In this study, Ni-supported catalysts were synthesized using the wet impregnation method to obtain 5%Ni/Al2O3(SA-5239), 5%Ni/Al2O3(SA-6175), 5%Ni/SiO2, 5%Ni/MCM41, and 5%Ni/SBA15. The catalysts were tested in dry reforming of methane at 700 °C, 1 atm, and a space velocity of 39,000 mL/gcat h, to study the interaction of Ni with the supports, and evaluation was based on CH4 and CO2 conversions. 5%Ni/Al2O3(SA-6175) and 5%Ni/SiO2 gave the highest conversion of CH4  and CO2 . The catalysts were characterized by some techniques. Ni phases were identified by X-ray diffraction patterns. Brunauer–Emmett–Teller analysis showed different surface areas of the catalysts with the least being 4 m2/g and the highest 668 m2/g belonging to 5%Ni/Al2O3(SA-5239) and 5%Ni/SBA15, respectively. The reduction profiles revealed weak NiO-supports interaction for 5%Ni/Al2O3(SA-5239), 5%Ni/MCM41, and 5%Ni/SBA15; while strong interaction was observed in 5%Ni/Al2O3(SA-6175) and 5%Ni/SiO2. The 5%Ni/Al2O3(SA-6175) and 5%Ni/SiO2 were close with respect to performance; however, the former had a higher amount of carbon deposit, which is mostly graphitic, according to the conducted thermal analysis. Carbon deposits on 5%Ni/SiO2 were mainly atomic in nature.A promising method to reduce global warming has been methane reforming with CO Therefore, good use of the two agents CO2 and natural gas, , will ultimately reduce the earth warming effect being caused by them.The abundant natural gas in large reserves positions methane as a good candidate, ahead of any other, for the chemistry of one-carbon-atom-containing compounds . Consequ2/CO in comparison with steam reforming of methane  (CAS 13621) was used as the active metal for all the supports, and the percentage loading was maintained at 5 wt.% in all samples.All of the Nickel (Ni)-supported catalysts that were investigated in this research work were prepared using the wet impregnation technique. Some of the supports were purchased from chemical manufacturing industries, while the others were synthesized. Alained in ,33. HydrCalculated amounts of the active metal were weighed and dissolved in 30 mL of distilled water and subsequently followed by the addition of the supports in separate crucibles. Each of the mixtures , in a separate crucible, was placed over a hot plate with the temperature set at 80 °C for 3 h with continuous stirring. Thereafter, the slurry was placed in a furnace for drying at 120 °C overnight. The dried samples were calcined at 550 °C for 3 h in the same furnace.The Brunauer–Emmet–Teller technique was used in calculating the specific surface area of the samples with the aid of a device that analyzes the surface area and porosity, i.e., a Micromeritics Tristar II 3020 . To perform nitrogen physisorption measurements, 0.20–0.30 g of the samples were weighed and subjected to degassing at 200 °C for three hours prior to the analysis.α radiation source and a nickel filter, operated at 40 kV and 40 mA. The step size and scanning range of 2θ for analysis were set to 0.01° and 5–80°, respectively. Standard powder XRD cards (JCPDS) were used to document the available phases.X-ray powder diffraction patterns for the catalyst samples were recorded with a Rigaku (Miniflex) diffractometer,  employing a Cu K2/Ar gas at 40 mL/min. A thermal conductivity detector (TCD) was used to observe the H2 consumption. Temperature-programmed desorption of carbon dioxide (CO2-TPD) and CO pulse chemisorption measurements were obtained using automatic chemisorption equipment (Micromeritics AutoChem II 2920) with a TCD. At the start, a 70 mg sample was heated at 200 °C for 1 h under a helium (He) flow to remove adsorbed components. Then, CO2 adsorption was carried out at 50 °C for 60 min in the flow of a He/CO2 gas mixture (90:10 volume ratio) at 30.0 mL/min. Afterwards, an increase in temperature up to 800 °C at 10 °C/min was registered as the CO2 desorption signal by the TCD.The reduction behavior of the fresh catalysts was determined with the AutoChem II (Micromeritics). A sample weight of 75.0 mg was analyzed. Heating of samples was carried out under pure Ar at 150 °C for half an hour, thereafter cooled to 25 °C. Afterwards, samples were heated to 1000 °C at 10 °C/min by allowing the flow of 10% HCarbon deposition over the used catalysts was measured by doing thermal analysis under atmospheric condition with the aid of TGA-51  equipment. A certain amount from the spent catalyst (10 mg) was subjected to heat treatment within the temperature range 25–1000 °C. The ramping temperature was maintained at 20 °C/min.2/He mixture at 40 mL/min. The spent catalyst was first pretreated in the presence of high-purity Argon at 150 °C for 30 min, and subsequently cooled to room temperature.TPO was performed in an oxidative atmosphere to determine the kind of carbon deposited over the surface of the catalyst using the Micromeritics AutoChem II over a temperature range of 50–800 °C, under the flow of a 10% OCatalysts’ morphology was studied using the JEOL JSM-7100F  field emission SEM.2 flow of 40 mL/min at 700 °C for 60 min. N2 gas was then let into the reactor for 20 min to remove the remnants of H2 while the system was maintained at the reaction temperature (700 °C). Thereafter, feed gases of CH4, CO2, and N2 were injected at the molar ratio of 6:6:1, respectively, at a 65 mL/min total flow rate. The temperature, pressure, and reaction variables were inspected through the reactor panel. A GC GC-2014 Shimadzu, unit having a TCD and two columns, Porapak Q and Molecular Sieve 5A, were connected in series/bypass connections in order to completely analyze the reaction products. The following equations were used to calculate the CH4 and CO2 conversions, respectively.The DRM was carried out in a stainless steel fixed-bed tubular micro-reactor (ID = 9 mm) at atmospheric pressure. The reactor system was procured from Process Integral Development (PID Eng. & Tech). Before the start of the DRM reaction, 0.10 g of catalyst was activated by a H4 and CO2 conversion. From the results of the investigation, 5%Ni/Al2O3(SA-6175) and 5%Ni/SiO2 gave the highest CH4 conversion, averaging about 78 and 75%, respectively. The same trend was observed for the CO2 conversion, with 5%Ni/Al2O3(SA-6175) having 84%, while 5%Ni/SiO2 had 82% conversion. CO2 conversion was observed to be higher than CH4 conversion for all the catalysts.In this research, Ni-based catalysts were synthesized using the wet impregnation method and tested for DRM. Different supports were used in the synthesis, aimed at obtaining support that would give the best metal–support interaction. The synthesized catalysts were evaluated in terms of CH2O3 (SA-5239), 5%Ni/SBA15, and 5%Ni/MCM41, relative to 5%Ni/Al2O3(SA-6175) and 5%Ni/SiO2. The TPO profile showed similar behavior among 5%Ni/Al2O3(SA-5239), 5%Ni/Al2O3(SA-6175), and 5%Ni/SBA15, which had a sharp single peak that appeared at different temperatures. The former two were mostly characterized by amorphous carbon, while the latter has a graphitic carbon deposit. Two distinct peaks were observed for 5%Ni/SiO2 and 5%Ni/MCM41. The carbon deposit in 5%Ni/SiO2 was mostly atomic, while that of 5%Ni/MCM41 was comprised mainly of amorphous carbon. 5%Ni/MCM41 had the lowest carbon deposit, while 5%Ni/Al2O3(SA-6175) had the highest deposit, probably due to its high activity, according to the TGA.Both fresh and spent catalysts were put through some characterizations. According to the BET method, the catalysts possess isotherms belonging to the type IV and V class, which are mesoporous in nature. The TPR profiles showed that Ni interacted weakly with the supports in 5%Ni/Al


Drosophila Antennapedia homeodomain protein, translocate various cargos including peptides and proteins across cellular barriers. This mode of delivery has been harnessed by our group and others to deliver antigenic proteins or peptides into the cytoplasm of antigen processing cells (APC) such as monocyte-derived dendritic cells (MoDC). Antigens or T cell epitopes delivered by CPP into APC in vivo generate antigen-specific cytotoxic T cell and helper T cell responses in mice. Furthermore, mice immunised with these peptides or proteins are protected from a tumour challenge. The functional properties of CPP are dependent on the various cargos being delivered and the target cell type. Despite several studies demonstrating superior immunogenicity of TAT and Antp-based immunogens, none has compared the immunogenicity of antigens delivered by TAT and Antp CPP. In the current study we demonstrate that a cytotoxic T cell epitope from the mucin 1 (MUC1) tumour associated antigen, when delivered by TAT or Antp, generates identical immune responses in mice resulting in specific MUC1 T cell responses as measured by in vivo CTL assays, IFNγ ELISpot assays and prophylactic tumour protection.Cell penetrating peptides (CPP), including the TAT peptide from the human immunodeficiency virus transactivator of transcription (HIV-TAT) protein and penetratin from  Fundamental to an effective vaccine is the delivery of antigens to antigen presenting cells (APC) and the ensuing processing and presentation of epitopes to cytotoxic T cells or helper T cells in the context of a relevant MHC haplotype to activate T cells and induce an immune response ,2. VacciDrosophila Antennapedia homeodomain are the two most widely investigated CPP [CPP offer a unique approach for the transport of peptides and proteins into the cytoplasm of APC. The TAT peptide (RKKRRQRRR) from the HIV transactivator of transcription protein and penetratin  from ated CPP . Using dated CPP . TAT, inated CPP ,21. We hated CPP ,15,16,18ated CPP . Yet to b CD8 8-mer epitope SIINFEKL from the model antigen ovalbumin (OVA) , or to the H-2Kb CD8 9-mer epitope SAPDTRPAP from the human tumour associated antigen mucin 1 (MUC1) . These studies showed that the tandem fusion peptide of Antp with SIINFEKL was immunogenic in mice, whereas TAT fused to SIINFEKL was not. In contrast, the immunogenicity of the MUC1 cytotoxic T cell epitope fused in tandem to either TAT or Antp CPP was identical.We have compared the efficiency of TAT and penetratin linked to either the H-2Kb and TATMUC1Kb at varying concentrations were added to DC2.4 cells and cell death was measured quantitatively by lactate dehydrogenase (LDH) levels, a stable cytosolic enzyme that is released upon cell lysis. Cells exposed to Triton-X-100 were used as a positive control. None of these peptide antigens induced detectable levels of cell death when used at up to 200 µg/mL (not shown).To establish the toxicity of Antp and TAT peptides on cells, AntpSIIN, TATSIIN, AntpMUC1KTo compare the processing and presentation of AntpSIIN and TATSIIN, BMDC (bone marrow derived dendritic cells) were pulsed with varying peptide concentrations then incubated with B3Z T cells for 18 h. The recognition of the SIINFEKL epitope on the MHC class I molecule by its specific TCR was assessed via a colorimetric assay. Untreated DC and DC with Antp or TAT were used as negative controls. DC pulsed with AntpSIIN strongly presented SIINFEKL to B3Z T cells . Surprisin vivo, mice were immunised with 25 µg AntpSIIN or TATSIIN and 20 h later were injected i.v. with purified OT-1 splenocyte T cells labelled with CFSE. 60 h after i.v injection, CD3+ splenocyte T cells were analysed by flow cytometry for antigen specific proliferation. Mice immunised with AntpSIIN demonstrated substantial T cell proliferation in vivo, seen as a sequential diminution of CFSE fluorescence in the daughter cells. However mice immunised with TATSIIN showed no proliferation greater than the control group , incorporating a linker of 4 amino acids preceding SIINFEKL in the native OVA sequence, was used in the B3Z assay. When the peptide was incubated with DC2.4 cells in vitro it was processed and stimulated the SIINFEKL-specific B3Z T cells. A control peptide (PEPCD8) of equal length to TATSIIN, incorporating only the amino acids preceding SIINFEKL in the native OVA sequence, was not efficiently presented to B3Z cells by DC2.4 cells . Both AntpMUC1Kb and TATMUC1Kb displayed similar uptake kinetics with rapid internalisation seen within 5 min which remained constant for at least 360 min  depletes ATP-dependent mechanisms in the cell and blocks cell membrane activity while Cytochalasin D (10 µg/mL) affects the contraction of actin containing microfilaments and blocks phagocytosis. Both Cytochalasin D and NaN3/2-deoxyglucose blocked the uptake of both AntpMUC1Kb and TATMUC1Kb by DC in vitro  or TATMUC1Kb (83% ± 2%), with no difference in efficacy observed (p > 0.05).The capacity of C57BL/6 mice to generate SAPDTRPAP antigen specific killing rol mice A. In conin vivo immune responses, the capacity of AntpMUC1Kb and TATMUC1Kb antigens to induce CD8+ T cell responses in vivo was determined using IFN-γ ELISpot analysis. C57BL/6 mice were immunised i.d. with 25 µg AntpMUC1 Kb or TATMUC1Kb on days 0, 10 and 17 and IFN-γ was measured 14 days later. Mice immunised with either AntpMUC1Kb or TATMUC1Kb generated equally strong IFN-γ secreting cell responses to the SAPDTRPAP (MUC1 Kb) epitope .The capacity of AntpMUC1Kb or TATMUC1Kb immunisation confers tumour protection in vivo, C57BL/6 mice (n = 8) were injected i.d. with PBS, 25 µg AntpMUC1Kb or 25 µg TATMUC1Kb on days 0, 10 and 17. Seven days later, mice were challenged s.c. with 2 × 105 B16-MUC1 tumour cells. Immunisation of mice with either AntpMUC1Kb or TATMUC1Kb delayed tumour growth . In addition 4 of 8 AntpMUC1immunised mice and 3 of 8 TATMUC1 immunised mice were tumour free at day 28.To assess whether AntpMUC1Kr growth . On day et al. were the first to demonstrate that immunisation with DCs pulsed with TAT peptide conjugated OVA induced antigen specific CTLs in vivo, whereas DC pulsed with full length OVA protein failed to promote antigen specific cytotoxicity [in vivo against the growth of the OVA expressing tumour cell line E.G7-OVA [+ specific IFN-γ responses to immunisation with AntpSIIN alone or AntpSIIN pulsed DC, and protection against the growth of OVA expressing tumours. Moreover, similar IFN-γ responses were observed following a single immunization [Peptide and protein based vaccines based solely on CTL epitopes have limited efficacy, as APC are not efficient in the uptake and processing of exogenous antigens via the MHC class I pathway ,2,3. Thutoxicity . SeveralE.G7-OVA . Additionization . Recentlnization .b CTL epitope from the model antigen OVA SIINFEKL or the MUC1Kb SAPDTRPAP epitope linked in tandem to the TAT peptide or to Antp. When linked to the TAT peptide, SIINFEKL (TATSIIN) failed to be processed and presented to T cells either in vitro or in vivo  peptide and furin sensitive (RVKR) or furin insensitive (VRVV) linkers to multiple CTL epitopes [It was interesting that the TATSIIN peptide, sequence RKKRRQRRRSIINFEKL without a linker between TAT and SIINFEKL, was not processed and presented by DC to T cells epitopes . Furin iepitopes . To demob, AntpMUC1A2 and AntpMUC1FP peptide and proteins into the cytoplasm of peritoneal macrophages, whereas proteins and peptides without the internalising sequence were not efficiently taken up [The uptake and intracellular processing pathways of Antp and TAT, when linked to a single CTL epitope, has been extensively characterised. Using primary bone marrow-derived mouse DCs, AntpSIIN was shown to be endocytosed in an ATP dependent manner with the involvement of negatively charged receptors. Further investigation revealed the majority of peptide was taken up via phagocytosis and/or macropinocytosis in a caveolae independent manner . Examinataken up . Flow cyb or TATMUC1Kb peptides generated potent cellular responses, measured by in vivo CTL killing and IFN-γ ELISpot assays, however neither AntpMUC1Kb nor TATMUC1Kb immunisation induced an antibody response  generated strong immune responses. Fusion of the MUC1Kb SAPDTRPAP epitope to either TAT or Antp demonstrated that both are rapidly and efficiently internalised by BMDC in an ATP dependant manner. Furthermore, mice immunised with either AntpMUC1Kb or TATMUC1Kb generated strong in vivo IFN-γ T cell responses and showed delayed B16-MUC1 tumour growth. Most importantly, these studies indicated that TAT and Antp both function equivalently for delivery of cytotoxic T cell epitopes to APC, provided a suitable linker is used.In conclusion, this study demonstrated that the TAT protein linked to SIINFEKL, without an intermediate linker sequence, failed to be processed and presented to T cells either Peptides  were synC57BL/6 and OT-I mice, aged 6–10 weeks, were purchased from the Biological Research facilities of the Walter and Eliza Hall Institute . All mice were housed in the facilities at Burnet Institute (Heidelberg Campus) or RMIT University.4) were seeded in a volume of 100 µL complete RPMI media (10% (v/v) heat inactivated fetal calf serum, 4 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin sulphate and 100 µM β-mercaptoethanol) in a flat bottom 96-well microtitre plate and allowed to adhere overnight at 37 °C. Next day media was removed and replaced with 200 µL complete RPMI with peptides at specified concentrations. Cells were incubated for 24 h at 37 °C and subsequently 100 µL media was removed to determine cytotoxicity with the CytoTox 96 Non-Radioactive Cytotoxicity Assay according to manufacturer’s instructions . 50 µL/well of reconstituted substrate mix was added to the enzymatic assay plate containing samples and incubated for 30 min at RT. 50 µL/well of stop solution was added and absorbance read at 490 nm.DC2.4 cells  to lyse erythrocytes. Cells were washed and cultured at 5 × 105 cells/mL in 24 well plates with complete RPMI-1640 medium with 10 ng/mL of recombinant mouse granulocyte macrophage colony-stimulating factor (GM-CSF) . At day 6, cells were observed to be >80% CD11c+ by flow cytometry.Bone marrow cells from C57BL/6 female mice were collected by flushing the tibias of hind legs and treated with ACK lysis buffer  for 30 min at 4 °C. Cells were washed and FITC-anti-mouse (Fab’)2  was added in BSA/PBS for a further 30 min at 4 °C. For intracellular staining, cells were fixed with 2% paraformaldehyde for 10 min at room temperature, washed and permeabilised with 0.25% v/v saponin/PBS for 10 min. Cells were then stained as above in saponin/PBS. Kinetics of uptake was also assessed by adding peptides at a fixed concentration for specified times (5 to 360 min) at 37 °C. DC were resuspended in PBS and analysed by flow cytometry . Results are expressed as percent intracellular stain–percent surface stain. Isotype controls were used to measure negative control (background) staining.Day 6 cultured C57BL/6 BMDC were pulsed with 5, 20, 100 or 200 µM peptides for 1 h at 37 °C in serum free media. Due to cell fixation causing artifactual uptake of CPP peptides, all uptake experiments were performed by measuring surface and intracellular expression by flow cytometry, with results expressed as percent intracellular stain–percent surface stain. For surface staining, DC were washed with 0.5% 3)  and 2-deoxyglucose (2DG) (Sigma) or 10 µg/mL cytochalasin D (Sigma), which blocks phagocytosis, for 45 min at 37 °C. Cells were then pulsed with AntpMUC1Kb or TATMUC1Kb for 60 min at 20 µg/mL at 37 °C. Uptake of antigen was measured as described in BMDC were pre incubated with and without biochemical inhibitors; 10 mM sodium azide (NaNEscherichia coli lacZ reporter gene linked to the nuclear factor of activated T cells. Recognition of the SIINFEKL peptide in the context of class I by the T-cell receptor (TCR) results in activation of the enzyme and conversion of a chromogenic substrate that can be measured by absorbance spectrophotometry. DC (2 × 105 cells) were pulsed with peptides at various concentrations in 96-well microtitre plates  for 24 h at 37 °C. Cells were then washed and 105 B3Z cells were added for 18 h at 37 °C. Next day, cells were washed with sterile PBS and incubated with chlorophenol red-β-galactoside  . After 4 h incubation at 37 °C the absorbance was read at 560 nm to detect chlorophenol red released by β-galactosidase.The B3Z mouse T-cell hybridoma line contains a gene construct of w/v BSA/PBS and labelled with 5 μM carboxyfluorescein succinimidyl ester (CFSE) (Molecular Probes) for 10 min at 37 °C. Labelling was stopped with a 5× volume of ice cold complete RPMI and cells were washed extensively in PBS. CFSE labeling was confirmed by flow cytometry. 107 CFSE labelled OT-I T cells were then injected intravenously (i.v.) in 200 µL PBS into C57BL/6 mice immunised i.d. at the base of the tail 20 h prior. Splenocytes were collected 60 h following CFSE injection and stained with APC-conjugated anti-CD3 (BD Pharmingen) for 30 min at 4 °C in 2% FCS/PBS. Cells were washed and CFSE dilution was determined by flow cytometry and analysed by Weasel curve fitting software .Splenocytes from OT-I mice were isolated and purified as described in section 4.4. Purified OT-I T cells were resuspended in 0.1% 7 cells/mL in serum free RPMI and divided into 2 populations, pulsed or unpulsed. Pulsed splenocytes were incubated with 1 µg/mL SIINFEKL or SAPDTRPAP peptide for 1 h at 37 °C. Cells were washed and resuspended in 0.1% w/v BSA/PBS and pulsed splenocytes were labelled with a high concentration of CFSE (5 µM) whereas unpulsed splenocytes were labelled with a concentration of CFSE (0.5 µM) for 10 min at 37 °C. Labelling was stopped with 5× volumes of ice cold complete RPMI and cells were washed extensively in PBS.Splenocytes from naïve C57BL/6 mice were isolated and resuspended to 106 CFSEhigh cells and 5 × 106 unpulsed CFSElow cells were mixed and a total of 107 CFSE labelled cells in 200 µL PBS was injected i.v. into mice immunised 8 days prior or into naïve mice. After 20 h, splenocytes were isolated and analysed by flow cytometry.Peptide-pulsed 5 × 10Specific lysis was calculated as:5/well) in 100 µL of complete medium were incubated with 20 µg/mL recall antigens for 18 h in IFN-γ ELISpot. Recall antigens were SIINFEKL , ISQAVHAAHAEINEAGRKG , OVA and SAPDTRPAP (MUC1Kb). Concanavalin A (ConA) (1 µg/mL) or cells alone were used as positive and negative controls respectively. Triplicate wells were set up for each condition. Cells were discarded after washing (PBS) and 1 µg/mL biotinylated anti-mouse IFN-γ antibody (Mabtech) was added for 2 h at RT. The plates were washed with PBS and 1 µg/mL streptavidin-alkaline phosphatase (Mabtech) was added at room temperature for 2 h. Spots of activity were detected using a colorimetric AP-conjugate substrate kit . Cytokine spots were counted with an AID ELISpot Reader system . Data were presented as mean spot-forming units (SFU) per 5 × 105 cells ± standard error of the mean (SEM).To determine the effector immune response, splenocytes from C57BL/6 mice immunised i.d. on days 0, 10 and 17 were isolated 14 days after final immunisation and assessed by ELISpot for IFN-γ secretion. MultiScreen filter plates  were coated with 70 µL 5 µg/mL anti-mouse IFN-γ antibody (AN18)  overnight at 4 °C. Plates were washed six times with sterile PBS and blocked with 200 µL complete RPMI media for 2 h at 37 °C. Spleen cells , non-specific binding was blocked with 2% BSA/PBS for 1 h at RT. Serial dilutions of sera were added (in 2% BSA/PBS) and incubated for a further 2 h at RT. Plates were washed and bound antibody was detected using horseradish peroxidase-conjugated sheep anti-mouse IgG . Plates were washed as described above and HRP-streptavidin was added for 1 h at RT. Responses were detected with TMB substrate solution and stopped with 1 M HCl. Absorbance was read at 450 nm.The MUC1 peptide corresponding to a single VNTR repeat of MUC1, Cp13-32 (C-PAHGVTSAPDTRPAPGSTAP), was coated onto PVC microtiter plates at 10 µg/mL in 0.2M NaHCO5 MUC1-B16 melanoma cells suspended in 100 µL PBS. Expression of MUC1 on MUC1-B16 cells was confirmed by flow cytometry prior to challenge. The growth of tumours was monitored by measuring the two perpendicular diameters using callipers and the results were expressed as their product.Groups of 8 C57BL/6 mice were injected intradermally (i.d.) at the base of tail with 25 µg of various peptides or PBS on days 0, 10 and 17. Seven days following the last immunisation, mice were shaved on the abdominal area and challenged subcutaneously (s.c.) with 2 × 10p < 0.001 to indicate a highly significant difference, and p < 0.05 to indicate a significant difference.Assays were set up in triplicate. Mean values were compared using the two-tailed unpaired t-test and ANOVA. Two p-value thresholds were used for protection and immunogenicity assays:


Malaria transmission-blocking anti-malarial drugs, such as primaquine, offers an effective strategy for reducing the incidence of falciparum malaria. However, this drug induces haemolytic anaemia among glucose-6-phosphate dehydrogenase (G6PD) deficient individuals. The distribution of G6PD deficiency in Brazzaville, Republic of Congo and the association of G6PD deficiency with haemoglobin levels and blood cell counts were investigated.Plasmodium falciparum diagnosis was conducted by microscopy and nested PCR. Sanger sequencing was used to assess G6PD deficiency by detecting 202G>A (rs1050828) and 376A>G (rs1050829) single nucleotide polymorphisms.A total of 212 febrile children were recruited for this study. p < 0.05).Two hundred and twelve children were successfully genotyped for G6PD variants. Overall, 13% (27/212) of the children were G6PD deficient and 25% (25/100) females were heterozygous (11 BA− and 14 A+A−). The remaining 160 children had a normal G6PD genotype. The mean red blood and mean platelet counts were significantly lower in hemizygous male (G6PD A−) participants than in normal male (G6PD A+ or B) participants (This study gives an update on G6PD deficiency among Congolese children. Understanding the distribution of G6PD deficiency in other geographical regions is recommended before primaquine is adopted in the malaria control programme. Glucose-6-phosphate dehydrogenase (G6PD) is the first enzyme of the pentose phosphate pathway  and the The geographical distribution of the various G6PD variants, as defined by the G6PD SNP (s) present, varies extensively. The most prevalent variants include G6PD B , G6PD A+ (moderately deficient) and G6PD A− (severely deficient). Of this, G6PD B, the wild-type variant, is the most common variant globally . G6PD A+Plasmodium falciparum uncomplicated malaria in Africa [The prevalence of G6PD deficiency is highly heterogeneous with the prevalence in the tropics ranging from for 3% to 35% . Globalln Africa .Plasmodium vivax and Plasmodium ovale relapse provided the G6PD status is known with exception of pregnant women, children < 6 months years and women breastfeeding children below 6 years old [Even though G6PD deficiency is largely asymptomatic, acute haemolytic anaemia can be triggered among G6PD deficient individuals by an infection, ingestion of fava beans or certain drugs , 13. Oneears old .P. falciparum transmission to the vectors [P. falciparum gametocytes offers an attractive strategy for reducing malaria transmission in the Republic of Congo and other malaria-endemic areas [In the context of falciparum malaria control, a single dose of PQ in combination with artemisinin-based combination therapy (ACT) is recommended for reducing  vectors , 15. Higic areas , 17. HowPlasmodium falciparum is the predominant cause of Plasmodium species infection [This study was conducted in Brazzaville, the capital of the Republic of Congo. Malaria transmission is perennial in this area. nfection , 19. Twonfection . The incPfmsp1) was used to genotype P. falciparum isolates as described elsewhere [P. falciparum infection.The QIAamp DNA blood Mini Kit  was used to extract genomic DNA from archived EDTA blood following the manufacturer’s instructions. DNA samples were stored at − 20 °C. The merozoite surface protein 1 gene (lsewhere . This waG6PD) as described by Nguetse et al. [2, 200 µM dNTPs, 100 Nm of each primer and 1U Taq DNA polymerase. The primers (forward primer 5′-GCCCCTGTGACCTCCCGCCA-3′ and reverse primer 5′-GCAACGGCAAGCCTTACATCTGG-3′) used here amplified a G6PD fragment containing the 202G>A (rs1050828) and 376A>G (rs1050829) single nucleotide polymorphisms. The thermal cycler conditions were 94 °C for 5 min followed by 30 cycles at 94 °C for 30 s, 67 °C for 1 min and 72 °C for 1 min and a final extension step at 72 °C for 5 min. The amplicons were separated by gel electrophoresis using a 1.2% agarose gel. The PCR products were purified using Sephadex™ G-50 fine DNA grade  and subsequently Sanger sequenced by the 3130 xl Genetic Analyzer  in the forward and reverse direction using the foregoing primers and the BigDye Terminator v.1.1 cycle sequencing kit . The BioEdit software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html) was used to align the forward and reverse sequences using G6PD (NG_009015.2) as a reference, visually inspect the DNA sequence chromatograms to resolve discordant base calls and identify alleles G and/or A at position 202 and alleles A and/or G at position 376 of G6PD.A single-step PCR approach was used to amplify a 968 bp fragment of the G6PD gene  samples for analyses and failure to obtain nucleotides sequences despite repeat attempts, respectively. The baseline characteristics of the participants are shown in Table p < 0.05). In contrast, the presence or absence of P. falciparum infection did not associate with G6PD genotype (p > 0.05) of male and female febrile children (Table p < 0.05). No significant differences were found in the different female G6PD genotype groups.Sanger sequencing was used to determine the G6PD genotype and infer the G6PD phenotype of the participants based on the allele (s) present at nucleotide positions 202G>A (rs1050828) and 376A>G (rs1050829), as described in “en Table . The meaG6PD deficient alleles (SNPs) and the hemizygous nature of the X-chromosome in males.This study investigated the prevalence of G6PD deficiency in febrile children from Brazzaville, Republic of Congo. This study shows that 13% of the children were carriers of the severe G6PD deficiency genotype . This finding is consistent with studies conducted in the other Central African countries where the prevalence of G6PD deficiency ranges from 9 to 22% . PreviouG6PD deficiency has a heterogeneous impact on the severity of malaria. Ruwenda et al. and Nguetse et al. showed that G6PD deficient males and heterozygous females were less susceptible to severe malaria , 22. A sp > 0.05).A similar heterogeneity has also observed in the protection of G6PD deficiency against uncomplicated malaria. High incidence of uncomplicated malaria was observed in heterozygous females and hemizygous males with G6PD A− compared to participants with the wild-type genotype . Other sAnalysis of the association between haemoglobin (Hb) levels and G6PD deficiency in this study showed that mean Hb levels were similar in G6PD deficient children, G6PD heterozygous female children and G6PD normal children. This is congruent with findings from studies conducted in Angola, Ghana and Burkina Faso , 35. LowG6PD SNP(s). PQ is highly efficacious against P. falciparum gametocytes [P. falciparum transmission [G6PD deficiency is also an important factor to be considered prior to the administration of some drugs. This is because G6PD is a key enzyme involved in the production of the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) required by erythrocytes to withstand oxidative stress . Primaquetocytes , 17 and smission , 15. Of Taken together, this study provides an update on G6PD deficiency in children from Brazzaville, Republic of Congo. Understanding the distribution of G6PD deficiency in other geographical regions is recommended to inform the use of malaria intervention(s) such as primaquine that induces acute haemolytic anaemia in G6PD deficient individuals.


A high Ni(II) adsorption capacity of 35.50 mg·g-1 in 100 ppm Ni(II) solution with 10 mg modified sewage sludge biochar (MSBC) was achieved. The adsorption kinetic and isotherm were fitted well by the pseudo-second-order model and the Langmuir model, respectively. The optimal pH was found around a neutral pH of 7. The adsorption mechanisms of Ni(II) onto MSBC were described as the synergistic effects of electrostatic attraction, ion exchange, inner-sphere complexation and co-precipitation. The initial rapid adsorption phenomenon could be attributed to electrostatic attraction and ion exchange, and then inner-sphere complexation and co-precipitation acted as a crucial role in the following step. The remarkable performance of MSBC provides an effective waste utilization approach to simultaneous sewage sludge recycle and Ni removal from aqueous solution.A novel approach was employed to load α-Fe Nickel (II) ion (Ni (II)), a toxic heavy metal ion, is discharged into natural waters mainly from various chemical industries, including textile dyeing, electroplating, and metal finishing . Once thSo far, numerous attempts were made to eliminate Ni contamination, including chemical precipitation , phytore2O3 [2 existed on the surface of α-Fe2O3 [2+ was found possibly via the formation of Fe-O-Cu complexes [2O3 and α-FeOOH are potential for enhancing the Ni adsorption from aqueous solution like Cu2+. Until now, no systematic study concerning the adsorption performance of biochar with α-Fe2O3 and α-FeOOH in Ni (II) removal from aquatic environment has been published yet. To develop sewage sludge biochar for efficient Ni removal, we prepared sludge biochar (SBC) and then modified sludge biochar (MSBC) with α-Fe2O3 and α-FeOOH for Ni adsorption in aqueous solution. BET, XRD, SEM-EDS and FTIR were employed to characterize the prepared biochar. Dosages and pH were tested for the influences of adsorption parameters. In addition to the adsorbent regeneration, sorption kinetics and isotherms were assessed for the adsorption mechanisms in aqueous solution. This study could provide an efficient approach for simultaneously Ni removal and sludge application.In recent years, special attention was paid to α-Fe2O3 , 22 and 2O3 , 24 modi2O3  and it w α-Fe2O3 . In addiomplexes , indicat3·6H2O, ≥99%), iron(II) sulfate heptahydrate , Ni nitrate(≥99%), sodium hydroxide (≥96%) and hydrochloric acid (36%) were purchased from Sinopharm Chemical Reagent Co., Ltd. . All reagents used in the experiments were of analytical grade and solutions were prepared with deionized water. The sewage sludge was provided by Tangxunhu Municipal Wastewater Plant in Wuhan, China. The obtained sludge was oven dried at 60 °C for 24 h and then went through slow pyrolysis at 500 °C with a heating rate of 10 °C·min-1 for 2 h to produce the sludge biochar (SBC). The obtained SBC was milled and then sieved (<0.074 mm) for further experiments.Iron(III) chloride hexahydrate (FeCl3·6H2O (40 g) and FeSO4·7H2O (22.2 g) were dissolved in 1200 mL deionized water and then 30 g SBC was added into the solution. The mixture was stirred at room temperature (25 ~ 30 °C) for 30 min. Subsequently, 10 M NaOH solution was added into the mixture until the pH reached 10 and stirred for 2 h. After precipitation, the mixture was filtrated and the residual was washed with deionized water and ethanol several times and dried at 70 °C for 24 h. The obtained MSBC powder (0.074 mm) was kept for further experiments.The modified sludge biochar (MSBC) was fabricated based on SBC with the following procedure. FeCl-1 region. The software named OMNIC was applied to analyze the FTIR spectra. The surface morphologies of the powder samples were analyzed by a JSM-7100F SEM .The surface area, pore width and pore volume of SBC and MSBC were measured with a Micromeritics ASAP 2020 analyzer (USA). The crystalline structure analysis was conducted with D8 X-ray diffraction spectra . Fourier-transform infrared spectroscopy (FTIR) spectra were obtained with IRprestige-21  using the KBr pellet method and examined in the 4000–400 cm2EDTA solution. The Ni loaded MSBC was soaked in 0.1 M Na2EDTA solution and shaken for 2 h. And then, the Ni-desorbed MSBC was employed for further recycle experiments. The concentrations of Ni(II) solutions were measured by a 700P atomic absorption spectroscopy . The amount of adsorbed Ni(II) was calculated according to a previous study [To measure the adsorption capacities of SBC and MSBC adsorbents, 10–100 mg of these adsorbents were added to 50 ml of Ni(II) solution (100 ppm) at 25 °C for 24 h. Various pH values (2–7) were set to find the optimal pH for Ni(II) removal. The pH was adjusted to the desired values by using 0.01 M NaOH and HCl. Kinetic experiments were conducted by adding 50 mg MSBC adsorbent to 50 ml of Ni(II) solution (100 ppm) at 25 °C for various time intervals . The vials were sealed and shaken in a constant temperature shaker for 18 h to obtain the adsorption equilibrium. Correspondingly, an adsorption isotherm study was conducted by adding 50 mg of MSBC with 50 ml of Ni(II) solution (20–1000 ppm) at 25 °C. Finally, the desorption experiment was carried out using Naus study . SPSS 19BET). As reported in previous studies, SBET is critical for the adsorption of heavy metals including Ni [BET of MSBC (34.99 m2·g-1). The higher SBET of MSBC was of significant benefits to the adsorption capacity for Ni removal , which were in line with the data in a previous study [2 could be reasonably considered as the dominating crystalline phase in both of SBC and MSBC. Furthermore, several peaks at 27.91°, 34.79°, 39.12°, 40.25°, 45.90°, 54.95° and 19.80°, 36.50°, 55.80°, 60.00° for MSBC revealed the formation of α-Fe2O3 (PDF#16–0653) and α-FeOOH (PDF#26–0792), respectively. The formation processes could be described by reactions +. According to pzc values of biochar were found within the range of 7.3–7.8 [The increase from pH 2 to pH 7 caused the increase in the adsorption capacity of Ni(II) by both SBC and MSBC . The varsolution . It was solution . Therefo biochar . A previ biochar . Thus st 7.3–7.8 . Overallt represents the adsorbed amount of Ni(II) at a certain time. The adsorption amount of Ni(II) gradually increased and reached an equilibrium level within 18 hours. It was noticed that the adsorption of Ni(II) achieved nearly 50% of the maximum adsorption capacity at 100 ppm concentration of Ni(II) within 2 min. Several previous studies reported the similar adsorption process when biochar was utilized to adsorb heavy metal ions [1 represents the pseudo-first-order rate constant (min−1), k2 is the pseudo-second-order rate constant (g/mg·min−1) of adsorption, qe and qt are the amounts of Ni(II) adsorbed on MSBC (mg·g-1) at equilibrium and at time t, respectively.The adsorption kinetics of MSBC on Ni(II) removal are shown in tal ions . The raptal ions . The psee-qt) versus time (t) for the pseudo-first-order kinetic model did not yield good convergence (R2 0.9435). In this regard, the correlation coefficient (R2 = 0.9995) of the pseudo-second-order model was remarkably higher than that of pseudo-first-order model. Furthermore, the equilibrium adsorption amount (qe) of the pseudo-second-order model (24.45 mg·g-1) was close to the experiment data (24.55 mg·g-1). Therefore, the kinetic analysis revealed the chemical adsorption of Ni onto MSBC was the dominating mechanism.The above parameters were obtained through curve fitting and presented in e). Once the Ce reached above 800 ppm, no further increase was observed for Qe. The Langmuir and Freundlich models, frequently used in various adsorption studies [2 value of 0.9930, while that of the Freundlich model was 0.9821. Actually, the adsorption path of the Langmuir model was believed to be monolayer sorption on a homogeneous sorption surface [2 value of the Langmuir model was slightly higher than that of the Freundlich model. The calculated values of Ni(II) adsorption capacity were 35.97 mg·g-1 (Qm) and 10.50 mg·g-1 (KL), respectively for the Langmuir and Freundlich models. Comparing to the adsorption capacity of MSBC  from the saturated biochar with less negative effects[2EDTA was employed as a desorption agent. The adsorption and desorption efficiencies of MSBC for five cycles are shown in The regeneration capability of the biochar is vital for further application from an economic point of view. Desorption attempts could be applied to recycle the metal ions and regenerate the saturated adsorbents. Thermal activation and acid solution were reported as regeneration methods based on previous literatures , 44. Hige effects. In this+, Mg2+ and Ca2+ were found after the adsorption treatment, convincingly demonstrating the ion exchange between Ni(II) and aforementioned metal ions. Comparison analysis was achieved between the FTIR spectra of MSBC before and after Ni(II) adsorption . In addition, the peak at 602 cm-1 disappeared after adsorption, which suggested the involvement of Fe-OH. The interaction between α-FeOOH and Cu2+ was found possibly via the formation of Fe-O-Cu complexes [3(PO4)2 (PDF#38–1473) and Ni(OH)2 (PDF#38–0715) co-precipitations were identified by XRD analysis with the typical 2 theta of 24.12°, 30.03°, 38.20°, 53.17°, 63.89° and 22.61°, 45.83°, 59.93°, 61.32°, respectively (3(PO4)2 and Ni(OH)2 may be due to reactions (3(PO4)2.The mechanism of Ni(II) adsorption on adsorbents was believed to be the synergistic effect of physical and chemical adsorptions . The higsorption . Signifi α-Fe2O3 . Accordi α-Fe2O3 . Meanwhiomplexes . The forectively . The foreactions  in MSBC In conclusion, Ni adsorption was mainly attributed to electrostatic attraction, ion exchange, inner-sphere complexation and co-precipitation. Fast adsorption in the first several minutes was due to electrostatic attraction and ion exchange. And then the crucial mechanisms were inner-sphere complexation and co-precipitation.2O3 and α-FeOOH particles onto the surface of SBC. The maximum Ni adsorption capacity of MSBC reached 35.50 mg·g-1. The adsorption process could be fitted by the pseudo-second-order model and adsorption isotherm was well described by the Langmuir model. The initial rapid adsorption phenomenon could be attributed to electrostatic attraction and ion exchange, and then inner-sphere complexation and co-precipitation acted as a crucial role in the following step. Although the mechanism of Ni(II) adsorption was believed to be the synergistic effect of physical and chemical adsorptions, the dominant role was proved to be chemical adsorption. The results in this study indicate that MSBC could act as an excellent adsorbent for Ni removal.Modified sewage sludge biochar (MSBC) was technically produced by distributing both α-FeS1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S1 Data(ZIP)Click here for additional data file.


Many non-coding and regulatory RNA elements have evolved to exploit transient or metastable structures that emerge during transcription to control complex folding pathways or to encode dynamic functions. However, efforts to engineer synthetic RNA devices have mostly focused on the thermodynamically stable structures. Consequently, significant challenges and opportunities exist in engineering functional RNAs that explicitly take advantage of cotranscriptionally generated transient or metastable structures. In this work, we designed a short RNA sequence that adopts a robust metastable structure when transcribed by an RNA polymerase. Although the metastable structure persists for hours at low temperature, it refolds almost completely into the thermodynamically stable structure upon heat denaturation followed by cooling. The synthetic RNA was also equipped with the Broccoli aptamer so that it can bind its ligand and become fluorescent only in the thermodynamically stable structure. We further demonstrated that the relaxation to the thermodynamically stable and fluorescent structure can be catalyzed by a short trigger RNA in a sequence-specific manner. Finally, the RNA architecture was redesigned to sense and respond to microRNA sequences. In summary, we designed RNA nanomachines that can detect an RNA sequence, amplify signal and produce an optical output, all encoded in a single RNA transcript, self-powered by a metastable structure. As RNA is transcribed in the 5′ to 3′ direction, local secondary structures may form cotranscriptionally which can affect the global folding pathway and kinetics . While translation is still suppressed in plasmid-containing cells due to the Sok-RNA, those cells that lack the plasmid  express Hok and are killed.In fact, natural RNA elements that actively exploit such cotranscriptionally generated structures have been reported. For example, the splicing . In anotsplicing . This issplicing . Similar Sok-RNA ,7. As thTetrahymena ribozyme), or sophisticated and dynamic regulation of RNA functions (as in the cases of MS2 and hok/sok systems).These and other observations ,8,9 suggin vitro , but after heat denaturation and cooling, the RNAs equilibrate equally to the two structures with similar thermodynamic stability ..in vitroAnother interesting example from the Sugimoto group studied the relaxation of a cotranscriptionally formed hairpin structure to a G-quadruplex (G4) using fluorescent probes Figure  11). Th. Th11). In this work, we sought to design more sophisticated functions that fully exploit the metastability of cotranscriptionally generated RNA structures. First, we aimed to design a robust metastable structure that can be generated simply by transcription. Second, we wanted the system to fully relax to a thermodynamically stable structure upon appropriate triggers. Finally, the designed RNA should exhibit a prototypical function beyond structural transformation.2, 1 mM dithiothreitol (DTT), 2 mM spermidine, 100 mM KCl.Enzymes, ribonucleotide triphosphates (NTPs) and gel loading buffers were purchased from New England Biolabs (NEB) unless noted otherwise. DFHBI-1T was purchased from Lucerna. All other chemicals used were of molecular biology grade or equivalent, or of the highest quality available, and purchased from Nacalai Tesque or Wako Pure Chemical Co. The 1 × reaction buffer used in this work has the following composition: 40 mM Tris–HCl, pH 7.9, 10 mM MgClIn vitro transcription was performed in a volume of 20 μl containing the 10 × supplied buffer (2 μl), 12.5 mM (each) NTP mix (3.2 μl), 40 mM MgCl2 (1 μl), RNase Inhibitor, Murine (0.8 μl), DNA template (400 ng) and T7 RNA polymerase (1 μl). The reactions were kept at 37°C for 3 h. SK-miR-21 was prepared similarly except with 200 ng DNA template, without additional MgCl2, and 1 h reaction time. After in vitro transcription, DNase I (2.5 μl) was directly added and incubated at 37°C for 15 min to remove DNA template. RNAs were then purified by column , and concentrations were calculated based on absorbance at 260 nm.Linear dsDNA templates that encode the T7 promoter sequence followed by the desired transcripts  were prewMT8 RNA samples  were mixed with RNA Loading Dye, (2×) and heated to 95°C for 3 min followed by cooling on ice. The samples were separated on an 8% polyacrylamide gel (acrylamide: bis-acrylamide 19: 1) in Tris-borate-EDTA (TBE) and 8 M urea at 40 V/cm for 25 min. The gel was stained with SYBR Gold (Invitrogen) and imaged by LuminoGraph II (ATTO).2O, the samples were diluted with 1 × reaction buffer to 50, 100 or 200 ng in 4.6 μl. The samples were mixed with 4.6 μl of 2× native gel loading buffer , no sodium dodecyl sulphate (NEB) and 50% sucrose) and gently loaded onto the precooled gel. The RNAs were separated at 38 V/cm for 25 min at 4°C and was stained with SYBR Gold and imaged by LuminoGraph II.An electrophoresis cell, native 8% acrylamide gel (acrylamide: bis-acrylamide 19: 1), and 1 × TBE buffer were cooled at 4°C for at least 1 h before sample loading. wMT8 RNA was transcribed and column purified as described above. Two 9 μl aliquots of wMT8 RNA (430 ng) diluted in 1.11 × reaction buffer were prepared. One aliquot was heated to 75°C for 5 min and slowly cooled to 37°C using a thermal cycler while the other aliquot was kept on ice. After adding 1 μl HColumn purified metastable RNAs (0.5 μM) and DFHBI-1T (5 μM) were diluted in the reaction buffer. An aliquot of each solution was renatured by heating to 75°C for 5 min and slowly cooling to 37°C in a thermal cycler. RNA samples (10 μl) were transferred to a 386-well black microplate and fluorescence was measured by an Infinite M1000 PRO microplate reader (Tecan) with excitation at 469 nm, emission at 501 nm and 5 nm bandwidth. The samples were stored at 4°C for 17 h and measured similarly.Column purified metastable RNA (1.11 μM) was diluted in 1.11 × reaction buffer with DFHBI-1T (11.1 μM). Nine microliters of the RNA/DFHBI-1T mixture was mixed with 1 μl of a 10 × trigger RNA solution (0–1 μM) in a PCR tube. For the positive controls representing the 1 μM fully thermodynamically stable structures, the metastable RNA (1 μM) was mixed with 1.5 μM each of the stem III binding oligonucleotides  and renawMT8 DNA template solution , 2.5 × reaction buffer (4 μl), 25 mM (each) NTP mix (0.8 μl), 50 μM DFHBI-1T (2 μl), RNase Inhibitor, Murine (0.4 μl), and T7 RNA polymerase (0.5 μl) were mixed on ice . To this solution was added 1 μl of 500 nM c4+8 or c4+8sc solution. Water (1 μl) was added for the uncatalyzed reaction. Broccoli-DFHBI-1T fluorescence of these samples were monitored at 37°C using a real-time PCR instrument .We based our initial design Figure  on the bWe started with a design shown in Figure in vitro transcription from appropriate DNA templates using T7 RNA polymerase. The reactions were stopped after 3 h and treated with DNase I. The RNAs were then purified by silica columns and diluted to 500 nM in the reaction buffer supplemented with 5 μM DFHBI-1T. An aliquot from each sample was renatured by heat to convert the metastable RNA to the thermodynamically stable structure. The Broccoli fluorescence measurements of the renatured and untreated RNAs showed higher fluorescence in the renatured samples in all RNA designs  showed a major band of the expected size (129 nt) with few minor bands attributable to prematurely truncated products . The untHaving established that L+U, wMT8 and L+U-mm6 adopt robust metastable structures after transcription and that the thermodynamically stable structure activates the Broccoli aptamer, we set out to explore means other than heating/renaturation to trigger the structural transformation. An intriguing possibility was to use an RNA trigger to transiently destabilize a stem in the branched structure to create a toehold from which an intramolecular strand exchange reaction can be initiated. For example, the short trigger RNA (c4+10) shown in Figure First, the three robust metastable RNAs (1 μM) were mixed with DFHBI-1T (10 μM) and the trigger RNA c4+10 (100 nM) in the reaction buffer, and the Broccoli fluorescence was monitored over time at 37°C . InteresNtoe+Ninv where Ntoe is the number of bases that hybridize with the loop sequence (toehold), and Ninv is the number of bases that hybridize with (invade) the 3′ side of stem I. Fixing Ninv to 10 bases, the size of the toehold (Ntoe) was varied from 0 to 9. Metastable wMT8 (1 μM) was mixed with DFHBI-1T (10 μM) and a trigger RNA (100 nM), and the Broccoli fluorescence was monitored over time at 37°C  probably due to spontaneous ‘breathing’ of the stem near the loop region. While relaxation of the metastable wMT8 to the thermodynamically stable structure was mostly complete within 300 min (for Ntoe = 2–5), slower catalysis and lower turnover was observed for Ntoe = 8 and 9 likely due to slower release of the trigger RNA. Overall, toehold size between 3 and 5 showed faster initial rate and higher conversion among the tested trigger RNAs.Interestingly, slow catalysis was observed even without the toehold  and c4+8 (50 nM). The results  with a built-in energy source into single RNA molecules opens a new path for design of biomolecular machines. Future goals include designing self-powered RNA nanomachines with more than one metastable state, with multiple sensors, or with other computational or output functions.gkz364_Supplemental_FileClick here for additional data file.


With 5 wt % F8BT in PCBM, the short circuit current (JSC) and fill factor (FF) of PSC both significantly increased from 17.21 ± 0.15 mA·cm−2 and 71.1 ± 0.07% to 19.28 ± 0.22 mA·cm−2 and 74.7 ± 0.21%, respectively, which led to a power conversion efficiency (PCE) improvement from 12.6 ± 0.24% to 15 ± 0.26%. The morphology investigation suggested that doping with F8BT facilitated the formation of a smooth and uniform ETL, which was favorable for the separation of electron-hole pairs, and therefore, an improved performance of PSC.The commonly used electron transport material -phenyl-C61 butyric acid methyl ester (PCBM) for perovskite solar cells (PSC) with inverted planar structures suffers from properties such as poor film-forming. In this manuscript, we demonstrate a simple method to improve the film-forming properties of PCBM by doping PCBM with poly (F8BT) as the electron transport layer (ETL), which effectively enhances the performance of CH Organometal halide perovskite-based solar cells (PSCs) have attracted increasing attention throughout the world because of their high power conversion efficiency (PCE), low cost and easy fabrication techniques ,2,3. ThePlanar structure PSC has great potential because of its simple solution-based fabrication technique. According to the order of preparation, the planar structure can be divided into two types: n-i-p and p-i-n ,10. For 2 [4N [SC). Therefore, this doping method can be optimized by using dopants with good film-forming properties and electrical conductivities.In a p-i-n PSC device, PCBM was usually deposited on the surface of the perovskite layer as the ETL. It can enhance electron and hole separation by smoothing the perovskite surface and passivating surface traps ,17. Thus2 , LiF [212 [4N , zwitter2 [4N , perylen2 [4N  and PFN 2 [4N , the eff2 [4N , fluoren2 [4N , graphdi2 [4N  and olea2 [4N . On the 2 [4N . Howeverco-benzothiadiazole) (F8BT) are commonly used polymers in organic optoelectronic devices [SC), fill factor (FF) and PCE of PSC all increased, from 17.21 ± 0.15 mA·cm−2, 71.1 ± 0.07% and 12.6 ± 0.24% to 19.28 ± 0.22 mA·cm−2, 74.7 ± 0.21% and 15 ± 0.26%, respectively. The influence of PFO in the performances of PSCs was also studied. The charge carrier dynamics were analyzed as well.Fluorene-based polymers such as poly (PFO) and poly, PbI2, PCBM and 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline (BCP) were purchased from Xi’an Polymer Light Technology Corp. . Dimethyl sulfoxide (DMSO), chlorobenzene and Dimethyl Formamide (DMF) were all purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd.  and used as received. NiOx nanoparticles were synthesized in our lab according to Reference [PFO and F8BT were synthesized in South China University of Technology according to Reference . CH3NH3Peference .3 precursor solution was prepared by dissolving 1.4 mmol MAI and 1.4 mmol PbI2 in 1 mL GBL and DMSO (7:3/v:v). Before spin-coating, the MAPbI3 precursor solution was stirred at 65 °C for 12 h in a N2 atmosphere glove box.MAPbI2 flow, 4 min of ozone plasma at a power of 70 W was applied to remove any organic residues. Immediately after, NiOx nanoparticle solution was spin-coated onto the ITO glass at 4000 rpm for 30 s and then baked at 130 °C for 20 min. The substrate was then transferred into a N2 protected glovebox with the contents of water and oxygen less than 1 ppm. Following this, the MAPbI3 precursor solution was spin-coated. Specifically, the spin-coating process was composed of two stages: 900 rpm for 15 s and then 4000 rpm for 25 s. To perform toluene washing during the spin-coating of MAPbI3 precursor solution at a delay time of 15 s from the beginning of the second stage, 400 μL of toluene was dripped. After thermally annealing at 100 °C for 10 min, the mixed PCBM solution with F8BT or PFO in chlorobenzene (20 mg/mL) was spin-coated at a speed of 1000 rpm for 20 s. A control device was also fabricated with PCBM as the ETL. Then, the substrates were annealed at 70 °C for 40 min. Finally, BCP and Ag were thermally evaporated in sequence at a basic pressure of 4 × 10−4 Pa. The active area was 0.077 cm2 defined by a shadow mask.The indium tin oxide (ITO)-coated glass substrates were cleaned sequentially in detergent, deionized water, acetone and ethanol under sonication for 20 min, respectively. After being dried by NTo check the reproducibility of PSCs, multiple batches of the devices were made. The average values in performances were obtained accordingly, which were then used to evaluate the experimental error.−2, NewPort, 94043A SOLAR SIM, Irvine, CA, USA) at a scan rate of 200 mV·s−1. The incident-photon-to-current efficiency (IPCE) measurement was performed through an Enlitech QE-R Quantum Efficiency Measurement System . The crystal structures of the MAPbI3 films were characterized by using a Bruker D8 ADVANCE X-ray diffraction (XRD) device . The morphology of the MAPbI3 layer was obtained using a Hitachi S-4800 field emission scanning electron microscope  and a Bruker atomic force microscope (AFM). The thicknesses of the NiOx, MAPbI3 and PC61BM films were determined using a Bruker DektakXT Stylus Profiler. The photoluminescence (PL) spectra were measured using an Edinburgh FLS980 fluorescence spectrophotometer  with an excitation at 550 nm. The impedance characteristics of the devices were measured via a Wayne Kerr 6500B analyzer . The current density–voltage (J–V) characterization of PSC was carried out using a Keithley 2400 source meter under a simulated AM 1.5 illumination  in GBL and DMSO (7:3/v:v). Mirror-like dark-brown MAPbI3 film was obtained after thermal annealing at 100 °C for 10 min. The perovskite crystal size was evaluated from the scanning electron microscope (SEM) image as shown in x substrate.The device configuration of PSC incorporating the energy level of each layer is pictured in OC of 1.03 ± 0.01 V, a JSC of 17.21 ± 0.15 mA·cm−2 and a FF of 71.1 ± 0.07%. After doping PCBM with 5 wt % F8BT, the device demonstrated a clear increase of PCE to 15 ± 0.26%, with a VOC of 1.04 ± 0.01 V, a JSC of 19.28 ± 0.22 mA·cm−2 (12% enhancement) and a FF of 74.7 ± 0.21%. VOC was almost unchanged after doping. However, the enhancement of JSC was significant after doping with F8BT in PCBM, which caused the increase of PCE. The increase of JSC was further justified by the IPCE data as shown in SC values from the IPCE spectrum were 17.77 and 18.44 mA·cm−2 for the control device and the PCBM: 5 wt % F8BT-based device, respectively, which were close to the JSC values from the J–V scan. When increasing the F8BT doping ratio to 10 wt.%, the PCE of the device began to decrease with loss in JSC. It should be noted that the typical structure of p-i-n type PSC includes ITO/HTL/perovskite/ETL/cathode. The performance of PSC can be influenced by each layer and the interfaces between them. Therefore, there are many factors that can be considered to improve the performance. In this work, to emphasize the effect of the film-forming property of PCBM on the performance of PSC, a basic device structure was used. That is why the performance in this manuscript was moderate compared with the reports. We except better performance with higher PCE if combining with other optimizing approaches.SC and FF to 16.3 ± 0.24 mA·cm−2 and 64 ± 0.98%, respectively. A possible explanation is that PFO is a hole-dominated material with a very low electron mobility [In the case of doping PCBM with 5 wt % PFO, the PCE of PSC decreased to 10.8 ± 0.27% because of the decrease of Jmobility . The dopSC as reported recently [We noted that the optimal F8BT doping ratio was 5 wt % in our study, whilst doping with only 1.5 wt % PS caused a decrease of Jrecently . Thereforecently , which mTo gain more insights into the PCBM: F8BT films, atomic force microscopy (AFM) was used to examine the impact of the F8BT additive on the film property of the PCBM layer. The 5 μm × 5 μm AFM topography images of PCBM films (with or without F8BT) are shown in For a photovoltaic device, efficient light absorption of perovskite was the primary factor for high solar cell performance. Thus, the light absorption of the films of perovskite and perovskite /ETL were examined, as shown in 3. When in contact with PCBM, the light intensity of perovskite/PCBM decreased because of the transfer of electrons from perovskite to PCBM. It decreased further when PCBM was doped with F8BT, indicating the more effective electron transfer [3 increased, indicating a worse electron transfer from perovskite to ETL.The PL spectra in transfer . Howeverrec). Higher Rrec values represent less recombination and more effective dissociation of carriers at the interfaces [rec values of PSCs increased from 157 Ω·cm2 to 272 Ω·cm2, indicating more effective dissociation of carriers at the interfaces of PSCs, which is good for the performances of PSCs. To further understand the effect of F8BT or PFO on the device performances, electrochemical impedance spectroscopy (EIS) measurements for the PSCs based on different ETLs was carried out with their Nyquist plots shown in terfaces . After dSC of PSC decreased even though the surface roughness of ETL had improved.From the above experimental results, we can see that the doping of PCBM with F8BT resulted a smoother surface, which was favorable for the separation of electron-hole pairs. However, the PCE of PSC based on PCBM: 10 wt % F8BT decreased compared with the control device. That might be due to the fact that the lowest unoccupied molecular orbital (LUMO) of F8BT was higher than that of PCBM, which is bad for the collection of electrons . In addiSC and FF, thus a great enhancement of PCE. With 5 wt % F8BT in PCBM as the ETL, a 19% enhancement of the PCE of PSC was achieved. The morphology study shows that doping with F8BT improved the uniformity of the ETL, which is favorable for the separation of electron-hole pairs. Therefore, the collection of electrons was improved. Our method has shown to be an effective and simple way to realize highly efficient PSCs.Highly efficient PSCs were fabricated by doping PCBM with F8BT as the ETL, which led to significant improvements of J


These new Ba-containing materials were designed to present a more open framework to enhance oxygen conduction. The citrate-nitrate soft-chemistry technique was used to synthesize such structural perovskite-type materials, followed by annealing in air at moderate temperatures (1150 °C). The subtleties of their crystal structures were investigated from neutron powder diffraction (NPD) data. They crystallize in the orthorhombic Pbca space group. Interstitial O3 oxygen atoms were identified by difference Fourier maps in the NaCl layer of the K2NiF4 structure. At variance with the parent compound, conspicuous oxygen vacancies were found at the O2-type oxygen atoms for x = 0.2, corresponding to the axial positions of the InO6 octahedra. The short O2–O3 distances and the absence of steric impediments suggest a dual oxygen-interstitial mechanism for oxide-ion conduction in these materials. Conductivity measurements show that the activation energy values are comparable to those typical of ionic conductors working by simple vacancy mechanisms (~1 eV). The increment of the total conductivity for x = 0.2 can be due to the mixed mechanism driving both oxygen vacancies and interstitials, which is original for these potential electrolytes for solid-oxide fuel cells.The title compounds exhibit a K There are several types of fuel cells, of which solid-oxide fuel cells (SOFC) exhibit the highest performance; they are promising for stationary applications [Fuel cells are electrochemical devices capable of transforming the chemical energy stored in a fuel directly into electricity. The direct combustion of a fuel, e.g., hydrogen, involves a direct transfer of electrons from H to the oxidant, e.g., oxygen, taking advantage of the combustion heat as the only profit of the direct chemical reaction. The efficiency is restricted to the Carnot cycle  in the cications ,4. In a 1−xYxO2−δ, Ce1−xGdO2−δ, La1−xSrxGa1−yMgyO3−δ) [The design of these materials should have strict requirements such as an oxygen-ion transport with low activation energy, low electrical resistance, and proper electrochemical kinetics. The commercial and most studied ionic conductors are based on fluorite and perovskite-type structures (ZrMgyO3−δ) ,6,7. AllMgyO3−δ) ,12,13,14xY3−xFe5O12+δ composition, where the oxide-ion conduction is based on an excess oxide-ion concentration mechanism [Various alternative frameworks have been described, such as apatites  or garneechanism .2NiF4-type structure have been described to exhibit ionic conduction. These layered structures contain an open interlayer space where an appreciable number of interstitial oxygen atoms can be hosted; therefore, they seem to be a suitable alternative to be used as electrolytes in SOFCs [Recently, rare-earth perovskites with Kin SOFCs ,18,19,202BO4 is constituted by single layers of perovskite consisting of corner-sharing BO6 octahedra, which alternate with a NaCl-type layer of AO atoms. There are two types of oxygens; the equatorial O1 oxygen atoms link the BO6 octahedra in the basal plane, while the axial O2 oxygen atoms connect the perovskite layers with the AO layer. The interstitial atoms, responsible for the oxide-ions conduction, are found in the sodium chloride type layer, only coordinated by the A cations [1+xSr1−xInO4+δ  and SrIn1−xBxO4+δ  oxides as possible solid electrolytes for SOFCs [1.2Sr0.8InO4.11(1), exhibiting extremely low activation energy of only 0.51 eV for the conduction mechanism via interstitials at low temperatures (T< 650 °C), significantly smaller than those of other electrolytes working with a vacancy mechanism, typically of 1 eV.The atomic arrangement A cations . In prevor SOFCs ,23. The 2NiF4-type materials, the strategy to induce the accommodation of oxygen interstitials in the NaCl layers is the creation of a positive charge unbalance at La, Sr positions; the mentioned La1.2Sr0.8 stoichiometry implies the nominal incorporation of 0.1 oxygen atoms in the interlayer space, assuming trivalent La and In and divalent Sr ions.In these layered K1.2Sr0.8−xBa0.2InO4±δ , where the introduction of Ba instead of Sr ions aims to expand the NaCl-type layer of the structure in which the ionic conduction takes place. Through a neutron powder diffraction (NPD) study we unveiled a dual defect mechanism for conduction, since both oxygen vacancies concerning the axial octahedral positions, as well as interstitial oxygen atoms where found in the interlayer space.Based on these results, in the present work we describe the synthesis and characterization of a new family of oxides with composition La1.2Sr0.8−xBaxInO4+δ  oxides were prepared via a citrate-nitrate route. Stoichiometric amounts of analytical grade Sr(NO3)2 , La(NO3)3·6H2O , Ba(NO3)2 (99 at.% Merck), and In(NO3)3·9H2O (99.99 at.% Alfa Aesar) were dissolved under stirring in 250 mL of 10% citric-acid (Panreac) aqueous solution with several droplets of concentrated HNO3 (J.T. Baker) on a porcelain capsule. This mixture was slowly evaporated on a hot plate with a magnetic stirrer at 300 °C, leading to organic resins where a random distribution of the involved cations was obtained. The resins were dried at 120 °C in a laboratory stove (Heraeus) and slowly decomposed on a muffle at temperatures up to 600 °C for 12 h (JH Hornos). Subsequent treatment at 800 °C for 2 h ensured the total elimination of all the organic materials and nitrates. Final heating at 1150 °C for 12 h gave rise to well-crystallized, homogeneous samples. Laα radiation (λ = 1.5418 Å). Neutron powder diffraction (NPD) data were collected in the High-Resolution Powder Diffractometer for Thermal Neutrons (HRPT) at the Swiss Spallation Neutron Source in the Paul Scherrer Institute (SINQ-PSI) Switzerland with a neutron wavelength λ = 1.494 Å. About 2 g of the sample was contained in a vanadium can. The measurements were carried out at 25 °C (RT). The counting time for each pattern was 3 h. The crystal structures were analyzed by the Rietveld method [The identification and characterization of the final products were carried out by X-ray diffraction (XRD) for phase identification and to assess phase purity using a Bruker-axs D8 diffractometer , controlled by DIFFRACTplus software, in the Bragg–Brentano reflection geometry with CuKd method , using tSolid bars (0.08 × 0.3 × 0.8 cm) were subjected to 2-probe DC conductivity measurements using Pt ink (PSI supplies) as the current collector. Conductivity values were acquired with an Agilent 34972A Data Acquisition Unit at a temperature range between room temperature and 1000 °C under atmospheric air .1.2Sr0.8−xBaxInO4 series. The patterns correspond to layered perovskites that can be indexed in the orthorhombic Pbca space group (Card 04-017-3962 ICDD). A small amount of La2O3 was found (Card 01-073-2141 ICSD) in all patterns.The samples were obtained as polycrystalline, yellowish powders. Pbca structural model, as proposed by Titov et al. [3) and 0.3 (437.15(7) Å3) with respect to the parent La1.2Sr0.8InO4 (432.42(4) Å3, [2+ vs. Sr2+.A neutron diffraction study was essential to reveal the structural features of the three materials. The crystal structures can be defined in the v et al. . The A-t2(4) Å3, ), as it 6 octahedra. Additionally, difference Fourier maps allowed the localization of extra interstitial oxygen atoms at 8c  sites, x ≈ y ≈ z ≈ 0.25, as positive peaks , as expected from the metal charge misbalance between La3+, Sr2+ and Ba2+. A minor impurity phase of La2O3 was detected in all the patterns and included in the refinement as a second phase. 1.2Sr0.6Ba0.2InO4.10(2) and La1.2Sr0.5Ba0.3InO4.10(2). Once introduced in the structural model as O3 atoms, a significant decrease of the R6 octahedra alternating with (La/Sr/Ba)–O layers with NaCl structure.6 octahedra are significantly tilted by θ = 9.16° and 7.83° for x = 0.2 and 0.3, respectively  with reasonable distances of 2.50 Å (in average) range.The InOectively , obtaine6 octahedra as well as interstitial oxygens conspicuously located in the NaCl layers. As it is plausible that all these materials combine an excellent ionic conductivity with a sufficient ionic transport of oxide anions, as demonstrated for the parent La1+xSr1−xInO4+δ materials [2+ ions prone to form weak covalent bonds to oxygen. In this respect, a last issue concerns the mechanism that can be induced from the structural features determined by neutron diffraction for both perovskite oxides. In principle, it is believed that the ionic transport required for the performance of the electrolyte materials for SOFC works with a vacancy mechanism, the pre-existing vacancies being filled by neighboring oxygen atoms upon conduction of oxide ions. However, in this case, the neutron data consistently indicate, for both compounds at RT, that the layered perovskites contain both oxygen vacancies located at the axial oxygen atoms of the InOaterials , we coul1.2Sr0.8InO4.11 [B is the Boltzmann constant in (J/K).The total conductivity was measured to compare our samples with the parent La8InO4.11 . The res1.2Sr0.8−xBaxInO4+δ  compared with that of commercial electrolytes. The parent sample (x = 0.0), as reported in Reference [1−xSrxGa1−yMgyO3−δ, Ce1−xGdO2−δ and Zr1−xYxO2−δ, at low temperatures. In that case [1.2Sr0.6Ba0.2InO4.10 increases by 1 order of magnitude its conductivity, and at low temperatures, the tendency to be competitive with the typical electrolytes remains. In eference , shows ahat case , NDP dat1.2Sr0.6Ba0.2InO4.10 is among the few oxides containing a measurable concentration of permanent vacancies as well as adjacent interstitial atoms in the interlayer space, which together can participate in the conduction mechanism to ensure a fast ionic transport.In other words, the layered perovskite of La1+xSr1−xInO4+δ layered perovskites have recently been described as suitable candidates for electrolyte materials in SOFCs [1.2Sr0.8InO4+δ, which contains a substantial amount of interstitial oxygen atoms that easily move in the interlayer plane, here we designed a unique series containing Ba2+ ions. Its larger ionic size drives the expansion of the unit cell, where a better oxygen conduction is expected. A neutron diffraction study reveals novel and unexpected features, since oxygen vacancies at the axial O2 octahedral positions were detected, besides the interstitial oxygen atoms located in the NaCl-type layer. Difference Fourier maps from NPD data at RT clearly show prominent positive peaks corresponding to the O3 interstitial oxygen atoms; the short distances with the oxygen vacancies make possible a dual jump mechanism where both point defects participate. Moreover, the strongly anisotropic, cigar-shaped ellipsoids for O2 mimic the displacement of axial oxygen atoms in the interlayer space, also anticipating an excellent ionic motion for oxide ions. The low activation energies measured for x = 0.2 and x = 0.3 compounds endorses this hypothesis. The presence of Ba ions in this novel series, besides the forecasted expansion of the framework, induces a second beneficial effect: the basic character of these cations, more prone to form weaker chemical bonds, favor the formation of oxygen vacancies in a crystal structure involving very labile atoms where oxygen conduction is promoted in the –O (NaCl-type) layers.Lain SOFCs ,23. Base


The papillomavirus (PV) protein E2 is one of only two proteins required for viral DNA replication. E2 is the viral transcriptional regulator/activation protein as well as the initiator of viral DNA replication. E2 is known to interact with various cellular DNA replication proteins, including the PV E1 protein, the cellular ssDNA binding complex (RPA), and topoisomerase I. Recently, we observed that cellular DNA polymerase ε (pol ε) interacts with the PV helicase protein, E1. E1 stimulates its activity with a very high degree of specificity, implicating pol ε in PV DNA replication. In this paper, we evaluated whether E2 also shows a functional interaction with pol ε. We found that E2 stimulates the DNA synthesis activity of pol ε, independently of pol ε’ s processivity factors, RFC, PCNA, and RPA, or E1. This appears to be specific for pol ε, as cellular DNA polymerase δ is unaffected by E1. However, unlike other known stimulatory factors of pol ε, E2 does not affect the processivity of pol ε. The domains of E2 were analyzed individually and in combination for their ability to stimulate pol ε. Both the transactivation and hinge domains were found to be important for this stimulation, while the E2 DNA-binding domain was dispensable. These findings support a role for E2 beyond E1 recruitment in viral DNA replication, demonstrate a novel functional interaction in PV DNA replication, and further implicate cellular pol ε in PV DNA replication. Papillomaviruses (PV) encode only two proteins required for viral DNA replication: the viral hexameric DNA helicase, E1, and the multifunctional transcriptional regulator and replication initiator protein, E2 ,4,5. AltE2 has three distinct domains, each of which can be separated by function. The amino-terminal 200 amino acids comprise the transactivation domain. Residues within the transactivation domain (TAD) are important for both transcriptional activation and repression as well as DNA replication . ResidueE2 has been shown to interact with or modulate the activity of several cellular proteins. These include RPA, topoisomerase I (topo I), and p53, among others ,18,19. OWe recently reported that HPV E1 helicase binds to, stimulates, and confers processivity to pol ε . These fHPV 11 glutathione S-transferase (GST)-E2 truncated constructs were created using the Takara In-Fusion cloning kit . HPV 11 E2 transactivation domain consists of amino acids 1–202. HPV 11 E2 hinge domain consists of amino acids 203–286. HPV 11 E2 DNA-binding domain consists of amino acids 287–365. We PCR amplified these regions of the E2 ORF individually and pair-wise and integrated them into pGex6p as per the manufacturer’s instructions. Construct cloning was verified by DNA sequencing. The HPV 11 maltose binding protein (MBP)-TAD-H construct was also created using the Takara In-Fusion cloning kit, in a similar manner to the creation of the GST-E2 proteins, but into the pMALC2 expression vector , rather than into pGex6p. All clones were verified by DNA sequencing.Escherichia coli using GST-affinity. Briefly, the various GST-E2 constructs were transformed into E. coli strain BL21 (DE3). The cultures were grown to A595 = 0.4 at 37 °C, the temperature reduced to 18 °C, further grown to A595 = 0.6, and isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to 0.3 mM to induce expression. After 16 h of induction at 18 °C, cells were collected by centrifugation (5000× g for 20 min at 4 °C), washed with cold phosphate buffered saline (PBS) and again subjected to centrifugation (5000× g for 20 min at 4 °C). Cells were re-suspended in 25 mL of ice cold Buffer A (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM EDTA, 2 mM DTT, 20% sucrose and 1 mM PMSF). The cell suspensions were subjected to 1500 pounds per square inch for 5 min in a 4 °C ParrTM cell disruption chamber, and the suspension released slowly to lyse. Lysates were then sonicated for five 20 s pulses on ice. NP40 was added to 0.1% final concentration and the lysates were subjected to centrifugation for 20 min at 20,000× g at 4 °C. One mL of pre-equilibrated glutathione Sepharose  was added to the lysates and incubated for three hours at 4 °C. The resin was washed twice with 40 mL of Buffer A and poured into column format. The resin was washed with 5 mL Buffer A containing 0.1 mM PMSF, 5 mL of Buffer B (50 mM Tris-HCl (pH 8.8), 1 mM EDTA, 2 mM DTT, 10% glycerol) with 1 M NaCl, and then 5 mL of Buffer B with 0.2 M NaCl. Protein was then eluted with 6 mL of Buffer B containing 0.2 M NaCl and 10 mM glutathione. Fractions were evaluated using SDS-PAGE and fractions containing the GST-E2 construct were pooled.GST-E2 proteins were purified from E. coli using maltose binding protein (MBP) affinity. Briefly, transformed BL21 (DE3) E. coli was grown to A595 = 0.4 at 37 °C. The temperature was reduced to 18 °C, further grown to A595 = 0.6, and isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to 0.4 mM to induce expression. Cells were grown for 2 h at 18 °C. Cells were collected by centrifugation (5000× g for 20 min at 4 °C), washed with cold PBS, and again collected by centrifugation (5000× g for 20 min at 4 °C). Cells were frozen using liquid nitrogen and stored at −80 °C. Before use, cells were thawed on ice and resuspended in 25 mL MBP column buffer (20 mM Tris-HCl (pH 7.5), 200 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 5% glycerol, 0.01% NP40). The cell suspension was subjected to 1500 psi for 5 min in a cold ParrTM cell disruption chamber, and the suspension released slowly to lyse. Lysates were then sonicated for five 20 s pulses on ice and subjected to centrifugation for 20 min at 20,000× g at 4 °C. Supernatants were collected, diluted 1:6 in MBP column buffer, and applied to 1 mL of pre-equilibrated amylose resin  Resin was washed with 20 mL MBP column buffer containing 1 M NaCl, and further washed with 20 mL MBP column buffer containing 0.2 M NaCl. MBP-TAD-H was eluted using 2 mL MBP column buffer containing 10 mM maltose. Fractions were evaluated using SDS-PAGE and protein peak fractions were pooled.The MBP-TAD-H protein was purified from 2, 1 mM DTT, 1 mM PMSF) was added to the cell pellet, which was then resuspended, incubated on ice for 10 min, and then subjected to Dounce homogenization by 20 strokes. Lysates were subjected to centrifugation  for 10 min at 4 °C. The supernatant was diluted with Buffer Q  and 10 mM 2-mercaptoethanol) until the ionic strength was equivalent to Buffer Q with 50 mM NaCl. This was applied to a 10 mL Q Sepharose column. After washing with 50 mL Buffer Q containing 50 mM NaCl, the column was eluted with 10 mL Buffer Q containing 500 mM NaCl. The peak protein-containing fractions were pooled and applied to a 1 mL affinity column . After E. coli and purified as described [DNA polymerases δ and ε were prepared as full complexes using multiple baculovirus infection expression and purified as described . Recombiescribed . The humescribed .2, 4 mM ATP, 200 μM each of CTP, UTP and GTP, 25 μM dATP, 100 μM each of dCTP, dGTP and dTTP and 0.5 mM DTT), and 32P-dATP . The replication factors RPA, RFC, PCNA, pol ε, pol δ, HPV 11 EE-E1, and HPV 11 GST-E2 were individually titrated into the reactions to establish optimal DNA synthesis activity and were used at the following concentrations: RFC (3 ng/μL), PCNA (10 ng/μL), RPA (70 ng/μL), Pol ε (1 ng/μL unless otherwise indicated), HPV E1 (60 ng/μL unless indicated otherwise), HPV 11 E2 (20 ng/μL unless indicated otherwise). After incubation at 37 °C for 1.5 h (unless indicated otherwise), reactions were terminated (with 2% SDS (wt/vol), 50 mM EDTA, 5% glycerol (vol/vol) and 0.2 mg/mL proteinase K for 15 min at 37 °C), and DNA products were then denatured )and subjected to electrophoretic separation on a 25 cm × 20 cm 1% agarose in 50 mM NaOH and 1 mM Na2EDTA at 40 V for 16 h at room temperature Gels were fixed and subjected to phosphorimager analysis. Quantifications were performed using ImageJ software . All replicates of each experiment were quantified by boxing the entire lane, and a background (no sample lane) was subtracted from the experimental values. All experiments were performed at least three independent times. Experiments were performed with polymerases prepared using baculovirus expression both in our own laboratory as well as with polymerase samples prepared in the laboratory of Dr. Jerard Hurwitz ; similar results were obtained with preparations from both sources. As a result of isotope variation, quantifications across multiple experiments were plotted as percent synthesis, with pol ε in the presence of replication factors  set to 100% as an internal standard for each experiment.The primed M13 polymerase assays was carried out at described . Briefly32P-ATP (3000 cpm/pmol) using T4 polynucleotide kinase as per instructions (Thermo Scientific). A second “marker” was presented in each gel created by carrying out a polymerase reaction containing pol ε and Replication Factors (RF) (as described above) to create a standard spectrum “smear”. Precise molecular weight/banding was not required for our analyses/quantification but, to provide information on relative strand length, these markers were run on a parallel gel run identically to the reaction gels and were only present to demonstrate approximate strand length.The molecular weight marker was created by end-labeling the GeneRuler 1 kb ladder  with γ-t tests were performed using GraphPad software  Analyses were conducted with a 95% confidence interval.Unpaired Student’s PV E2 has interactions with various cellular proteins, including functional interactions with topo I ,19,26. Bt test (p = 0.26).We observed that HPV E2 stimulated the DNA synthesis activity of pol ε in the absence of pol ε’s processivity cofactors RFC, PCNA, and RPA . To evalOther factors that stimulate pol ε, such as RF  as well as HPV E1, increase pol ε’s processivity and its ability to synthesize long DNA products. Because E2 was observed to stimulate pol ε, we evaluated whether E2 could confer processivity to pol ε . Single-p value less than 0.0001 (see Methods and E2 is comprised of three distinct domains: the transactivation domain (TAD), the DNA binding domain (DBD), and the flexible hinge domain (H) that connects the other two domains ,30. ThroE2 plays a critical role in the initiation of PV DNA replication and exhibits other, likely important, interactions with cellular DNA replication proteins. To date, the complete role of E2 in PV DNA replication remains not entirely understood. E2 is mostly considered to be a transcriptional activator and DNA replication initiator protein which helps E1 bind to the PV origin ; howeverIn this paper, we have demonstrated a novel functional interaction between PV E2 and cellular pol ε. E2 was observed to stimulate the DNA polymerase activity of pol ε, independently of DNA replication cofactors RFC, PCNA, and RPA . The stiBecause E2 has three distinct domains, we analyzed which of these domains was important for the interaction with pol ε. By creating various combinations of the three E2 domains as fusion proteins and testing them for stimulation of pol ε, it was found that the DBD is the only domain of E2 that is fully dispensable for pol ε stimulation; the hinge and TAD domains are both important for stimulation . It has We have only recently implicated pol ε in PV DNA replication, and the interactions described here are new and remain relatively unexplored. The results shown here suggest that E2 may be playing a role during the initiation of PV DNA replication, possibly assisting pol ε in primer acquisition. As the polymerase departs from the PV origin, it needs to associate with its processivity factors, PCNA and E1, possibly through some sort of “hand-off” mechanism. Any potential competition between binding of pol ε to E2 versus the processivity factors is presumably not a problem if E2 stays primarily associated with the origin (or is displaced from the replication complex). Although we have previously suggested a possible role for E2 in elongation thanks to its binding and stimulation of topoisomerase I , topoiso


Chlamydia trachomatis is an obligate intracellular pathogen that replicates inside a parasitic vacuole called the inclusion. The nascent inclusion is derived from the host plasma membrane and serves as a platform from which Chlamydia controls interactions with the host microenvironment. To survive inside the host cell, Chlamydia scavenges for nutrients and lipids by recruiting and/or fusing with various cellular compartments. The mechanisms by which these events occur are poorly understood but require host proteins such as the SNARE proteins  Receptor). Here, we show that SNAP-23 and Syntaxin 4, two plasma membrane SNAREs, are recruited to the inclusion and play an important role in Chlamydia development. Knocking down SNAP-23 and Syntaxin 4 by CRISPR-Cas9 reduces the amount of infectious progeny. We then demonstrate that the loss of both of these SNARE proteins results in the dysregulation of Chlamydia-induced lipid droplets, indicating that both SNAP-23 and Syntaxin 4 play a critical role in lipid droplet homeostasis during Chlamydia infection. Ultimately, our data highlights the importance of lipid droplets and their regulation in Chlamydia development. Chlamydia trachomatis is the most common agent of bacterial sexually-transmitted infections with an estimated worldwide incidence of ~130 million cases per year Where, A = EGFP intensity of HeLa C1 well containing D/D solubilizer, A1 = EGFP intensity of HeLa well containing D/D solubilizer, B = EGFP intensity of HeLa C1 well containing ethanol, B1= EGFP intensity of HeLa well containing ethanol.5 cells per well in a 24-well plate 24 h prior to infection. Cells were infected with C. trachomatis L2 constitutively expressing mCherry at a MOI of 0.5 for flow cytometry or with WT C. trachomatis L2 at a MOI of 1 for immunofluorescence analysis. The infection was synchronized by centrifugation at 1,000 xg for 1 h at 4°C. The cells were transitioned to 37°C and 5% CO2 for 20 h. The cells were incubated with 5 μM NBD-C6-ceramide for 30 min at 37°C. The cells were washed with HBSS and incubated with 0.34% FA-free BSA in serum-free DMEM for 6 h to allow for back-exchange. For microscopy analysis, the coverslips were washed several times with HBSS, stained with 1 μg/ml of Hoechst (Invitrogen) for 5 min at 37°C and mounted for live cell imaging with HBSS. Images were acquired and processed using a Nikon TiE inverted immunofluorescence microscope with a 60x oil immersion lens and Nikon NIS Elements AR 4.2 software. Contrast enhancement and image cropping were conducted using ImageJ (NIH). For flow cytometry, the cells were trypsinized into suspension and fixed in 4% PFA in HBSS for 10 min at RT. Each sample was resuspended in FACS Buffer  and analyzed on LSRFortessa flow cytometer (BD Biosciences).HeLa cells were seeded onto coverslips or onto wells at a density of 1x104 cells per well in a 24-well plate. To pre-treat cells with OA, cells were incubated with 200 μM of OA diluted in PBS containing 10% FA-free BSA for 24 h. DMSO was used as the control. The cells were then infected with C. trachomatis L2 constitutively expressing mCherry at a MOI of 1 by centrifugation at 1,000 xg for 1 h at 4°C or mock-infected with DMEM. For OA treatment at the time of infection, 200 μM of OA or DMSO was added after centrifugation. The cells were then transitioned to 37°C and 5% CO2 for 24 h. The cells were stained with 50 μM BODIPY 493/503 (ThermoFisher) for 30 min at 37°C and washed several times with HBSS. The cells were trypsinized into a single cell suspension and fixed in 4% PFA in HBSS for 10 min at RT. Each sample was then resuspended in FACS Buffer and analyzed on a LSRFortessa flow cytometer (BD Biosciences). For each cell line, a ratio was obtained for the BODIPY intensity in the corresponding control cells with that of the treated cells (i.e. not infected:infected or DMSO:OA). This ratio for the EV control cells was considered 100% and all values were normalized to the EV ratio for each experiment. Each experiment was independently performed at least 3 times in triplicate.HeLa cells were seeded at a density of 6x104 cells per coverslip in a 24-well plate 24 h prior to infection. The cells were infected with C. trachomatis L2 at a MOI of 0.5 by centrifugation at 1,000 xg for 1 h at 4°C. The cells were then transitioned to 37°C and 5% CO2. At 30 h pi, cells were fixed with 3% PFA and 0.025% of glutaraldehyde in PEMS buffer  for 20 min at RT. The coverslips were washed with IF buffer containing 100 mM glycine. The cells were permeabilized with PBS containing 0.2 M glycine, 30 mg/mL BSA and 0.01% saponin for 45 min at RT and then blocked with blocking buffer  for 30 min at RT. Coverslips were washed with IF buffer and then incubated with anti-IncA antibody (1:500) diluted in PBS containing 0.01% saponin and 1 mg/mL of BSA for 1h at RT. The coverslips were then washed several times with IF Buffer and incubated with goat anti-rabbit IgG Alexa Fluor 647-conjugated secondary antibody , 1 μg/mL Hoechst (Invitrogen) and 12.5 μM BODIPY 493/503 (ThermoFisher) in PBS containing 0.01% saponin and 1 mg/mL of BSA for 1 h at RT. Coverslips were mounted with Prolong Glass Antifade reagent (Invitrogen). Images were acquired with a Nikon A1R+ confocal microscope with 60x oil immersion lens and Nikon NIS Elements software. Contrast enhancement and image cropping, as well as 3D reconstruction were conducted using ImageJ (NIH).HeLa cells were seeded at a density of 4x105 cells per well and incubated at 37°C with 5% CO2 for 24 h, 48 h, 72 h and 96 h. At each timepoint, the cells were collected and resuspended in FACS Buffer. CFSE intensity was measured on a LSRFortessa flow cytometer (BD Biosciences).HeLa cells were stained with 5 mM carboxyfluorescein succinimidyl ester (CFSE)  and seeded in 6-well plates at 1x106 cells per dish 24 h prior to infection. The cells were infected with C. trachomatis L2 at a MOI of 5 or mock infected with HBSS by absorption for 2 h at 37°C rocking and then transitioned to 37°C and 5% CO2 for 2 h. Both cell populations were then transfected with 2 μg of 3xFLAG-SNAP-23 DNA supplemented with 7 μg of empty pcDNA3.1+ plasmid at a 1 μg of DNA:1 μl of Continuum transfection reagent (Gemini Bio-products) ratio and incubated for an additional 20 h at 37°C and 5% CO2. At 24 h pi the cells were lysed in ice-cold lysis buffer  supplemented with 250 U/ml of Turbonuclease (Accelagen), 10 mM NaF, 5.4 mM Na3VO4, and protease inhibitor cocktail (APExBIO) for 1 h on ice. Samples were clarified by centrifugation at 20,000 xg for 30 min at 4°C and then incubated with 50 μl of settled Protein G Plus agarose  for 30 min at 4°C. Pre-cleared samples containing 6 mg of total protein were then incubated with 30 μg of mouse anti-FLAG antibody  overnight at 4°C rotating (8 rpm). Antibody-antigen complexes were captured with 20 μl of settled Protein G Plus agarose at 4°C for 2 h, washed with lysis buffer, and eluted with SDS-PAGE sample buffer. Eluates and lysates (40 μg) were analyzed by Western blot.HeLa cells were seeded onto 150 mm tissue culture dishes at a density of 3.6x10p values < 0.05.A two-tailed student t-test was used to determine statistical differences between the means of two populations from at least three independent experiments. Statistical significance was assumed at Click here for supplemental data file.www.microbialcell.com.All supplemental data for this article are available online at