The RING domain proteins BRCA1 and BARD1 comprise a heterodimeric ubiquitin (E3) ligase that is required for the accumulation of ubiquitin conjugates at sites of DNA damage and for silencing at DNA satellite repeat regions. Despite its links to chromatin, the substrate and underlying function of the BRCA1/BARD1 ubiquitin ligase remain unclear. Here, we show that BRCA1/BARD1 specifically ubiquitylates histone H2A in its C-terminal tail on lysines 127 and 129 in vitro and in vivo. The specificity for K127-129 is acquired only when H2A is within a nucleosomal context. Moreover, site-specific targeting of the BRCA1/BARD1 RING domains to chromatin is sufficient for H2Aub foci formation in vivo. Our data establish BRCA1/BARD1 as a histone-H2A-specific E3 ligase, helping to explain its localization and activities on chromatin in cells.  •BRCA1/BARD1 is a histone-H2A-specific ubiquitin ligase•BRCA1/BARD1 ubiquitylates histone H2A on lysines 127 and 129•Specificity for H2A is provided within in a nucleosomal context•Heterodimeric RING domains of BRCA1/BARD1 are sufficient for E3 function in vivo The ubiquitin ligase (E3) activity of BRCA1 is its only known biochemical activity in vitro. Kalb et al. report that the heterodimeric complex formed by BRCA1/BARD1 ubiquitylates histone H2A in nucleosomes specifically at its C-terminal tail. Moreover, in vitro and in vivo assays identify H2A lysines127 and 129 as the target lysines. These results help to explain the localization and activity of BRCA1/BARD1 on chromatin in cells. Breast cancer-associated protein 1 (BRCA1) is a key mediator in the DNA damage response, which is linked to a wide range of functions that serve to maintain genomic stability. Cellular BRCA1 forms a heterodimer with BRCA1-associated RING domain 1 (BARD1) , which pIn vitro studies have shown that the RING domains of BRCA1/BARD1 possess a ubiquitin ligase (E3) function , but a bRecently, We investigated the interaction of BRCA1/BARD1 with chromatin and found that it involves a highly specific histone H2A-ubiquitin ligase that modifies previously uncharacterized lysines in the C-terminal tail of H2A. We discuss these observations in light of the known biological functions of BRCA1.Although BRCA1 binds to DNA in a sequence-independent manner in vitro , in cellWe examined BRCA1/BARD1 E3 activity on individual histone proteins and reconstituted nucleosome substrates in vitro and compared it with the activity of the RING1B/MEL-18 subunits of PRC1 A and 1B.2SerGly2-BRCA1 RING (residues 2–109) fusion protein (hereafter referred to as BDfBC) was fused to mCherry and Lac Repressor (LacI) sequences and transfected into U2OS 2-6-3 cells containing 200 copies of a transgene with a 256 × lac operator sequence (lacO) integrated at a single locus in the genome. Expression of BDfBC-mCherry-LacI in 2-6-3 cells coincided with the appearance of a single predominant mCherry nuclear focus that colocalized with conjugated ubiquitin (using FK2 antibody) or H2Aub  might be the predominant residue for BRCA1/BARD1-dependent ubiquitin conjugation in vitro.To determine which of the lysines in the H2A C-terminal tail is the preferred site of ubiquitin conjugation by BRCA1/BARD1, we ubiquitylated chromatin purified from chicken erythrocytes in vitro and analyzed the products by mass spectrometry. We were able to identify ubiquitylation of the C-terminal peptide KAK , ubiquitin (FK2), or FLAG epitope D. This cWe next expressed the separate BRCA1 and BARD1 RING domains (BC-R/BD-R) in DT40 cells with different mutant FLAG-H2A proteins. In untransfected cells expressing wild-type FLAG-H2A, western blots revealed a single band corresponding to H2AubK119 band 1; E. Upon tGiven that H2Aub comprises only 5%–10% of all H2A in cells, the amount of H2Aub that was modified at lysine residues other than K118-119 was extremely low D and 3F.To date, neither the substrate nor the function of the BRCA1/BARD1 ubiquitin ligase has been well established. We have established that BRCA1/BARD1 specifically ubiquitylates histone H2A in chromatin in vitro and in vivo. Our data support a role for BRCA1/BARD1 as a histone-H2A-specific ubiquitin ligase that ubiquitylates the C-terminal tail of H2A at the previously uncharacterized lysine residues K127-129.Several pieces of evidence link BRCA1-dependent E3 activity with ubiquitylation of histones in chromatin. First, purified BRCA1/BARD1 ubiquitylates individual histone proteins in vitro, albeit with little specificity . We showSecond, the RING domains of BRCA1/BARD1 that confer its E3 activity are structurally related to the RING1B/MEL18 RING domain subunits of the histone-H2A-specific ubiquitin ligase PRC1 . Of noteWe note that BRCA1/BARD1 and RING1B/MEL18 are heterodimeric E3 ligases , whereasThird, our data support recent evidence indicating that small interfering RNA (siRNA)-mediated knockdown of BRCA1 results in derepression of satellite DNA with an accompanying loss of H2Aub in this region . Our finBRCA1 has been linked to ubiquitin conjugates on chromatin at sites of DNA breaks. However, previous attempts to visualize the ubiquitylated products of BRCA1/BARD1 E3 activity at sites of DNA damage have been hampered by its complex recruitment to sites of DNA breaks involving a ubiquitin-mediated signaling pathway and several different ubiquitin ligases . Here, wPrevious studies led us to believe that the position of ubiquitin within a nucleosome is more important for its function than the exact location of the modification within the histone tail. Indeed, it has been shown that ubiquitin fused to the very C-terminal amino acid of histone H2A contributes to the repression of satellite DNA. The exact role(s) of ubiquitin, however, is still enigmatic.The contribution of BRCA1 E3 activity to the DNA damage response is unclear. Mice expressing the enzymatically dead mutant BRCA1(I26A) are no more tumor prone than those expressing wild-type protein . On the Although the role of monoubiquitylated H2A at DNA breaks is still unclear, our data establish the potential of BRCA1/BARD1 as a specific H2A ubiquitin ligase on nucleosome substrates. Moreover, the identification of a previously uncharacterized form of H2Aub that might be uniquely associated with the E3 activity of BRCA1 raises the possibility of generating antibodies directed against H2AubK127-129 for use as a diagnostic tool to identify biochemically active BRCA1.2. HeLa cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal calf serum. HEK293 FlpIn T-Rex cells (Invitrogen) were grown in DMEM containing 10% tetracycline-free fetal bovine serum.DT40 chicken cells were propagated in standard media supplemented with RPMI (Invitrogen) at 37°C, 6% COCells were prepared as described in the X. laevis histones were expressed and purified from E. coli and reconstituted into nucleosomes as described in the 125I-ubiquitin, substrate and 1 mM in 1x ub buffer . Histones were separated by PAGE using 12% Bis-Tris gel (Invitrogen) and MES buffer, and Coomassie-stained H2Aub bands were excised. In-gel digestion with pepsin (pH 1.3) was performed and peptides were analyzed by nanoLC-LTQ/Orbitrap in a data-dependent tandem mass spectrometry mode. For nucleosomes ubiquitylated in vitro, in-gel digestion was also performed with pepsin and analyzed by NextGen Bioscience.R.K., D.L.M., C.L., and K.H. conceived and designed experiments. R.K., D.L.M., and C.L. performed experiments. J.T.J.H. performed mass spectrometry and analyzed the results. R.K. and K.H. wrote the manuscript.


The distribution of Co was controlled by means of the synthesis protocol to yield three different morphologies, namely, Co3O4 nanoparticles located on the surface of CeO2 particles, coexistent Co3O4 and CeO2 nanoparticles, or Co oxide structures homogeneously distributed within CeO2. The effect of the different morphologies on the properties of Co3O4–CeO2 was investigated with regard to the crystallite phase(s), particle size, surface area, and catalytic activity for CO oxidation. The material with Co3O4 nanoparticles finely dispersed on the surface of CeO2 particles had the highest catalytic activity.Co This synthetic route proved to be very efficient for silica-based mixed-metal oxides , because a variety of alkoxysilanes (RO)3Si(CH2)3L′ with a coordinating group L′ are available. and sodium tert-butoxide in 1,2-dimethoxyethane (DME) according to literature procedures.[−3 mbar for at least 5 h to remove H2O. Almost all H2O of Co(OAc)2⋅4 H2O was then removed, as shown in Figure 2 to differentiate it from Co(OAc)2⋅4 H2O. F127 and acetaldoxime were used as received.All solvents and solid chemicals were dried by standard methods. All operations that involved metal alkoxides were carried out under moisture-free and oxygen-free argon using standard Schlenk or glovebox techniques. Cerium ocedures., 21 The p-Carboxybenzaldehyde oxime [2⋅4 H2O (1 mmol) were put into a Schlenk tube and dried at <10−3 mbar for 5 h to remove water. Then DME (5 mL) was added, and the mixture was stirred until a clear purple solution was formed, followed by the addition of a solution of CeB (1 mmol) in DME (5 mmol). During further stirring for 30 min the color of the solution changed to brown, which indicated the formation of the Ce-POBC-Co single-source precursor.; 2 mmol) and Co(ORoute 1: AO (8 mmol) of was added to a solution of CeB (4 mmol) in DME (20 mL), and the mixture was stirred for 30 min. After the addition of F127 (0.025 mmol), stirring was continued for 1 h. No water was added during this stage. The sol was then deposited onto glass sheets (20×30 cm), which had been cleaned with 10 % NaOH, iPrOH, and acetone, then dried at 100 °C. The as-deposited films were exposed to ambient humidity at room temperature for 24 h. The obtained solid was then scraped off with a razor blade. The obtained gel powder (labeled 1) was put into the Teflon liner of the autoclave (60 mL) and EtOH (30 mL) was added, followed by the addition of Co(OAc)2 (1 mmol) and stirring for 10 min. Then the autoclave was sealed and heated at 200 °C for 6 h. The product was washed at least three times with EtOH and H2O and finally dried at 105 °C overnight. The sample was labeled 1-STE. Part of the sample was further heat-treated in air at 500 °C for 2 h (heat rate: 2 °C min−1) and was labeled 1-STE-AC.Route 2: AO (8 mmol) was added to a solution of CeB (4 mmol) in DME (20 mL) and the mixture was stirred for 30 min, followed by the addition of F127 (0.025 mmol) and stirring for 1 h. Then Co(OAc)2 (1 mmol) was added, and the mixture stirred for an additional 30 min. A gel powder (labeled 2) was obtained as described for route 1, put into autoclave with EtOH (30 mL), and solvothermally treated at 200 °C for 6 h. The product was washed at least three times with EtOH and H2O and finally dried at 105 °C overnight. The sample was labeled 2-STE. Part of the sample was further heat-treated in air at 500 °C for 2 h (heating rate: 2 °C min−1) and was labeled 2-STE-AC.Route 3: As the ratio of Ce/Co in the SSP is 1:1, an additional Ce source was needed to obtain a Ce/Co ratio of 4:1 in the final material. Firstly, SSP (1 mmol) was prepared as described above. In another Schlenk tube, AO (6 mmol) was added to a solution of CeB (3 mmol) in DME (15 mL) and the mixture was stirred for 30 min. Then the two solutions were combined, F127 (0.025 mmol) was added, and the resulting mixture was stirred for 30 min. The sol was treated similarly to that of route 2. The as-synthesized gel was labeled 3, the solvothermally treated sample 3-STE, and the calcined sample 3-STE-AC.−1 under synthetic air. IR spectra were recorded using a Bruker Tensor 27 instrument with an ATR Micro Focusing MVP-QL instrument with a ZnSe crystal and using OPUS software version 4.0 for analysis.TGA was performed using a Netzsch Iris TG 209C instrument with a platinum crucible and a heating rate of 10 °C minNitrogen-sorption measurements were performed using an ASAP 2020 instrument (Micromeritics). The samples were degassed under vacuum at 80 °C for at least 5 h prior to measurement. The total surface area was calculated according to Brunauer, Emmett, and Teller (BET) and the pore-size distribution (from the desorption branch) according to Barrett, Joyner, and Halenda (BJH).Kα radiation (λ=1.5406 Å).XRD measurements were performed using a Philips X′Pert diffractometer with CuHigh-resolution transmission electron microscopy HRTEM was performed using a TECNAI F20 instrument operated at 200 kV. The samples were ultrasonically dispersed in EtOH for 30 min before the measurements and then deposited onto copper grids covered with porous carbon films.Kα radiation (λ=0.1542 nm) using a microfocus source (Incoatec IμS High Brilliance) and a 2D position sensitive detector . Before the measurements, the powder samples were dispersed in EtOH and ultrasonically deagglomerated for 30 min. Two positions of sample-to-detector distance allowed us to cover a range of the scattering vector q from 0.1 to 20 nm−1. All SAXS patterns were radially averaged to obtain the scattering intensities, which were dependent on the scattering vector q=4π/λsinθ, in which 2θ is the scattering angle and λ the X-ray wavelength. A power-law background with slope q−4 (large objects>100 nm) was subtracted for each sample.Small-angle X-ray scattering was performed with Cu−1) at 200 °C for STE samples or 400 °C for STE-AC for 30 min (heating rate 10 °C min−1). The sample was then cooled to 30 °C under a flow of synthetic air before 5 vol % CO, 12.5 vol % O2, and 82.5 vol % He mixture  were introduced. Then the system was heated to 250 °C with a ramping rate of 2.5 °C min−1. The concentrations of CO and CO2 in the outlet stream were monitored by gas chromatography using a HP-PLOT Q column and a flame-ionization detector.CO oxidation reactions were performed using a continuous-flow fixed-bed quartz reactor under atmospheric pressure. The sample (10 mg), mixed with quartz powder to avoid mass- and heat-transfer limitations, was loaded into the reactor and pretreated with synthetic air (50 mL min


As a key link between innate and adaptive immune responses, the interferon (IFN) system is the first line of defense against viral infection. IFN, and in particular, IFN-α, has been used clinically as an effective therapeutic agent for viral infections. However, different subtypes of IFN-α demonstrate distinct antiviral activity. Therefore, it is important to identify IFN-α subtypes with high antiviral activity for the development of genetically engineered antiviral drugs.Escherichia coli. The cytopathic effect inhibition assay showed that all the 13 recombinant mink IFN-α subtypes inhibited the propagation of vesicular stomatitis virus in WISH cells, with IFN-α2 and IFN-α12 demonstrating the highest activities. Furthermore, recombinant mink IFN-α2 and IFN-α12 significantly suppressed the propagation of canine distemper virus in Vero cells, with IFN-α2 demonstrating the highest activity.In this study, we cloned the genes for 13 IFN-α subtypes from peripheral blood lymphocytes of the mink. The homologies of the 13 mink IFN-α genes were 93.6–99.3% and 88.8–98.4% at the nucleotide and amino acid sequence levels, respectively. In contrast to human and canine IFN-α subtypes, most mink IFN-α subtypes contained two N-glycosylation sites. We expressed and purified 13 mink IFN-α subtypes in We identified the mink IFN-α2 subtype as a promising candidate for the development of effective antiviral drugs. Interferon (IFN) was first identified by Isaacs in 1957 . The IFNIFNs are key cytokines with antiviral, antitumor, and immunomodulatory activities. Based on gene sequences, chromosome location, and receptor specificity, the members of the IFN family are classified into I, II, and III subtypes. Subtype I includes IFN-α, β, ω, ε, κ, δ and ζ -8, and tNeovison vison) is one of the major fur animals and its health problems have become an important issue for the fur industry. Infection by the canine distemper virus, Parvovirus, and Aleutian mink disease virus among minks has caused serious economic losses to the fur animal industry [IFN is a conserved molecule that has been found in humans, mouse, sheep, rabbits, dogs, weasels and other mammals, as well as in fish, turtles and insects. IFNs have been used in both human and veterinary medicine, not only for the treatment of viral infections but also for cancer. Mink (industry . TherefoBy reverse transcription polymerase chain reaction (RT-PCR), we successfully amplified the predicted 564 bp mink IFN-α (MiIFN-α) cDNA. Restriction enzyme analysis confirmed that we had correctly subcloned MiIFN-α cDNAs into recombinant plasmids. DNAStar software analysis demonstrated that the cDNAs of 13 subtypes of MiIFN-α were 564 bp long and encoded 187 amino acids. Due to the different amino acids components, there were slight variations in molecular weight among the 13 MiIFN-α subtypes. The GenBank registration number and molecular weight of the cloned 13 MiIFN-α subtypes are listed in Table http://www.CBS.DTU.DK/Services/SignalP) analysis predicted that the N terminal 23 amino acids represented the signal peptide. The homologies of 13 MiIFN-α subtypes were 93.6–99.3% and 88.8–98.3% at the nucleotide and amino acid sequence levels, respectively  predicted that IFN-α1–3 contained one glycosylation site at 54NYTN57, whereas IFN-α4–13 contained two glycosylation sites. In addition to the glycosylation site at 54NYTN57, IFN-α4–9 and IFN-α13 contained a glycosylation site at 101NTTL104, whereas IFN-α10–12 contained a glycosylation site at 101NMTL104. Further analysis demonstrated that the number and position of cysteines were not the same among the 13 subtypes. IFN-α1–3, IFN-α5, IFN-α7, IFN-α10, IFN-α11 and IFN-α13 contained 8 cysteine residues at the same positions of 5, 16, 24, 52, 92, 109, 122 and 160. IFN-α4, IFN-α6, IFN-α8, IFN-α9 and IFN-α12 contained only 7 cysteine residues with a cysteine residue at position 16 replaced by serine  by an N-glycan, a process known as N-glycosylation . AnalysiMustelidae. The homology between IFN-α1–13 and the published sequences for ferret (GenBank registration number: EF368207) were 95.0–97.3% and 91.5–94.2% at the nucleotide and amino acid sequence levels, respectively. The amino acid sequence analysis showed that fox (EF990625), raccoon (EF543192), and canine (EF28625) IFN-α each contained only one potential N-glycosylation site at 101NMTL104, which is the same site in MiIFN-α10–12. The homologies between mink, fox, raccoon and canine IFN-α were 79.1–80.9% and 68.8–71.4% at the nucleotide and amino acid sequence levels, respectively. Nucleotide evolution tree analysis showed significant differences in the nucleotide sequences among the 13 subtypes of MiIFN-α and those of other species of animals and human IFN-αs -polyacrylamide gel electrophoresis (PAGE) analysis showed the presence of protein bands at 19 kDa  in 50 VSV for 24 h resulted in significant cell lesions. However, pretreatment with 13 subtypes of MiIFN-α attenuated VSV-induced cell lesions, with MiIFN-α2 and MiIFN-α12 demonstrating the strongest effects. Further analysis showed that the effective antiviral concentrations of MiIFN-α2 and MiIFN-α12 in WISH cells were 0.18 × 10 4 IU/mg and 0.3 × 10 3 IU/mg, respectively.To examine the antiviral activity of recombinant MiIFN-αs, we first used the WISH/ vesicular stomatitis virus (VSV) system. Compared to the cells without virus infection, challenge of WISH cells with 100 TCID50 CDV-mediated cell lesions. These data indicate that MiIFN-α2 exhibits the strongest antiviral activity against VSV and CDV.To confirm that the antiviral activity of recombinant MiIFN-α is not limited to VSV, we employed the canine distemper virus (CDV)/Vero system. We selected MiIFN-α2 and MiIFN-α12, because these two subtypes exhibited the strongest antiviral activity against VSV. The results showed that recombinant MiIFN-α2 and MiIFN-α12 significantly inhibited CDV-induced cell lesions. Further analysis showed that 0.145 μg purified MiIFN-α2 protein and 0.28 μg purified MiIFN-α12 could suppress 100 TCIDThe fur animal industry is expanding in the Northeast and Western parts of China. However, the health of fur animals has been compromised by viral infections, such as those with CDV, Parvovirus, and Aleutian mink virus. IFN was first recognized on the basis of its anti-viral activity and only later shown to act primarily as an immunomodulator. IFN-α has been identified from different species, but the biological activity of MiIFN-α has not been tested until now. To determine whether MiIFNα also exhibits antiviral activity similar to IFNs from other species, we examined the ability of recombinant MiIFN-αs to inhibit the replication of VSV.Using RT-PCR, we successfully amplified the predicted 564 bp mink IFN-α cDNA. The homologies of 13 MiIFN-α subtypes were 93.6–99.3% and 88.8–98.3% at the nucleotide and amino acid sequence levels, respectively. It is surprising that all the subtypes have the same size, whereas size differences have been reported in other species. In 14 pig IFN-αs, multiple sequence alignment revealed a C-terminal deletion of 8 residues in six IFN-α subtypes  . The antet al. cloned 12 IFN-α genes from the giant panda and found that IFN-α8, IFN-α4, and IFN-α10 had higher activity whereas IFN-α11 had low biological activity in 293 cells  [et al. cloned 5 dog IFN-α genes and found that rCaIFN-α8 demonstrated high anti-VSV activity. In addition, the anti- canine adenovirus (CAV)-1 activity of rCaIFN-α8 in Madin-Darby canine kidney (MDCK) cells was 33–666 times higher than the anti-VSV activity, but rCaIFN-α8 had no effects on CHV-1 [IFN has a broad-spectrum of antiviral effects and represents an ideal choice for the development of antiviral drugs. IFN was the first cytokine approved by the US Food and Drug Administration for clinical application. Among the members of the IFN family, IFN-α has relatively higher antiviral activity. IFN-α has been identified from different species, including birds, rodents, and primates . IFN-α i1A gene) . Further1A gene) . Taira eon CHV-1 . Taken tEffective induction of IFN expression plays an important role during the immune response to viral infection. IFN-α is a multi-gene family consisting of different subtypes with high homology and similar function. However, recent studies have demonstrated that there are functional differences among the subtypes, probably due to the different amino acid sequences. Indeed, our 13 cloned MiIFN-αs demonstrated a variety of constituent amino acids in each subtype. MiIFN-α4–13 had two N-glycosylation sites, whereas MiIFN-α1–3 had only one N-glycosylation site. IFN-α1–3, IFN-α5, IFN-α7, IFN-α10, IFN-α11, and IFN-α13 had 9 cysteine residues, whereas IFN-α4, IFN-α6, IFN-α8, IFN-α9, and IFN-α12 contained only 8 cysteine residues. Antiviral activity analysis showed that only IFN-α1, IFN-α2, IFN-α3, IFN-α8, IFN-α9, and IFN-α12 had antiviral function. It will be interesting to investigate whether N-glycoslylation sites are essential for the antiviral activity of IFN-α.E. coli. Most of the purified recombinant MiIFN-α subtypes demonstrated antiviral activity against VSV and CDV, and MiIFN-α2 exhibited the highest antiviral activity. Therefore, the MiIFN-α2 subtype is a promising candidate for the development of effective antiviral drugs for the fur animal industry.In summary, in this study for the first time, we have cloned 13 subtypes of the MiIFN-α gene and successfully expressed them in E. coli JM109/BL21, and TRIzol were purchased Invitrogen (USA). RPMI 1640 medium was purchased from GIBCO. Lymphocyte isolation reagent was from the Chinese Academy of Medical Sciences. ExTaq polymerase, AMV reverse transcriptase, and Concanavalin A (Con A) were purchased from Sigma (USA). The Ni-NTA protein purification system and anti-6xHis monoclonal antibodies were purchased from Invitrogen. PCR primers were synthesized by Shanghai Ying-Jun Biotech.VSV was provided by Dr. Haidong Zhi from the Institute of Harbin Veterinary Research, Chinese Academy of Agricultural Sciences (CAAS). Canine distemper virus (CDV) strains were from commercial CDV-3 vaccine strains. WISH and Vero cells were purchased from the Chinese Academy of Sciences Shanghai Cell Bank. Plasmid pProEX HTb, Six-month-old minks were purchased from a fur animal farm in the Jilin Province of China and housed in boxes at the animal house of the Central Laboratory for Animal Diseases of the Institute of Special Animal and Plant Sciences. All animal work and experimental procedures were performed according to the regulations for the administration of affairs concerning experiental animals, which was approved by the state council on October 21, 1988 and promulgated by decree No. 2 of the State Science and Technology Commission on November 14, 1988.15 primer, and PCR was performed with cDNA and primers to amplify IFN-α cDNA [http://www.cbs.dtu.dk/Services/SignalP/). The glycosylation sites were predicted using the online 1.0 NetNGlyc Server.Peripheral blood lymphocytes (PBMCs) were separated under sterile conditions from healthy minks, stimulated with 25 μg/mL ConA for 24 h, centrifuged, and resuspended in 1 mL TRIzol reagent. Total RNA was isolated by the phenol/chloroform method as described previously ,23. cDNAN-α cDNA . The priBamH I and Hind III, ligated into the pProEX HTb vector, and transformed into competent E. coli /BL 21 cells. Positive clones were validated by PCR and restriction enzymes digestion and sequenced by Shanghai Ying-Jun BioTech. The recombinant plasmid was named pHTb/MiIFN-α.The PCR products were separated by 1.5% agarose gel electrophoresis. The gel purified DNA was cut with E. coli BL21 competent cells and selected with ampicillin. Positive clones were inoculated in 5 ml liquid Luria broth (LB) media and cultured at 37°C. When the optical density at 600 nm (OD600) reached 0.5, 1 mmol/L IPTG was added to induce protein expression. For purification, the bacteria were centrifuged at 10,000 rpm for 10 min. Following several cycles of freezing and thawing, the bacterial pellet was mixed with 1 mg/mL lysozme and lysed by sonication on ice. After collection by centrifuging at 4°C at 10,000 rpm for 10 min, the pellet was washed with buffer  and dissolved in 8 mol/L urea buffer. Then 50% NI –NTA (4:1) was added and incubated on ice with slow mixing using a magnetic stirrer blender. Finally, the mixture was loaded onto a chromatography column (Novagen) and eluted by serial washing . The eluents were separated by 12% SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked with 5% milk in TBST (0.1 phosphate buffer contain 0.5% Tween20) and incubated with mouse anti-6xHis monoclonal antibody (1:300) at 4°C overnight, followed by incubation with goat anti-mouse IgG-HRP (1:2000) at room temperature for 1 h. Finally, the membrane was developed using the TMB Chromogenic Reagent .pHTb/MiIFN-αs were transformed into 2 for 12–18 h. Diluted recombinant IFN-α was added to the cell monolayer and incubated at 37°C with 5% CO2 overnight. Next, the cells were challenged with 100 TCID50 VSV, and the plates were incubated at 37°C for 1–2 days. VSV-induced cytopathic effects were assessed by microscopic examination, and IFN-α concentrations were expressed as the inverse dilution that provided protection of 50% of the cells from VSV-induced cytopathic effects (CPE50).The antiviral activity of 0.5 mg/mL recombinant IFN-α was determined by cytopathic inhibition assay. WISH cells were plated in 96-well plates and incubated at 37°C with 5% CO50 was determined by reference for Reed-Muench method. Vero cells were plated in 24-well plates until the formation of a monolayer. Diluted recombinant IFN-α was added to the cell monolayer and incubated at 37°C with 5% CO2 overnight. Next, the cells were challenged with 100 TCID50 CDV, and the plates were incubated at 37°C for 1–2 days. The CDV-induced cytopathic effects were assessed by microscopic examination, and IFN-α concentrations were expressed as the inverse dilution that provided protection of 50% of the cells from CDV-induced cytopathic effects (CPE50).In addition, the antiviral activity of recombinant IFN-α was determined by the inhibition of CDV propagation in Vero cells. The titer of CDV TCID


Introduction. Luteolin, a falconoid compound in many Chinese herbs and formula, plays important roles in cardiovascular diseases. The underlying mechanism of luteolin remains to be further elaborated. Methods. A model of hydrogen peroxide- (H2O2-) induced H9C2 cells apoptosis was established. Cell viabilities were examined with an MTT assay. 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) and flow cytometry were used to detect ROS level and apoptosis rate, respectively. The expressions of signaling proteins related to apoptosis were analyzed by western blot and mRNA levels were detected by real-time polymerase chain reaction (PCR). Quercetin was applied as positive drug. Results. Incubation with various concentrations of H2O2  for 1 h caused dose-dependent loss of cell viability and 100 μM H2O2 reduced the cell viability to approximately 50%. Treatments with luteolin and quercetin protected cells from H2O2-induced cytotoxicity and reduced cellular ROS level and apoptosis rate. Moreover, luteolin could downregulate the expressions of Bax, caspase-8, cleaved-caspase-3, and p53 in apoptotic signaling pathway. Further study showed that the expressions of Akt, Bcl-2, and Mdm2 were upregulated by luteolin. Conclusion. Luteolin protects H9C2 cells from H2O2-induced apoptosis. The protective and antiapoptotic effects of luteolin could be mediated by regulating the Akt-P53/Mdm2 apoptotic pathway. Heart failure (HF) is the common condition resulting from a variety of cardiovascular diseases. The morbidity and mortality of HF remain high even though great efforts have been made to manage it . The fivP53, known as the tumor suppressor gene, could induce apoptosis by blocking cellular DNA damage repair. Accumulating evidence shows that p53 plays a pivotal role in the progression of AS, MI, and IR , 7. ExprMurine double minute 2 (Mdm2), which is induced by p53, interacts with p53 and inhibits the apoptotic effect of p53. P53 level is regulated by the feedback between Mdm2 and p53 , 10. A mTraditional medicine has been making great contributions in treating varieties of human diseases including heart failure . Luteoli2O2 culture and the effect of luteolin on p53/Mdm2 apoptotic signaling pathway was investigated.In this study, we explored the antiapoptotic effects of luteolin in H9C2 cell lines. Cellular apoptosis was induced by H2O2 (3 wt.%) were purchased from Sigma (USA). The Annexin V-PI kit was obtained from BD (USA). cDNA Synthesis kit, Green PCR Master Mix, penicillin, streptomycin, and 0.05% trypsin were purchased from Invitrogen (USA). Dulbecco's modified Eagle's medium (DMEM) was purchased from HyClone (USA). Fetal bovine serum (FBS) was purchased from Corning (USA). Luteolin and quercetin were purchased from National Institutes for Food and Drug Control (China). Luteolin and quercetin were dissolved in DMSO for in vitro assay as storage concentration (100 mM).MTT, DCFH-DA, and Hμg/mL) in a humidified incubator with 5% CO2 at 37°C. Cells were nearly 80–90% confluent and treated with 5, 10, and 20 μM luteolin and 10 μM quercetin for 6 h. Then cells were divided into several groups: normal control group, H2O2-induced model group, luteolin-treated group, and quercetin-treated positive control group. Cells in model group were cultured in DMEM for 6 h, followed by treatment with H2O2 for 1 h. Cells in luteolin and quercetin groups were pretreated with luteolin and quercetin for 6 h before exposed to H2O2.H9C2 cardiomyocyte cell line was obtained from China Infrastructure of Cell Line Resources . The H9C2 cells were cultured in high glucose DMEM supplemented with 10% FBS and a mixture of penicillin (100 U/mL) and streptomycin  in DMEM at 37°C for 1 h. Cells viabilities were determined by MTT assay. Cells were treated with MTT solution dissolved in DMEM (0.5 mg/mL) for 4 h. The supernatants were removed, followed by the addition of 150 μL DMSO to each well to dissolve the precipitate. The absorbance of each well was then measured with a microplate reader  at a wavelength of 492 nm.H9C2 cells were seeded in 96-well plates at a density of 102O2 for 1 h, cells were incubated with luteolin  and quercetin (10 μM) in DMEM supplemented at 37°C for 6 h. After treatment, MTT assay was taken to measure cell viability.H9C2 cells were seeded as described above. Before being treated with H5 cells/mL in 6-well plates. When the confluence reached 70–80%, cells were pretreated with/without luteolin (10 μM) and quercetin (10 μM) for 6 h and then exposed to H2O2 (100 μM) for another 1 h. After incubation, cell morphology was photographed under an inversion microscope .H9C2 cells were seeded at a density of 10μM) at 37°C for half an hour. Fluorescent intensity was examined under a fluorescence microscope  with excitation at 485 nm and emission at 525 nm. Image J was used to analyze the data.The cellular production of ROS was measured by detecting the fluorescent intensity of DCFH-DA, an oxidant-sensitive probe. After incubations at different conditions, H9C2 cells were washed three times with PBS, followed by incubation with DCFH-DA (10 μL binding buffer and incubated with Annexin V (10 μg/mL) and PI (10 μg/mL) in the dark for 15 minutes at room temperature. Then cells were detected with a flow cytometer .The Annexin V-PI method was used to detect the apoptosis rate of H9C2 cells by flow cytometry. Cells were harvested with 0.05% trypsin, washed three times with cold PBS (4°C), and collected by centrifugation at 1000 rpm for 5 minutes. After that cells were resuspended in 200 μg of total RNA using an RT kit  following the manufacturer's instructions. The mRNA quantity of Mdm2 gene was assessed by quantitative real-time PCR . The primers used for amplification are as follows: β-actin F, CCCATCTATGAGGGTTACGC; β-actin R, TTTAATGTCACGCACGATTTC; Mdm2 F, TTGATGATGGCGTAAGTGA; Mdm2 R, AGGCTGTAATCTTCTGAGTC.The total mRNA of cells was extracted using TRIzol  and cDNA was synthesized from 2 β-actin  served as an internal control.The collected cells were prepared with RIPA buffer  and proteins were extracted according to the manufacture's instruction. Protein contents were measured with BCA Bradford protein assay . After addition of loading buffer, cell lysates were separated by 10% SDS-PAGE and transferred to NC membranes . After being blocked with 5% nonfat dry milk for 2 h, the membrane was incubated with different primary antibodies  overnight at 4°C and washed with TBST three times. The membrane was then incubated with HRP-conjugated secondary antibodies  for 1 h at room temperature. After being washed with TBST for three times, the proteins were detected with an enhanced chemiluminescence agent  and quantified by densitometry using an image analyzer . The antibodies included rabbit monoclonal antibodies against cleaved-caspase-3 (1 : 1000), Bcl-2 (1 : 1000), Bax (1 : 1000), Akt (1 : 1000), rabbit multiclonal antibodies against caspase-8 (1 : 1000), mouse monoclonal antibody against p53 (1 : 1000), and secondary antibodies .P < 0.05.Data were expressed as the mean ± SD. Analysis was undertaken by one-way ANOVA. Differences between groups were considered as statistically significant when 2O2 for 1 h. Cell viability was measured by MTT assay. The result showed that H2O2 induced dose-dependent decrease of cell viability . The cellular survival rate in 100 μM H2O2-treated group was 52.48 ± 0.0416% and these cells could be applied as apoptotic model to assess effects of luteolin. Therefore, 100 μM H2O2 was applied to cells to induce apoptosis in the subsequent experiments.H9C2 cell lines were incubated with different concentrations of Hiability . When ceμM) and quercetin (10 μM) groups were similar to that in control group, indicating that quercetin and luteolin have no cytotoxicity on H9C2 cells. The effects of luteolin/quercetin on H2O2-induced cytotoxicity were also detected. As shown in μM, 10 μM, and 20 μM)/quercetin (10 μM) pretreatment provided significant protective effects on H2O2-mediated cytotoxicity in a dose-dependent manner (##P < 0.01). Cells treated with 10 μM luteolin had significant higher viability than those treated with 5 μM luteolin and cell viabilities were similar between cells treated with 10 μM and 20 μM luteolin. Therefore, 10 μM luteolin was applied to treat cells to assess its effects in the subsequent experiments.Cell viabilities were assessed by MTT assay. As shown in 2O2-induced cellular injury, cell morphology was examined under an inversion microscope. Normal cells in control group showed long fusiform shapes with clear structure (2O2 (100 μM) for 1 h resulted in cell shrinkage, alterations of cell shape, and wider intercellular gap. Pretreatment with luteolin and quercetin protected cells from the morphological changes induced by H2O2. These results demonstrated that luteolin pretreatment had significant protective effects against H2O2-induced cytotoxicity in H9C2 cells.To further evaluate the protective effect of luteolin against Htructure . Treatin2O2 could promote cellular damage. To determine whether pretreatment with luteolin could alleviate H2O2-induced early-stage oxidative stress, the treated cells were detected with DCFH-DA assay. Fluorescent intensity was examined under a fluorescence microscope. As shown in 2O2-treated H9C2 cells compared with that in the control group. After pretreatment with luteolin (10 μM)/quercetin (10 μM), intracellular ROS was significantly reduced compared with that in H2O2-treated cells.ROS generated by H2O2-induced injury. To analyze the effects of luteolin on apoptosis in H9C2 cells, Annexin V-PI method was used to detect the apoptosis rate of H9C2 cells. As shown in 2O2 group compared to control group . Pretreatment with luteolin/quercetin (10 μM) for 6 h significantly reduced the percentage of apoptotic cells to 9.8 ± 0.85%/16.4 ± 3.96% (##P < 0.01), suggesting that luteolin could protect H9C2 cells from apoptosis induced by H2O2.Apoptosis is considered to be one of the pathogenesis of H2O2, apoptosis is induced and expression of p53 is enhanced [2O2 group increased slightly compared to control group (P < 0.05). Pretreatment with luteolin could remarkably elevate expression of Mdm2 (P < 0.05).Mdm2, an E3 ubiquitin ligase, can be induced by increased cellular level of p53 and in turn negatively regulate p53 stability. When cells are exposed to Henhanced , as well2O2-induced apoptosis. Expressions of key molecules in apoptotic signaling pathway were measured by western blot. Bax, caspase-8, cleaved-caspase-3, and p53 are proapoptotic proteins. Akt and Bcl-2 have antiapoptotic effects. As shown 2O2 group, expressions of Bcl-2 and Akt decreased, whereas the expressions of Bax, caspase-8, cleaved-caspase-3, and p53 increased compared to control group, indicating that apoptotic signaling pathway was activated by H2O2. Pretreatment with luteolin reversed the expressions of these proteins back toward normal levels. The results showed that protection of luteolin against H2O2-induced cell apoptosis may be mediated through Akt-p53 apoptotic signaling pathway.We further investigated the mechanism of luteolin in protecting cells from H2O2 in H9C2 cardiomyocytes and explored the antiapoptotic mechanism. Our study showed that one of the antiapoptotic mechanisms of luteolin was mediated through Akt-p53/Mdm2 signaling pathway.Luteolin is a plant flavonoid that exists in a variety of plants and has been shown to have antitumor, antioxidant, antiapoptotic, and anti-inflammatory effects –15. LuteStudies have shown that apoptosis of cardiac myocytes is an essential process in the progression of heart failure . A study2O2 could induce apoptosis in H9C2 cells [2O2 has proapoptotic effect on H9C2 cells. Pretreatment with luteolin could increase cell viability and reduce intracellular ROS level and apoptosis rate suggesting that luteolin possesses antioxidant and antiapoptotic properties in cardiomyocytes [2O2-treated group, indicating that quercetin also has antiapoptotic effects. We further explored the mechanisms by which luteolin exerts antiapoptotic effects.Previous study showed that HC2 cells . In thismyocytes , 25. Que2O2 induced apoptosis by downregulating Bcl-2 and upregulating expressions of proapoptotic proteins, such as Bax, caspase-8, and cleaved-caspase-3. In cells that were pretreated with luteolin/quercetin, expression of Bcl-2 was upregulated and expressions of Bax, caspase-8, and cleaved-caspase-3 were downregulated compared to apoptotic model cells, indicating that luteolin protects cells from apoptosis by modulating anti-(pro)apoptotic proteins.Extrinsic and intrinsic signaling pathways can both lead to apoptosis. In the extrinsic pathway, caspase-8 activates caspase-3 after it is activated by intracellular and extracellular stimuli . In the Apoptosis is an orderly regulated process and p53 is one of the most important modulators in apoptotic signaling pathways , 30, 31.2O2-treated cells. Expression of p53 was upregulated and expression of Akt was downregulated by stimulus of H2O2, leading to cellular apoptosis. Pretreatment with luteolin rescued cells from apoptosis by increasing expressions of Mdm2 and Akt and suppressing expression of p53. Pretreatment with quercetin only decreased the expression of p53 and had no significant effects on the expressions of Mdm2 and Akt. The results indicated that the antiapoptotic effect of luteolin is mediated by regulating Akt/Mdm2/p53 signaling pathway in H9C2 cells.The activity and stability of p53 are regulated by a variety of enzymes and Mdm2 is one of the most important regulators. Mdm2 inhibits the transactivation ability of p53 by interacting with p53 transactivation domain and decreases the stability of p53 by promoting p53 ubiquitination . Overexp2O2 could induce cellular apoptosis. ROS level was increased and Akt-p53/Mdm2 apoptotic signaling pathway was activated in H2O2-treated cells. Pretreatment with luteolin could protect H9C2 cells from H2O2-induced apoptosis. The antiapoptotic effect of luteolin was mediated by upregulating expression of Bcl-2 and downregulating apoptotic proteins, including Bax, caspase-8, and cleaved-caspase-3. Furthermore, luteolin could suppress expression of p53 by activating Mdm2 and Akt. Our results illustrate that Akt-p53/Mdm2 signaling pathway is a potential target of luteolin in the prevention of HF.Treatment of H9C2 cells with H


The lanthanide-functionalized multifunctional hybrid nanoparticles combining the superparamagnetic core and the luminescent europium complex were successfully designed and assembled via layer-by-layer strategy in this work. It is noted that the hybrid nanoparticles were modified by a hydrophilic polymer polyethyleneimine (PEI) through hydrogen bonding which bestowed excellent hydrophilicity and biocompatibility on this material. A bright-red luminescence was observed by fluorescence microscopy, revealing that these magnetic-luminescent nanoparticles were both colloidally and chemically stable in PBS solution. Therefore, the nanocomposite with magnetic resonance response and fluorescence probe property is considered to be of great potential in multi-modal bioimaging and diagnostic applications.The online version of this article (doi:10.1186/s11671-016-1497-3) contains supplementary material, which is available to authorized users. This ne3O4 NPs to achieve multifunctional capabilities. Compared with these fluorochromes, lanthanide-based complexes are of particular attractiveness for their large Stokes shift and sharp line-like emission bands @PEI , thus to reduce the effect of UV damage on living biological samples 1,10-phenanthroline propyl isocyanate was purchased from Jinan Xinna Medicine Science and Technology Co. Other chemicals and solvents were obtained from Aladdin Chemicals Company and were of analytical grade.1,10-Phenanthroline-5,6-dione and ammonium acetate were obtained from Shanxi Xinhua Co. FeCl−1 wave number range using a Nicolet 360 FTIR spectrometer with the KBr pellet technique. Transmission electron microscope (TEM) images were taken on a JEM-100CX II apparatus, and the fluorescence images were taken on an Olympus FluoView 500 laser scanning confocal microscope . Dynamic light-scattering (DLS) measurements were performed at 25 °C using a Malvern Zetasizer Nano ZS. Magnetic measurements were carried out at room temperature with a Lake Shore-735 vibrating sample magnetometer (VSM) magnetometer. The steady-state luminescence spectra and the lifetime measurements were measured on an Edinburgh Instruments FSL920 fluorescence spectrometer, with a 450-W Xe arc lamp as the steady-state excitation source or an Nd-pumped OPOlette laser as the excitation source for lifetime measurements. The solid-state quantum yield was measured using a Model F-3029, Quanta-Phi 6 Integrating Sphere connected with a Horiba Jobin Yvon Fluorolog-3 spectrophotometer.NMR spectra were recorded on a Varian Mercury-300B spectrometer. CHN elemental analyses were measured on an Elementar Vario EL analyser; the contents of Eu(III) ions were obtained by inductively coupled plasma-atomic emission spectroscopy (ICP) using an IRIS Advantage ER/S spectrophotometer. The solid-state absorption spectra were recorded with a Shimadzu UV-3100 spectrophotometer. Fourier transform infrared (FTIR) spectra were conducted within the 4000–400 cmδH  7.65–7.68 , 8.37–8.40 , 8.98–9.00 . Anal. Calcd C, 73.07; H, 3.87; N, 17.94. Found C, 73.37; H, 3.57; N, 17.82.1,10-Phenanthroline-5,6-dione  and ammonium acetate  were dissolved in 5 mL glacial acetic acid. While the mixture was stirred, a solution of 4-hydroxybenzaldehyde  in glacial acetic acid was added dropwise to the mixture. After heating for 3 h, the mixture was then poured in 200 mL water. The solution was neutralized with ammonia to pH = 7 and was then cooled to room temperature. The precipitate was filtered off and washed with large portions of water, then the crude product was purified by recrystallization from a mixture of EtOH and water solution (yield 65 %). p  prepared was mixed with 25 mL of 95 % ethanolic solution of Eu(DBM)3(H2O)2 , and then the suspension was sonicated for 20 min. After sonication, the reaction system was stirred at room temperature overnight. The yellow precipitate formed was filtered and washed with the mixed solution of ethanol and water ((v/v) 1:1) and dried in vacuum, to obtain 70.0 mg of Eu(DBM)3Lp. Anal. Calcd C, 67.78; H, 4.01; N, 4.93. Found C, 67.59; H, 4.35; N, 4.85.Solid L3·6H2O (0.54 g), sodium acrylate (1.5 g), and NaOAc (1.5 g) were dissolved in a mixture of 5 mL ethylene and 15 mL diethylene glycol under vigorous stirring. The obtained homogeneous yellow solution was transferred to a Teflon-lined stainless-steel autoclave, sealed, and heated at 200 °C. After reaction for 10 h, the autoclave was cooled to room temperature. The obtained Fe3O4 NPs were washed several times with ethanol and water and then dried in vacuum for 12 h. The PXRD analysis of the Fe3O4 NPs is shown in Additional file 3O4 crystal (JCPDS no. 88-0315) and no other peaks are detected, indicating that the products are pure-phase Fe3O4.FeClacrylate .5 g, and3O4@SiO2 nanospheres were prepared according to a previously reported method. Typically, the Fe3O4 NPs was treated with diluted HCl solution by ultrasonication for 10 min. The magnetite NPs were washed and homogeneously dispersed in a mixture of ethanol, deionized water, and concentrated ammonia aqueous solution, followed by the addition of tetraethyl orthosilicate . After being stirred for 6 h, the Fe3O4@SiO2 nanospheres were separated, washed, and then dried in vacuum.The core-shell Fep was homogeneously dispersed in an excess of 3-(triethoxysilyl)propyl isocyanate (2.5 mL) under ultrasonication. Then, the mixture was stirred under argon at 80 °C for 72 h. The mixture was added slowly to 20 mL of cold hexane, and a white-yellow precipitate was formed. The precipitate was filtered off, washed, and then dissolved in ethanol. The solution was filtered, and the ethanol was removed by rotary evaporation. The obtained compound was dissolved in a small portion of dichloromethane (DCM). This DCM solution was added dropwise to 30 mL of cold hexane to reprecipitate the compound. The purified product was filtered off and dried in vacuo. Yield 60 %; δH : 1.08–1.15 , 1.51–1.55 , 3.02–3.07 , 3.71–3.76 , 7.31–7.33 , 7.79–7.86 , 8.24–8.26 , 8.91 , 9.04 , 13.73 .A batch of 200 mg (7.45 mmol) of L3O4@SiO2 in 50 mL of dry toluene mixed with 40 mg of Lp-Si was stirred and refluxed for 8 h. The solid obtained was then separated by centrifugation, washed with ethanol, and dried at room temperature.Two hundred milligrams Fe3O4@SiO2-Lp was refluxed with 30 mg of Eu(DBM)3(H2O)2 in ethanol, then the obtained magnetic-luminescent NPs were collected by centrifugation after 24 h, and the excess of unbound complex was thoroughly washed away with ethanol. After drying at 120 °C for 3 h, Fe3O4@SiO2–[Eu(DBM)3Lp] was obtained.A batch of 200 mg of Fe3O4@SiO2-[Eu(DBM)3Lp] was homogeneously dispersed in ethanol under ultrasonication; PEI ethanol solution was then slowly added to the solution. After being fiercely stirred at room temperature for 6 h, the microspheres were separated, washed with ethanol, and then dried in vacuum at 60 °C for 6 h.A batch of 200 mg of Fe3O4 magnetic NPs, the Fe3O4@SiO2 composites, the Fe3O4@SiO2-[Eu(DBM)3Lp] magnetic-luminescent dual-functional NPs, and Fe3O4@SiO2-[Eu(DBM)3Lp]@PEI nanocomposite are shown in Fig. 3O4 NPs are spherical and remarkably uniform with an average size about 100 nm 3Lp] NPs which are shown in Fig. 3O4@SiO2 nanospheres. And TEM images of the PEI-modified nanocomposite are shown in Fig. TEM images of the Fe nm Fig. . Clearly2 coating, the value zeta potential at physiological pH of 7.4 decreased from −12.7 mV for the starting Fe3O4 NPs to −28.3 mV. The zeta potential value changed to 9.4 mV upon Eu(III) complex conjugation and increased steeply to 30.2 mV after modifying with PEI, confirming the pronounced increase in the surface amino group density. Thus, the changes in the potential value of the NPs suggested that the surface modifications of SiO2, Eu(III) complex and PEI to the NPs were successful.The above electron microscope observation results can be further confirmed by zeta potential measurements of the products, which are sensitive mainly to the outer surface of the NPs . The mea3O4@SiO2-[Eu(DBM)3Lp] could significantly increase the water solubility and stability of the nanocomposite, which was essential for them in biological applications  3Lp] NPs, and Fe3O4@SiO2-[Eu(DBM)3Lp]@PEI nanocomposite in the solid state. The Fe3O4@SiO2-[Eu(DBM)3Lp] NPs showed a wide absorption band at 320–410 nm, which was similar with that of Eu(DBM)3Lp corresponding to π-π* transitions of the ligand. However, a blueshift (15 nm) could be noticed compared to those of Eu(DBM)3Lp, indicating that the complex was grafted onto the matrix. After PEI modification, the functionalized nanocomposite displayed a peak at 230 nm that was typical for PEI. More importantly, comparing with that of Fe3O4@SiO2-[Eu(DBM)3Lp] NPs, the wide absorption band of the Fe3O4@SiO2-[Eu(DBM)3Lp]@PEI nanocomposite exhibited redshift optical absorption (10 nm), which indicated that hydrogen-bonding interaction occurred between the PEI molecules and the surface ligands of the Fe3O4@SiO2-[Eu(DBM)3Lp] nanocomposite , and the PEI-modified nanocomposite were 68.7, 51.0, 32.3, and 27.4 emu g−1 3Lp]@PEI nanocomposite is less than the magnetite NPs as magnetic core, it may be believed to possess enough strong magnetic attraction for effectively magnetic targeting and separation.Magnetic characterization at 300 K with a VSM showed that the saturation magnetization values of Fe3+ complex [Eu(DBM)3Lp] and the Fe3O4@SiO2-[Eu(DBM)3Lp]@PEI nanocomposite as solid at room temperature. The excitation spectra which were both obtained by monitoring at 612 nm exhibited a broad excitation band (BEB) between 250 and 450 nm. In the emission spectra, only characteristic emission of Eu(III) arising from the transition 5D0 → 7FJ  was detected with the transition 5D0 → 7F2 (red emission) as the dominant group, which indicated that an efficient energy transfer from the ligands to Eu(III) could take place not only in the Eu(III) complex but also in the complex incorporated in the matrix [3Lp] with the Fe3O4@SiO2-[Eu(DBM)3Lp]@PEI nanocomposite at different excitation wavelengths from the UV to visible range . From these comparisons, one could note that both the precursor and the nanocomposite exhibited significant luminescent efficiency at different excitation wavelengths, which demonstrated the potential utility of this novel material in bioimaging [Figure e matrix . Figure oimaging .Fig. 5ExΦ) of the Fe3O4@SiO2-[Eu(DBM)3Lp]@PEI nanocomposite was measured to be 2.70 % lower than that of the free Eu(DBM)3Lp (14 %), which might be due to the absorption of the matrix. The excited-state lifetime of the Fe3O4@SiO2-[Eu(DBM)3Lp]@PEI NPs is 0.32 ms, which was close to that of the free Eu(DBM)3Lp (0.48 ms), indicating that the assembled nanocomposite was capable of eliminating background emission from a biological background for sensitive optical-imaging applications. The shortened lifetime might have given rise to the quenching of O-H oscillators on the matrix surfaces and the non-radiative dissipation of energy on the high-energy N-H vibrations from the modified PEI molecule, which also made the quantum yield of Fe3O4@SiO2-[Eu(DBM)3Lp]@PEI nanocomposite be lower than that of Eu(DBM)3Lp [The quantum yield ((DBM)3Lp .3O4@SiO2-[Eu(DBM)3Lp]@PEI nanocomposite emitted bright-red light originating from the characteristic emission of Eu(III) as shown in the digital photographs of Fig. 3O4@SiO2-[Eu(DBM)3Lp]@PEI nanocomposite possessed excellent magnetic responsiveness, luminescent property, and water solubility, which were important in terms of the practical manipulation.Direct proof of magnetic-luminescent properties of the final product could be found in the following designed experiments. Upon UV light irradiation, the well-dispersed aqueous Fe3O4@SiO2-[Eu(DBM)3Lp]@PEI by using a fluorescence microscope. One of the advantages of using a fluorescence microscope as a tool is that the dispersive and luminescent properties at a micro level can be monitored. In the current study, observations using a fluorescence microscope were carried out on the Fe3O4@SiO2-[Eu(DBM)3Lp]@PEI nanocomposite dispersed in PBS solution. When the nanocomposite was sonicated with a concentration of 0.1 wt.%, a stable dispersion was formed. Inside this dispersion, shinning red spots were observed as shown in Additional file 3O4@SiO2-[Eu(DBM)3Lp] were dispersed in PBS solution at the same condition; however, great agglomeration could be seen from the photographs  complexes enables us to directly capture the Fe3O4 NPs into hydrophilic and biocompatible magnetic-luminescent dual-functional nanocomposites. The silica shells formed via the sol-gel method played significant roles in terms of trapping Fe3O4 NPs; the Eu(III) complex grafted made the NPs potential in a time-resolved imaging, and the PEI surface layer endowed the final material with hydrophilic and modifiable properties. The nanocomposite obtained was characterized by various techniques, and the results showed the desired properties as designed. Furthermore, this approach of functionalizing magnetic-luminescent nanocomposite via hydrogen-bonding method may be applied to fabricate other nanocomposites in order to obtain broader optical properties and potential applications.In summary, a simple and versatile strategy has been developed to transform Fe


Caldicellulosiruptor lactoaceticus 6A, an anaerobic and extremely thermophilic bacterium, uses natural xylan as carbon source. The encoded genes of C. lactoaceticus 6A for glycoside hydrolase (GH) provide a platform for xylan degradation. The GH family 10 xylanase (Xyn10A) and GH67 α-glucuronidase (Agu67A) from C. lactoaceticus 6A were heterologously expressed, purified and characterized. Both Xyn10A and Agu67A are predicted as intracellular enzymes as no signal peptides identified. Xyn10A and Agu67A had molecular weight of 47.0 kDa and 80.0 kDa respectively as determined by SDS-PAGE, while both appeared as homodimer when analyzed by gel filtration. Xyn10A displayed the highest activity at 80°C and pH 6.5, as 75°C and pH 6.5 for Agu67A. Xyn10A had good stability at 75°C, 80°C, and pH 4.5–8.5, respectively, and was sensitive to various metal ions and reagents. Xyn10A possessed hydrolytic activity towards xylo-oligosaccharides (XOs) and beechwood xylan. At optimum conditions, the specific activity of Xyn10A was 44.6 IU/mg with beechwood xylan as substrate, and liberated branched XOs, xylobiose, and xylose. Agu67A was active on branched XOs with methyl-glucuronic acids (MeGlcA) sub-chains, and primarily generated XOs equivalents and MeGlcA. The specific activity of Agu67A was 1.3 IU/mg with aldobiouronic acid as substrate. The synergistic action of Xyn10A and Agu67A was observed with MeGlcA branched XOs and xylan as substrates, both backbone and branched chain of substrates were degraded, and liberated xylose, xylobiose, and MeGlcA. The synergism of Xyn10A and Agu67A provided not only a thermophilic method for natural xylan degradation, but also insight into the mechanisms for xylan utilization of C. lactoaceticus. O-methyl-glucuronyl, acetyl, and arabinofuranosyl substituents http://www.cazy.org/). Thermostable hemicelluloytic enzymes, with a number of advantages over mesophilic enzymes, have thus gained worldwide industrial and biotechnological interest.Xylan, the main hemicellulose component of plant cell wall, is a heteropolymeric polysaccharide consisted mostly of linear backbone of β-1,4-D-xylopyranoside units which are commonly decorated with 4-Caldicellulosiruptor lactoaceticus 6A, an anaerobic and extremely thermophilic, cellulose and hemicelluloses degrading bacterium, was isolated from an alkaline hot spring in Iceland C. lactoaceticus 6A provide a platform for degrading natural polysaccharides at higher temperature.C. lactoaceticus 6A GHs was studied. The genes of endo-β-1,4-xylanase Xyn10A and α-glucuronidase Agu67A were identified and cloned in the genome of C. lactoaceticus 6A, and heterologously expressed, purified and biochemically characterized. The synergistically hydrolytic properties of the two enzymes on MeGlcA decorated xylan and XOs were also investigated.In present study, thermophilic degradation of MeGlcA decorated xylan using C. lactoaceticus genome sequence analysis, Calla_1331 and Calla_1259 were annotated as putative GH10 endo-β-1,4-xylanase (Xyn10A) and α-glucuronidase (Agu67A), respectively. Both Xyn10A and Agu67A had no signal peptide, indicating they are intracellular enzymes. Xyn10A only contained a GH10 motif  and identity (65.1%) with Thermoanaerobacterium saccharolyticum GH10 xylanase [GenBank: ADQ57411.2], and similarity (71.5%) and identity (57.8%) with GH10 xylanase from Alicyclobacillus sp. A4 [GenBank: ADK91076.1]. However, Xyn10A showed much lower similarity (27.0–41.8%) and identity (14.4–23.5%) with other characterized thermophilic GH10 xylanases, indicating that Xyn10A was a novel thermophilic GH10 xylanase. The amino acid residues Glu161 and Glu266, predicted acting as proton donor and catalytic nucleophile, were also conserved in Xyn10A T. saccharolyticum xylanase, and then clustered with Alicyclobacillus sp. xylanase as a single evolutionary clade which was distinct from other thermophilic bacteria and fungi. On the contrary, Xyn10A was far isolated from other reported Caldicellulosiruptor sp. xylanase indicating the distinct interspecific-diversity of xylanases.Through 10 motif , the calas found . In aminlactoaceticus agu67A gene was predicted to encode a 693-amino-acid GH67 α-glucuronidase with Mw of 80,343 Da and a pI of 8.31. Agu67A had a conserved GH67 N-terminus (14–136 amino acids), a GH67 middle domain (146–467 amino acids), and a GH67 C-terminus (468–692 amino acids) motifs without signal peptide , Thermoanaerobacterium xylanolyticum LX-11 [GenBank: AEF17768.1] , Geobacillus stearothermophilus [GenBank: ABI49940.1] , Bacillus halodurans C-125 [GenBank: BAB04780] , and Paenibacillus sp. Strain JDR-2 [GenBank: ABV90485.1] , respectively. Analysis of Neighbor-Joining (N-J) tree revealed Agu67A was located in the group of thermophilic bacteria . Proteins were heat-treated at 65°C for 30 min followed by Ni-affinity chromatography, and further purified through Superdex 200 gel filtration. The recombinant Xyn10A displayed as a single band with Mw of about 47.0 kDa by SDS-PAGE analysis, which was in agreement with the predicted Mw based on the amino acid sequence  Trichoderma reesei (90.0 kDa) Glaciecola mesophila KMM 241 (43.0 kDa) Cohnella laeviribosi HY-21 (42.0 kDa) B.halodurans TSEV1 (40.0 kDa) Remersonia thermophila CBS 540.69 (42.0 kDa) For functional analysis of the recombinant enzymes, both sequence . Size exsequence . Other xC. polysaccharolyticus (158.0 kDa) Bacillus stearothermophilus No. 236 (161.0 kDa) B.stearothermophilus T-6 (150.0 kDa) SDS-PAGE analysis of the purified Agu67A indicated a single protein at approximately 80.0 kDa, which was also consistent with the theoretical Mw . HoweverBoth Xyn10A and Agu67A were observed with clearing hydrolytic activity zones on the agar plates containing beechwood xylan or XOs . In contO-acetyl-4-O-methylglucuronoxylan in hardwoods with higher degree of polymerization , and as arabino-4-O-methylglucuronoxylan in softwoods It was reported that most of the α-glucuronidases are only active on MeGlcA linked short xylo-oligomers, while few studies have confirmed its high activity against polymeric substrates Xyn10A displayed the highest activity at 80°C and maintained more than 50% of its activity at 65–85°C . MoreoveCertain properties of Xyn10A were compared with some other thermophilic xylanases from bacteria and fungi as shown in 3+ and Zn2+ showed obviously effects on Xyn10A activity. It was strongly activated by 1 mM or 5 mM NH4+, Na+, K+, Mg2+, Ni2+; 1 mM Fe2+; and 5 mM Ca2+. Furthermore, the addition of 1 mM Ca2+ exhibited a moderate elevation to the activity. On the contrary, other concentrations of ions, including 1 mM or 5 mM Co2+, Mn2+, Cu2+, and 5 mM Fe2+, Fe3+, Zn2+, significantly inhibited the xylanase activity. Reductant, detergents, and metal chelator also had influence on the enzyme activity. 1 mM or 5 mM DTT, 0.1% or 0.5% Triton X-100, 5 mM EDTA, along with 0.1% β-ME markedly as well as 1 mM EDTA slightly increased the activity. However, the activity was strongly interrupted by 0.1% or 0.5% SDS and 0.5% β-ME. In addition, both 5% and 10% glycerol showed the highest positive impact on xylanase catalytic activity. But it was noteworthy that the activity was almost completely inhibited by three tested organic reagents .In addition, the influences of various additives including metal ions and reagents on Xyn10A activity were also investigated . Most ofBacillus sp. NG-27 extracellular GH10 endo-xylanase revealed a metal binding site located at the C-terminal end of the catalytic domain 2+-binding site was seen and the xylanase activity had a concentration-dependent manner with the presence of Mg2+. Some metals, such as Cu2+ and Zn2+ had strong affinities toward sulphydryl groups hence strongly inhibited the activity Thermotoga thermarum xylanase, the enzyme activity was found to be greatly stimulated by Ca2+, Mn2+ and Co2+T. saccharolyticum NTOU1 XynFCB activity was also enhanced by the metal ions NH4+, K+, Na+, and Ca2+, while strongly inhibited by Cu2+ and Zn2+2+, β-ME and EDTA enhanced the Alicyclobacillus sp. A4 XynA4-2 activity, whereas 1 mM Zn2+, Cu2+, and SDS resulted in severe or complete inhibition Actinomadura sp. strain Cpt20 xylanase was also found to be enhanced by Ca2+ and Co2+, but was inhibited by Fe2+, Zn2+, Cu2+ and Mg2+, yet almost unaffected by K+, Na+ and Mn2+Streptomyces rameus L2001 xylanase activity In the previous reports, a large number of xylanases were also affected to some extent by these metals which suggested its possible function as a cofactor for maintaining structure stability and aiding enzyme-substrate reaction C.polysaccharolyticus Agu67A was most active at 60°C and pH 5.5 with specific activity reaching 10.8 IU/mg and 11.6 IU/mg for (4-O-methyl-α-D-glucurono)-D-xylan and birchwood xylan, respectively B. stearothermophilus T-6 α-glucuronidase were 65°C and pH 5.5–6.0, respectively Optimal temperature and pH for Agu67A were 75°C  and 6.5 In order to assay the relationship between Xyn10A capacity and substrate abundance, different concentrations of beechwood xylan were hydrolyzed by constant amount of enzyme. Xylose, xylobiose, xylotriose, xylotetraose, as well as higher polymeric XOs, accumulated with the increasing of substrates . Meanwhidata not shown). Xyn10A primarily degraded XOs into xylobiose and xylose, while Agu67A specifically digested aldobiouronic acid to MeGlcA and xylose. As a result, a slightly synergistic effect of xylose and xylobiose equivalents was seen when they coordinately acted together.Moreover, to explore their effects on XOs hydrolysis, the two enzymes were incubated separately or combination with XOs, and the products were analyzed. TLC analysis revealed the efficient break down of XOs into xylose and xylobiose in the presence of Xyn10A or two combined enzymes . As aldoFurthermore, to evaluate the synergistic activity of Xyn10A and Agu67A, single or mixed enzymes were incubated with beechwood xylan and the products of hydrolysis were analyzed accordingly. As shown by the TLC analysis , Xyn10A In addition, to further evaluate the synergistic activity of Xyn10A and Agu67A, the products of beechwood xylan hydrolyzed for 2 h were determined by HPLC . Peaks rT. thermarum, in which xylose and xylobiose were the major end products of beechwood xylan after 5 h hydrolysis Alicyclobacillus sp. A4 XynA4-2 contained 92.7% xylose and 7.3% xylobiose C. laeviribosi HY-21 iXylC Streptomyces sp. CS428 Xyn428 from beechwood xylan Many GH10 xylanases were detected to degrade xylan polymer into a mixture mostly of xylose and XOs with DP lower than five C. polysaccharolyticus, the release of either xylose or xylobiose tended to be slightly higher from birchwood xylan when incubated with two enzyme mixture than with single xylanase at 65°C Aspergillus tubingensis observed to liberate minor amounts of MeGlcA from birchwood xylan, and the amounts of MeGlcA and short oligo-saccharides (xylobiose and xylotriose) were improved in the two enzymes combination system Schizophyllum commune α-glucuronidase was able to remove the MeGlcA groups from polymeric glucuronoxylan, and 76% of MeGlcA side groups were cleaved from the backbone of glucuronoxylan together with a xylanase Given the possibility that endoxylanases might breakdown xylan into XOs substituted with MeGlcA, microorganisms form α-glucuronidase to remove these side chains hence ensuring the effectiveness of xylan degradation. Nevertheless, a majority of the α-glucuronidases only has the capacity to act on small model xylan or MeGlcA branched XOs C. lactoaceticus 6A is an extremely thermophlic plant biomass-degrading bacterium, with capability of utilizing xylan as carbon source C. lactoaceticus revealed only three genes encoding endo-β-1,4-xylanase . Both Calla_1781 and Calla_0206 contained one GH10 domain and two to three carbohydrate-binding modules (CBMs). Interestingly, all of the three xylanases were predicted to be intracellular based on the absence of signal peptides. Specifically, no genes encoding putative β-xylosidase were annotated throughout the genome. Hence, other xylan-specific enzymes, as well as their novel synergistic roles need to be further investigated to give a deeper understanding of the mechanisms involved in xylan deconstruction process. In previous study, Calla_1781 was also expressed in E. coli BL21 (DE3) whereas no activity was detected (data not shown). Consequently, Calla_1331 (namely Xyn10A), the first characterized xylanase without CBMs, might played a prominent role in efficient degradation of xylan.C.lactoaceticus were obtained and characterized. Xyn10A and Agu67A showed optimum temperature of 80°C and 75°C, respectively. Xyn10A also had good thermostability (75°C and 80°C for 6 h) and broad pH stability (4.5–8.5). Xyn10A could hydrolyze branched xylan and produce xylose, xylobiose, and MeGlcA decorated XOs. Agu67A was active on MeGlcA decorated XOs, and produced MeGlcA and equivalents XOs. The synergistic activity of Xyn10A and Agu67A was detected with both MeGlcA branched xylan and XOs as substrates, and produced xylose, xylobiose, and MeGlcA. The synergistic function of Xyn10A and Agu67A provided a promising way for degrading natural xylan at high temperature. The characterization of the two intracellular GHs also offered an opportunity to systematically evaluate the mechanisms for xylan utilization of C. lactoaceticus.In this study, two novel thermostable xylanolytic enzymes endo-β-1,4-xylanase Xyn10A and α-glucuronidase Agu67A from C. lactoaceticus DSM 9545 was purchased from DSMZ . E. coli Top10  and plasmid pET-28b  were used for gene cloning, and E. coli BL21 (DE3) was used for protein expression. Beechwood xylan was purchased from Sigma-Aldrich , and XOs  was a kind gift from Longlive Bio-Technology Co. . D-xylose, locust bean gum (LBG), soluble starch, Avicel, carboxymethyl cellulose (CMC), and chemicals for buffer preparations were obtained from Kepujia Reagent Co. . All other chemicals were of analytical grade unless otherwise stated.C. lactoaceticus 6A was extracted from 5 mL culture using TIANamp Bacteria DNA Kit . Based on the whole genome of strain 6A xyn10A encoding a hypothetical endo-β-1,4-xylanase [GenBank: YP_004798927.1] and gene agu67A encoding an α-glucuronidase [GenBank: YP_004798856.1] were predicted and primers were designed as follows: xyn10A-F , xyn10A-R , agu67A-F (5′-GCCGCGCGGCAGCATGATTTTATCAAGTAGCAGTAAC-3′), and agu67A-R (5′-GCGGCCGCAAGCGTTTATGGATATATCACTCTTC-3′). The PCR mixture contained genomic DNA 1 µL, forward primer 1 µL, reverse primer 1 µL, 2×Pfu PCR MasterMix (TianGen) 12.5 µL, and ddH2O 9.5 µL. PCR conditions were as follows: 94°C 5 min, 30 cycles of 94°C 30 s, 55°C 30 s and 72°C 2 min, followed by one cycle of 72°C 5 min. The target PCR products were purified with TIAN gel Midi Purification Kit (TianGen).The genomic DNA of xyn10A amplification were then digested with Nhe I and Hind III  and inserted into pET-28b at the corresponding sites, obtaining the plasmid pET-28b-xyn10A. The purified PCR products of agu67A amplification were then digested with T4 DNA polymerase (Takara) and sub-cloned into pET-28b EK/LIC vector, yielding the plasmid pET-28b-agu67A. Both two plasmid were transferred into E.coli Top10 competent cells by heat shock and grown overnight at 37°C on Lysogeny Broth (LB) agar broth containing kanamycin (50 µg/mL). Positive recombinants were screened by using colony PCR and sequenced with T7 primers from both strands.The purified PCR products of E. coli BL21 (DE3) by heat shock and grown overnight at 37°C on LB agar plates supplemented with kanamycin (50 µg/mL). Seed culture was prepared by growing one single colony separately at 37°C on a rotary shaker (220 rpm) for overnight in 5 mL LB liquid medium containing kanamycin (50 µg/mL). The pre-cultures were then diluted individually 100-fold in fresh LB with antibiotic and cultured at 37°C on a rotary shaker (220 rpm). Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM when the culture reached an optical density of 0.4-0.6 at 600 nm, and incubations were further continued for an additional 16 h at 16°C. The cells were harvested by centrifugation at 4000 rpm for 15 min at 4°C, and re-suspended in binding buffer . The cell suspensions were lysed by sonication and centrifugation at 10,000 g for 15 min at 4°C. The supernatants were heated at 65°C for 30 min and centrifuged at 10,000 g for 15 min at 4°C to remove E. coli heat labile proteins. The supernatants were loaded individually onto a His-Tag Ni-affinity resin  pre-equilibrated with binding buffer for five times. Thereafter, the columns were washed with binding buffer for three times to remove the unbound proteins. Finally, the binded target proteins were eluted from the column with elution buffer . The purity of the target proteins were verified by sodium dodecyl sulfate-polycrymide gel electrophoresis (SDS-PAGE) as described by Laemmli The recombinant plasmids were then extracted using TIANprep Mini Plasmid Kit (TianGen) and transformed individually into w/v) in 100 µL citrate buffer (pH 6.5) at 80°C for 2 min. The amount of reducing sugar generated by Xyn10A was determined by using the para-hydroxybenzoic acid hydrazide (PHBAH) method with xylose as a standard To get initial reaction velocity for activity assay, preliminary experiments were conducted at different conditions. Appropriate amount of Xyn10A or Agu67A was incubated with excess substrates, and the hydrolysis was terminated at different time to get a linear curve of production versus reaction time. Xylanase activity was assayed by incubating 0.78 µg purified recombinant enzyme with beechwood xylan . Both two enzymes were checked for the ability to hydrolyze various substrates using the Congo red assay w/v) substrate and 0.8% (w/v) agar with citrate buffer (pH 6.5). Each of 10 µL enzymes was spotted onto the plates and incubated at 60°C for 12 h. After incubation, all the plates were stained with 0.1% (w/v) Congo red for 15 min and washed with 1 M NaCl for 3 times. Meanwhile, substrate specificity of the enzymes was also examined by measuring the produce of reducing sugar from those substrates at 80°C and pH 6.5 for 40 min. All the experiments were performed in triplicate.The substrate specificity of Xyn10A and Agu67A were screened respectively with different polysaccharide substrates including beechwood xylan, XOs, LBG, soluble starch, Avicel, and CMC. All of the tested substrates were at a fixed concentration of 1.0%  at 80°C. The optimal temperature and pH of Agu67A was investigated with MeGlcA decorated XOs as substrates as described above. In optimal temperature assay, the assay was conducted at pH 6.5 with temperature of 40-95°C, while the pH profile (4.0–8.5) was conducted at 75°C. To detect the thermostability of Xyn10A, purified Xyn10A was incubated in phosphate buffer (pH 8.5) at 75, 80 and 85°C, respectively, and residual activity under optimal conditions was compared at the moment of 0.5, 1, 2, 3, 4 and 6 h. The pH stability of Xyn10A was measured by assaying the relative activity under optimal conditions after the enzyme was pre-incubated in different pH buffers ranging from 4.0–8.5 at room temperature for 10 h.4+, Na+, K+, Mg2+, Fe2+, Ca2+, Fe3+, Zn2+, Co2+, Mn2+, Cu2+, Ni2+), or 1 mM and 5 mM of chemicals  or 0.1% (v/v) and 0.5% (v/v) of different solvents  or 5% (v/v) and 10% (v/v) organic reagents  respectively in the reaction mixture and incubating at 80°C for 5 min. The residual enzyme activity was determined at optimal conditions and the xylanase activity without addition of metal ions or chemical reagents was defined as 100%. All the experiments were performed in triplicate.In addition, the effects of various additives on the recombinant xylanase activity were assayed by adding 1 mM and 5 mM of various metal ions (NHw/v) of beechwood xylan under optimal conditions for 4 hours. Besides, Xyn10A , or Agu67A , or a Xyn10A and Agu67A mixture  was incubated with XOs  for 4 hours under optimal conditions. To further evaluate the synergistic activity of Xyn10A and Agu67A, Xyn10A , or Agu67A , or a Xyn10A  and Agu67A  mixture was incubated with beechwood xylan  for 0.25, 1, 2, 6 and 12 hours in the same way. Each of the control reaction was performed under the same experimental condition except adding the heat denatured enzyme. After reaction, hydrolysis products were centrifuged at 12,000 g for 10 min. Supernatants were spotted on Silica Gel GF254 TLC plates  and then developed in 1-butanol/acetic acid/H2O  system for 2 hours after drying w/v) and sulfuric acid  followed by heating at 75°C for 10 min 1), xylobiose (X2), xylotriose (X3), and xylotetraose (X4) were used as standards. At the same time, the hydrolysis products of XOs and beechwood xylan were determined by reducing sugar assay using PHBAH method as mentioned above. And the products from the hydrolysis of XOs and 2 h's hydrolysis of beechwood xylan were also detected by HPLC. All the experiments were performed in triplicate.The capacity of Xyn10A and Agu67A to hydrolyze beechwood xylan and XOs was assessed by detecting the hydrolysis products using thin-layer chromatography (TLC), reducing sugar assay, and HPLC analysis. Xyn10A  was incubated with different concentrations (0.1–2.0%,


Myocardial black blood (BB) T2* relaxometry at 1.5T provides robust, reproducible and calibrated non-invasive assessment of cardiac iron burden. In vitro data has shown that like T2*, novel native Modified Look-Locker Inversion recovery (MOLLI) T1 shortens with increasing tissue iron. The relative merits of T1 and T2* are largely unexplored. We compared the established 1.5T BB T2* technique against native T1 values at 1.5T and 3T in iron overload patients and in normal volunteers.A total of 73 subjects  were recruited, comprising 20 healthy volunteers (controls) and 53 patients . Single mid-ventricular short axis slices were acquired for BB T2* at 1.5T and MOLLI T1 quantification at 1.5T and 3T.0.282 at 1.5T (R2 = 0.717), and y =406 · x0.294 at 3T (R2 = 0.715). Measures of reproducibility of T1 appeared superior to T2*.In healthy volunteers, median T1 was 1014 ms (full range 939–1059 ms) at 1.5T and modestly increased to 1165ms (full range 1056–1224 ms) at 3T. All patients with significant cardiac iron overload  had T1 values <939 ms at 1.5T, and <1056 ms at 3T. Associations between T2* and T1 were found to be moderate with y =377 · xT1 mapping at 1.5T and at 3T can identify individuals with significant iron loading as defined by the current gold standard T2* at 1.5T. However, there is significant scatter between results which may reflect measurement error, but it is also possible that T1 interacts with T2*, or is differentially sensitive to aspects of iron chemistry or other biology. Hurdles to clinical implementation of T1 include the lack of calibration against human myocardial iron concentration, no demonstrated relation to cardiac outcomes, and variation in absolute T1 values between scanners, which makes inter-centre comparisons difficult. The relative merits of T1 at 3T versus T2* at 3T require further consideration. Since a biological pathway for body iron surplus elimination is non-existent in humans, excess iron from gut absorption or transfusion is stored in a stable hemosiderin form but once this storage capacity is exhausted free iron can be left unbound. This free ferrous iron can consequently catalyse cellular Fenton reactions and create highly reactive hydroxyl radicals causing mitochondrial dysfunction,  apoptosiCardiovascular magnetic resonance (CMR) T2* assessment of myocardial iron burden at 1.5 Tesla (T)  plays a Against this background of substantial gains in clinical management of transfusion dependent patients over the last 15 years are new advances in hardware and software technology. Increasingly 3T scanners are entering clinical practice and these may offer advantages over conventional 1.5T magnets for iron burden quantification. Unfortunately, 3T CMR may suffer from increased artefacts making myocardial T2* assessment challenging, particularly in patients with a high degree of iron overload , 15. AnoA total of 53 consecutive subjects were prospectively recruited over a 6 month period from September 2012 to February 2013 from referrals for cardiac siderosis screening or follow-up. A wide dynamic range of iron loading was present in the patient group and was a realistic reflection of our population. Patients had successive CMR scans at both 1.5T and at 3T. A group of 20 matching healthy volunteers was also scanned. Exclusion criteria were: 1) claustrophobia, 2) metallic implants or permanent pacemaker and 3) inability to hold recumbent position for >15 minutes. The study protocol was approved by the local ethics committee, and all subjects gave written informed consent.The BB T2* and MOLLI T1 CMR protocol was performed on a 1.5T Sonata scanner, and then a 3T Skyra scanner .At 1.5T, a four-element cardiac phase-array coil was used. After routine localizer acquisitions, an ECG gated single breath hold multi-echo sequence was acquired at a single mid-ventricular short axis slice with a 10 mm thickness at eight separate echo times . The BB sequence used a flip angle of 35°, a matrix of 128 X 256 pixels a field of view (FOV) of 40 cm, repetition time of 20 ms between each radiofrequency (RF) pulse and a sampling bandwidth of 810 Hz per pixel. Furthermore, for the black blood preparation, double inversion pulses were applied at the R wave trigger and the inversion time (TI) was set to extend into diastole.The sequence used for myocardial T1 evaluation has been reported elsewhere . In brieThe myocardial T2* quantification with truncation has been described elsewhere . In brieT2* was subsequently transformed into its reciprocal R2* according to the equation:Curve fitting was performed with the truncation method . The conAn identical mid-ventricular septum region of interest (ROI) was used for T1 quantification. Delineation of the epicardial and endocardial borders was done on the T1 maps. The interventricular septum was subsequently isolated for quantification. Careful consideration was given not to include blood-pool or artefacts in the septal ROI. T1 maps were automatically derived as described previously, –27 by fiWhere SI is the signal intensity at a specific inversion time (TI), and T1 can be approximated:When required, T1 was subsequently transformed into its reciprocal R1 using a similar equation:No gadolinium contrast agent was administered. A single, blinded, experienced observer performed analysis for BB T2* at 1.5T, and MOLLI T1 at 1.5T and 3T.A group of 20 patients was randomly selected for inter-observer, intra-observer and inter-study variability measurements. These patients had levels of myocardial siderosis that covered the entire range of the disease (from absent to severe iron overload). For intra-observer variability, the same investigator blindly reassessed myocardial T1 and T2* on 2 occasions a month apart. For inter-observer variability, two highly experienced investigators separately and blindly reported T1 and T2* for each patient. Finally, these 20 patients underwent a repeated cardiac T1 scan at 1.5T and at 3T one hour after completing the previous scan. The same investigator randomly and blindly re-evaluated T1 in these subjects to obtain inter-study variability.U test. Association between T2*, T1, R2* and R1 values at different field strengths were evaluated with a linear or non-linear regression when appropriate. Coefficients of variation (CoV) and intra-class correlation (ICC) with 95 % confidence interval (CI) analysis were conducted to assess inter- and intra-observer variability in addition to inter-study variability of T1 values. For ICC, an alpha two-way mixed model with absolute agreement analysis was used. Finally, Bland-Altman plots were generated to further describe the reproducibility of T1 measurements at 1.5T and at 3T  and the full range of values was 939–1059 ms. In accord with previous intellectual considerations defining the lower limit of normal of T2* to represent the onset of significant iron loading, so that low values should rarely be seen in the normal population, we used the lowest T1 observed in normals of 939 ms at 1.5T as the lower limit of normality for the scanner and sequence that was used in this report. The median T1 at 3T was longer at 1165 ms [1119–1194] and the full range of values was 1056–1224 ms. Using the same argument as above, we therefore set the lower limit of normal for significant iron loading as being the lower limit of the normal range at 3T which was 1056 ms.0.282 (R2 = 0.717). All patients with cardiac iron overload (T2* <20 ms at 1.5T) had T1 values at 1.5T below the lower limit of normal T1 values in the control cohort (939 ms). Only four patients had T2* values >20 ms at 1.5T and had T1 values at 1.5T below the lower limit of T1 values found in the present normal cohort. As shown in Fig. 2 = 0.772).The relation between T1 and T2* at 1.5T is shown in Fig. 0.294 (R2 = 0.715). All patients with a significant degree of iron overload (T2* <20 ms at 1.5T) had a T1 values at 3T <1056 ms . A total of seven patients with normal T2* values at 1.5T had T1 values below 1056 ms at 3T. The association between R1 at 3T and R2* at 1.5T is shown in Fig. 0.293 (R2 = 0.715).The relation between T1 at 3T and T2* at 1.5T is shown in Fig. The intra-observer, inter-observer and inter-study variability of T1 at 1.5T and at 3T is shown in Table These data show reasonable correlations between cardiac T1 and cardiac T2* measures across a wide range of cardiac iron loading, but with significant scatter, and there is reasonable agreement with our previous data comparing T1 and T2* at 1.5T in thalassaemia patients,  and withThe advantages of T1 CMR include: A) At low iron levels, T1 is less affected by non-iron influences that affect T2* measurements, making T1 appear to be more suitable for clinical conditions where low iron levels may be responsible for disease pathogenesis; B) T1 has good inter-study reproducibility, which appears superior to T2* which may be of benefit for longitudinal patient follow-up and clinical trials. Counterbalancing views to these advantages are: A) The dominant pathologies associated with iron dysfunction by prevalence are those involving substantial iron overload  and for these conditions there is rather little obvious merit in defining iron levels moving within the non-significant range, when movements of T2* are robustly identifiable in the low-risk mild to moderate iron overload range of cardiac T2* between 10–20 ms. Nonetheless, some more unusual pathologies are apparently associated with iron dysfunction at low iron levels, , 30 for It has also been suggested that cardiac T1 might have improved utility for identifying patients with cardiac iron loading through a better delineation of the lower limit of normal for cardiac iron loading . It is dAnother potential advantage of T1 measurement of iron concentration which is identified in this paper, is the use of T1 at 3T. There has been substantial increase in the use of 3T for MR of the brain and musculoskeletal system, and a number of centres no longer have 1.5T scanners available causing problems for thalassaemia patients. The two most likely solutions to this problem are to calibrate T2* at 3T against 1.5T, on which progress has already been made, , 15, 35 A problem associated with the current implementation of T1 mapping is the variation of absolute values of T1 between sequences and scanners. This is illustrated in this paper where the median value of T1 at 1.5T in normal subjects was 1014 ms which compares with the mean of 968 ms from Sado et al.  Sado useSome further technical issues need to be considered in the use of T1 for iron assessment. Diffuse fibrosis raises native T1, whereas myocardial iron lowers native T1. There is debate about the prevalence of myocardial fibrosis in thalassaemia, , 42 but Further issues with the validation of T1 also exist, that are troublesome. Clearly this paper and others have shown that T1 can be used to measure tissue iron concentration, but in humans the only comprehensive validation in the heart has been performed using T2*. Myocardial T2* measured at 1.5T is firmly established as the gold standard measurement of myocardial iron concentration,  and thisNative T1 mapping at 1.5T appears to be promising as a tool to evaluate myocardial iron. However, considerable further work is required in standardisation and transferability between scanners and centres, as well as in understanding whether T1 can be used as a substitute for T2* in the absence of direct studies of calibration and linkage with cardiovascular outcomes. The established practice worldwide of using T2* at 1.5T is unlikely to change in the near future without persuasive additional arguments. However, T1 measurements at 3T may prove useful where T2* measurements can be problematic.