CAG repeat tracts on the activities of key BER enzymes, as well as on repeat instability. We found that DOB crosslinked with DNA polymerase β and inhibited its synthesis activity in CAG repeat tracts. Surprisingly, we found that DOB also formed crosslinks with DNA ligase I and inhibited its ligation activity, thereby reducing the efficiency of BER. This subsequently resulted in the accumulation of DNA strand breaks in a CAG repeat tract. Our study provides important new insights into the adverse effects of an oxidized abasic lesion on BER and suggests a potential alternate repair pathway through which an oxidized abasic lesion may modulate TNR instability.Oxidative DNA damage and base excision repair (BER) play important roles in modulating trinucleotide repeat (TNR) instability that is associated with human neurodegenerative diseases and cancer. We have reported that BER of base lesions can lead to TNR instability. However, it is unknown if modifications of the sugar in an abasic lesion modulate TNR instability. In this study, we characterized the effects of the oxidized sugar, 5’-(DOB) in  CAG/CTG), Friedreich’s ataxia (GAA/TTC), fragile X syndrome (CGG/CCG), and myotonic dystrophy (CTG/CAG)  ATP (6000 mCi/mmol) and Cordycepin 5’-triphosphate 3’-[α-32P] (5000 mCi/mmol) were purchased from Perkin Elmer Inc. . Deoxynucleotide 5’-triphosphates (dNTPs) were from Fermentas . Micro Bio-Spin 6 chromatography columns were purchased from Bio-Rad Laboratories . All other chemical reagents were purchased from Thermo Fisher Scientific  and Sigma-Aldrich . Uracil-DNA glycosylase (UDG) was from New England Biolabs . Recombinant human pol β was expressed in Escherichia coli and purified as described previously [Oligonucleotides containing the DOB lesion were synthesized as previously described . All otheviously . Pol β KCAG)3 flap or a 3’-primer with no repeats and a downstream primer containing a 5’-(CAG)2 flap with the template strand containing (CTG)5 or (CTG)2, respectively, at a molar ratio of 1:3:3. Schematic representations of how these substrates are generated during BER are shown in o-nitrobenzyl protecting groups are indicated in 32P at the 5’-end of the upstream strand or 3’-end of the downstream strand as indicated.Oligonucleotide substrates were prepared as described previously . BrieflyE. Coli BL21(DE3). Two one-liter flasks containing lysogeny broth (LB) medium were inoculated with one colony each of the transformed BL21(DE3) cells and were then incubated overnight without shaking. The cultures were then incubated at 37°C at 225 rpm until OD600 reached 0.6, and protein expression was induced with 1 mM IPTG for 3.5 hours. Bacterial cells were harvested by centrifugation at 2600 rpm for 45 minutes at 4°C. Cells were lysed with a French press cell disruptor  at 150,000 PSI in lysis buffer containing 30 mM 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid (HEPES), pH 7.5, 30 mM KCl, 1 mM dithiothreitol (DTT), 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.5% inositol. The cell lysates were centrifuged at 12,000 rpm for 30 minutes at 4 °C. The supernatant was loaded onto a 10-ml Sepharose Q column operated by an AKTA Fast Protein Liquid Chromatography system (FPLC) . The flow-through was collected and dialyzed into buffer containing 30 mM HEPES, pH 7.5, 30 mM KCl, 0.5% inositol, and 1 mM PMSF, and subsequently loaded onto a 5-ml CM sepharose column , with fractions eluted using a linear gradient of KCl from 30 mM to 2 M. Peak fractions were combined and dialyzed into buffer containing 30 mM HEPES, pH 7.5, 0.5% inositol, 1.7 M (NH4)2SO4, and 1 mM PMSF. Samples were then loaded onto a 2 ml phenyl sepharose column. Fractions were eluted using a linear gradient of (NH4)2SO4 from 1.7 M to 0 M. The peak fractions were combined and dialyzed in buffer containing 30 mM HEPES, pH 7.5, 30 mM KCl, 0.5% inositol, and 1 mM PMSF. Samples were then loaded onto a 1 ml Mono-S column , and eluted using a linear gradient of KCl (30 mM to 2 M). Purified FEN1 was aliquoted and frozen at -80 °C until further use.Recombinant human FEN1 was purified as described previously . BrieflyE. Coli BL21(AI). Six one-liter flasks containing LB medium were inoculated with one colony each of the transformed BL21(AI) cells and incubated overnight without shaking. The cells were then incubated at 37°C at 225 rpm until OD595 reached 0.6, and LIG I expression was induced by 1 mM IPTG for 24 hours at 15–18 °C. Cells were then harvested by centrifugation at 2500 rpm for 30 minutes at 4 °C. The supernatant was discarded, and cell pellets were lysed in lysis buffer containing 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1% Nonidet P-40, 1 mM PMSF, and 1 cOmplete tablet of protease inhibitors  and subjected to French Press at 150,000 PSI. The cell lysates were centrifuged at 12,000 rpm for 30 minutes at 4 °C. The supernatant was then loaded onto a 20 ml P11 phosphocellulose column. Fractions were eluted using a linear gradient of NaCl (50 mM to 600 mM). The peak fractions were combined and dialyzed in buffer containing 30 mM HEPES, pH 7.0, 30 mM KCl, 0.1% inositol, and 1 mM PMSF. Samples were loaded onto a 10 ml Q sepharose column and eluted using a linear gradient of KCl (30 mM to 2 M). Peak fractions were combined and dialyzed in buffer containing 50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 7 mM 2-mercaptoethanol, 10 mM imidazole, 0.5% inositol, and 1 mM PMSF. Samples were then loaded onto a 4-ml nickel-nitrilotriacetic acid (Ni-NTA) column, with fractions eluted using a linear gradient of imidazole (10 mM to 600 mM). Purified LIG I was aliquoted and frozen at -80 °C until further use.Recombinant human LIG I was expressed in In vitro BER of the DOB lesion, AP, and the abasic site analog, THF . All reactions were done in triplicate.log, THF  at the 52 and 50 μM dNTPs in a total volume of 20 μl. For the pol β DNA synthesis assay, the 20 μl BER reaction mixtures were incubated at 37 °C. Aliquots were removed at time points ranging from 2–30 minutes, and the reactions were quenched by the addition of 20 μl of 2 x stopping buffer containing 95% formamide and 10 mM EDTA. For the FEN1 5’-flap cleavage activity assay, samples were preincubated with 2.5 nM pol β for 1–5 minutes to allow for crosslink formation prior to incubation with FEN1 for 15 minutes at 37 °C. Reactions were quenched with 200 nM NaBH4 on ice for 30 minutes. Reaction mixtures were then denatured at 95 °C for 10 minutes and separated by 15% urea-denaturing polyacrylamide gel electrophoresis. Substrates and products were detected and analyzed using a Pharos FX Plus PhosphorImager.The activities of pol β DNA synthesis and FEN1 flap cleavage on all substrates were measured by incubating 50 nM substrates with the indicated concentrations of pol β and FEN1 in reaction buffer containing 30 mM HEPES, pH 7.8, 50 mM KCl, 0.5% (w/v) inositol, and 0.1 mg/ml BSA, with 5 mM MgCl2 and 4 mM ATP. A single strand containing either a 5’-DOB or 5’-THF was preincubated with LIG I for 30 minutes prior to addition of the nicked ligatable substrate. Reaction mixtures (20 μl) were incubated for 15 minutes at 37 °C. Reactions were quenched by the addition of 20 μl of 2 x buffer containing 95% formamide and 10 mM EDTA. Reaction mixtures were then denatured at 95 °C for 10 minutes and separated by 15% urea-denaturing polyacrylamide gel electrophoresis. Substrates and products were detected and analyzed using a Pharos FX Plus PhosphorImager.LIG I activity was tested on a substrate of random DNA sequence containing a central nick with a phosphate at the 5’-end of the downstream strand in reaction buffer containing 30 mM HEPES, pH 7.8, 50 mM KCl, 0.5% inositol, and 0.1 mg/ml BSA, with 5 mM MgClCAG)2 flap substrates and (CAG)3/(CAG)2 double-flap substrates in reaction buffer containing 30 mM HEPES, pH 7.8, 50 mM KCl, 0.5% inositol, 0.1 mg/ml BSA, 10 mM MgCl2 and 1 mM 2-mercaptoethanol in the absence and presence of 100 mM NaBH4. The LIG I-DOB crosslink was captured by incubating 1 μM of LIG I with 1 μM 5’-DOB containing single strand CAG repeat DNA in reaction buffer containing 30 mM HEPES, pH 7.8, 50 mM KCl, 0.5% inositol, 0.1 mg/ml BSA, 10 mM MgCl2 and 1 mM 2-mercaptoethanol in the absence and presence of 20, 50, or 100 mM NaBH4. The native sugar was generated by pre-incubating the uracil-containing substrate with UDG for 30 minutes. The photolyzed DOB-containing substrates were subjected to photolysis at 365 nm UV light for 20 minutes to remove the protecting groups for creating a DOB lesion immediately prior to the crosslink reaction. Reaction mixtures (20 μl) were incubated for 1 hr at 37 °C, and the reactions were terminated by adding an equal volume of SDS loading buffer containing 100 mM Tris-HCl, pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol, 0.2% (w/v) bromophenol blue, and 200 mM dithiothreitol. The samples were then heated at 95 °C for 3 min, and crosslink products were separated by 10% SDS-PAGE. Substrates and products were detected and analyzed using a Pharos FX Plus PhosphorImager.The pol β-DNA crosslink was captured by incubating 1 μM of pol β wild-type or K72A mutant protein with 2.5 nM DOB- or AP-containing nicked 5’-. Significant differences in the data were examined by standard two-way analysis of variance with Tukey’s multiple comparison posttests. The significant difference was designated at CAG repeat substrates containing DOB and compared this to substrates containing a native sugar (AP) or THF at the 5’-end of the downstream flap (2-flap (CAG repeat duplex. A (CAG)3/(CAG)2 double-flap substrate (CAG)5 hairpin. Pol β DNA synthesis activity on these substrates was tested at a time interval ranging from 2–30 minutes.The DOB lesion residing in the 1-nt gap of duplex DNA forms a covalent crosslink with the deoxyribosephosphate (dRP) lyase domain of pol β, resulting in irreversible enzyme inactivation . Howeveream flap . These seam flap . Fig 1 i (2-flap  was usedubstrate  was usedP<0.05). For the double-flap substrate, pol β mainly inserted 1 nucleotide on the DOB substrate  were present simultaneously  of pol β as it does on a 1 nt-gapped substrate [CAG repeat flap crosslinked with Lys72 and/or other lysines in pol β, thereby inhibiting its DNA synthesis and dRP lyase activities during BER in CAG repeat tracts. In contrast, pol β was only transiently crosslinked at K72 by the native AP site. It should be noted that with a relatively high concentration of pol β, we successfully detected the DOB-pol β crosslink product in a SDS-PAGE gel. This suggests that a high level of pol β proteins was required to generate a sufficient amount of pol β-DOB crosslink products to be detected by SDS-PAGE. It should be also noted that in the results, the multiple crosslink species in the gels were observed. This may be due to the formation of a series of secondary structures by the CAG repeats that migrated differently in a SDS-PAGE gel.For the nicked-flap substrate with a DOB, 2.5 nM pol β mainly inserted up to 2 nucleotides 2-flap (CAG)3/(CAG)2 double-flap substrate (CAG)5 hairpin. For the nicked-flap substrate, in the absence of pol β, FEN1 cleavage on the substrates with different types of sugar lesions resulted in a similar amount of the 26 nt product (23.1%-38.4%) (CAG repeats during BER.We then sought to determine whether DOB inhibition of pol β mediated DNA synthesis affects FEN1 5’-flap cleavage. We therefore tested FEN1 cleavage activity on the substrate containing a nick with a downstream (G)2-flap  formed dubstrate , which mCAG)2-flap (CAG)3/(CAG)2 double-flap substrate (CAG)2-flap (P<0.05). The results indicate that BER efficiency is decreased by the oxidized abasic site, leading to the accumulation of unligated DNA strand break intermediates. For the (CAG)3/(CAG)2 double-flap substrate (P<0.05). This suggests that DOB also inhibited repair product formation with the double-flap substrate, possibly by preventing the formation of a ligatable nick suitable for ligation by LIG I. Our results also showed that DOB significantly decreased the amount of the repaired products in the absence of pol β compared to that from a native sugar or THF lesion 2-flap (CAG)3/(CAG)2 double-flap substrates was observed 2-flap , as wellubstrate , containG)2-flap , the oxiubstrate , the amolap P<0.0. The reslap P<0.0. The res se Figs . Taken tThe inhibition of the formation of the BER product by DOB suggested that LIG I was also inhibited by the lesion. Since LIG I preferentially binds to a nick, it cannot directly interact with the oxidized sugar at the 5'-end of a flap. However, the enzyme may interact and react with a single strand containing a 5’-DOB after the flap is cleaved by FEN1. To test this possibility, we examined LIG I activity on a nicked substrate in the presence of increasing concentrations (0–1000 nM) of the downstream strand of the damaged strand containing either a 5’-DOB , panel ACAG repeat tracts by crosslinking with the polymerase , which contains the same functional 1,4-dicarbonyl group as DOB crosslinks with the dRP lyase domain of pol β . HoweverCAG repeat tracts, inhibits pol β mediated DNA synthesis and LIG I activity through crosslinking with the enzymes during BER. This subsequently leads to inefficient BER, resulting in the accumulation of DNA strand breaks in TNR tracts. We suggest that this can further lead to error-prone DNA repair, such as DNA recombination, causing TNR instability.In conclusion, in this study, we provide the first evidence that an oxidized abasic site, DOB, when present in S1 File(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 FigDOB-containing substrates were prepared by exposure of o-nitrobenzyl protected lesions to 365 nm UV for 20 minutes.(PDF)Click here for additional data file.S2 Fig32P-labeled at the 5’-end of the upstream strand and are illustrated above each gel. The experiments were repeated at least three times, and only the representative gels were shown in the figures. Two-way ANOVA with Tukey’s multiple comparison posttests was used to determine statistically significant differences. "*" denotes P < 0.05, compared to the DOB-containing substrates.Pol β DNA synthesis activity on a nicked flap (left panel) and double-flap (right panel) with a 5’-phosphate was examined as described in the “Methods”. Lanes 1 and 7 indicate the substrate only. Lanes 2–6 and lanes 8–12 indicate the substrate incubated with pol β (2.5 nM) at the time interval of 2, 5, 10, 15, and 30 minutes. Substrates were (PDF)Click here for additional data file.S3 Fig4. Lane 4 illustrates the reaction with the substrate, 5 U UDG, 10 nM APE1 and 2.5 nM pol β without NaBH4. Lane 5 indicates the reaction with the substrate, 5U UDG, 10 nM APE1, 2.5 nM pol β and 340 mM NaBH4. Lane 6 indicates the reaction with the substrate, 5 U UDG, 10 nM APE1, and 2.5 nM pol β that was pre-incubated with the unphotolyzed nick-flap substrate in the presence of 340 mM NaBH4. Lane 7 illustrates the reaction with the substrate, 5 U UDG, 10 nM APE1 and 2.5 nM pol β that was pre-incubated with the photolyzed nick-flap substrate (pol β precrosslinked with DOB) in the presence of 340 mM NaBH4. Lane 8 indicates the reaction with the substrate, 5 U UDG, 10 nM APE1 and 2.5 nM pol β that was pre-incubated with the unphotolyzed double-flap substrate in the presence of 340 mM NaBH4. Lane 9 indicates the reaction with the substrate, 5 U UDG, 10 nM APE1 and 2.5 nM pol β that was pre-incubated with the photolyzed double-flap substrate (pol β precrosslinked with DOB) in the presence of 340 mM NaBH4. Substrates were 32P-labeled at the 3’-end of the damaged strand and are illustrated above each gel. The experiments were repeated at least in triplicate, and only the representative gel was shown in the figures. The quantification results were shown below the gel.Pol β dRP lyase activity was measured as described in (PDF)Click here for additional data file.


Human infection with avian influenza A virus (H7N9) was first reported in China in March 2013. Since then, hundreds of cases have been confirmed showing severe symptoms with a high mortality rate. The virus was transmitted from avian species to humans and has spread to many neighboring areas, raising serious concerns over its pandemic potential. Towards containing the disease, the goal of this study is to prepare a virus-like particle (VLP) that consists of hemagglutinin (HA), neuraminidase (NA) and matrix protein 1 (M1) derived from the human isolate A/Taiwan/S02076/2013(H7N9) for potential vaccine development.Full length HA, NA, and M1 protein genes were cloned and expressed using a baculoviral expression system, and the VLPs were generated by co-infecting insect cells with three respective recombinant baculoviruses. Nanoparticle tracking analysis and transmission electron microscopy were applied to verify the VLPs’ structure and antigenicity, and the multiplicity of infection of the recombinant baculoviruses was adjusted to achieve the highest hemagglutination activity. In animal experiments, BALB/c mice and specific-pathogen-free chickens receiving the VLP immunization showed elevated hemagglutination inhibition serum titer and antibodies against NA and M1 proteins. In addition, examination of cellular immunity showed the VLP-immunized mice and chickens exhibited an increased splenic antigen-specific cytokines production.The H7N9 VLPs possess desirable immunogenicity in vivo and may serve as a candidate for vaccine development against avian influenza A (H7N9) infection.The online version of this article (doi:10.1186/s12896-016-0321-6) contains supplementary material, which is available to authorized users. Human infection with avian influenza A (H7N9) was first reported in China in March 2013, and since then hundreds of human cases have been confirmed. The disease is associated severe respiratory illness, and the high mortality rate has garnered increasing attention globally [Similar to other influenza A viruses, H7N9 viral capsids are comprised of three primary proteins, including hemagglutinin (HA), neuraminidase (NA), and matrix protein 1 (M1). Upon co-expression, these three proteins self-assemble into nanoparticles, which are responsible for packaging viral genomes for disease transmission . PreviouS. frugiperda Sf9 (ATCC CRL-1711) and Sf21  insect cells were maintained as suspension cultures in Grace’s medium  and supplemented with 10% FBS  and 1% P/S/A antibiotics .E. coli strain DH10Bac, and recombinant bacmid was purified using the HiPure Plasmid Midiprep kit (Invitrogen). Sf9 cells were used for transfection with the recombinant bacmid. Right before transfection, 8 μl of the Cellfectin II Reagent (Invitrogen) was diluted in 100 μl of Grace’s medium (without antibiotics and serum), and 1 μg of bacmid DNA was diluted in 100 μl of Grace’s medium (without antibiotics and serum). Subsequently, the diluted bacmid DNA and diluted Cellfectin II were combined and incubated for 30 min at room temperature. The DNA-lipid mixture was then added onto the cells dropwise and incubated at 27 °C for 4 h. Following removal of transfection mixture, fresh cell medium was added to the cells. After 72 h, recombinant baculoviruses were harvested from the supernatant and designated as rBac-H7, rBac-N9, and rBac-M1, respectively. The recombinant baculoviruses were subsequently amplified in Sf9 cells, and virus titers were determined by plaque assays in Sf21 cells.Recombinant baculoviruses were prepared by using the Bac-to-Bac baculovirus expression system (Invitrogen). Briefly, three separate recombinant plasmids were constructed by inserting full HA, NA, and M1 genes of A/Taiwan/S02076/2013(H7N9)  into the pFastBac-1 vector using primers listed in Table Sf9 cells were infected with rBac-H7, rBac-N9, and rBac-M1 at a multiplicity of infection (MOI) of 2. Three days later, the monolayer of cells was washed and fixed with 80% acetone at −20 °C for 20 min. Rabbit polyclonal antibody against H7/HA peptide , rabbit polyclonal antibody against N9/NA peptide , and mouse polyclonal antibody against a recombinant M1 protein were applied at 1:10,000, 1:2,000, and 1:1,000 dilutions respectively and incubated for 1 h. After PBS wash, cells were further incubated with FITC-conjugated anti-rabbit or anti-mouse IgG . Fluorescence signal was observed under a fluorescence microscope.H7, N9 and M1 recombinant proteins were harvested from lysed Sf9 cells that were infected with rBac-H7, rBac-N9, or rBac-M1 (MOI = 2). After 72 h, the cells were washed and lysed with the I-PER insect cell protein extraction reagent (Thermo Scientific). Recombinant proteins were purified using the Glycoprotein Isolation Kit, ConA (Thermo Scientific) according to the manufacturer’s instructions.H7N9 VLP was harvested and purified from the culture supernatant of Sf9 cells co-infected with rBac-H7, rBac-N9, or rBac-M1. Briefly, 72 h after co-infection, the cell culture supernatant was centrifuged at 3,000 × g for 20 min, and the VLPs were pelleted by centrifugation at 70,000 × g for 2 h at 4 °C. The pellet was resuspended in TEN buffer . The resultant solution was layered onto a sucrose gradient solution (20–50% in TEN buffer) and centrifuged at 100,000 × g for 2 h. Particles from each gradient fraction were separately collected for the HA test and TEM analysis (described below), and the fractions demonstrating the highest HA activity and appropriate morphology were pooled as purified VLPs.The protein concentration was quantified by the Bradford protein assay  according to the manufacturer’s recommendations. H7N9 recombinant proteins and VLPs were analyzed by 10% SDS-PAGE, and probed with anti-H7/HA, anti-N9/NA, and anti-M1 antibodies in the IFA section. To detect the protein signals, the membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch Laboratories) at a 1:2,000 dilution for another hour and developed using ECL Western blotting detection kit (Bio-rad).Purified VLPs were absorbed onto a plasma-discharged copper grid for 2 min and fixed with 4% paraformaldehyde for 5 min. After PBS washes and blocking with 1% BSA , the grid was incubated with either anti-H7/HA or N9/NA antibodies (1:200) for 1 h followed by incubation with 6 nm gold-conjugated goat anti-rabbit antibodies (1:20) (Jackson ImmunoResearch Laboratories). After PBS washes, 2% phosphotungstic acid was applied for negative staining. Particles were observed under a transmission electron microscope (TEM) (JEOL JEM-1400).Particle concentration, size distribution, and surface zeta potential of the expressed H7N9 VLPs were measured by nanoparticle tracking analysis using NanoSight NS-500  based on the manufacturer’s instructions. The purified influenza virus A/PuertoRico/8/34(H1N1)  was inclFour different conditions of co-infection were tested. rBac-HA , rBac-NA , and rBac-M1  were combined respectively and used for co-infection in Sf9 cells. After 72 h, the cell culture supernatant was centrifuged at 3,000 × g for 20 min, and the VLPs were pelleted by centrifugation at 70,000 × g for 2 h at 4 °C. The pellet was resuspended in TEN buffer, and the resultant solution was layered using a sucrose gradient solution (20–50% in TEN buffer) via centrifugation at 100,000 × g for 2 h. Particles from each gradient fraction were separately collected for a hemagglutination test (described below).n = 6 per group), receiving H7N9 VLPs, HA/NA/M1 free proteins (only for chickens), or PBS. Briefly, 10 μg of VLPs or pre-mixed HA/NA/M1 proteins at a 1:1:1 ratio were emulsified with the complete Freund’s adjuvant and used for the primary immunization using an intramuscular route. For the booster dose, the same amount of the antigen was mixed with the incomplete Freund’s adjuvant. Mouse serum was collected before immunization and 14 days post-immunization (dpi), and mice were sacrificed at 28 dpi. Chicken serum was collected before immunization and at 14, 19, and 26 dpi, and all the chickens were sacrificed at 40 dpi. Mice and chickens were sacrificed by CO2 inhalation.Female 6-week-old BALB/c mice were purchased from BioLASCO . Two-week-old specific-pathogen-free (SPF) chickens were obtained from JD-SPF Biotech . Animals were randomly divided into different experimental groups (H7N9 VLPs and inactivated H7N9 virions were used as HA antigens in this study. Inactivated H7N9 virions were prepared as previously described . HA and 6 splenocytes were plated in 96-well U-bottom plates (Corning) and were either mock-stimulated or stimulated with 10 μg of H7N9 VLPs in the presence of brefeldin A  for 5 h at 37 °C. Cells were then washed, incubated with 2.4G2 antibody, and labeled with anti-CD3e-FITC, anti-CD4-PerCP-Cy5.5, and anti-CD8a-PerCP-Cy5.5. The cells were then fixed and permeabilized using a Cytofix/Cytoperm Kit (BD Biosciences) and stained with anti-IL4-PE, anti-IFNγ-APC and anti-TNF-PE. All antibodies for mouse experiments were purchased from BD Biosciences. Samples were read on the BD FACSCalibur and analyzed using FlowJo software .Mice spleens were harvested at 28 dpi, and splenocytes were isolated for intracellular cytokine staining assays. Briefly, spleens were minced and passed through 70-μm cell strainers (Corning) to obtain single-cell suspensions. RBCs were lysed using an RBC lysis buffer (eBiosciences), and cells were resuspended in RPMI 1640 medium  containing 10% FBS. Viable cells were determined by trypan blue staining. 104 lymphocytes was determined. For the quantification of cytokine expression, the stimulated splenocytes were lysed, and total RNA was isolated by TriSolution reagent . Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed using iScript (Bio-rad) and iQ SYBR Green Supermix Kit (Bio-rad) with previously described primers for chicken IL-4, IFN-γ and GAPDH [-ΔΔCt method [Chicken spleens were harvested at 40 dpi, and splenocytes were isolated and stimulated with H7N9 VLPs in the presence of brefeldin A as described above. For the flow cytometric analysis, cells were washed and labeled with anti-chicken CD4 or CD8 antibodies . The amount of CD4+ or CD8+ T cells gated on 2 x 10nd GAPDH . Meltingt method .An amount of 100 ng purified NA or M1 protein was coated onto the flat-bottomed 96 well microplate  overnight at room temperature. After blocking with 5% skim milk , the test chicken sera were serially diluted and incubated for 1 h. Following washes, 100 μl of peroxidase-conjugated goat anti-chicken IgY (H + L) (Jackson ImmunoResearch Laboratories) diluted 1:2,000 in blocking buffer was dispensed into each well and incubated for another 1 h. After additional three washes, the wells were incubated with 100 μl of SureBlue Reserve TMB Microwell Peroxidase Substrate , and color was allowed to develop in the dark for 10 min. The reaction was stopped by the addition of 100 μl of TMB stop solution (KPL). All the incubation steps were performed at room temperature. The optical density (OD) at 450 nm was read using an automated plate reader (Thermo Scientific).p values smaller than 0.05 were considered significant.Data were analyzed by unpaired t tests or ANOVA followed by Dunnett’s multiple comparison tests using GraphPad Prism . The 6 plaque-forming unit (PFU)/mL, 3 × 107 PFU/mL, and 1 × 108 PFU/mL, respectively, by plaque assays , NA , and M1 (759 nt) genes of A/Taiwan/S02076/2013(H7N9) were cloned into three separate recombinant baculoviruses. As indicated in Fig. ,683 nt, 8) was obtained from the co-infection condition at an MOI of 2 for each of the recombinant baculovirus. This heightened HA activity was particularly pronounced in fractions containing 30–40 wt.% of sucrose , NA (68 kDa), and M1 (25 kDa) proteins. Each protein on the VLPs was observed to be largely identical in size to the protein from cells infected individually by rBac-H7, rBac-N9, and rBac-M1 Fig. . This re9 particles per 1 μg of the VLPs, yielding a molecular weight of roughly 5.42 × 108 Da per particle. Protein quantification showed that the HA protein contributed to approximately 10% of total proteins in collected H7N9 VLPs  with a two-week interval Fig. . Blood wTo examine splenic virus-specific T cell response in H7N9 VLP-immunized mice, intracellular cytokine staining was conducted on the harvested splenocytes on the day of sacrifice. In VLP-vaccinated mice, production of IL-4 and TNF were shown to be elevated in splenic CD4 and CD8 T cells respectively Fig. . AlthougTo examine the immune-potentiating effect of the H7N9 VLPs in avian species, two-week-old SPF chickens were administered with a primary and a booster vaccination of 10 μg of VLPs on day 0 and day 14 Fig. . To furtAnti-NA and anti-M1 titers in the serum of the vaccinated chickens were also quantified by ELISA with the blood samples collected on day 26 post immunization. For the anti-NA titer, immunization with free protein was found to elevate the titer to a median value of 2,434, whereas immunization with the VLP resulted in a median titer of 3,957 Fig. . SimilarTo analyze antigen-specific T cell responses in chickens, chicken spleens were harvested on day 40 post immunization. Following splenocyte isolation and stimulation with H7N9 VLPs, the splenocytes were lysed and real-time RT-PCR was performed to quantify IFN-γ and IL-4 mRNA expression levels Fig. . It was p = 0.03). While the immunization with the free protein formulation also increased the mean value of CD4/CD8 ratio, no statistical significant was observed (p = 0.31). The results corroborate a more robust and consistent immune response upon exposure to H7N9 virions following VLP immunization.CD4/CD8 ratio was also analyzed using flow cytometric analysis Fig. . On day Since the identification of the avian influenza A (H7N9) infection in human in March 2013, the viral threat has prompted a global effort to develop vaccine candidates as humans have little immunity to this reassorted virus –20. ThisVLPs are a versatile system that presents a compelling alternative to conventional vaccine formulations as they possess the morphological semblance to natural viral particles. In the present study we applied nanoparticle tracking analysis and transmission electron microscopy to validate the structure and antigenicity of the VLPs. These analyses showed virus-like features consistent with other VLP formulations reported in previous studies , 11, 21.We first validated the potency of the VLPs in a mouse model. Given that neutralizing antibodies against the globular head of hemagglutinin protein are the primary mediators of most vaccine-induced protection against influenza, a hemagglutination inhibition assay was used to examine the VLPs as a vaccine candidate against H7N9. An HI antibody titer of 1:40 is the accepted correlate of protection for human HA split inactivated vaccines . ImmunizDespite previous studies that examined different formulations of H7N9 VLPs in mouse models , 25, 26,In the present study, we purified our VLPs and made sure that no detectable baculovirus titer was present in any of our batches. The purification is critical as residual baculoviral proteins were previously shown to trigger innate immune responses through TLR9 and other pattern recognition receptors , 28. TowEven though the free protein formulation, which is a mixture consisting of free HA, NA, and M1 proteins at a 1:1:1 ratio, also elicited humoral and cellular responses in our study, the responses were either weaker or more variable as compared to those elicited by the VLPs. The finding fortifies the notion that the H7N9 VLPs may serve as a compelling vaccine candidate in poultry farming, which typically employs inactivated or subunit vaccines for disease management –31.Our study has prepared H7N9 VLPs co-expressing HA, NA, and M1 as a vaccine candidate against avian influenza. The platform elicited more potent humoral immune responses and more consistent cellular immune responses as compared to the free protein formulation. Although future studies involving viral challenges are warranted, the platform presents a viable candidate for avian influenza management in both human and animal settings.


Mycobacterium tuberculosis and Mycobacterium bovis are attenuated in mice lacking the P2X7 receptor, an ion channel activated by extracellular ATP. Therefore, P2X7 receptor is a potential target for therapeutic intervention. In vitro, hypervirulent mycobacteria cause macrophage death by a P2X7-dependent mechanism that facilitates bacillus dissemination. However, as P2X7 receptor is expressed in both bone marrow (BM)-derived cells and lung structural cells, several cellular mechanisms can operate in vivo. To investigate whether the presence of P2X7 receptor in BM-derived cells contributes to TB severity, we generated chimeric mice by adoptive transfer of hematopoietic cells from C57BL/6 or P2X7−/− mice into CD45.1 irradiated mice. After infection with hypervirulent mycobacteria (MP287/03 strain of M. bovis), P2X7−/−>CD45.1 mice recapitulated the TB resistance observed in P2X7−/− mice. These chimeric mice showed lower lung bacterial load and attenuated pneumonia compared to C57BL/6>CD45.1 mice. Lung necrosis and bacterial dissemination to the spleen and liver were also reduced in P2X7−/−>CD45.1 mice compared to C57BL/6>CD45.1 mice. Furthermore, an immature-like myeloid cell population showing a Ly6Gint phenotype was observed in the lungs of infected C57BL/6 and C57BL/6>CD45.1 mice, whereas P2X7−/− and P2X7−/−>CD45.1 mice showed a typical neutrophil (Ly6Ghi) population. This study clearly demonstrates that P2X7 receptor in BM-derived cells plays a critical role in the progression of severe TB.Tuberculosis (TB) remains a serious public health problem despite the great scientific advances in the recent decades. We have previously shown that aggressive forms of TB caused by hypervirulent strains of  Mycobacterium tuberculosis complex, resulting in an estimated 10.4 million new cases of active tuberculosis (TB) in 2015 -derived macrophages. We observed that eATP induces the P2X7-mediated killing of intracellular H37Rv bacilli and the P2X7-mediated release of viable hypervirulent Beijing 1471 and MP287/03 bacilli    Figure . Recapit−/− and P2X7−/−>CD45.1 mice. Accordingly, the areas of alveolar space were significantly lower in infected C57BL/6 and C57BL/6>CD45.1 mice compared to infected P2X7−/− and P2X7−/−>CD45.1 mice  stained tissue sections revealed a more severe disease in infected mice expressing the P2X7 receptor in BM-derived cells Figure A. On daye Figure B. In addy Figure C. Compat+ cells compared to P2X7−/− and P2X7−/−>CD45.1 mice, respectively  and lung structural cells   , 29, 30.+GR1int phenotype and expresses intermediate levels of Ly6G , and CD45.1 (B6.SJL-Ptprca Pepcb/BoyJ) male mice  were bred at the Animal Facility of the Biomedical Science Institute, USP. Six- to eight-week-old mice were infected and maintained in microisolator cages at the Biosafety Level 3 Mice Facilities at the Faculty of Pharmaceutical Sciences, USP, under controlled temperature and humidity and were fed ad libitum.Specific pathogen-free C57BL/6, P2X7M. bovis isolate (MP287/03—SB0295 spoligotyping). Mycobacteria were cultured in Middlebrook 7H9 medium  with 0.4% sodium pyruvate , 0.05% Tween 80 (Sigma-Aldrich), and 10% ADC . Frozen aliquots of 108 bacilli/ml, at –80°C, were thawed and cultured in complete medium for 7 days at 37°C. The bacilli were sonicated for 1 min, homogenized and maintained for 10 min at rest to prevent bacterial clumps, which were monitored by microscopic examination. The bacterial concentrations were determined by spectrophotometry at 600 nm.Dr. José Soares Ferreira Neto  provided the bovine The mycobacterial burden was quantified by sequential dilutions and the culture of tissue homogenates  in Middlebrook 7H10 medium (Difco) with 0.4% sodium pyruvate and 10% OADC . Three weeks after incubation at 37°C, the CFU numbers were determined.After anesthetizing mice with xylazine  and ketamine , a volume of 60 µl of the mycobacterial suspension (~100 bacilli) was introduced in the trachea through a short midline incision, which was then sutured with sterile silk .−/− mice by flushing with PBS. A single-cell preparation was obtained by carefully cycling through a 26-gauge needle. Recipient CD45.1 mice were irradiated with a dose of 12 Gy from a 137Cs source. After irradiation, 2 × 107 BM cells from C57BL/6 and P2X7R−/− mice in a volume of 200 µl PBS were transferred i.v. under anesthesia. The chimeric mice were housed for at least 12 weeks before infection and were fed with water containing antibiotic (0.1 mg/ml of ciprofloxacin) in the first 4 weeks after BM transplantation.Bone marrow cells were harvest from femur of C57BL/6 or P2X7Lung relative mass was calculated (infected mouse lung weight/control mouse lung weight). The superior lobes of the right lungs were fixed with 10% buffered formalin, photographed, and embedded in paraffin. Serial 4–5 µm sections were stained with HE dye to analyze the tissue alterations and by the Ziehl–Neelsen method to detected BAARs. The samples were examined with a Leica microscope (Germany), and images were captured with a Coolpix P995 Nikon camera (Japan).The reduction in the percentages of pulmonary intralveolar space was determined as described elsewhere , 39. Eig6) were stained using appropriate combinations of FITC-, PercP-, Pecy7-, and APC-labeled monoclonal antibodies to CD11b (M1/70), CD45.1 (A20), Ly6G (1A8) , and CD45.2 (104) (eBioscience). Cells were fixed with 2% paraformaldehyde and analyzed by flow cytometry  using the FlowJo software.The left lungs were dissected and digested with Collagenase type 4  at 37°C for 40 min. A syringe plunger (BD Bioscience) was used to disperse the cells. Cell suspensions were then filtered with a cell strainer  and incubated with ACK lysing Buffer (Thermo Fisher Scientific) at room temperature for 1 min to deplete erythrocytes. Cells (1 × 10p < 0.05 (5%).Data were statistically analyzed by Mann–Whitney test with the GraphPad Prism 5 software  and were considered significantly different when All procedures were in accordance with the national regulations of the National Board of Health and Brazilian College of Animal Experimentation , with respect to their ethical guidelines for mouse experimentation and welfare. The protocol was approved by the Animal Care Committee of the Biomedical Science Institute, University of São Paulo, with permit number 153/11.CCB, EA, and MD-L designed and conceived the experiments, analyzed the data, and wrote the manuscript. CCB, EA, AC, ES, and RN performed the experiment. MH, JA, and EL contributed with reagents, materials, and analysis tools.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Herein, we demonstrate the clinical results of closed reduction in a hyperextended supine position with transsacral-transiliac (TSTI) and iliosacral (IS) screw fixations for Denis zone III sacral fractures. Sixteen consecutive patients with Denis zone III sacral fractures who were treated between January 2009 and September 2016 were evaluated. All patients were treated using percutaneous TSTI/IS screw fixation during closed reduction performed with patients placed in a hyperextended supine position with body manipulation. The clinical and radiological results were evaluated, and the neurological outcomes were retrospectively assessed using Gibbon's classification system. The clinical outcomes were evaluated using the German Multicenter Study Group Pelvic Outcome Scale (POS). The sacral kyphotic angle was reduced by 18.06° ± 15.26° , and fracture translation was reduced by 5.93 ± 4.95 mm . The mean initial Gibbon's score was 3.00 ± 1.32. Among 15 patients with a follow-up duration of over 12 months, the mean reduction loss in the sacral kyphotic angle was 5.87° ± 10.40° and was 1.00 ± 3.00 mm for the fracture translation. The final Gibbon's score was 1.80 ± 1.21, and 73.3% of patients had good results based on the POS score. Although closed reduction in a hyperextended supine position with percutaneous posterior screw fixation is associated with some surgical limitations and technical difficulties, the procedure is minimally invasive and highly effective for stabilizing Denis zone III sacral fractures. Denis zone III sacral fractures are generally due to high-energy traumas such as falls from heights, traffic accidents, and crush injuries, with most of these fractures occurring in patients with polytrauma . Zone IIHistorically, the standard treatment for Denis zone III sacral fractures did not involve surgery or was limited to sacral laminectomy , 5. HoweThe purpose of the present study was to review our experiences and demonstrate the clinical results of closed reduction in a hyperextended supine position with percutaneous TSTI/IS screw fixation methods for Denis zone III sacral fractures.Patients with Denis zone III sacral fractures classified as AO/OTA type C pelvic injuries, treated between January 2009 and September 2016, were selected from our trauma database. Patients who had undergone surgical treatment and had been followed up for a minimum of 12 months were selected for analysis. The patients' medical records were reviewed retrospectively, and the fractures were classified by the sacral level according to the method of Roy-Camille (as modified by Strange-Vognsen and Lebech) and the fracture morphology , 15.The following injury data were collected: demographics, mechanism of injury, associated injury, and the ISS. An initial clinical neurological examination was performed if the patient's condition permitted and was graded according to the classification system described by Gibbons et al , 16. PreDuring the 8-year study period, a standardized operative technique using closed reduction and posterior pelvic ring fixations (TSTI and/or IS screw fixation) was performed. In patients with combined sacral fractures and anterior pelvic ring injuries, staged and/or one-stage reconstruction was performed. Injuries of the anterior part of the pelvic ring were treated using an anterior extraperitoneal or percutaneous approach. Posterior pelvic ring fixation was performed through the percutaneous approach.For posterior screw fixation, the patients were positioned supine on a radiolucent table and in hyperextension with handmade pillows placed vertical to the sacral fracture line . Under fThe patient was allowed to sit with the torso upright starting on postoperative day 1. Nonweight bearing activities were allowed 4 weeks after definitive surgery. Weight-bearing activities began 5 weeks after definitive surgery.After definitive pelvic ring fixation, a follow-up CT was performed within 1 week to indirectly evaluate the decompression quality. If a remnant fracture fragment in the sacral canal and/or poor indirect decompression quality was found, a secondary direct sacral decompression was considered. One orthopedic trauma surgeon (H.I.) performed all operative procedures. Early and late complications associated with the surgical treatment were recorded.The sacral kyphotic angulation of the Denis zone III sacral fracture was measured from sagittal CT reformations by measuring the angle between the posterior sacral cortices, superior and inferior to the level of the transverse fracture. Fracture translation was also measured from the sagittal CT reformations by measuring the displacement of the anterior cortex of the sacrum above and below the transverse fracture . All meaLower extremity sensory and motor function and rectal examinations were performed pre- and postoperatively to identify injuries to the lower lumbosacral plexus. Neurological deficits from cauda equina injuries were classified according to the method of Gibbons et al . ImproveThe clinical outcomes were evaluated at the final follow-up using the clinical criteria of the German Multicenter Study Group Pelvis Outcome Scale (POS) . These cData was enrolled through Microsoft Excel 2016 , followed by a statistical analysis using IBM SPSS Statistics, version 19 . Data are given in terms of arithmetic mean and standard deviation. The initial, postoperative, and final follow-up data were analyzed using paired t-test. Data differences were considered significant for values of p < 0.05.Sixteen patients  with Denis zone III sacral fractures classified as AO/OTA type C pelvic injuries were identified. The mean ISS was 25.94 ± 13.88 (range: 9–50). Thirteen of the 16 patients showed associated injuries, including 9 cases of associated injuries of the head and trunk with an Abbreviated Injury Scale Score ≥3. Other injuries of the spine and extremities were present in 10 patients, while two patients experienced pelvic fracture and urinary tract injuries. At the time of initial examination, neurological deficits were observed in 12 out of 16 patients, with a mean Gibbons' grade of 3.13 ± 1.25 (range: 1–4).The transverse fractures involved the following levels of the sacrum: S1 (n=1), S2 (n=7), S2-3 (n=2), S3 (n=3), S3-4 (n=1), S2 + S3-4 (n=1), and S2 + S3 (n=1). The fracture patterns were classified using the Roy-Camille classification system and the fracture morphology. Roy-Camille type 2 fractures occurred in 12 patients and type 3 fractures occurred in 4 patients. Further, H-shaped (n=3), T-shaped (n=4), U-shaped (n=7), and Y-shaped fractures (n=2) were found. A Morel-Lavallée lesion was observed in 1 case, while no cases of open fracture were found.Posterior percutaneous internal fixation was performed between 0 and 8 days after the injury (median: 1.31 ± 2.73 days). The operative methods for posterior pelvic ring stabilization included 12 cases of 2- or 3-TSTI screw fixation only Figures , 1 bilatAdditional operations due to remnant bone fragments and neurological deficits were performed in 5 patients, including delayed direct sacral decompression (n=5). Posterior implant removal was routinely performed in 14 patients after the fractures had healed.The mean preoperative and postoperative sacral kyphotic angles were 39.44° ± 20.57° (range: 13–89°) and 21.38° ± 7.39° , respectively. The mean postoperative reduction angle of the sacral kyphosis was 18.06° ± 15.26° (range: 2–57°). The kyphotic angle was improved with a significant difference  9.93 to 26.19) The mean preoperative and postoperative translations were 8.69 ± 8.03 mm (range: 0–35 mm), 2.75 ± 3.97 mm (range: 0–16 mm), respectively. The mean postoperative translation reduction was 5.93 ± 4.95 mm (range: 0–19 mm). The fracture translation was also improved with a significant difference .Fifteen patients were followed for more than 12 months. At the final follow-up , the mean sacral kyphotic angle and reduction loss were 27.93° ± 13.01° (range: 13–60°) and 5.87° ± 10.40° (range: -1-32°), respectively. The kyphotic angle was decreased with a significant difference between post-OP and final follow-up . The mean sacral translation and reduction loss were 3.80 ± 6.71 mm (range: 0-24 mm) and 1.00 ± 3.00 mm (range: -2-8 mm), respectively. The translation was maintained with a no difference . The mean Gibbons' grade was 1.80 ± 1.21 (range: 1–4) and 8 out of 12 patients (66.71%) with neurological symptoms showed improvement in neurological status. The Gibbons grade was improved with a significant difference . The mean POS score was 2.93 ± 1.28 (range: 1-4). The clinical results as indicated by the POS score were good and poor in 73.3% (11/15) and 26.7% (4/15) of the patients, respectively .All patients showed bone union. No case of deep and/or superficial wound infection was noted. Screw loosening was observed in 3 patients; one patient had greater reduction loss  are required, as the screws need to provide resistance against the vertical load shear force and the rotational force of the sacral vertebral body. However, it has been reported that ilio-sacro-iliacal corridors for intraosseous implants were not inserted in 18–25% and 10–12% of S1 and S2 cases, respectively; moreover, a high frequency of this issue has been reported for female patients . Thus, oIn conclusion, the current study showed that closed reduction in a hyperextended supine position with TSTI/IS screw fixation is effective for treating Denis zone III sacral fractures in terms of the fracture reduction, loss of reduction, neurological recovery, and clinical outcomes. Thus, despite some limitations, we believe that our procedure is an effective and appropriate method for treating Denis zone III sacral fractures.


Rickettsia typhi is an intracellular bacterium that causes endemic typhus, a febrile disease that can be fatal due to complications including pneumonia, hepatitis and meningoencephalitis, the latter being a regular outcome in T and B cell-deficient C57BL/6 RAG1-/- mice upon Rickettsia typhi infection. Here, we show that CD4+ TH1 cells that are generated in C57BL/6 mice upon R. typhi infection are as protective as cytotoxic CD8+ T cells. CD4+- as well as CD8+-deficient C57BL/6 survived the infection without showing symptoms of disease at any point in time. Moreover, adoptively transferred CD8+ and CD4+ immune T cells entered the CNS of C57BL/6 RAG1-/- mice with advanced infection and both eradicated the bacteria. However, immune CD4+ T cells protected only approximately 60% of the animals from death. They induced the expression of iNOS in infiltrating macrophages as well as in resident microglia in the CNS which can contribute to bacterial killing but also accelerate pathology. In vitro immune CD4+ T cells inhibited bacterial growth in infected macrophages which was in part mediated by the release of IFNγ. Collectively, our data demonstrate that CD4+ T cells are as protective as CD8+ T cells against R. typhi, provided that CD4+ TH1 effector cells are present in time to support bactericidal activity of phagocytes via the release of IFNγ and other factors. With regard to vaccination against TG Rickettsiae, our findings suggest that the induction of CD4+ TH1 effector cells is sufficient for protection. Rickettsia typhi usually is a relatively mild disease. However, CNS inflammation and neurological symptoms are complications that can occur in severe cases. This outcome of disease is regularly observed in T and B cell-deficient C57BL/6 RAG1-/- mice upon infection with R. typhi. We show here that CD4+ T cells are as protective as cytotoxic CD8+ T cells against R. typhi as long as they are present in time. This is evidenced by the fact that neither CD8+ nor CD4+ T cell-deficient C57BL/6 mice develop disease which is also true for R. typhi-infected C57BL/6 RAG1-/- mice that receive immune CD8+ or CD4+ at an early point in time. Moreover, adoptive transfer of immune CD4+ T cells still protects approximately 60% of C57BL/6 RAG1-/- mice when applied later in advanced infection when the bacteria start to rise. Although CD8+ T cells are faster and more efficient in bacterial elimination, R. typhi is not detectable in CD4+ T cell recipients anymore. We further show that immune CD4+ T cells activate bactericidal functions of microglia and macrophages in the CNS in vivo and inhibit bacterial growth in infected macrophages in vitro which is in part mediated by the release of IFNγ. Collectively, we demonstrate for the first time that CD4+ T cells alone are sufficient to protect against R. typhi infection. With regard to vaccination our findings suggest that the induction of R. typhi-specific CD4+ TH1 effector T cells may be as effective as the much more difficult targeting of cytotoxic CD8+ T cells.Endemic typhus caused by  Rickettsiae (R.) are small obligate intracellular bacteria that are transmitted to humans by arthropod vectors. R. prowazekii and R. typhi represent the two members of the typhus group (TG) of Rickettsiae [R. typhi is usually milder and the lethality is estimated to be <5% [kettsiae , 2 and akettsiae . Many pakettsiae . Fatal ckettsiae , 5 and akettsiae . The couo be <5% , 8 if uno be <5% , 9.R. typhi and the bacteria are transmitted to humans by fleas, predominantly the rat flea Xenopsylla cheopis, from these animals [R. typhi in France. While 0.54% of the homeless in Marseille were seropositive in the years 2000–2003, seropositivity increased to 22% in the years 2010–2013 [Epidemic and endemic typhus generally occur worldwide. Epidemic typhus that is transmitted from human-to-human by the body louse sporadically appears in low-income countries of South America and Africa but also in upper-middle economies such as Peru  and Alge animals , 15. End animals –22 and A animals –26 where animals  and Texa animals , 29) as  animals , 31, Gre animals , Spain [ animals –36 inclu animals , 38) and animals , 40. Esp animals . Another010–2013 .R. prowazekii, the closest relative of R. typhi. R. prowazekii can cause the so-called Brill-Zinsser disease years to decades after primary infection which appears with similar symptoms as the primary infection and is usually accompanied by meningitis and neurological symptoms [R. prowazekii [R. typhi because we recently showed that R. typhi persists in mice [R. prowazekii persists irrespective of antibiotic treatment [Rickettsiae are considered potential bioweapons.A vaccine against rickettsial infections is not available but clearly desired for several reasons. It is known that some rickettsial species persist and can re-appear. This is true for symptoms –46. Streowazekii . Similar in mice . Moreovereatment . In addiRickettsiae although animal models of rickettsial infections have been established. Current studies mainly focused on immunity against spotted fever group (SFG) Rickettsiae that represent the vast majority of Rickettsiae but phylogenetically differ from TG Rickettsiae. Especially immunity against R. conorii and R. rickettsii of this group has been studied in mice. Generally, BALB/c and C57BL/6 mice have been reported to be resistant against rickettsial infections [+ T cells that can directly kill infected cells play an important role in defense against Rickettsiae. CD8+ T cells showed enhanced cytotoxic activity in C3H/HeN mice upon infection with R. conorii and R. australis [+ T cells led to enhanced susceptibility of C3H/HeN mice to R. conorii and R. australis [R. typhi [+ immune T cells protected C3H/HeN mice against a lethal dose of R. conorii [+ T cells [-/- mice succumbed to the infection with a normally sublethal dose of R. conorii. Furthermore, neutralization of IFNγ led to severe disease in C3H/HeN mice upon R. conorii infection [+ T cells and macrophages (MΦ) [-/- mice that lack adaptive immunity survive the infection with R. conorii at least for 20 days [R. typhi [-/- mice, however, R. typhi, re-appears several months after infection and then grows predominantly in the brain [R. typhi.Vaccine development requires understanding of protective immune responses as well as of a possible contribution of immune reactions to pathology. To date little is known about immune response against fections –54 whilefections , 54. In ustralis . Furtherustralis , 56 as wnfection . Similarnfection  which cages (MΦ) , 60. Nev 20 days  which isR. typhi , suggesthe brain . Animalshe brain . These f+ T cells are sufficient to protect against R. typhi in the C57BL/6 RAG1-/- infection model by activating MΦ via IFNγ and other factors.In the present study we show that CD4All experimentations and procedures were approved by the Public Health Authorities  and performed according to the German Animal Welfare Act.-/- [-/- lacking CD8+ cytolytic cells [-/- lacking CD4+ T cells [C57BL/6, C57BL/6 RAG1-/- , C57BL/6ic cells , 64 and  T cells , 66 miceR. typhi  was cultured in L929 mouse fibroblasts (ATCC CCL-1) in RPMI1640  supplemented with 10% FCS , 2 mM L-glutamine  and 10 mM HEPES  without antibiotics (standard culture medium). 1×107 γ-irradiated (1966 rad) L929 cells were seeded in 175 cm2 culture flasks . One day later cells were infected with R. typhi and incubated for 5 to 7 days. For the preparation of bacterial stocks, infected L929 cells were resuspended in 1.5 ml PBS. 200 μl silicium particles  were added and cells were vortexed thoroughly for 1 min. The crude lysate was strained through a 2 μm cell strainer . Bacteria were centrifuged at 4300×g for 5 min at room temperature and frozen in FCS with 7.5% DMSO in liquid nitrogen in Cryo.S tubes . Thawed bacterial stocks were centrifuged at 6200×g for 5 min at room temperature, washed twice with PBS and analyzed for bacterial content by quantitative real-time PCR (qPCR) and immunofocus assay as described previously [eviously  to deterR. typhi stocks were thawed and washed in PBS as described above. 2×106 sfu were administered in 50 μl PBS subcutaneously (s.c.) into the base of the tail. Blood samples were obtained by submandibular bleeding.The state of health was evaluated by a clinical score. The following parameters were assessed: body mechanics/motion  and weight loss ; 1: mild (>10%), 2: severe (>15%)) giving a maximum score of 5. Mice were considered healthy with a score ≤1, moderately ill with a score of 2–3 and severely ill with a score of 4–5. Mice were sacrificed reaching a total score of ≥4 or showing weight loss of >20%.6 cells were used for DNA preparation according to the manufacturer´s instructions.DNA was prepared from purified bacteria, cell cultures and organs employing the QIAamp DNA Mini Kit . 10 mg tissue were homogenized in 500 μl PBS in Precellys ceramic Kit tubes  in a Precellys 24 homogenizer . 80 μl tissue homogenizate or up to 1×10R. typhi, bacteria in cell cultures and organs from infected mice was performed by amplification of a 137 bp fragment of the PrsA gene as previously described [Quantification of purified escribed .Cytokines were detected in cell culture supernatants with LegendPLEX assay  according to the manufacturer´s instructions. Cell culture supernatants were diluted 1:2–1:10.2O) were added to 100 μl cell culture supernatant in microtiter plates . A serial dilution of sodium nitrite (NaNO2) in cell culture medium was used as a standard (cmax 125 μM). The absorbance was measured at 560 nm with a Dynex MRXII spectrophotometric microplate reader .50 μl Griess 1 reagent (0.5 g sulfonamide in 50 ml 1M HCl) and 50 μl Griess 2 reagent  for 5 min at RT. Cells were afterwards washed two times in PBS. Brain and spinal cord cells were strained through a 30 μm CellTrics cell strainer  and directly used for stainings. Cells were fixed and permeabilized with Cytofix/Cytoperm and Perm/Wash solutions  according to the manufacturer´s protocol. Fc receptors were blocked with 5% CohnII human IgG fraction  in Perm/Wash for 15 min at 4°C followed by the addition of either mouse anti-R. typhi (BNI52) [+ and CD8+ T cells were stained extracellularly in blood with anti-mouse CD4-PE  and anti-mouse CD8-PerCP-Cy5.5  and in brain with anti-mouse CD4 PerCPCy5.5  and anti-mouse CD8-Alexa488 . In the brain, cells were additionally stained with rat anti-mouse CD45-AF647  to identify CD45high infiltrating cells. KLRG1 and CD11a were detected by extracellular staining employing anti-mouse KLRG1-PE  and anti-mouse CD11a-eFluor450 . Intracellular IFNγ and Granzyme B were detected with anti-mouse IFNγ-PE/Dazzle  and anti-mouse Granzyme B-PacificBlue  in spleen cells restimulated with 10 ng/ml PMA and 500 ng/ml Ionomycin in 200 μl in 96well plates in the presence of 1 μl GolgiStop  for 4h and permeabilized with cytofix/cytoperm  according to the manufacturer´s protocol. For the analysis of surface expression of MHC molecules and CD80 bmMΦ were stained with anti-mouse CD11b-BV421 . R. typhi was detected by intracellular staining with anti-R. typhi BNI52 (1 μg/ml), anti-mouse IgG3-FITC  and anti-FITC-Alexa488 . MHC molecules and CD80 were stained extracellularly with anti-mouse MHCI (H2-Kb)-PE , anti-mouse MHCII (I-A/I-E)-APC  and anti-mouse CD80-PE/Dazzle . Analysis was performed employing a Accuri C6  or LSRII flow cytometer  and FlowJo software .Brain from naïve and infected mice were homogenized. In addition, blood samples were analyzed. Erythrocyte lysis was performed for blood by incubating the cells in erythrocyte lysis buffer   or mouse6 bone marrow cells were cultured in petri dishes  in IMDM medium  supplemented with 2 mM L-glutamine, 5% horse serum and 10% M-CSF-containing cell culture supernatant from L929 fibroblasts. Medium was exchanged every 3 days. Cells were harvested for experimentation after 10–12 days of culture. Virtually 100% of the cells were CD11b+F4/80+ MΦ.bmMΦ were generated from tibia and femur of the hind legs. 2×105 bmMΦ were infected with 10, 25 or 50 copies of R. typhi per cell in 24well plates. Control cells were left untreated or stimulated with LPS (500 ng/ml). Supernatants and cells were harvested at 24h and 48h post infection. Bacterial content and the expression of MHCI, MHCII and CD80 on the cell surface was analyzed by flow cytometry at 24h and 48h. Cytokines and NO were detected in the supernatant of cultures that were infected with 50 copies R. typhi/cell at 48h by LegendPLEX assay and Griess reaction. For the analysis of bacterial growth 2×105 bmMΦ were infected with 5 copies R. typhi per cell. Free bacteria were washed out 3h afterwards and cells were further incubated for 96h. At indicated points in time qPCR was performed to quantify R. typhi.For the analysis of cytokine and NO production and the expression of MHCI, MHCII and CD80 on the cell surface 2×10+ and CD8+ T cells were isolated from C57BL/6 mice on day 21 post R. typhi infection employing the MagniSort Mouse CD4 and CD8 T cell enrichment kits from eBioscience  using an EasySep Magnet from Stemcell Technologies . Procedures were performed according to the manufacturer´s instructions. Purity was analyzed by flow cytometry and found to be >98% for each cell population in all preparations. CD4+ T cells were generally absent in CD8+ T cell preparations and vice versa. 1×106 CD4+ or CD8+ T cells were injected i.v. in 100 μl PBS into C57BL/6 RAG1-/- mice 63 days after R. typhi infection, which is the point in time when bacteria become detectable by qPCR in the brain and approximately 20–40 days prior to the usual onset of disease. R. typhi-infected control animals received PBS instead of T cells. Additional C57BL/6 RAG1-/- control mice were not infected but received either CD4+ or CD8+ immune T cells.Immune CD46 bmMΦ were infected with 5 copies R. typhi per cell in 24well plates. Free bacteria were washed out after 3h and cells were further incubated for 24h at 37°C to allow bacterial entry and replication. Immune CD4+ T cells were isolated as described above from R. typhi-infected C57BL/6 wild-type mice 7 days post infection. Control CD4+ T cells were purified from the spleen of mice that received PBS instead. 1.5×106 purified CD4+ T cells were added per well and cells were further incubated for 72h. IFNγ was neutralized by simultaneous addition of anti-IFNγ . In another experimental setup 1×106 bmMΦ were infected with 5 copies R. typhi per cell. After 3h hours the medium was exchanged by cell culture medium with or without recombinant IFNγ  or a combination of recombinant IFNγ that was pre-incubated with anti-IFNγ (1 μg/ml) for 15 min at RT for neutralization. Supernatants and cells were harvested 96h after infection. Cytokines and NO were quantified in the supernatants by LegendPLEX and Griess assay. Bacterial content was determined in the cell pellet by PrsA qPCR.1×102O. Deparaffinized sections were boiled for 30 min in 10 mM citrate buffer  for antigen retrieval. Staining was performed using a Ventana Benchmark XT apparatus . Antibodies were diluted in 5% goat serum  in Tris-buffered saline pH7.6 (TBS) and 0.1% Triton X100 in antibody diluent solution . Rabbit anti-mouse CD3 , rabbit anti-mouse IBA1  and rabbit anti-mouse iNOS  were used. Anti-rabbit or anti-rat Histofine Simple Stain Mouse MAX peroxidase-coupled antibodies  were used as secondary antibodies. Detection was performed with ultraview universal DAB detection kit . Sections were covered with Tissue-Tek embedding medium . Images were taken with a BZ9000 Keyence microscope .For immunohistochemistry (IHC) tissues from infected mice were fixed in 4% formalin in PBS and embedded in paraffin. Deparaffinization of the sections was performed using standard methods. Sections were first heated at 63°C for 30 min in a heating cabinet followed by treatment with Xylol for 30 min and EtOH . Each step was performed for 3–5 min. Slides were finally washed in HStatistical analysis was performed with GraphPad Prism 5 software . Student´s T test, Mann-Whitney U test, or One-way ANOVA test followed by Kruskal-Wallis and Dunn´s post test were performed as indicated in the figure legends.+ T cells, are important in the elimination of intracellular pathogens. Therefore, we first analyzed the CD8+ T cell response in R. typhi-infected C57BL/6 mice during the course of infection. For these analyses spleen cells were re-stimulated in vitro with PMA/Ionomycin followed by flow cytometric analysis of IFNγ expression and the expression of Granzyme B as a cytotoxic effector molecule. In addition, CD8+ T cells from the spleen of the mice were analyzed for the expression of KLRG1 as a marker for terminally differentiated non-replicative T cells [+CD11a+ T cells began to rise on day 3, peaked on day 7 and declined until day 15  while CD8+ T cells were present in the brain of only 2 out of 5 recipient mice at this point in time (36.20±2.20%)   althoughhe blood . These d-/- mice that were infected with R. typhi but received PBS instead of T cells succumbed to the infection between day 70 and 120, showing neurological symptoms such as tremor, ataxia and paralysis followed by body weight loss. These were evaluated by a neurological score  as well as of infiltrating MΦ (CD45highCD11b+) was detectable in the brain of R. typhi-infected control C57BL/6 RAG1-/- mice  compared to naïve mice (microglia: 625.6±162.8) where infiltrating MΦ were virtually absent (MΦ: 68.19±10.42) as expected. Counts of microglia were not signicantly altered in CD4+ T cell recipients (4285±774.4) while an enhanced infiltration of MΦ, although not significant, was observed (5082±995.3) compared to infected control mice. In contrast, mice that had received CD8+ T cells showed significantly reduced numbers of microglia (1663±397.4) and numbers of infiltrating MΦ were unchanged compared to infected control mice (2272±767.4) (We have previously shown that bacteria . Therefo2±767.4) .R. typhi-infected C57BL/6 RAG1-/- mice at the time of death [-/- control mice (8.375±1.118%) compared to naïve mice (0.6533±0.6533%), these differences were not significant at this point in time (+ T cells (11.1±3.156%). In contrast, the transfer of CD4+ T cells led to a significantly increased proportion of iNOS-expressing MΦ (28.10±6.713%). Furthermore, the presence of CD4+ T cells led to a significantly increased frequency of iNOS-expressing microglia (3.132±0.717%). These cells normally do not express iNOS in R. typhi-infection of C57BL/6 RAG1-/- mice [R. typhi-infected control mice (0.8054±0.2214%) compared to 0.5350±0.2074% in naïve mice. Furthermore, enhanced frequencies of iNOS-expressing microglia were not present in mice that had received CD8+ T cells (1.254±0.2734%) . This enzyme is usually expressed by activated MΦ and important for the killing of intracellular bacteria by catalyzing the generation of nitric oxide (NO) . We prevof death . Here, a in time . The fre-/- mice . In line0.2734%) .+ T cell recipients in histological stainings. R. typhi-infected control mouse, a CD4+ and a CD8+ T cell recipient 7 days post transfer. iNOS-expressing cells were hardly detectable in control mice and animals that received CD8+ T cells. In CD4+ T cell recipients locally accumulating iNOS-expressing cells were found at the ventricle borders as well as in the parenchyma and the pia mater of the cerebellum . As expected, LPS significantly induced the release of several cytokines as well as the production of NO (R. typhi-infected bmMΦ hardly released any cytokines. Only very low amounts of IL-6 (28.4±15.4 pg/ml), TNFα (11.1±4.6 pg/ml) and IL-10 (10.9±3.5 pg/ml) were detectable. These were negligible compared to LPS-induced levels of these cytokines . Furthermore, the release of bactericidal NO was not induced in R. typhi-infected bmMΦ cultures  and IL-2 (1219.7±130.8 pg/ml). In addition, low levels of TNFα (85.2±26.7 pg/ml), IL-6 (11.8±6.8 pg/ml), IL-10 (288.6±28.4 pg/ml) and bactericidal NO (3.9±2 μM) were detectable in cultures containing immune CD4+ T cells  compared to cultures with control CD4+ T cells (17320±13458 R. typhi PrsA copies)  . This ef copies) . To furt copies) . CollectR. typhi shows a neurotropism in C57BL/6 RAG1-/- mice that lack adaptive immunity. In these mice, R. typhi re-appears months after infection predominantly in the CNS and causes severe CNS inflammation and lethal paralysis. Employing this model and adoptive transfer we describe here that CD8+ and CD4+ T cells enter the CNS and that both T cell populations are protective against this infection and R. typhi-induced disease.We recently described that + TH1 T cell response that is characterized by IFNγ expression in response to R. typhi infection. In addition, functional cytotoxic CD8+ T cells expressing IFNγ and Granzyme B, an effector molecule that is crucial for target cell killing by rapid induction of apoptosis [+ and CD8+ T cells as well as Granzyme B expression in CD8+ T cells peaked on day seven post infection which was consistent with the expression of CD11a and KLRG1 on CD8+ T cells, demonstrating that these cells were antigen-experienced effector cells. In line with these findings KLRG1 was expressed on CD8+ T cells with similar kinetics in C3H/HeN mice upon R. typhi infection [+ T cell responses were observed in R. conorii- and R. australis-infected C3H/HeN mice peaking at day 10 post infection [R. typhi in C57BL/6 mice declined until day 15 but did not reach basal levels again until day 35. This is consistent with the observation that R. typhi persists in these mice [C57BL/6 wild-type mice mount a classical CD4poptosis , were genfection . Furthernfection . T cell ese mice  and sugg+ T cells are sufficient for protection against R. typhi infection. Neither CD4+ T cell deficient C57BL/6 MHCII-/- nor CD8+ T cell-deficient C57BL/6 MHCI-/- mice developed disease and survived the infection. We further performed adoptive transfer of immune T cells isolated from C57BL/6 wild-type mice into R. typhi-infected C57BL/6 RAG1-/-. Transfers were performed at a point in time when the bacteria already start to dramatically increase in the brain, the organ with the highest bacterial load in these animals [R. typhi-infected C57BL/6 wild-type mice on day 21 post infection. These mice are highly resistant to the infection with R. typhi and other rickettsiae. Pure T cells from these mice were used for transfer. Because T cells are not target cells for the bacteria it is highly unlikely that significant amounts of bacteria were co-transferred and might have established a superinfection. In line with that, the bacteria were not detectable in the organs of non-infected animals that received either immune CD4+ or CD8+ T cells. Moreover, both immune CD8+ and CD4+ T cells entered the brain in R. typhi-infected C57BL/ RAG1-/- mice but not in non-infected animals, efficiently eliminated the bacteria and were protective. The survival rate of CD4+ T cell recipients, however, was lower than that of CD8+ T cell recipients, indicating that CD4+ T cells are less efficient in protecting animals with already advanced infection. Generally, CD8+ T cells were absent in CD4+ recipients and vice versa  the T cell response calms down. In this situation a certain threshold of either activated CD4+ or CD8+ T cells as observed in R. typhi-infected C57BL/6 mice . Immune CD4+ and CD8+ T cells were isolated from C57BL/6 mice on day 21 post R. typhi infection and adoptively transferred into R. typhi-infected C57BL/6 RAG1-/- mice on day 55 post infection (n = 5 for each group). R. typhi-infected control animals received PBS instead of T cells (n = 5). All CD4+ and CD8+ T cell recipients survived the infection (B).CD4(TIF)Click here for additional data file.S2 Fig-/- mice that received either immune CD4+ or CD8+ T cells from C57BL/6 mice (x-axis) was determined by qPCR on day 7 post transfer  and on day 210 post infection when the experiments were terminated . R. typhi was generally not detectable at all in these animals, excluding that contaminating bacteria that might have been present in the T cell preparations contributed to the infection. Furthermore, these mice did not show symptoms of disease at any point in time.The bacterial load in the brain, spinal cord, spleen and lung (y-axis) of non-infected control C57BL/6 RAG1(TIF)Click here for additional data file.


A novel MOF decorated with O– groups was elaborately constructed and showed excellent performance for Pb2+ removal. ii)-based MOF decorated with O– groups for the removal of Pb2+. Benefiting from its multiple porosity, sufficient adsorption sites and strong affinity, the activated MOF material exhibits an ultrahigh Pb2+ uptake capacity (616.64 mg g–1), surpassing all those of reported MOF adsorbents. Moreover, it can selectively capture Pb2+ with high efficiency (>99.27%) against background ions. Even in the presence of a high concentration of competitive ions, such as Ca2+ or Mg2+, effective removal (>99.21%) can also be achieved in a short time. The excellent removal performance demonstrates the strong electrostatic attraction and coordination interaction between the highly accessible O– groups and Pb2+. The possible adsorption mechanism was systematically verified by zeta potential, FT-IR and XPS studies. Our work reveals the enormous potential of functionalized MOFs as an appealing platform to construct sorbent materials.Heavy metal ions are highly toxic and widely spread as environmental pollutants. New strategies are being developed to efficiently remove these toxic ions. Herein, we use the intrinsic advantages of metal–organic frameworks (MOFs) and develop a porous Zn( Many efforts have been dedicated to purify wastewater to get more available clean water. However, an efficient, cost-effective, robust and handy technology for the decontamination of water is urgently needed. The adsorption technique for removing heavy metal ions has gained extensive attention considering the easy operation, eco-friendliness and cost-effectiveness.2+ removal. Therefore, there is an increasing interest in developing more efficient absorbents for the removal of Pb2+ from aqueous solution.Lead Pb+, a prev2+, UO2+, Cd2+, Hg2+, etc. from aqueous solution.2+ and Hg2+ from water with high uptake capacities (286 mg g–1 for Cd2+ and 439.8 mg g–1 for Hg2+).Metal–organic frameworks (MOFs), constructed by metal ions or metal clusters and organic ligands through coordination bonds, are considered as a favorable platform for adsorption applications,– groups was designed and applied to the removal of Pb2+ from aqueous solution  porous framework {[Zn3L3(BPE)1.5]·4.5DMF}n ethylene, DMF = N,N-dimethylformamide) functionalized with O– groups for the removal of Pb2+. The activated MOF material [Zn3L3(BPE)1.5]n (1a) exhibited an ultrahigh uptake capacity (616.64 mg g–1), a very high affinity (Kd 106 mL g–1) and a high removal efficiency for Pb2+. The adsorption mechanism was revealed by zeta potential, FT-IR and XPS studies. To the best of our knowledge, we are the first to introduce functional groups with negative charges into the pores of MOFs for the removal of heavy metal ions with excellent performance.Herein, a functionalized MOF decorated with negatively charged Osolution . Based o2L was synthesized according to the literature method.–1. Simultaneous inductively coupled plasma optical emission spectrometry (ICP-OES) on a PerkinElmer Optima 8000 instrument was used to determine the metal ion concentration in aqueous solution.The ligand H3)2·6H2O , H2L , BPE  and 6 mL DMF were placed in a 10 mL vial. The mixture was stirred until complete dissolution, and then it was kept in an oven at a temperature of 100 °C for 24 h. Orange block crystals of 1 were obtained, washed with DMF, and dried at room temperature. Yield: 8.8 mg . Anal. calcd for C73.5H70.5N13.5Zn3O19.5: C, 53.46; H, 4.30; N, 11.45. Found: C, 53.38; H, 4.07; N, 11.07.Zn(NO1a was prepared by heating MOF 1 at 140 °C under vacuum (24 h) to remove the encapsulated DMF guests before the adsorption studies. As confirmed by PXRD, TG and elemental analysis, 1a retained the same framework of 1, but without DMF solvent molecules in the cavity. Anal. calcd for C60H39N9Zn3O15: C, 54.50; H, 2.97; N, 9.53. Found: C, 54.21; H, 3.22; N, 9.15.MOF λ = 0.71073). The structure was solved by a direct method and expanded with the Fourier technique. All of the calculations were performed with the SHELXL-97 package.1, two azoxy groups of the L2– ligand were found to be disordered over two positions. All H atoms in 1 were placed in geometrically idealized positions and constrained to ride on their parent atoms. Moreover, the diffused electron densities resulting from these residual solvent molecules were removed from the data set using the SQUEEZE routine of PLATON and refined further using the data generated.3L3(BPE)1.5]·4.5DMF}n was derived from thermogravimetric characterization. The crystal data for 1 are summarized as follows: C60H39N9O15Zn3, Mr = 1322.17, monoclinic, space group C2/c, a = 19.604(4) Å, b = 28.079(6) Å, c = 32.670(7) Å, α = 90°, β = 94.47(3)°, γ = 90°, V = 17 929(7) Å3, Z = 8, Dc = 0.980 g cm–3, F(000) = 5376 and μ = 0.846 mm–1, 84 732 reflections collected, 15 810 unique (Rint = 0.0546). R1 = 0.0623, wR2 = 0.1871 and S = 1.093. Crystallographic data have been submitted to the Cambridge Structural Database with the deposition number CCDC ; 1536031.Single X-ray diffraction intensities of crystals were collected on a CCD diffractometer at 153 K. All diffractometers were equipped with graphite monochromated Mo-Kα radiation (1a and 80 mL of Pb2+ standard solution. The Pb2+ solution was prepared by dissolving Pb(NO3)2 in deionized water and diluting to the desired concentration. To study the adsorption kinetics, adsorption experiments were performed at pH 6.0 (10 ppm Pb2+ solution) under continuous stirring. The Pb2+ concentrations of samples were measured at given time intervals using an ICP-OES spectrometer. The adsorption isotherm experiments were investigated by adding 10 mg MOF 1a into 80 mL Pb2+ solutions with different concentrations to reach adsorption equilibrium within 180 min. The adsorption capacity qt (mg g–1) and the removal efficiency were obtained from the following equations:C0 (mg L–1) and Ce (mg L–1) are the initial and equilibrium Pb2+ concentrations, respectively; Ct (mg L–1) is the Pb2+ concentration at time t (min); V (L) is the volume of the solution, and m (g) is the dry weight of adsorbent.All of the adsorption experiments were carried out at 25 °C using 10 mg of MOF 2+ loaded MOF in 80 mL of HNO3 solution (0.1 mmol L–1) at 25 °C for 24 h. Then it was washed three times with water and dried under vacuum at 140 °C for 24 h. The desorbed Pb2+ was evaluated by the same method used in the adsorption studies.Furthermore, the desorption test was carried out by immersing the Pb1 crystallizes in the C2/c monoclinic space group. The framework is composed of paddlewheel dinuclear Zn2(COO)4 secondary building units that are bridged by L2– ligands and further pillared by BPE to construct an interpenetrating 3D framework . As expected, the pores in MOF 1 are decorated by a large number of O– sites, resulting in the functionalized pores. These functionalized pores will certainly be favorable for the capture of heavy metal ions.The single-crystal X-ray diffraction study indicates that MOF ramework . Althougework  of the DMF guest molecules at 40–222 °C 1.5]n.Thermogravimetric analysis (TGA) of C Fig. S1. So, thed Fig. S1, which s1a, gas sorption isotherms were investigated  and CO2 (3.30 Å which results in easier diffusion into the micropores). Similar N2 and CO2 sorption behaviors have been observed in many other flexible MOF materials.1a is calculated to be 82.5 m2 g–1 based on the CO2 adsorption isotherm. This is probably due to a higher diffusion barrier imposed on the 1D pore for the flexible MOF after desolvation and the corresponding structural contraction.To examine the permanent porosity of d Fig. S2. Unexpec1a were sharp, indicating the crystalline nature of the desolvated phase. The low-angle Bragg’s reflection in 1 vanished in 1a. According to the literature, there is a slight shrinkage of the framework of 1a after guest molecule removal from 1.1a in DMF for 24 h generated 1′, the low-angle Bragg’s reflection reappeared and the PXRD pattern was almost the same as that of 1 .As illustrated in Fig. S3,1 Fig. S3, which iScheme S1.34,35,41K Fig. S3. It can 1 was immersed in water (24 h), and the single crystallinity of the resulted H2O-exchanged 1 (1–H2O) was not good. However, by immersing 1–H2O in DMF for 24 h, 1–H2O–DMF with better crystal quality was obtained and the unit cell parameters were very similar to those of 1  at 153 K (Table S23 for 1 (153 K) to 17 753 Å3 for 1–CHCl3 . These results further confirmed that the MOF is dynamic in nature and the transformation of the flexible framework is reversible,1 are different from those of 1, but return to 1 after immersion in DMF for 24 h  was isolated and washed with water to remove the residual Pb2+ on the exterior of 1a–Pb. Then it was examined by energy-dispersive X-ray spectroscopy (EDS) and this confirmed the existence of Pb  was calculated as follows:C0 (mg L–1) and Ce (mg L–1) are the initial and equilibrium concentrations of Pb2+; V is the volume of the treated solution (mL); m is the dry weight of adsorbent (g). Kd represents an important aspect of the sorbent performance. In general, a material with a Kd value above 104 mL g–1 is considered to be an excellent adsorbent.Kd value of 1a for Pb2+ is 2.3 × 106 mL g–1, surpassing that of various reported benchmark materials, for examples, biochars (103 to 104 mL g–1),5 mL g–1),4 to 5.1 × 105 mL g–1),5 mL g–1),4-LDH (2.6 × 105 mL g–1),3 to 1 × 104 mL g–1).52To assess the sorbent’s affinity for Pb1a by varying the Pb2+ concentrations from 5 ppm to 200 ppm at pH 6.0. As shown in qe (equilibrium adsorption capacity) increased with the increasing Pb2+ concentrations and finally reached the maximum value of 616.64 mg g–1. In addition, we also considered the removal efficiency at different Pb2+ initial concentrations (BPE)0.5]n ,2+. Results show that the activated [Zn(ADC)(BPE)0.5]n displayed a lower uptake capacity of 473.92 mg g–1. Meanwhile, we calculated the theoretical uptake capacity of 1a based on the molecular formula, pore volumes and one O– group per L2– adsorbing one Pb2+ ion by a coordination interaction. The calculated theoretical uptake capacity is 470.11 mg g–1, which is obviously lower than the experimental equilibrium value (616.64 mg g–1) of 1a. The higher experimental adsorption capacity for 1a could be attributed to the various adsorption modes formed by the O– groups, which offered not only coordination interactions but also electrostatic attractions between the O– groups and Pb2+. To the best of our knowledge, the Pb2+ uptake capacity of 1a is the highest among the reported MOF adsorbents  and Cd2+ (soft acid), and bind difficultly with Na+, Mg2+, K+, Ca2+, and Mn2+ (hard acids). Compared to the borderline acid Pb2+, the relatively low removal efficiencies for the borderline acids Co2+ and Ni2+ are attributed to their fairly high hydration energies .2+ and Ni2+ to detach water molecules and further interact with the negatively charged O atoms. So the MOF material has a strong preference and fairly high selectivity for Pb2+ against other metal ions, highlighting its potential in selectively removing Pb2+ from real wastewater.The selective removal of heavy metal ions will facilitate environmental protection and allow the reuse of heavy metals.her ions . In the 2+ and Mg2+ are common cations that always ubiquitously co-exist in the natural water environment, and the same positive-charge and high level concentration might lead to competitive adsorption with Pb2+.2+, which has a comparable ionic size and charge density to Pb2+, and often co-exists with Pb2+,2+, Mg2+ and Cd2+ on the adsorption of Pb2+. As shown in 2+ removal efficiencies were obtained in the presence of competing ions of Ca2+ and Mg2+. With the molar ratios of Ca2+/Pb2+ and Mg2+/Pb2+ ranging from 1 to 50, there were only little decreases for Pb2+ removal efficiencies, changing from 99.82% to 99.21% with Ca2+ and 99.65% to 99.31% with Mg2+. A large removal capacity (96.43%) was still obtained at a molar ratio of 10 for Cd2+/Pb2+. When the ratio was increased to 50, the Pb2+ removal efficiency reduced to 56.31%. The concentration of Cd2+ in the natural water environment can hardly reach such a high ratio, thus Cd2+ could not affect the Pb2+ removal performance in a practical application.Ca1a has a strong preference and an ultrahigh selectivity for Pb2+ sorption. The particular selectivity can be ascribed to the following reasons: (1) the densely populated O– groups of the functionalized MOF provided strong interactions between O– and Pb2+; (2) adsorbents preferentially adsorb divalent cations with a lower hydration energy in general, as metal ions have to detach a large share of hydrated water before they enter the smaller channels of the adsorbents.2+ (1505 kJ mol–1), Mg2+ (1830 kJ mol–1) and Cd2+ (1755 kJ mol–1), the lowest Gibbs free energy of hydration was observed for Pb2+ (1425 kJ mol–1), indicating Pb2+ preferential adsorption than other divalent cations;2+, Mg2+ and Cd2+ are 0.100 nm, 0.072 nm and 0.095 nm, respectively, while the radius of Pb2+ is 0.119 nm. The larger radius would endow 1a with quite good selectivity for Pb2+ adsorption.All of these results indicate that 1a, we performed in situ PXRD measurements to evaluate the crystalline structure. In 1a in water for 300 min, and even soaking in Pb2+ solution for more than 300 min, it still holds a high crystalline form and retained the structure of 1a, indicating the good chemical stability of 1a in water and Pb2+ solution. On further immersion of 1a in aqueous solution of pH 3.0–10.0 (300 min), its high crystalline form was also preserved , we can affirm that the adsorbent still maintains the single crystallinity even after loading Pb2+.Stability and reusability are two important factors in the application of an adsorbent. To verify the chemical stability of  Fig. S12. Meanwhi Fig. S14. The dif Table S1. By comp1a exhibited a favorable cycle performance for Pb2+ adsorption, and the removal efficiency remained almost unchanged in the first two cycles and only slight fading was observed in the following cycles. The slight decrease may result from the loss of the adsorbent during cycling. Similar results were also reported in previous studies.From the consideration of economical and practical purposes, it is of great significance to study the desorption and regeneration of an adsorbent. As shown in 2+, zeta potential measurements were performed. When the pH was above 4.0, the surface of 1a was negatively charged, and the density of the negative charge increased remarkably with increasing pH value  scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>C and C–N vibrations,–1, 1573 cm–1, and 1363 cm–1, respectively, after loading Pb2+. This might give further evidence of coordination interactions between Pb2+ and O atoms.To further investigate the mechanism of Pb1a and Pb2+. As shown in 2+ is undoubtedly loaded on 1a. A more detailed structure of the Pb species could be obtained in the high resolution XPS spectrum of Pb 4f. In 5/2 and Pb 4f7/2. Compared with the Pb2+ binding energies of purified Pb(NO3)2 at 144.5 eV for Pb 4f5/2 and 139.6 eV for Pb 4f7/2,1a–Pb, which reveals the formation of strong affinities between Pb2+ and 1a.5/2 (143.3 eV) and Pb 4f7/2 (138.4 eV) peaks further confirms that the coordination interaction, and not merely the electrostatic interaction, accounts for the mechanism of Pb2+ sorption.XPS spectra were also employed to provide more information on the interactions between ii)-based MOF functionalized with O– groups was elaborately constructed for the removal of Pb2+ from aqueous solution. The activated MOF showed excellent performance for Pb2+ removal with a record-high uptake capacity of 616.64 mg g–1 among MOF adsorbents, a high removal efficiency in a wide range (>90% at 1–75 ppm) and an exceptional distribution coefficient value of 2.3 × 106 mL g–1, making the activated MOF one of the most promising materials for eliminating Pb2+ pollution from water. Notably, the activated MOF can selectively capture Pb2+ with a high efficiency even in the presence of a high concentration of other metal ions. Also, the adsorbent can be readily regenerated and recycled without a significant decrease of the removal efficiency. The densely populated and highly accessible O– groups with their remarkable affinity for Pb2+ are responsible for the impressive results. This work therefore lays a foundation for introducing charged groups into MOFs as a new platform for efficiently removing contaminants from aqueous solution.In summary, a novel Zn(There are no conflicts to declare.Supplementary informationClick here for additional data file.Crystal structure dataClick here for additional data file.


The MOFs were characterized using various techniques and further used as precursor materials for the synthesis of metal/metal oxide nanoparticles inserted in a carbon matrix through a simple thermal conversion method. The newly synthesized carbon materials were characterized by scanning electron microscopy, transmission electron microscopy, energy-dispersive X-ray spectroscopy analysis, powder X-ray diffraction and BET analysis. The results showed that the MOF-derived carbon composite materials maintained the morphology of the original MOF upon carbonization, and confirmed the insertion of metal/metal oxide particles in the carbon matrix.Three isostructural metal-organic frameworks, (MOFs), [Fe(OH)] ( Metal/metal oxide nanoparticles have demonstrated distinct properties from their bulk materials and have shown promising applications in many fields such as catalysis , magnetietc.  (2), and [In(OH)] (3)) of 1,4-naphthalene dicarboxylate  using a simple, one-step thermal cracking method. Further, the MOFs and their metal/metal oxide embedded carbon hybrid materials were characterized using various physico-chemical techniques, and their gas sorption properties have been studied.Though a few articles have been published on the synthesis of the dispersed metal/metal oxide nanoparticles in porous carbon matrices from MOFs, the synthesis of metal/metal oxide nanoparticle-embedded porous carbon hybrid functional materials using a simple, one-step thermal conversion is still a challenge. Hence, herein we report three new metal/metal oxide embedded porous carbon hybrid functional materials from three MOFs ([Fe(OH)]  or Al(NO3)3·9H2O  or In(NO3)3·xH2O , H2-1,4-NDC , and H2O (10 mL) was placed in a 23 mL Teflon autoclave and then heated at 180 °C for 1 day. After filtering off and washing with distilled water, powder samples of MOFs were obtained. 1: color, white; yield, 0.104 g; elemental analysis, found/calcd.: C, 39.60/40.13; H, 4.16/4.21% for 1·4H2O. 2: color, white; yield, 0.117 g; elemental analysis, found/calcd.: C, 49.24/49.00; H, 3.67/3.77% for 2·2H2O. 3: color, white; yield, 0.113 g; elemental analysis, found/calcd.: C, 38.12/38.64; H, 2.65/2.70% for 3·1.5H2O.All reagents were commercially available and used as received without further purification. In a typical synthesis of the MOFs, the mixture of FeCl2 gas with a heating rate of 5 °C·min−1 to carbonize the MOFs. After reaching the target temperature (800 °C), the temperature was maintained at 800 °C for 5 h, then cooled down to room temperature with a cooling rate of 1 °C·min−1. The final black-colored powder products were further characterized. Conversely, the synthesis of metal/metal oxide nanoparticles within a porous carbon matrix involves a direct carbonization method using the above synthesized MOFs as precursors. The MOFs (0.200 g) were taken in a silica boat and then placed in a tube furnace and heated from room temperature to 800 °C under N2-1,4-NDC (0.5 mmol) with corresponding metal salts (1.0 mmol) yielded the new MOFs. The new MOFs were initially characterized by elemental analysis, which agrees well with theoretical values. The as-synthesized MOFs may contain guest H2O molecules which are removed by heating of these MOFs to 180 °C for 12 h under vacuum (~10−3 torr); the phase purity of the bulk materials were independently confirmed by powder X-ray diffraction (PXRD) measurements  MOF material [1–3) have the same type of structural architecture as Al-1,4-NDC .It is to be noted that the diffraction peaks of compound 1–3) were investigated via scanning electron microscopy (SEM) and images are presented in 1, 2 and 3, respectively).The shape and microstructure of the resulting MOFs . These TGA observations have further been confirmed through various in situ temperature PXRD measurements for all the compounds  on heating to 400 °C  during calcination at 600 °C for one hour  were calcined at 800 °C under nitrogen gas flow for 5 h. The final products were characterized with the aid of various techniques. The carbonized products from 1, 2 and 3 are designated as 1c, 2c and 3c, respectively. Compounds 1c–3c were initially characterized using PXRD measurements. The PXRD pattern matching of 1c with Fe and Fe3C  was carried out via Raman spectroscopy. Raman spectra of the obtained carbon samples are shown in −1 and 1600 cm−1, respectively, which is due the disordered carbon structures and the stretching vibrations in opposite directions of two carbon atoms in a graphene sheet. The relative ratios of the G band to the D band (GI/DI) were found to be 1.11, 1.15 and 1.18 for 1c, 2c and 3c, respectively, suggesting that in all samples the graphene sheets were not well-developed and the local carbon structures contained both graphitic and disordered carbon atoms [Further investigation of the local structure of the synthesized carbon materials (on atoms .1c exhibited a strong interaction with a magnetic field (2c and 3c (3c revealed that the surface of nanoporous carbon samples was embedded with indium oxide nanoparticles (Since compound ic field , we werec and 3c  suggestearticles b,d. 2c showed the presence of aluminum oxide nanoparticles inside the carbon matrix (3c at two different SEM image locations clearly confirms the presence of In2O3 in the carbon framework of 3c (The composition of the carbon matrix and the metal oxide nanoparticles were confirmed through EDS analysis, and the results are given in n matrix . On the rk of 3c .1c, 2c and 3c (2 gas during the carbonization process, which makes the resulting carbon materials more porous than the parent MOF materials. Nitrogen gas adsorption analysis was used to further characterize the nanoporous structure of c and 3c . The shaIn summary, Fe-, Al-, and In-MOFs based on a 1,4-NDC linker have been prepared and characterized. Using these MOFs as precursor materials, metal/metal oxide nanoparticle-inserted nanoporous carbons were prepared via simple direct carbonization without using any additional carbon sources. The PXRD, SEM, TEM, EDS and nitrogen sorption measurements confirm the dispersion of metal/metal oxide nanoparticles in the resulting nanoporous carbon materials. It can be expected that these newly synthesized carbon composite materials have promising prospects for application in supercapacitors.