-ray diffraction (XRD) examination  until the formation of nanocomposite structure with an amorphous matrix surrounding β-Ti nanocrystals after 70 h milling. Then, differential scanning calorimetry  was employed to determine the semi-solid temperature interval of the as-milled powder. Afterwards, the as-milled powder was semi-solid sintered by the following two-step method under continuous heating to semi-solid interval (1100 °C) under an argon atmosphere by a Dr. Sintering SPS-825 system. In the first step, samples were heated from room temperature to 1050 °C at a heating rate is 100 °C/min and the sintering pressure was 50 MPa. In the second one, when the samples were heated to 1100 °C from 1050 °C at 50 °C/min under 50 MPa, the sintering pressure was relieved immediately and the sample were hold at 1100 °C for 5 min without pressure. For comparison, additional Ti62Nb12.2Fe13.6Co6.4Al5.8 specimens were sintered at 950 °C using the same parameters. The detailed experimental procedures can be seen in ref. In present experiments, the process started by preparing Ti−4 s−1.Instantaneous values of sintering parameters, such as temperature, punch displacement, and time, were recorded every 2 seconds by the attached software. The cooling rate of the semi-solid sintered alloy was determined by calculating the slope of the temperature curve of the as-sintered alloy during the cooling process. All sintered bulk samples had a cylindrical shape with a dimension of Ф20 × 10 mm. A Philips XL-30 FEG scanning electron microscopy  and a Tecnai G2 F30 field emission gun transmission electron microscopy  coupled with energy dispersive X-ray (EDX) analysis were used for microstructure investigation. In order to evaluate the mechanical properties under compression for comparison with the reference alloys, cylindrical specimens of 3 mm in diameter and 6 mm in length were tested in a universal testing machine (MTS testing system) under quasistatic loading at a strain rate of 5 × 10How to cite this article: Liu, L. H. et al. A new insight into high-strength Ti62Nb12.2Fe13.6Co6.4Al5.8 alloys with bimodal microstructure fabricated by semi-solid sintering. Sci. Rep.6, 23467; doi: 10.1038/srep23467 (2016).


GATA3 mutations are frequent in breast cancer and have been classified as driver mutations. To elucidate the contribution(s) of GATA3 alterations to cancer, we studied two breast cancer cell lines, MCF7, which carries a heterozygous frameshift mutation in the second zinc finger of GATA3, and T47D, wild-type at this locus.The transcription factor GATA3 is a favorable prognostic indicator in estrogen receptor-α (ERα)-positive breast tumors in which it participates with ERα and FOXA1 in a complex transcriptional regulatory program driving tumor growth. Immunofluorescence staining and subcellular fractionation were employed to verify cellular localization of GATA3 in T47D and MCF7 cells. To test protein stability, cells were treated with translation inhibitor, cycloheximide or proteasome inhibitor, MG132, and GATA3 abundance was measured over time using immunoblot. GATA3 turn-over in response to hormone was determined by treating the cells with estradiol or ERα agonist, ICI 182,780. DNA binding ability of recombinant GATA3 was evaluated using electrophoretic mobility shift assay and heparin chromatography. Genomic location of GATA3 in MCF7 and T47D cells was assessed by chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq).GATA3 localized in the nucleus in T47D and MCF7 cells, regardless of the mutation status. The truncated protein in MCF7 had impaired interaction with chromatin and was easily released from the nucleus. Recombinant mutant GATA3 was able to bind DNA to a lesser degree than the wild-type protein. Heterozygosity for the truncating mutation conferred protection from regulated turnover of GATA3, ERα and FOXA1 following estrogen stimulation in MCF7 cells. Thus, mutant GATA3 uncoupled protein-level regulation of master regulatory transcription factors from hormone action. Consistent with increased protein stability, ChIP-seq profiling identified greater genome-wide accumulation of GATA3 in MCF7 cells bearing the mutation, albeit with a similar distribution across the genome, comparing to T47D cells.GATA3 deregulates physiologic protein turnover, stabilizes GATA3 binding across the genome and modulates the response of breast cancer cells to estrogen signaling.We propose that this specific, cancer-derived mutation in  Nearly 31,000 point mutations and small insertions or deletions (indels) in at least 170 previously reported and novel cancer genes have been implicated in the development of breast tumors [GATA3, with a 10% frequency of alterations, among the top three (together with p53 (TP53) and phosphoinositide-3-kinase (PIK3CA)) mutation driver genes in breast cancer [Accumulation of somatic mutations is responsible for development of breast cancer, as 85% of affected women have no family history of the disease  lead to loss of interaction with a cofactor, FOG2, or altered DNA-binding affinity [GATA3 mutations are spread throughout the gene, breast cancer mutations cluster around ZnF2 and C-terminal domain [GATA3 mutations from eight breast tumors has demonstrated loss or reduction of DNA binding ability, aberrant nuclear localization, decrease in transcription activation, and alterations in invasiveness, but not proliferation [Haploinsufficiency of GATA3 in humans results in HDR syndrome, a rare condition inherited as autosomal dominant trait, characterized by hypoparathyroidism, deafness, and renal dysplasia . Genomicmeshifts . Mutatioaffinity ,21. Intel domain ,23. Analferation . HoweverGATA3 on biochemical properties and genomic location of the protein. We utilized two luminal breast cancer cell lines, MCF7 harboring a heterozygous frameshift mutation in ZnF2, and T47D carrying wild-type version GATA3. We observed that mutant GATA3 was expressed at elevated levels relative to wild-type protein and it accumulated in nuclei. Surprisingly, the mutation led to enhanced protein stability following challenge with estrogen receptor agonist or antagonist. This increased stability led to increased levels, but not to global redistribution, of GATA3 binding in the genome as determined by ChIP-seq. The data collectively support the hypothesis that the carboxyl terminus of GATA3 contains protein regulatory information that ensures appropriate turnover following ligand binding by ERα.The aim of the present study was to evaluate the effect of a breast cancer-specific mutation in 2. Protein stability was evaluated in the normal growth medium and cells were treated with 1 μM cycloheximide (CHX) and/or 1 μM MG132 (MG) for up to eight hours. For estrogen starvation assays, cells were grown for 72 hours in MEM medium containing 5% FBS and then for 24 hours in phenol red-free MEM supplemented with 5% charcoal-dextran stripped FBS. Cells were treated with 50 nM 17β-estradiol (E2) for 24 hours. The effect of ERα inhibitor, ICI 182,780 (ICI) was tested in normal growth medium. ICI was added at 100 nM concentration and cells were harvested 24 hours later. MG  was dissolved in DMSO, CHX  in water, ICI  and E2  in ethanol.Human breast carcinoma cell lines MCF-7 and T47D were obtained from the American Type Culture Collection  and cultured in DMEM/F-12 medium supplemented with 10% FBS at 37°C in 5% CO2, 10 mM KCl, 0.5% NP-40, 0.5 mM DTT and protease inhibitors. The cytoplasmic fraction was separated by centrifugation at 2500 rpm for 10 min. The pellet was resuspended in nuclear extraction buffer containing 0.1, 0.2, 0.4 or 0.8 M NaCl, 25 mM HEPES, pH 7.4, 0.15 mM spermidine, 0.5 mM spermine, 5% glycerol, 1 mM EDTA and protease inhibitors. Samples were rotated for 30 min at +4°C and spun down in Optima Max centrifuge  at 38,000 rpm for 45 min at +4°C. The nuclear fraction was collected and remaining pellet was dissolved in lysis buffer .Cells were grown in 10 cm tissue culture dishes until they were 70-80% confluent. The cells were washed with PBS, collected by scraping and resuspended in buffer containing 0.15 M NaCl, 10 mM HEPES, pH 7.4, 1.5 mM MgClWhole cell lysates were obtained using 8 M urea lysis buffer . Protein extracts (15 μg) were resolved on SDS–PAGE gels and immunoblotted using the following antibodies: GATA3 , FOXA1 , ERα  and actin . Signal intensity was analyzed using rectangular volume tool in Quantity One Analysis Software  with global background subtraction.Cells were grown on glass coverslips in six-well tissue culture dishes. They were fixed with 4% formaldehyde in PBS for 10 min, washed with PBS, and permeabilized with 0.1% Triton X-100 for 2 min, washed with PBS, and blocked with 5% BSA in PBS. The coverslips were incubated with the anti-GATA3 antibody  for one hour, washed with PBS, incubated with the secondary antibody  for one hour, washed with PBS, and mounted on glass slides with mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI). The slides were examined and photographed using a Zeiss Axiovert 200 M microscope equipped with an Axiocam MR digital camera controlled by AxioVision software .E.coli BL21 (DE3) CodonPlus RIL cells, and the cells were cultured at 37°C. The bacterial cell lysate was centrifuged at 15,000 rpm for 20 min. The supernatant was mixed gently by the batch method with Ni-NTA beads  at +4°C for 30 min. The beads were washed with 5 mM imidazole-containing buffer and GATA3-DBD was eluted with 500 mM imidazole-containing buffer. The fractions containing GATA3-DBD were subjected to MonoS column  chromatography. The binding domain was eluted with a 4-column volume linear gradient of 100–600 mM NaCl. The protein was further purified by Superdex 75 column  in a buffer containing 20 mM Tris–HCl pH 7.5, 0.3 M NaCl, 10% glycerol, 2 mM 2-mercaptoethanol, and 1 μM zinc sulfate. For the purification of GATA3 mutant (D336fs) DBD, the Ni-NTA beads were washed with the 20 mM imidazole-containing buffer. The fractions eluted from Ni-NTA beads were dialyzed against 20 mM Tris–HCl pH 7.5, 0.3 M NaCl, 10% glycerol, 2 mM 2-mercaptoethanol, and 1 μM zinc sulfate buffer, and concentrated with Amicon ultra-centrifuge filter .DNA binding domain (DBD) of GATA3 (amino acids 261 to 371) was cloned into the pET-15b vector to produce a hexahistidine tagged fusion protein. The expression vector was transformed into the GATAAGACG or GATA3 recognition motif-lacking oligonucleotide AATGTCAAACTTTTAAGACG) in 10 μl of a reaction buffer . After 10 min incubation at 37°C, the samples were analyzed by polyacrylamide gel electrophoresis, and the bands were visualized by ethidium bromide staining. In the competitive DNA binding assay, wild-type and mutated GATA3 DBDs were used individually or mixed in equimolar proportion. The reactions were performed with 15 μM of 20 bp GATA3 motif-containing oligonucleotide and 23 bp GATA3 motif-lacking DNA (CACTTTTTAACGTAATTTACTCT).GATA protein  was incubated with 30 μM of 20 bp dsDNA . The column was eluted with a 10 ml linear gradient of NaCl concentration from 0.1 to 1 M in 20 mM Hepes, pH 7.9 containing 20% glycerol, 0.2 mM EDTA, 0.1 mM PMSF, and 0.5 mM DTT. Separated fractions were analyzed by Western blot directed against anti-GATA3.GATA3 antibody was generated in rabbits using recombinant 6x histidine tag-fused GATA3 full-length wild-type protein. ChIP was performed as previously described  with theDNA immunoprecipitated by GATA3 antibody in four to five individual reactions performed at the same time was pooled for T47D and MCF7 cells separately and purified using MinElute PCR Purification kit (Qiagen). Total 100 μg of ChIP or input DNA, quantified with Qubit Fluorometer  and dsDNA High Sensitivity Assay kit (Life Technologies), was used for library construction with the help of TruSeq RNA Sample Preparation kit . The library was prepared following the manufacturer’s instructions, starting with the end repair step, and amplified with twelve PCR cycles. Two sets of libraries (ChIP and input) were prepared for each of the cell lines from samples immunoprecipitated on separate occasions. The libraries were sequenced on a Genome Analyzer IIx (Illumina) as single end 36mers.http://picard.sourceforge.net). Replicate libraries were in good agreement and were merged prior to downstream analysis. All alignments were extended at the 3’ end to a length of 180 bases (the average expected genomic fragment size for these libraries). ‘bedGraph’ files were generated from these uniquely-mapped, non-duplicated, extended reads for visualization of aggregate genomic coverage. Peak calling for regions of enriched GATA3 binding was performed with HOMER  [To ensure that low quality reads were excluded from the analysis, the raw sequence reads were filtered to remove any entries with a mean base quality score < 20. Filtered reads were aligned to the human genome  via Bowtie (v0.12.8 with parameters –m 1 –v 2) ; only rettpb 0”)  using inhttp://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE51274).GATA3 ChIP-seq data have been deposited in NCBI’s Gene Expression Omnibus  and are GATA3 gene consists of six exons, encoding a protein of 444 amino acids, which contains two transactivation domains (TA1 and TA2) and two zinc fingers (ZnF1 and ZnF2) , MCF7 cells contained both the full length GATA3 as well as a truncated protein of approximately 37 kDa  with the ERα antagonist, ICI 182,780 (ICI). As expected, ERα expression was reduced in both cell lines Figure C. While The frameshift mutation present in GATA3 in MCF7 affects the second zinc finger, which is responsible for DNA binding . We emplTo further characterize the ability of endogenous GATA3 to bind DNA, we utilized heparin, a glycosaminoglycan structurally similar to nucleic acids. Nuclear extracts obtained from T47D and MCF7 cells were partially purified through ion exchange chromatography, applied to a heparin column, and eluted with a linear gradient of NaCl. The peak of full-length GATA3 from T47D eluted between 0.57 and 0.71 M NaCl Figure A. In MCF6 reads per library. Following quality control, mapping to unique positions in the human genome, and deduplication, 37-59% reads were retained  on asynchronous cultures. We first established a robust ChIP assay using polyclonal sera raised against full length recombinant GATA3 (which recognizes both full-length and mutant proteins). We assessed GATA3 enrichment at published positive control loci  using PCWe explored the similarities in ChIP-seq between the two cell lines in terms of location relative to genomic features and intensity. In spite of the difference in number of GATA3 enriched regions, peak distribution relative to the closest transcription start site (TSS) was similar in T47D and MCF7 cells  resulted in 4524 genes within 50 kb in T47D and 6934 in MCF7, including 3011 genes overlapping between both cell lines  recently reported a comprehensive study of human breast cancer: tumors from 507 patients were analyzed on multiple high information content platforms: whole exome sequencing, DNA copy number arrays, DNA methylation, mRNA array and sequencing, microRNA sequencing and reverse-phase protein arrays [GATA3 occurred in 58 cases (10.7%), predominantly in luminal A and B cancer subtypes, an additional 12 samples displayed copy number alterations . Strikingly, while mutations of GATA3 in the congenital disorder HDR syndrome are found throughout the protein [Large-scale genome sequencing projects have provided, and continue to provide, volumes of information on the mutational landscape of cancers. A current challenge for cancer biologists is to investigate the emerging genomic data in a mechanistic context, establishing the relationship of specific mutations to tumor biology and informing on clinical parameters including aggressiveness, response to therapy, and potential for metastasis. Here, we have initiated an attempt to address the mechanistic basis by which mutations in the transcription factor n arrays . Somatic protein , breast GATA3 in the MCF7 genome and showed that although both full-length and truncated proteins were expressed, the mutated protein was present in the cells at a higher level. The D336 frameshift does not affect the N-terminal and C-terminal sequences flanking ZnF1 that are required for nuclear localization [Careful scrutiny of the TCGA mutation data revealed that six mutations were localized in the second zinc finger and five of them were frameshifts, similar to the mutation in MCF7 , making lization  and GATAlization -22 suggelization , we obseChIP-seq was utilized to assess the degree of overlap of GATA3 across the two cell lines used in our study. Surprisingly, the number of binding sites detected in MCF7 was substantially higher than in T47D cells. In spite of the large difference in genomic occupancy, detailed analysis of genes associated with GATA3 binding failed to identify any major functional differences between binding profiles in T47D and MCF7 cell lines . Even though both T47D and MCF7 cell lines are classified as PGR and ERα positive, and belong to luminal A breast cancer subtype [Although the number of GATA3 peaks was considerably lower in T47D than in MCF7 cells, progesterone receptor gene was an example of a locus featuring a greater number of bound regions in T47D than in MCF7. Remarkably, lack of PGR expression, as determined by immunohistochemical staining, was a common denominator for all five patients in the TCGA database carrying a frameshift mutation in ZnF2 of GATA3  or ICI 182,780 (ICI). Figure S3. GATA3 enrichment determined by Real-Time PCR in ChIP reactions pooled for ChIP-seq library preparation. Figure S4. GATA3 enrichment in PCR-amplified ChIP-seq library in positive (ESR1 Enh1) and negative (ESR1 -6.9 kb) control regions located upstream from estrogen receptor transcription start. Figure S5. Real-Time PCR validation of GATA3 binding in T47D and MCF7 genome determined by ChIP-seq. Figure S6. Functional classification of genes located within 50 kb from a GATA3 ChIP-seq peak in T47D cells. Figure S7. Functional classification of genes located within 50 kb from a GATA3 ChIP-seq peak in MCF7 cells. Figure S8. GATA3 ChIP-seq peak presence in T47D and MCF7 cells within 10 kb from TSS of genes involved in normal mammary cell commitment and differentiation. Figure S9. GATA3 ChIP-seq peak presence in T47D and MCF7 cells within 50 kb from TSS of estrogen-responsive genes. Figure S10. GATA3 ChIP-seq peak presence in T47D and MCF7 cells within 50 kb from TSS of genes differentially expressed in different molecular subtypes of breast cancer. Figure S11. Progesterone receptor (PGR) expression in T47D and MCF7 cells treated with GATA3 siRNA  or infected with GATA3 adenovirus (Adv) (n=1). Figure S12. Frequency of GATA3 recognition motif, WGATAR, in GATA3 ChIP-seq peaks located within 10 kb.Quantification of Western blot signal intensity for full-length and truncated GATA3 protein in T47D and MCF7 cells treated with DMSO (Vehicle) or cycloheximide (CHX) over the course of eight hours. Click here for fileTable S2. Number of reads in merged samples for two replicates of GATA3 ChIP-seq in T47D and MCF7 cell lines.Number of reads in each sequencing run for GATA3 ChIP-seq in T47D and MCF7 cell lines. Click here for fileTable S4. GATA3 ChIP-seq peaks identified in MCF7 cell line. Peaks overlapping with T47D by at least 1 bp are indicated, as well as the nearest gene to the peak, within 10 kb or 50 kb from peak border.GATA3 ChIP-seq peaks identified in T47D cell line. Peaks overlapping with MCF7 by at least 1 bp are indicated, as well as the nearest gene to the peak, within 10 kb or 50 kb from peak border. Click here for fileGenes with GATA3 ChIP-seq peak within 10 kb from transcription start site (TSS).Click here for file


The different configurations of maternal and paternal chromatin, acquired during oogenesis and spermatogenesis, have to be rearranged after fertilization to form a functional embryonic genome. In the paternal genome, nucleosomal chromatin domains are re-established after the protamine-to-histone exchange. We investigated the formation of constitutive heterochromatin (cHC) in human preimplantation embryos. Our results show that histones carrying canonical cHC modifications are retained in cHC regions of sperm chromatin. These modified histones are transmitted to the oocyte and contribute to the formation of paternal embryonic cHC. Subsequently, the modifications are recognized by the H3K9/HP1 pathway maternal chromatin modifiers and propagated over the embryonic cleavage divisions. These results are in contrast to what has been described for mouse embryos, in which paternal cHC lacks canonical modifications and is initially established by Polycomb group proteins. Our results show intergenerational epigenetic inheritance of the cHC structure in human embryos. Following fertilization, the oocyte and sperm lose their distinct chromatin signature to form a functional embryonic genome. Here the authors find that, in human embryos, the paternal constitutive heterochromatin is inherited in the canonical configuration from the sperm and is propagated by the H3K9/HP1 pathway. Fertilization marks the fusion of two specialized gametes—oocyte and sperm. In mammalian zygotes, the maternal and paternal genomes exist in an asymmetric chromatin configuration. Extensive reorganization of chromatin to the embryonic configuration is crucial for the developmental potency3Constitutive HC assembles mostly on telomeric, centromeric and pericentric regions, remains condensed throughout the cell cycle and is important for genome stability and chromosome segregationThe H3K9/HP1 pathway underlies the formation of cHC. A central event is the trimethylation of histone H3 at lysine 9 (H3K9me3) by histone methyltransferases (HMTs) Suv39h1 and Suv39h2 refs , 8, 9. H5115Drosophila posterior sex combs , a Polyhomeiotic orthologue  and a Polycomb orthologue   ref. . The PRCIn this study, we addressed chromatin dynamics on cHC during human preimplantation embryo development. Our results identify striking differences with mouse: cHC in human embryos is not re-established by PRC1/2 action, but is transmitted and maintained by actors of the canonical H3K9/HP1 pathway. We show that human spermatozoa retain and transmit nucleosomes with cHC marks, such as H3K9me3, to the embryo. These paternal marks are subsequently bound by maternal HP1 and propagated over cell divisions. On the basis of this, we propose a model in which paternal cHC is transmitted intergenerationally.To determine the localization of cHC in human zygotes, pronuclear morphology was studied. Due to restrictions on the use of human embryos for research, we were limited to tripronuclear (3PN) zygotes and embryos resulting from 3PN zygotes to study embryos at Embryonic day (E) 1 and 2  in oocytes and embryos of eight developmental stages  oocytes that fail to fertilize after IVF, a sperm cell has penetrated, but failed to activate the oocyteNext we investigated if our observations for H3K9me3 on the paternal chromatin in post-S-phase human zygotes resulted from the To determine the fate of H3K9me3 on paternal chromatin in the zygote, we performed immunostaining of H3K9me3 on whole-mount embryos of all stages of preimplantation development . In nuclin vitro decondensation. In virtually all spermatozoa, H3K9me3 was prominently detected around the centromeres . We investigated α-satellite repeats located on chromosomes 7 and X and Sat II/III sequences of chromosomes 1, 9, 16 and Y. With the exception of the X α-satellite, all repeat sequences investigated were observed to localize partially or completely to the region enriched for H3K9me3 in the majority of sperm cells investigated . However, in contrast to mouse, both HP1α and HP1β were readily detected at the DAPI-rich knobs in paternal pronuclei, co-localizing with H3K9me3  and histone modifications H4K20me3 and H3K64me3 are additional markers of cHC. To investigate the extent of cHC formation on the paternal chromatin, we determined the dynamics of these additional markers by immunofluorescent analysis in spermatozoa, unfertilized oocytes with PCC, 3PN zygotes and all stages of preimplantation development. As described in mouse H3K9me3 . HP1β dy H3K9me3 . From E3H4K20me3 was detected around the centromeres in decondensed human spermatozoa, similar to H3K9me3. After fertilization, H4K20me3 was detected ubiquitously in the maternal chromatin, on DAPI-rich knobs at G2 phase in the paternal pronucleus and in the paternal pericentric regions in PCC and prometaphase chromosomes . From thH3K64me3 was observed at barely detectable levels in decondensed human spermatozoa. In unfertilized oocytes with paternal PCC, H3K64me3 was detected strongly in the maternal chromatin, but barely in the paternal chromatin. H3K64me3 levels appeared to increase after S-phase, as staining was observed on DAPI-rich knobs at G2 phase in the paternal pronucleus and prometaphase chromosomes . From thThese findings show that, in contrast to mouse, paternal cHC in human preimplantation stage embryos contains a full suite of canonical cHC markers, all of them grouped in the H3K9/HP1 pathway. Since we found that maternal HP1 binds sperm-derived H3K9me3 and that H3K64me3 is increased at H3K9me3-positive regions, our data also show that the modified histones contributed by the sperm cell enable further establishment and maintenance of cHC by the maternal machinery.The current work identifies three major mechanistic differences in paternal cHC build-up between mouse and human: (1) human paternal cHC is transmitted from the spermatozoon to the zygote in a canonical conformation, which is subsequently propagated by the H3K9me3/HP1 pathway, (2) in humans, paternal embryonic cHC remains under control of the H3K9/HP1 pathway and cHC is characterized by hallmarks as H4K20me3 and H3K64me3 much earlier in development than in mouse and (3) a PRC1/2-mediated back-up mechanism, as described in mouseOn the basis of our results, we propose an intergenerational model for cHC build-up in the early human embryo : nucleosIn mouse, only 1% of sperm DNA remains histone associated38In mouse, Polycombs are key to regulation of genes associated with development32In mouse embryos, the transcription of major satellites, especially from the paternal genome, has recently been reported to be crucial for rearrangement of pHCDuring mammalian spermiogenesis, histones are replaced by protamines. In humans, 5–15% of the DNA, encompassing specific genes, as well as (peri)centromeric DNA, appear to be protected against this removal and retain a nucleosomal structure .−1. Finally, the straws were cooled rapidly at −25 °C min−1 to −140 °C, before immersion in liquid nitrogen and storage in nitrogen vapour. After donation for research, thawing of embryos was performed at room temperature by consecutive washes in decreasing dimethyl sulfoxide concentrations in G-MOPS Plus medium (Vitrolife).Ovarian stimulation, oocyte retrieval, IVF procedures and assessment of embryo morphology were performed as described2 in air.Oocytes that failed to fertilize after IVF (0PN) were obtained 18 h post insemination at embryonic day (E) 1. Tripronuclear (3PN) embryos were used to study embryo development from E1 to E2. Surplus cryopreserved preimplantation embryos of good quality were used to study embryonic developmental stages from E3 to E5  and cultured at 37 °C in a humidified chamber in the presence of 5% CO2. Cells were plated on poly-D-Lysine-coated 12-mm coverslips, fixed with methanol at −20 °C for 10 min and then permeabilized with 0.2% Triton-X-100 in PBS.Human U2OS cells , tetra-acetyl-Histone H4 , H4K5ac , H4K8ac , H4K12ac , H4K20me3 (1:500 ref. ), H3K64mPrimary antibodies were detected by labelling with the appropriate secondary antibodies conjugated with Alexa Fluor 488, 555, 594 or 633 (Invitrogen).−1 DAPI for DNA counterstaining (Vector Laboratories)After removal of the zona pellucida by incubation with Acidic Tyrode’s Solution (Sigma), embryos were washed twice in G-MOPS, fixed for 15 min in 4% paraformaldehyde (PFA) in PBS (pH 7.4) and permeabilized with 0.2% Triton-X-100 in PBS for 15 min at room temperature. Embryos were incubated in blocking solution  and 5% normal goat serum) for 4 h at room temperature, followed by incubation with primary antibodies in blocking solution overnight at 4 °C. Embryos were washed three times for 20 min in 0.1% Tween-20 in PBS containing 2% BSA before application of secondary antibodies. These were diluted 1:200 in blocking solution and embryos were incubated for 1 h at room temperature, followed by three washing steps in 0.1% Tween-20 in PBS containing 2% BSA. Double antibody stainings were performed by mixing appropriate primary antibodies for simultaneous incubation, followed by detection with different secondary antibodies. Embryos were mounted on coverslips with Vectashield with 750 ng ml−1 Hoechst 33342 and imaged directly.For live staining of DNA in 3PN zygotes, zygotes were incubated in medium containing 1 μg ml−1 colcemid (Invitrogen) for 8–16 h to arrest cells at prometaphase. After zona pellucida removal, arrested embryos were incubated in hyposolution  for 5 min and subsequently transferred to a drop of fixative  on a glass slide. After horizontal drying for 1 h, slides were washed with 0.08% Photo-Flo (Kodak) and air driedTo obtain chromosome spreads, embryos were incubated with 1.5 μg mlFor each embryonic stage and antibody investigated, five to ten embryos were analysed unless otherwise stated.n=7); zygotes ; two-cell embryos , four-cell , eight-cell , 12–16 cell , morula  and blastocyst , HPRT1 , SOX2 , OCT4 , BMI1 , MEL18 , RING1A , RING1B , PHC2 , CBX2 , CBX7 , CBX8 , EZH1 , EZH2 , and EED . Assays were selected to be exon spanning and to recognize most of the validated (Ref Seq) splice variants of each gene of interest.For RT–qPCR of single oocyte/embryos, the Taqman PreAmp Cells-to-Ct Kit (Applied Biosystems) was used according to the manufacturer’s protocol with minor adjustmentsTo 12.5 μl of cDNA, 25 μl of Taqman PreAmp Master Mix and 12.5 μl of 0.2 × pooled Taqman Gene Expression Assays were added. After 10 cycles of preamplification , the preamplified cDNA (50 μl) was diluted with 100 μl of 0.5 × TE buffer. qPCR was performed on an ABI Prism 7000 Sequence Detecting System (Applied Biosystems) using 10 μl of 2 × Taqman Gene Expression Master Mix, 1 μl of Taqman Gene Expression Assay and 5 μl of nuclease-free water added to 4 μl of diluted preamplified cDNA. The two-step cycling parameters were as follows: one cycle of 2 min at 50 °C, followed by one cycle of 10 min at 95 °C to activate the polymerase and 40 cycles of 15 s at 95 °C and 1 min at 60 °C ref. . Results−1 hyaluronidase (Sigma). Thereafter, the oocytes were washed and kept until after injection in freshly prepared mem-alpha medium (Life Technologies), supplemented with 10% (v/v) fetal calf serum, 21 μM HEPES, 12.2 μM sodium lactate, 1 μM sodium pyruvate and 1 μM L-glutamin. Microinjection was performed using an inverted microscope equipped with an ICSI micromanipulation set-up (Narishige) and a piezo-actuated injector (Burleigh). A XYclone laser system (Hamilton Thorne) was used to breach the zona pellucida. Before installation, a small volume of mercury (Sigma) was inserted in an Straight Piezo Drill Micropipette (Humagen). Spermatozoa were used either directly after collection from the epididymis or, to prevent oocyte activation, after heat inactivation (incubation at 50 °C for 30 min (ref. 2 in air. Oocytes injected with heat-inactivated spermatozoa (n=60) were fixed 22 h after injection with 4% PFA as described above. Oocytes injected with normal spermatozoa were either fixed with 4% PFA 12–15 h after injection (n=60) or transferred to medium containing 1.5 μg ml−1 colcemid and processed for chromosome spreads 7–9 h later as described above (n=60).All institutional and national guidelines for the care and use of laboratory animals were followed and mouse experiments were approved by the local committee on animal experiments, DEC Consult. B6D2 F1 female mice (Harlan) were used as oocyte donors and superovulation was induced by intraperitoneal injection of 7.5 IU pregnant mare’s serum gonadotrophin (Intervet) followed by 7.5 IU human chorionic gonadotropin (Intervet) 48 h later60min ref. ). For ea−1, LEO Pharma BV) in decondensation buffer. Subsequently, the slides were fixed in 4% PFA for 15 min, air dried, washed in Photo-Flo and air dried. Slides were processed for immunofluorescence immediately as described above, starting with two washes in PBS-T. For each sperm sample, the optimal decondensation time was determined by sperm head morphology and ACA antibody accessibility. Preparations with optimal decondensation were used for further analysis. The presence of each histone modification under investigation was assessed in at least 100 sperm cells with clearly distinguishable ACA staining, from at least three separate donors.Sperm head decondensation was achieved as describedTo obtain sperm DNA in a chromatin fibre-like structure, 5 μl of spermatozoa suspension was added to 80 μl decondensation buffer, as described above and incubated for 10–20 min. Heparin was added to a concentration of 0.5% (v/v) and incubated for 10 min. Of this decondensed spermatozoa suspension, 5 μl was brought on a glass slide, spread out using the side of the pipet tip and air dried. Slides were fixed in 4% PFA for 15 min and processed for immunofluorescence as described above.in vitro decondensed spermatozoa, slides were first processed for FISH. The DNA probes used were Satellite DNA II/III probes for chromosomes 1  in 2 × standard saline citrate (SSC) was applied to each slide under a coverslip. Slides were denatured at 75 °C for 3 min and hybridization was performed in a humid box at 37 °C for 6 h. After hybridization, slides were washed in 2 × SSC/0.05% Tween-20 for 2 min at 42 °C, 0.4 × SSC for 6 min at 60 °C and in 2 × SSC/0.05% Tween-20 for 2 min at room temperature. The slides were rinsed once in PBS before proceeding to immunofluorescence of H3K9me3 as described above. Immuno-FISH was imaged simultaneously and for each probe co-localization for H3K9me3 and satellite DNA was measured in 10–12 randomly selected sperm cells from at least three separate donors.For immuno-FISH on .77 ref. ), 9 , 16.77 ref. , 65) and.77 ref. ), and al.77 ref. ) and X (.77 ref. ). ProbesFor immuno-FISH on chromosome spreads of unfertilized oocytes or 3PN zygotes (five to seven per probe combination), slides were first processed for immunofluorescence of H3K9me3 as described above. Chromosome spreads were subsequently imaged and positions of chromosomes were recorded in the form of the XY-position of the microscope table. These coordinates were used to find the same chromosomes after FISH. Subsequently, FISH was performed as describedin vitro decondensed spermatozoa were acquired using a Zeiss Axio Imager M2 confocal laser scanning microscope, equipped with four diode lasers , an Axiocam camera and Zen 2009 (Carl Zeiss) software. For embryos, we recorded Z-series of 5 μm slices and for chromosome spreads, we recorded Z-series of 0.5 μm slices. Images were processed with Image J (version 1.42n) and Adobe Photoshop CS3 software.Immunofluorescent images from whole-mount embryos, heterologous zygotes, chromosome spreads and immuno-FISH on Imaging for immuno-FISH on chromosome spreads was performed on a Zeiss Axio Imager M1 microscope (Carl Zeiss), equipped with a CoolCube 1m camera (MetaSystems) and Isis FISH Imaging System software . Images of immunofluorescence and FISH were obtained in two rounds of imaging and merged using Image J. With the ROI Manager in Image J, a selected area of one image was copied to the other images. Subsequently, selected areas were cropped and merged.For visualization of co-localization between H3K9me3 and satellite DNA, the distribution of fluorescence intensities was plotted using the Straight lines selection tool in Image J. Subsequently, using the Analyze—Plot Profile tool, the distribution of fluorescence intensities along the selected line was obtained. Sperm cells were divided into three categories according to their profiles for H3K9me3 and satellite DNA: (1) profiles where the peak intensities for H3K9me3 and FISH signal completely overlap, (2) profiles where the peak intensities are within a 1 μm distance and 3) profiles where the peak intensities are further apart.E.B.B. and A.H.F.M.P. conceived the project and designed the experiments. C.W., G.W.v.d.H., C.E., M.T. and M.A. performed the experiments and C.W., G.W.v.d.H., M.A., A.H.F.M.P. and E.B.B. analysed the data and evaluated the results. W.M.B. provided mouse material and facilities for heterologous ICSI. J.S.E.L. was responsible for IVF patients and informed consent for donated human embryos. C.v.d.W., G.W.v.d.H. and E.B.B. wrote the manuscript. All authors contributed to manuscript revision and critical discussion.How to cite this article: van de Werken, C. et al. Paternal heterochromatin formation in human embryos is H3K9/HP1 directed and primed by sperm-derived histone modifications. Nat. Commun. 5:5868 doi: 10.1038/ncomms6868 (2014).Supplementary Figures 1-7