T, was isolated from garden soil in Vienna (Austria). Cells were irregular cocci with a diameter of 0.6–0.9 µm and possessed archaella and archaeal pili as cell appendages. Electron microscopy also indicated clearly discernible areas of high and low electron density, as well as tubule-like structures. Strain EN76T had an S-layer with p3 symmetry, so far only reported for members of the Sulfolobales. Crenarchaeol was the major core lipid. The organism gained energy by oxidizing ammonia to nitrite aerobically, thereby fixing CO2, but growth depended on the addition of small amounts of organic acids. The optimal growth temperature was 42 °C and the optimal pH was 7.5, with ammonium and pyruvate concentrations of 2.6 and 1 mM, respectively. The genome of strain EN76T had a DNA G+C content of 52.7 mol%. Phylogenetic analyses of 16S rRNA genes showed that strain EN76T is affiliated with the recently proposed phylum Thaumarchaeota, sharing 85 % 16S rRNA gene sequence identity with the closest cultivated relative ‘Candidatus Nitrosopumilus maritimus’ SCM1, a marine ammonia-oxidizing archaeon, and a maximum of 81 % 16S rRNA gene sequence identity with members of the phyla Crenarchaeota and Euryarchaeota and any of the other recently proposed phyla (e.g. ‘Korarchaeota’ and ‘Aigarchaeota’). We propose the name Nitrososphaera viennensis gen. nov., sp. nov. to accommodate strain EN76T. The type strain of Nitrososphaera viennensis is strain EN76T ( = DSM 26422T = JMC 19564T). Additionally, we propose the family Nitrososphaeraceae fam. nov., the order Nitrososphaerales ord. nov. and the class Nitrososphaeria classis nov.A mesophilic, neutrophilic and aerobic, ammonia-oxidizing archaeon, strain EN76 Candidatus Nitrosopumilus maritimus’ SCM1 , and all strains in pure culture or enrichment are adapted to low concentrations of ammonia  by transferring 5 g soil into 50 ml freshwater medium (FWM) containing (l−1) 1 g NaCl, 0.4 g MgCl2 . 6H2O, 0.1 g CaCl2 . 2H2O, 0.2 g KH2PO4, 0.5 g KCl, 1 ml trace element mixture, 1 ml vitamin solution and 7.5 µM ferric sodium EDTA, 0.5 mM NH4Cl as energy source and 2 mM NaHCO3 as carbon source. Additionally 0.1 mM NaNO2 was supplied to the cultures. The medium was adjusted to pH 7.5 and cultures were incubated at 37 °C in the dark without shaking. Supplementation with antibiotics  and filtration of the cultures (0.45 µm pore size) were applied to reduce bacterial and fungal contaminants. Growth was followed by measuring ammonium consumption and nitrite production photometrically. Additionally, light microscopy and quantitative PCR were used as described previously  supplemented with 1 mM NH4Cl, 2 mM NaHCO3, 0.1 mM sodium pyruvate and 100 µg antibiotics ml−1 . The medium was buffered with HEPES/NaOH to pH 7.5. Larger volumes were cultured in glass bottles and shaken (150 r.p.m.) in darkness.The purified strain EN76T, the strain was cultivated in closed batch in 20 ml FWM containing 100 µg antibiotics ml−1 as described above. Substrates were dissolved in MilliQ water and sterile filtered (0.2 µm) before usage. To determine optimal growth parameters as well as the effect of different nitrogen and carbon substrates on the growth of EN76T, a design of experiments (DoE) strategy was applied, using the factors temperature, pyruvate concentration and ammonium concentration. Based on our preliminary knowledge of the strain’s growth requirements (4Cl. As nitrite production was shown to follow biomass production (max), which were eventually used to develop the model . Experiments were conducted in triplicate, except for the centre points of the initial two-level factorial screening design, which were set up in fivefold replicates. The two-level factorial design was applied in order to screen the design space rapidly. Due to a low model significance of data obtained from the initial two-level factorial screening design space, an augmented matrix was used in order to account for putative interactions of individual factors. Thus, the two-level factorial design space was extended by using a face-centred augmented matrix. Eventually, data points of all experiments (n = 51) were used to establish a response surface model (RSM). Data were analysed with the software Design-Expert 8. ANOVA, based on a stepwise regression elimination procedure, was used to develop the model. The desirability approach, as described elsewhere (max (variable) based on variation of quantitative factors, here c(ammonium), c(pyruvate) and temperature (within the design space). A score is given to each quantitative factor setting that can be used to maximize the variable. In this approach, desirability between 0 and 1 (corresponding to 0–100 %) can be assigned to a variable for optimization; factors identified as being outside a certain desirability function will not be considered for model generation. To verify the calculated optimal growth conditions identified by the established RSM model design space, one additional growth experiment (fivefold-replicated closed-batch cultures) was performed , grey butyl (Sigma Aldrich), blue butyl (Dunn Labortechnik), red isoprene (Sigma Aldrich), grey natural (Carl Roth) and grey–blue natural (VWR) rubber stoppers on the growth of strain EN76T was tested .For the cultivation of strain EN76For negative staining, cells were fixed with 2.5 % glutaraldehyde in 1× PBS, transferred to carbon-coated copper grids (200 mesh) and stained with 0.5 % uranyl acetate for 2 min as described previously . The cells were post-fixed with 1 % osmium tetroxide for 2 h at room temperature followed by dehydration in a graded ethanol series. After immersion in pure acetone, the slides were critical-point-dried with a CPD 300 unit (Leica). The slides were then mounted on stubs, gold-coated with an AGAR B7340 sputter-coater and imaged using an XL20 instrument (Philips) running the Microscope control program .For scanning electron microscopy (SEM), poly-4Cl and 0.15 mM sodium pyruvate until the late exponential growth phase. Cells were harvested by centrifugation and fixed as mentioned above for SEM preparation. After fixation, cells were washed twice in 100 mM PHEM buffer and covered with 1 % agar. Cells were post-fixed for 1 h in osmium tetroxide (1 %), washed three times , dehydrated in a graded ethanol series and embedded in resin with acetonitrile as solvent. Polymerization was achieved by incubating the resin blocks for 1 h at 40 °C and for 48–72 h at 60 °C .To determine the general morphology and properties of the potential S-layer protein of EN76Intact polar lipids and glycerol dibiphytanyl glycerol tetraether (GDGT) were analysed previously and described by T and 454 pyrosequencing on a 454/FLX-Titanium sequencer (Roche) have been described in detail previously  glycerol and stored at −80 °C. Growth could be restored after preservation for 12 months by carefully thawing cells on ice and removing the glycerol by centrifugation of cells prior to inoculation into fresh medium.Cells of strain EN76T is a mesophilic and neutrophilic organism, growing at 28–47 °C and pH 6–8.5 , strain EN76T and its close relative ‘Candidatus Nitrososphaera gargensis’ Ga9.2 (both associated with group I.1b) possess genes encoding urease and urea transporters  in amounts comparable to those of AOB under oxic conditions (4.6±0.6 amol N2O cell−1 h−1). However, in contrast to AOB, N2O production does not increase under reduced oxygen levels, and might occur via a hybrid formation mechanism  to stimulate growth  at a constant ammonium concentration of 2.5 mM. Based on the DoE data and the calculated model obtained after varying temperature, ammonium and pyruvate concentrations, we predicted the optimal growth conditions of EN76T (see Table S2). The highest maximum specific growth rate (μmax 0.024 h−1) should be reached at 41.83 °C, 1.05 mM pyruvate and 2.59 mM NH4Cl (with a desirability of 84 %). This corresponds to a generation time of 29.0 h. In order to verify μmax and the generation time predicted by the RSM experimentally, we grew the strain at 42 °C, 1 mM pyruvate and 2 mM NH4Cl and obtained a maximum specific growth rate (μmax) of 0.023 h−1 and a generation time of 30.1±0.6 h (based on nitrite production). Similar values for growth rate and generation time were obtained using cell counts of EN76T for the calculation . These experimentally determined values are close to the generation time (29.2 h) and a maximum specific growth rate (μmax 0.024 h−1) predicted by the model equation under the tested conditions (Table S2). The model equation was additionally verified by recalculating the generation time reported by Candidatus Nitrososphaera gargensis’ Ga9.2 have been reported to grow at 46 °C with an ammonium concentration of 1 mM (Strain EN76 3 years . In orde 3 years . As an eT was incubated in the dark. When cultivated in larger volumes (>100 ml), cultures were shaken at 150 r.p.m. Although the strain grows aerobically [21 % (v/v) O2 in the gas phase], it can grow at oxygen concentrations as low as 3 % (v/v) O2 in the gas phase . Inhibition of the activity of methanotrophic bacteria by black butyl rubber stoppers has been reported previously  and usually occurred as single cells, although clusters comprising several cells were sometimes observed. Cells were motile and possess archaella  were found in the genome of EN76T, supporting the conclusion that EN76T is probably motile. Crenarchaeota-like type-2 fla gene clusters have been found in ‘Candidatus Nitrososphaera gargensis’ Ga9.2 and the group I.1a-related strain ‘Candidatus Nitrosoarchaeum limnia’ SFB1, but not in the genome of ‘Candidatus Nitrosopumilus maritimus’ SCM1 , which have also been identified in genomes of other members of the Thaumarchaeota , closely related to strain EN76T, as a deep-branching group of the Crenarchaeota  based on 16S rRNA gene phylogeny  and Methanothermus fervidus DSM 2088T (79 %) represent the closest related cultivated strains of species with validly published names from the phyla Crenarchaeota and Euryarchaeota, respectively (T has a DNA base composition of 52.7 mol% G+C (Candidatus Nitrososphaera gargensis’ Ga9.2 (48.4 mol%) and higher than that of group I.1a strains such as ‘Candidatus Nitrosopumilus maritimus’ SCM1 (34.2 mol%), ‘Candidatus Nitrosoarchaeum koreensis’ MY1 (32.7 mol%) and ‘Candidatus Nitrosoarchaeum limnia’ SFB1 (32.4 mol%) .Nitrososphaera viennensis.Mesophilic to moderately thermophilic, acidophilic to neutrophilic, aerobic, autotrophic or mixotrophic, ammonia-oxidizing organisms. Cells are irregular coccoid. The major lipid is crenarchaeol and its regioisomer. The type species is Nitrososphaera viennensis .4Cl concentration for growth is 2.6 mM, but concentrations up to 15 mM are tolerated. Nitrite concentrations up to 10 mM are tolerated. Urea can be used as substrate. N2O is formed as a side product during ammonia oxidation. Mixotrophic growth is observed with bicarbonate and small carboxylic acids, i.e. pyruvate, α-ketoglutarate, oxaloacetate or glyoxylate, as carbon sources. The following substrates have a negative effect or inhibit growth under the conditions tested: ribose, glycerol, peptone, yeast extract, l-alanine, d-alanine, l-glutamine, l-aspartic acid, an amino acid mixture, Casamino acids, methylamine, trimethylamine, ethanolamine, methanolamine, nitrate, pyrimidine and purine.Irregular cocci with a diameter of 0.78±0.13 µm. Occur as single cells and as clusters of several cells. Cells exhibit archaella (12.0±1.8 nm) and archaeal pili (6.4±1.3 nm) as cell appendages, and clearly discernible areas of high and low electron density and tubule-like structures in the cytoplasm. Cells possess an S-layer with p3-symmetry. Grows at pH 6–8.5, with an optimum at pH 7.5. The temperature optimum is 42 °C; grows at 28–47 °C. Energy is produced by oxidizing ammonia to nitrite with oxygen as electron acceptor. Optimal NHT ( = DSM 26422T = JMC 19564T), was isolated from a garden soil in Vienna, Austria. The DNA base composition of the type strain is 52.7 mol% G+C.The type strain, EN76Nitrososphaeraceae .Nitrososphaera. The type genus is Nitrososphaera.The description is the same as for the genus Nitrososphaerales .Nitrososphaerales refers to the former group I.1b (or ‘soil group’) within the phylum Thaumarchaeota. Cultivated organisms of this order have an irregular coccoid cell shape and occur predominantly in terrestrial ecosystems. By contrast, cells of all known organisms affiliated with group I.1a  are rod-shaped. The order Nitrososphaerales comprises a highly supported distinct phylogenetic group based on 16S rRNA gene phylogeny .Thaumarchaeota might assimilate their cellular carbon via a modified version of this pathway  and only one copy of the proliferating cell nuclear antigen and lack genes for RNA polymerase G . Additio(Archaea . The typ


Weissella ceti strains WS74 and WS105, isolated from diseased rainbow trout in Brazil. The two genomes were sequenced with an Ion Torrent personal genome machine (PGM) using a fragment library. The genomes of strains WS74 and WS105 consist of circular chromosomes 1,389,513 bp and 1,390,396 bp long, respectively, both presenting a G+C content of 40.75%.We describe here the genome sequencing and annotation of  Weissella species belong to the group of lactic acid bacteria (LAB) and have been isolated from a broad variety of nutrient-rich environments  types  types .W. ceti strains WS74 and WS105 were thawed, streaked onto Man Rogosa and Sharpe (MRS) agar , and incubated at 25°C for 48 h. The DNA was extracted with a Maxwell 16 DNA purification kit using a Maxwell 16 instrument . Extracted DNA molecules were quantified with a Qubit 2.0 fluorometer , and the DNA integrity was assessed using TapeStation . The DNA was sequenced in a personal genome machine (PGM) Ion Torrent sequencing system  using a fragment library with an Ion PGM 200 sequencing kit.W. ceti WS74 yielded genome coverage of ~210×. An initial assembly of WS74 with Mira 4.0 , 3 pseudogenes, 1 tmRNA, 77 tRNAs, and 19 rRNAs. The genome of W. ceti WS105 has 1,390,396 bp with a G+C content of 40.75% and harbors 1,433 genes, which are composed of 1,336 CDSs, 1 tmRNA, 71 tRNAs, and 18 rRNAs. The genome sequences of the 2 strains of W. ceti described here open new doors for genome comparison of this bacterium, which will help in elucidating the mechanisms that drove the emergence of an LAB as a new fish pathogen.Automatic gene and pseudogene prediction and annotation were performed using the software PROKKA version 1.7  and RASTW. ceti strains WS74 and WS105 have been deposited at Genbank under the accession numbers CP009223 and CP009224, respectively.The whole-genome projects of


Myolipoma of soft tissue is an extremely rare benign tumor composed of mature adipose tissue and smooth muscle cells. It is found predominantly in women. The cytogenetic and molecular genetic features of myolipomas remain largely unexplored. Here we present the first cytogenetically analyzed myolipoma.Cytogenetic and molecular genetic analyses were done on a myolipoma.HMGA2 (in 12q14) was rearranged. Molecular genetic analysis showed that the translocation resulted in fusion of HMGA2 with the C9orf92 gene (from 9p22). The HMGA2-C9orf92 fusion transcript would code for a putative protein containing amino acid residues 1–94 of HMGA2 and 6 amino acid residues from the out-of-frame fusion with exon 4 of C9orf92.G-banding analysis of short-term cultured cells from the myolipoma yielded a karyotype with a single clonal chromosome abnormality: 46,XX,t. Fluorescence in situ hybridization experiments demonstrated that HMGA2 rearrangement in the present case of myolipoma is similar to what is found in other benign connective tissue tumor types, including lipomas, i.e., disruption of the HMGA2 locus leaves intact exons which encode the AT-hook domains but separates them from the 3´-terminal part of the gene. Whether any genetic features differentiate myolipomas from regular lipomas with HMGA2-involvement is a question that cannot be answered until more cases of the former tumor type are subjected to genetic analysis.The pattern of The online version of this article (doi:10.1186/s13000-016-0472-8) contains supplementary material, which is available to authorized users. Written informed consent was obtained from the patient. The consent included acceptance that his clinical details be published. The ethics committee’s approval included a review of the consent procedure. All patient information has been anonymized.The study was approved by the Regional Committee for Medical and Health Research Ethics, South-East Norway (REK Sør) The patient is a 66 years old female who underwent an abdominal CT-scan due to pain radiating into the left lower extremity. The scan revealed a well demarcated, lipogenic tumor in the left retroperitoneum measuring almost 20 cm in greatest diameter, invoking a suspicion of liposarcoma. The tumor was completely excised. The operation specimen showed a lipomatous tumor which was macroscopically well demarcated and without infiltrative growth. Microscopic evaluation showed a lipomatous tumor with areas with smooth muscle differentiation, without signs of malignancy Fig.  and b. TFresh tissue from a representative area of the tumor was received and cells from it were short-term cultured and analyzed cytogenetically as part of our diagnostic routine as described elsewhere . The karhttp://bacpac.chori.org/pHumanMinSet.htm). The “32k set” is mapped on the UCSC Genome browser on Human May 2004 (NCBI/hg17) assembly. Mapping data for the 32k human re-array are available in an interactive web format  and are obtained by activation of the ucsc browser track for the hg17 UCSC assembly from the “32k set” homepage (http://bacpac.chori.org/genomicRearrays.php). The BAC clones were selected according to physical and genetic mapping data on chromosome 12 as reported on the Human Genome Browser at the University of California, Santa Cruz website . In addition, FISH mapping of the clones on normal controls was performed to confirm their chromosomal location. The clones used were RP11-185 K16, (chr12:64103524–64274514), RP11-30I11 (chr12:64178505–64349708), RP11-662G15 (chr12:64288763–64498219), RP118B13 (chr12:64644968–64789255), RP11-745O10 (chr12:64752327–64926193), and RP11-263A04 (chr12:64908453–65103538). All of them map to chromosome subband 12q14.3 . A homemade breakapart HMGA2 probe was used. The 5´-end of the probe  was constructed from a pool of the clones RP11-185K16, RP11-30I11, and RP11-662G15. The 3´-end of the probe  was constructed from a pool of the clones RP118B13, RP11-745O10, and RP11-263A04. All of them map to chromosome subband 12q14.3 and cover the HMGA2 locus  was performed on metaphase plates as described previously . BAC clo4.3 Fig. . DNA wascus Fig. .Fig. 2CyTumor tissue adjacent to that used for cytogenetic analysis and histologic examination had been frozen and stored at −80 °C. Total RNA was extracted using miRNeasy Mini Kit according to the manufacturer’s instructions . Tumor tissue was disrupted and homogenized in Qiazol Lysis Reagent (Qiagen) using 5 mm stainless steel beads and TissueLyser II (Qiagen). Subsequently, total RNA was purified using QIAcube (Qiagen). Human Universal Reference Total RNA was used as control . According to the company’s information, it is a mixture of total RNAs from a collection of adult human tissues chosen to represent a broad range of expressed genes. Both male and female donors are represented.HMGA2-C9orf92 chimeric transcript (see below), RT-PCR was performed using the forward HMGA2-936F1 (5´-AGC CCT CTC CTA AGA GAC CCA G-3´) and the reverse C9orf92-316R1 (5´-TGA AGT TTT AAT CAA CAC AAG CAG C-3´) primers. Total RNA (1 μg) was reverse-transcribed in a 20 μL reaction volume using iScript Advanced cDNA Synthesis Kit for RT-qPCR according to the manufacturer’s instructions . The 25 μL PCR volume contained 12.5 μL Premix Ex Taq DNA Polymerase Hot Start Version (Takara Bio), 1 μL of cDNA, and 0.4 μM of each of the forward HMGA2-936F1 and reverse C9orf92-316R1 primers. PCR amplifications were run on a C-1000 Thermal cycler (Bio-Rad Laboratories) with an initial denaturation at 94 °C for 30 s, followed by 35 cycles of 7 s at 98 °C, 30 s at 58 °C, 1 min at 72 °C, and a final extension for 5 min at 72 °C. Three μL of the PCR products were stained with GelRed , analyzed by electrophoresis through 1.0 % agarose gel, and photographed. The remaining 22 μL PCR products were purified using the MinElute PCR purification kit (Qiagen) and sequenced at GATC Biotech . The BLAST software (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used for computer analysis of sequence data.The 3´-RACE methodology was described in detail elsewhere . To veriG-banding analysis of short-term cultured cells from the myolipoma yielded a karyotype with a single clonal chromosome abnormality: 46,XX,t9;12) [HMGA2 probe was split with one signal on der(12) and the other on der(9)  Fig. .HMGA2 from 12q14 (nt 1093 in the reference sequence with accession number NM_003483.4) was fused to exon 4 of the C9orf92 gene from 9p22 (nt 261 in the reference sequence NM_001271829.1).3´-RACE analysis amplified a single fragment Fig. . SubsequPCR with the primers HMGA2-936F1 and C9orf92-316R1 amplified a cDNA fragment from myolipoma but not from control Fig. . Direct C9orf92 (from 9p22) and HMGA2 (from 12q14) genes. The HMGA2-C9orf92 fusion transcript codes for a putative protein which contains amino acid residues 1–94 of HMGA2 protein (accession number NP_003474.1), corresponding to exons 1–4 of the gene, and 6 amino acid residues (AHKEDT) coming from an out-of-frame fusion with exon 4 of C9orf92.We describe the first cytogenetic and molecular genetic analysis of a myolipoma. The tumor had an acquired chromosomal translocation, t, which resulted in fusion of the http://www.ncbi.nlm.nih.gov/gene/100129385). Two transcript variants have been reported: Transcript variant 1 (reference sequence NM_001271829) which represents the shorter transcript and encodes a functional protein, and transcript variant 2 (reference sequence NR_073471) which uses an alternative 5' exon structure compared to variant 1. Transcript variant 2 is a non-coding RNA due to the presence of an upstream open reading frame (ORF) that is predicted to interfere with translation of the longest ORF (http://www.ncbi.nlm.nih.gov/gene/100129385). C9orf92 is low expressed in many normal human tissues as shown by RNA-sequencing (http://www.genecards.org/cgi-bin/carddisp.pl?gene=C9orf92&keywords=c9orf92).Information on C9orf92 is very scant and nothing is known about its cellular localization or function , or variants thereof, was reported before in lipomas [, and exon 9 of NFIB [HMGA2 with NFIB or MSRB3 [HMGA2-NFIB fusions found in pleomorphic adenomas also contain a stop codon located near but downstream of the fusion point [In lipomas, two different  of NFIB , 13, 15.of MSRB3 q14.3, anon point .NFIB gene codes for a transcription factor which recognizes and binds the palindromic sequence 5-TTGGCNNNNNGCCAA-3 of viral and cellular promoters (http://www.genecards.org/cgi-bin/carddisp.pl?gene=NFIB&keywords=NFIB). No functional information is at hand about the C9orf92 gene (http://www.genecards.org/cgi-bin/carddisp.pl?gene=C9orf92&keywords=c9orf92).The HMGA2-NFIB in pleomorphic adenoma and lipomas but HMGA2-C9orf92 in the only myolipoma examined, the pathogenetic pattern behind these changes is similar to that of HMGA2 rearrangements found generally in benign connective tissue tumors, i.e., disruption of the HMGA2 locus leaving intact exons 1–3 which encode the AT-hook domains and separates them from the 3´-untranslated region of the gene (3´-UTR) [HMGA2 was shown to regulate the transcription of the HMGA2 gene [Although the tp22;q14 ~ 15) thus appears to be heterogeneous at the molecular level, generating (3´-UTR) . The 3´-GA2 gene , 22.;q14 ~ 15HMGA2 rearrangements in the genesis of several benign connective tissue tumors. Further studies are necessary to find out whether myolipomas of soft tissue in any systematic way differ from the other tumor types in the exact manner in which HMGA2 is abrogated, in particular whether fusion with C9orf92 is a general feature of these rare neoplasms.The present case of myolipoma underscores the frequent and general role of


The effect of reaction temperature and time on the properties of the particles was investigated. The nanoparticles were characterized by transmission electron microscopy (TEM), electron diffraction (ED), energy dispersive spectroscopy (EDX), dynamic light scattering (DLS), thermogravimetric analysis (TGA), elemental analysis and X-ray diffraction (XRD) to determine morphology, size, polydispersity, crystal structure and elemental composition of the nanocrystals. TEM microscopy revealed that the morphology of the nanoparticles could be fine-tuned by modifying of the synthetic conditions. A cubic-to-hexagonal phase transition of the NaYF4:Yb3+/Er3+ nanoparticles at temperatures above 300 °C was confirmed by both ED and XRD. Upconversion luminescence under excitation at 980 nm was observed in the luminescence spectra of OM–NaYF4:Yb3+/Er3+ nanoparticles. Finally, the OM–NaYF4:Yb3+/Er3+ nanoparticles were coated with a silica shell to enable further functionalization and increase biocompatibility and stability in aqueous media, preventing particle aggregation.NaYF Hexagonal β-NaYF4 crystals sensitized by Yb3+ and activated by Er3+/Tm3+ ions are a highly efficient host material for green and blue upconversion  (2θ = 30.9) reflections, respectively [4:Yb3+/Er3+ nanoparticles prepared at lower temperatures exhibited both α- and β-phases, but the average α-phase crystallite size decreased from about 9 to 6 nm with increasing temperature F4 F4 ,b. The Er0.04)F4 . The spe4:Yb3+/Er3+ nanoparticles was elemental analysis. The amount of the OM shell on the NaYF4:Yb3+/Er3+ nanoparticles was determined from carbon and nitrogen concentrations according to the following equations: % coating according to C = (% C in sample × 100)/80.74, where 80.74 is % C in OM, and % coating according to N = (% N in sample × 100)/5.23, where 5.23 is % N in OM. The percentage of the OM shell on the nanoparticles prepared at 250–330 °C ranged between approximately 3 and 6 wt % according to both carbon and nitrogen analysis. In contrast, the no. 5 OM–NaYF4:Yb3+/Er3+ nanoparticles obtained at 350 °C  and red (660 nm) upconversion emissions corresponding to 2H11/2 and 4S3/2 → 4I15/2 and 4F9/2 → 4I15/2 transitions, respectively. These bands were induced by 4f–4f transitions of the Er3+ ions.To investigate the optical properties of the OM–NaYFcitation . The eneed state . This scEr3+ ion . The 2H1emission . Hence, I545/I660 ratio should be rather low and the total luminescence intensity high. The ratio of green to red emission intensities changed from 2.50 to 1.68. The light emitted by the no. 1 particles prepared at 250 °C had a low intensity due to their cubic crystal structure. However, the no. 2 particles prepared at 280 °C had a higher I545/I660 ratio. This can be explained by the different interatomic distances between the Yb3+ and Er3+ ions in the crystal structure, which changed from cubic to hexagonal. The crystals were not completely hexagonal and contained some cubic impurities, and the distances between Yb3+ and Er3+ therefore differed, increasing the green light intensity. The red light intensity remained constant due to insufficient energy transfer to Er3+ and energy losses between the ions. When the particles were prepared at temperatures above 300 °C, luminescence intensities were slightly higher, indicating that maximum luminescence efficiency was achieved by optimizing the interatomic distance between the absorbers and the emitters.Because the red light photons are important for prospective biomedical applications, the 4:Yb3+/Er3+ nanoparticles were prepared at 330 °C and reaction times ranged from 0.5 to 4 h. Other reaction conditions were held constant as described above. The size of the NaYF4:Yb3+/Er3+ nanoparticles of nos. 4 and 6–9 was again approximately 10 nm and the particle size distribution was rather narrow  and varying reaction time are shown in I545/I660 decreased with increasing reaction time.Visible upconversion spectra of the OM–NaYF4:Yb3+/Er3+ nanoparticles were easily dispersed in nonpolar organic solvents, such as hexane or toluene, due to the presence of aliphatic OM side chains on the surface. However, the nanoparticles must disperse in aqueous media for biological applications. To disperse the no. 4 NaYF4:Yb3+/Er3+ nanoparticles in water, they were coated with a thin silica shell using a microemulsion technique. TMOS and Igepal CO-520 were used as a precursor and an emulsifier, respectively. Compared with the initial 10 nm OM–NaYF4:Yb3+/Er3+ nanoparticles, the TEM micrograph  in biological experiments, the nanoparticles should emit photons at 660 nm to excite phthalocyanine, a typical photosensitizer [4:Yb3+/Er3+ nanoparticles were excited by near-infrared light at 980 nm, i.e., at the Yb3+ absorption maximum. Photons were emitted at 520, 545 and 660 nm in the fluorescence spectra of the OM–NaYF4:Yb3+/Er3+ nanoparticles. The NaYF4:Yb3+/Er3+ nanoparticles were successfully coated with a silica shell using the reverse microemulsion method, making them dispersible in water and promising candidates for applications in biology and medicine.OM–NaYFnsitizer . Upconve


Cryptococcus neoformans is the most common cause of fungal meningoencephalitis in AIDS patients. Depletion of CD4 cells, such as occurs during advanced AIDS, is known to be a critical risk factor for developing cryptococcosis. However, the role of HIV-induced innate inflammation in susceptibility to cryptococcosis has not been evaluated. Thus, we sought to determine the role of Type I IFN induction in host defense against cryptococci by treatment of C. neoformans (H99) infected mice with poly-ICLC (pICLC), a dsRNA virus mimic. Unexpectedly, pICLC treatment greatly extended survival of infected mice and reduced fungal burdens in the brain. Protection from cryptococcosis by pICLC-induced Type I IFN was mediated by MDA5 rather than TLR3. PICLC treatment induced a large, rapid and sustained influx of neutrophils and Ly6Chigh monocytes into the lung while suppressing the development of eosinophilia. The pICLC-mediated protection against H99 was CD4 T cell dependent and analysis of CD4 T cell polyfunctionality showed a reduction in IL-5 producing CD4 T cells, marginal increases in Th1 cells and dramatic increases in RORγt+ Th17 cells in pICLC treated mice. Moreover, the protective effect of pICLC against H99 was diminished in IFNγ KO mice and by IL-17A neutralization with blocking mAbs. Furthermore, pICLC treatment also significantly extended survival of C. gattii infected mice with reduced fungal loads in the lungs. These data demonstrate that induction of type I IFN dramatically improves host resistance against the etiologic agents of cryptococcosis by beneficial alterations in both innate and adaptive immune responses. Cryptococcus neoformans is the leading cause of mortality in AIDS patients in the developing world. It has been known that depletion of CD4 T cells is the most critical predisposing factor to cryptococcosis in HIV infected patients. What has not been clear is the effect of HIV-induced innate inflammation in susceptibility to cryptococcosis. We treated C. neoformans infected mice with poly-ICLC (pICLC), a dsRNA virus mimic, to study the role of virus-induced type I IFN in host defense against cryptococcosis. PICLC treatment induced type I IFN in C. neoformans infected mice via MDA5 and significantly prolonged the survival of mice with reduced fungal burden in the brain. PICLC also protected mice from cryptococcosis caused by C. gattii. PICLC treatment recruited large numbers of neutrophils and Ly6Chigh monocytes into the lung parenchyma and suppressed eosinophilia. PICLC-mediated protection against C. neoformans required CD4 T cells and was associated with suppressed Th2 and enhanced Th17 responses. IFNγ and IL-17A were also important for pICLC-induced protection of infected mice. Our study demonstrates that induction of type I IFN dramatically improves host resistance against cryptococci by beneficial alterations in both innate and adaptive immune responses as long as CD4 cells are not depleted.Meningoencephalitis due to  C. neoformans infection is primarily due to the depletion of CD4 T cells, which leads to defects in both innate and adaptive immunity AbB) on a C57BL/6 background, were purchased from Taconic Farms  under the NIAID Animal Supply Contract. Mda5-/- mice were obtained from The Jackson Laboratory. Ifnb-/- mice [WT C57BL/6 mice were purchased from DCT . -/- mice  were kinC. neoformans and C. gattii, respectively. Strains were stored in 25% glycerol at −80°C until use and were maintained on YEPD  agar plates at 30°C.The strains H99 and R265, both genome sequenced strains were chosen as representative strains of C. neoformans or C. gattii. Survival was monitored for up to 80 days post inoculation. Mice were observed daily for signs of disease and lethality. Mice with signs of irreversible disease  were euthanized. For quantification of fungal burden, organs (lung and brain) were weighted, homogenized, and diluted in 10-fold steps in PBS. Fungal CFUs were determined by plating serially diluted homogenates on YEPD agar plates. Lungs and brains from mice were subjected to histopathologic staining with hematoxylin and eosin (H&E) and Gomori methenamine silver (GMS).To induce respiratory infection, WT and knockout mice were anesthetized with isoflurane and inoculated with the designated number of yeast cells in 20 μl of phosphate-buffered saline (PBS) via intrapharyngeal (i.p.) aspiration  ,70. EachPICLC (Hiltonol), a synthetic analogue of viral dsRNA, was supplied by Oncovir Inc. Mice were inoculated intrapharyngeally with pICLC (5 μg in 20 μl/mouse) twice weekly up to 10 weeks, starting at the same day of infection. To examine preventive or therapeutic effect pICLC was administered twice weekly up to 10 weeks, starting 3 days prior or 1 day after cryptococcal exposure, respectively. Recombinant mouse IFNα and IFNβ were generous gifts from PBL InterferonSource . IFNα or IFNβ (1000 U) was administered intrapharyngeally twice weekly up to 10 weeks, starting at the same day of infection. Fluconazole (FLC) was used along with pICLC to assess antifungal combination therapy. FLC was administered intraperitoneally at a concentration of 10 mg/kg of body weight/day, starting 24 h after infection and continued for 28 days. For IL-17A neutralization experiments, mice were injected with 200 μg of αIL-17A mAb clone 17F3 every three days.The intravascular staining was performed as described previously . BrieflyThe Institutional Animal Care and Use Committee of the National Institute of Allergy and Infectious Diseases approved all animal studies (#A4149-01). Studies were performed in accordance with recommendation of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.http://exon.niaid.nih.gov [For intracellular cytokine staining, isolated lung leukocytes were stimulated with soluble αCD3 and αCD28 (both at 1 μg/ml) in the presence of brefeldin A and monensin for 5 hours. Lung cells were stained with various combinations of the following mAb clones: αTNF MP6-XT22, αCD8 53.67, αIL-17A TC11-18H10.1, αCD4 RM4-5, αCD45.2 104, αIL-5 TRFK5, αCD44 IM7, αIFNγ XMG1.2, αIL13 eBio13A, αFoxp3 FJK-16S, αLy6G 1A8, αCD11b M1/70, αCD11c N418, αCD103 2E7, αCD68 FA11, αLy6C HK1.4, αSiglec F E50-2440, αT-bet 4B10, αPD-1 29F.1A12, αRORγt B2D, and αGATA-3 TWAJ. All FACS antibodies were purchased from eBioscience  and Biolegend . Analysis of CD4 T cell cytokine polyfunctionality was performed using SPICE version 5.3, downloaded from .nih.gov .Lungs were homogenized in 1 ml PBS. The samples were then centrifuged at 16,000 g for 30 minutes at 4°C, and IFNα/β, IFNγ levels were determined in the supernatants using ELISA  according to the manufacturer’s recommendation. In some experiments, bronchoalveolar lavage fluid (BAL) was collected by lungs’ perfusion with 1 ml of cold PBS after partial tracheal resection using 22-gauge catheters. Mouse Cytokine Group I (23-Plex) was measured to quantify cytokine and chemokine concentrations in the supernatants obtained from mice, as described above, using Bio-Plex assay  following the manufacturer's instructions.P values of <0.05 were obtained, differences were considered statistically significant.Survival data from the animal experiments were analyzed using a two-group Wilcoxon test . An unpaired t test was used for evaluation of the CFU in tissue burden studies. Two-group comparisons were done with the Student’s t test. When S1 FigData are representative of 3 independent experiments with n = 3 mice/group. Scale bar = 50 μm.(TIF)Click here for additional data file.S2 FigC. neoformans H99 and treated with rIFNβ twice weekly starting on the day of infection or left untreated and monitored for survival. Data are representative of 2 independent experiments with n = 5 mice/group.WT mice were intrapharyngeally infected with 5000 CFU of (TIF)Click here for additional data file.S3 FigP < 0.05).BAL fluid was harvested from mice 7 days post-infection and KC (A), G-CSF (B), and CCL2 (C) were measured my multiplex analysis and IFNγ (D) was measured by ELISA. Data are representative of 3 mice/group and two independent experiments. Error bars represent the standard deviation Click here for additional data file.S4 FigC. neoformans H99. Lungs were harvested at day 20 post-infection and the percentage of each myeloid cell type that is negative for the intravascular stain was quantified. Data are representative of two independent experiments with n = 4–5 mice/group.Mice were intrapharyngeally infected with 5000 CFU of (EPS)Click here for additional data file.S5 FigC. neoformans H99 and lungs were harvested at day 20 post-infection. Data shows effector/memory CD4's that are negative for the intravascular stain. Data are pooled from 2 separate experiments with n = 5 mice/group.Mice were intrapharyngeally infected with 5000 CFU of (TIF)Click here for additional data file.S6 FigC. neoformans H99 and treated with pICLC twice weekly starting on the day of infection or left untreated and monitored for survival. Data are representative of 2 independent experiments with n = 5 mice/group.Mice were intravenously infected  with 5000 CFU of (TIF)Click here for additional data file.


In vitro studies show that Leishmania infection decreases the adhesion of inflammatory phagocytes to connective tissue by a mechanism dependent on the modulation of integrin function. However, we know little about the influence of this reduction in leukocyte adhesion on parasite dissemination from the infection site.L. amazonensis infection on the ability of inflammatory phagocyte populations to migrate from an inflammatory site to the draining lymph node. Uninfected or Leishmania-infected thioglycollate-elicited peritoneal exudate cells were transferred from C57BL/6 to BALB/c mice or from Ly5.1+ to Ly5.1- mice. The transferred cells were injected into the peritoneal cavity and tracked to the draining lymph node.In this work, we used a model of chronic peritonitis induced by thioglycollate to study the effect of +CD11c+ (myeloid dendritic cell) was lower after incubation with Leishmania  than after leukocyte incubation with medium alone (P < 0.01). There was no consistent decrease in the migration of CD11b+F4/80+ (macrophage) or SSChi GR-1+ (neutrophil) populations.Migrating cells corresponded to approximately 1% of the injected leukocytes. The proportion of migrating CD11bLeishmania changes the migratory pattern of dendritic cells in vivo. Such changes in dendritic cell migration may be associated with immunological events that maintain inflammation at the sites of infection.Coincubation with The online version of this article (doi:10.1186/1471-2334-14-450) contains supplementary material, which is available to authorized users. Leishmania promastigotes into the skin of a susceptible host during the blood meal of an infected sand fly [Leishmania inoculation site, various phagocytes have the potential to phagocytize and transport live amastigotes or killed parasites, which may also act as antigens, to the draining lymph node. These cells include dermal resident dendritic cells (DC), epidermal Langerhans cells, neutrophils and monocytes, which are released from the blood when the dermal capillary is disrupted and a reputed blood lake is formed [Leishmania infection [Leishmania impairs the adhesion of different mononuclear phagocytes to inflamed connective tissues and to connective matrix components in vitro and that this decrease in cell adherence correlates with the proportion of infected phagocytes and the parasite burden in the cell [Leishmaniasis is initiated by the inoculation of sand fly . Thereafsand fly , 2. At ts formed –6. Soon s formed , 7. The s formed , 7. In ps formed . At the nfection –10. In athe cell , 12.Leishmania burden may impair the exit of these cells from the inflammatory site. The capability of Leishmania-infected cells to migrate or to remain in the infection site may be important for defining the characteristics and lesion distribution in leishmaniasis. For example, most of cutaneous leishmaniasi ulcer develops at the infection site, sometimes months after an initial papule. In other instances, after cure of cutaneous lesion metastatic mucocutaneos ulcers emerges.Leukocyte exit from the inflammatory site towards the lymph node involves a sequence of steps, starting with the response of these cells to chemokines released by lymphatic endothelial cells –16. In rin vivo described by Bellingan and collaborators [Leishmania to the connective tissue observed in vitro corresponded to changes in the migratory pattern of these cells in vivo. The model described by Bellingan and colleagues is designed for studying macrophage exit through the lymphatic system during the resolution of a thioglycollate-induced peritonitis and has characteristics that make it suitable for use in our study: (1) the thioglycollate-induced peritoneal exudate contains a variety of phagocytes, including neutrophils, macrophages and dendritic cell populations [in vitro manipulation and reinjection; (3) the adherence of the PEC to the connective tissue is also decreased after cultivation with Leishmania [in vivo migration system is dependent on VLA-4 [Leishmania-infected phagocytes [In this study, we used the murine model of phagocyte migration borators  to examiulations , 20, 21;ishmania , 12; andon VLA-4 , a molecagocytes .th day of thioglycollate peritonitis [Leishmania infection on the migration of different populations of phagocytes to the draining lymph node. In this series of experiments we used Leishmania amazonensis. This species is endemic in parts of Brazil, usually associated with cutaneous lesions. However, some L. amazonensis strains have also been isolated from patients with visceral leishmaniasis [Leishmania. Subsequently, we examined the change in the migration rate of cells incubated with Leishmania in conditions favoring the maximal decrease (70%) in peritoneal exudate cell adhesion to connective matrix components observed in our studies in vitro [Bellingan and collaborators showed that the phagocyte exit from the peritoneal cavity to the parathymic lymph node is maximal in the 4itonitis . Hence, maniasis . Infectiin vitro , 12. Thead libitum. The animals were subjected to thioglycollate peritonitis and adoptive cell transfer via intraperitoneal injection (see below). At the end of the experiments, the animals were killed by lethal CO2 anesthesia, and the peritoneal exudate cells and parathymic lymph nodes were removed as described below. All experiments involving animals were conducted in accordance with Brazilian Federal Law on Animal Experimentation (Law 11794) [http://www.planalto.gov.br/ccivil_03/_ato2007-2010/2008/lei/11794.htm). This study was approved by the Committee of Ethics in the Use of Animals of the CPqGM-FIOCRUZ .Six- to eight-week-old BALB/c, C57BL/6 and C57BL/6 SJL mice (antigenic specificity Ly5.1) of both genders were obtained from the colony of the Gonçalo Moniz Research Center-FIOCRUZ . The animals were maintained under the controlled environmental conditions of humidity, temperature and a light–dark cycle, with commercial balanced mouse chow and water provided w 11794)   were grown in Schneider’s insect medium (Sigma) containing 10% FBS at 24°C. Stationary phase promastigotes were washed three times in HBSS, suspended in complete RPMI, and incubated with the phagocytes. Control phagocytes were cultured in medium alone.2+- and Mg2+-free Hanks’ balanced salt solution  containing 20 IU/ml heparin. The number of cells collected from each animal was estimated using a Neubauer chamber. Cell viability was assessed using the trypan blue exclusion technique, and cell populations were defined morphologically using cytospin preparations and by phenotyping using specific antibodies in flow cytometry. Parathymic lymph nodes were collected by dissection and were studied by flow cytometry.Peritonitis was induced in BALB/c, C57BL/6 and C57BL/6 SJL mice by injecting 3 ml of a 3% (wt/vol) thioglycollate  sterile solution. Three to four days after peritonitis induction, the PEC were collected by a standardized washing of the peritoneal cavity, performed twice with cold CaLeishmania phagocytosis rate by different phagocyte populations, parasites were labeled with PKH26 before coincubation with PEC. Briefly, 108 parasites were suspended in 2 ml of PBS, mixed with a 4 μM PKH26 solution in 2 ml of PBS and incubated for 5 min at room temperature in the dark under periodic agitation. Staining was terminated by the addition of 4 ml of fetal bovine serum (FBS), incubation at 37°C for 30 min and washing three times in HBSS. At the end of 24 h of co-culture, the cells were washed and subjected to flow cytometry for identification of the mononuclear phagocyte populations positive for PKH26.To estimate the b) mice. Three days after peritonitis induction, PEC were collected, washed twice in HBSS, suspended at 2 × 106 cell/mL in RPMI (Sigma) with 10% fetal bovine serum , 60 g/ml gentamicin and 2 mM glutamine (complete RPMI) and cultured alone or with 2 × 107 stationary phase L. amazonensis promastigotes for 18–24 h in non-adherent polypropylene tubes at 37°C and 5% CO2. The cells were washed and intraperitoneally transferred (1 × 107 cells) to BALB/c (H-2d) mice. On the following day, the recipient animals were killed, and the PEC and parathymic lymph nodes were removed. The proportion of H-2b+ (C57BL/6) phagocytes of different populations present in the peritoneal cavity and in the draining lymph node was studied by flow cytometry.Thioglycollate peritonitis was induced in C57BL/6  from C57BL/6 Ly5.1+ to C57BL/6 Ly5.1- recipient animals. After 24 h, the PEC and parathymic lymph nodes were removed and processed for flow cytometric analysis.To avoid the potential effect of the MHC mismatch between the mice strains, though the migration experiment was performed for only a short period, a similar set of experiments was performed using cell transfer-and-tracking experiments using C57BL/6 Ly5.1PEC were washed, counted and resuspended in FACS buffer containing an anti-Fc monoclonal antibody for 30 min on ice to block non-specific staining. Parathymic lymph nodes were cut into small fragments, digested with collagenase D  and diluted in RPMI 1640 medium supplemented with 3% FBS and 0.1% DNase I  for 10 minutes at 37°C with continuous agitation. Cell suspensions were filtered through gauze and washed twice in a phosphate-buffered saline (PBS) solution supplemented with 5% FBS and 5 mM EDTA containing 5 mg/ml DNase I [6/stain) were incubated with fluorescein-conjugated antibodies (anti-CD11b (M1/70) and anti-I-Ad (AMS-32.1) ; anti-F4/80 antigen ), phycoerythrin (PE) conjugates  and anti-H2b , and biotin-conjugated antibodies (anti-CD11c (HL3), CD45.1  followed by streptavidin Cy-5 or phycoerythrin conjugate . All antibody incubations were performed at 4°C for 20 min and were followed by three washes with FACS buffer. Unlabeled or isotype-matched stained cells were used as a control. Cells were analyzed on a FACScan flow cytometer using CellQuest software (Becton-Dickinson). The histograms and bitmaps of the distributions of cell populations were constructed using FlowJo Software . Fifty thousand events were analyzed per sample [hi CD11bhi were defined as myeloid dendritic cells.The cells  relative to the percentage of the same injected cell population remaining in the peritoneal cavity. The significance of the differences observed between the control and infected groups was tested using the paired Student’s t-test. The threshold for statistical significance was set at P < 0.05. All experiments were performed independently a minimum of three times, using a minimum of six animals in each experimental group.th day after thioglycollate injection, the number of cells present in the peritoneal cavity was 1.7 × 107, approximately five times higher than that observed in control animals injected with saline . To investigate the efficiency of Leishmania phagocytosis by the different mononuclear phagocytes present in PEC, we co-cultivated PEC with PKH26-labeled promastigotes for 24 h and examined the phenotypes of PKH26-labeled leukocytes by flow cytometry. The phagocytosis rate was high (68 ± 31% to 77 ± 22%) among the CD11b+ populations  mouse peritoneal donor cells were cultivated overnight with medium alone (control) or with L. amazonensis (infected) and then injected into the peritoneal cavity of BALB/c mice (see Methods). After 24 h, the recipient mice injected with donor leukocytes cultivated with medium alone (control) had 13.1 ± 3.8% of H2b+ (C57BL/6) cells in the peritoneal cavity. Among these cells, 2.8 ± 1.6% were neutrophils (SSChi GR-1+), 73.0±19.8% were macrophages (CD11b+ F4/80+) and 42.3±8.4% were myeloid DCs (CD11c+ CD11b+). In the lymph nodes (LNs) of these animals, 1.1 ± 0.4% of the cells were H2b+. Among these migrating cells, 3.8 ± 0.7% were neutrophils (SSChi GR-1+), 39.1±21.7% were macrophages (CD11b+ F4/80+) and 21.8±7.3% were myeloid DCs (CD11c+ CD11b+) , the proportion of the H2b+ cells in the peritoneum was 11.4 ± 6.8%. Among these cells, 1.7 ± 0.9% were neutrophils (SSChi GR-1+), 65.5±19.2% were macrophages (CD11b+ F4/80+) and 39.3±4.9% were myeloid DC (CD11c+ CD11b+). In the LNs, the proportion of migrating (H2b+) cells was 1.2 ± 0.6%. Among these cells, 4.4±2.8% were neutrophils (SSChi GR-1+), 34.7±21.8% were macrophages (CD11b+ F4/80+) and 8.0±3.1% were myeloid DCs (CD11c+ CD11b+)  or with L. amazonensis (infected) and were then injected into the peritoneal cavity of C57BL/6 Ly 5.1- mice (see Methods). After 24 h, the recipient mice injected with donor leukocytes cultivated with medium alone (control) presented 10.7 ± 1.6% of Ly 5.1+ cells in the peritoneal cavity. Among these cells, 2.1 ± 2.1% were neutrophils (SSChi GR-1+), 73.5±10.4% were macrophages (CD11b+ F4/80+) and 35.0±9.0% were myeloid DCs (CD11c+ CD11b+). In the LNs of these animals, 1.1 ± 0.4% of the cells were Ly 5.1+. Among these migrating cells, 3.4 ± 1.8% were neutrophils (SSChi GR-1+), 31.0±12.8% were macrophages (CD11b+ F4/80+) and 40.8±9.3% were myeloid DCs (CD11c+ CD11b+) , the proportion of the Ly 5.1+ cells in the peritoneum was 11.2 ± 0.6%. Among these cells, 2.1 ± 2.3% were neutrophils (SSChi GR-1+), 63.5±29.1% were macrophages (CD11b+ F4/80+) and 46.7±28.7% were myeloid DCs (CD11c+ CD11b+). In the LNs, the proportion of migrating (Ly 5.1+) cells was 1.2 ± 0.6%. Among these cells, 1.2 ± 0.9% were neutrophils (SSChi GR-1+), 26.2±18.6% were macrophages (CD11b+ F4/80+) and 17.3±12.4% were myeloid DCs (CD11c+ CD11b+)  myeloid DC populations was consistently decreased in the draining LN after co-incubation with Leishmania t Test, paired test, P<0.01, Figure In the group of animals injected with donor PEC cultivated with hi F4/80hi (macrophage) phenotype. The infection rate and phagocytosis of dead Leishmania were high among CD11b+ myeloid cells and were similar between CD11b+ CD11c- (macrophage/neutrophil) and CD11b+ CD11c+ (myeloid DC) populations. In contrast, Leishmania infection/phagocytosis was low among CD11b- cells, irrespective of CD11c expression. Among the CD11b- CD11c- cells, only 30 ± 19% phagocytized the parasite, and this total may include resident peritoneal macrophages [+ and GR-1+ cells were the most frequently infected cells present in primary lesions produced by Leishmania major infection of the mice hypoderm [+ CD11c-) and the dermal monocyte-derived dendritic cells (CD11b+ CD11c+) had the highest parasite burdens among the leukocytes found in the dermal-infiltrate after Leishmania infection in vivo [hi expression presented the highest levels of Leishmania infection compared with the CD11bneg and CD11blo populations [Our data showed a varied population of leukocytes in the peritoneum at 4 days after thioglycollate-induced peritonitis, with a predominance of cells with a monocyte/macrophage morphology. These data confirm previous observations by Cook and colleagues  and wererophages . These d in vivo . In a prulations . CD11b iin vitro co-incubation for 6–24 h with Leishmania decreases the adherence of the J774 cell line, human peripheral blood monocytes and thioglycollate-elicited inflammatory peritoneal exudate cells (PEC) to both connective tissue and connective matrix components, such as fibronectin. This decrease in phagocyte adhesion resulted from an impairment in integrin regulation rather than an inhibition of integrin expression. This decrease also correlated with the percentage of phagocytes containing amastigotes and with the parasite burden in the phagocytes to the extent that, with a 50% phagocyte infection rate and a parasite burden of 4 amastigotes per phagocyte, a 70% reduction in PEC adhesion to fibronectin was achieved [Leishmania in conditions that favor a high parasite burden also decrease phagocyte migration from an inflammatory site to the draining lymph node in vivo. Although a trend of a decrease in the migration rate of F4/80+ cells and of neutrophils was observed in some experiments, only the CD11c+ cell population presented a consistent decrease in migration in all experiments using the adoptive transfer of cells between C57BL/6 and BALB/c mice or between Ly5.1+ and Ly5.1- animals.In a previous study, we observed that achieved , 12. In L. major impaired their ability to migrate in response to a variety of chemokines, except to CCL21 [L. donovani-infected mice was impaired due to deficient CCR7 expression [Leishmania species. Alternatively, DC populations may present intrinsically different responses to Leishmania infection. In our previous work, we have shown that the decrease in phagocyte adherence to connective tissue was independent on the species of Leishmania. Furthermore, the parasite burden was directly associated with the decrease in phagocyte adherence to the connective tissue [Leishmania-infected cells [Leishmania deserve further comparative studies using sorted cell populations.Different dendritic cell subsets have been considered the leading transporters of antigens and pathogens from the periphery to the lymph nodes. For instance, Steigerwald and Moll showed that infecting DCs with to CCL21 . This chto CCL21 . Howeverpression . A possie tissue . In thise tissue . Therefoed cells , 17, 20.Leishmania concurs with studies by Jebbari and colleagues and Leon and colleagues that showed the permanence of infected dendritic cells at the inoculation site [Finally, this finding of the decrease in migration of DCs coincubated with ion site , 31. Theion site . Recentlion site . Hence, Leishmania impair DC migration from the inflammatory site to the draining lymph node. Such changes in dendritic cell migration pattern may be associated with immunological events that maintain inflammation at the sites of infection.Coincubation with


Mycobacterium bovis and the related mycobacterial species, Mycobacterium caprae. The genomes of three M. bovis  and one M. caprae (MB2) field isolates with different virulence, prevalence, and host distribution phenotypes were sequenced.Here we report the complete genome sequences of field isolates of  Mycobacterium bovis and is closely related to members of the M. tuberculosis complex (MTBC) such as M. caprae , genome sequences are available  is caused by infection with . caprae , 2. Presvailable . Here, wM. bovis spoligotypes SB0339  and SB0134 (MB3) and the M. caprae spoligotype SB0157 (MB2) with different virulence, prevalence, and host distribution were obtained from domestic or wild ungulates. The isolates were grown in 15 mL of Middlebrook 7H9 liquid media supplemented with 0.36% sodium pyruvate and 10% OADC  for 5 weeks. Chromosomal DNA samples were obtained as previously described  a set of bacterial antibiotic resistance-related Uniprot proteins selected using the GO annotation terms “antibiotic resistance” and “response to antibiotic” and a selection of proteins based on similarity to the proteins of ARDB M. tuberculosis was also present in these four genomes, while variability appeared to be concentrated in membrane proteins with repetitive sequence motifs, which bear frequent mutations that are probably related to selective pressures imposed by the host immune system.The sequenced genomes consisted of 4,275,214 (MB1), 4,288,871 (MB2), 4,259,788 (MB3), and 4,255,612 (MB4) bp. All genomes showed a high GC content: 64.82% (MB1), 65.36% (MB2), 65.10% (MB3), and 65.54% (MB4). The characteristic global genome stability of M. bovis and M. caprae will allow comparative analysis to other mycobacteria species to expand the study of the evolution and host specificity of Mycobacterium spp. and to find correlates with phenotypic variation with implications for TB disease risk assessment and control.The availability of genomes from field isolates of M. bovis MB1, MB3, and MB4 genome sequences were deposited in the European Nucleotide Archive under the accession numbers CDHF01000001 to CDHF01000049, CDHH01000001 to CDHH01000094, and CDHE01000001 to CDHE01000118, respectively. The M. caprae MB2 genome sequence was deposited in the European Nucleotide Archive under the accession numbers CDHG01000001 to CDHG01000059.The


Insulin (INS) gene mutations that cause permanent neonatal diabetes mellitus change single protein building blocks (amino acids) in the protein sequence. These mutations are believed to disrupt the cleavage of the proinsulin chain or the binding of the A and B chains to form insulin, leading to impaired blood sugar control. At least 10 mutations in the INS gene have been identified in people with permanent neonatal diabetes mellitus.To describe clinical features and laboratory manifestations of patients with INS gene mutation and to evaluate outcome of management.Clinical features, biochemical finding, mutation analysis and management outcome of 3 cases from 3 unrelated families were studied. All exons of INS gene were amplified from genomic DNA and directly sequenced.-3 2.63 ±0.85 mmol/l; BE 26.05±4.03 mmol/l, plasma glucose levels were 37.57±15.2 mmol/l, HbA1C of 9.9 ±2.5%. Mutation analysis of the INS gene showed: heterozygous for a novel missense mutation  in exon 2 of INS gene in one case; heterozygous for a novel INS splicing mutation, c.188-31G>A of the INS gene in two cases. After 8 months of insulin treatment, two patients with c.188-31G>A mutation have normal development with DQ 80-100%, HbA1C of 6.85 ±0.49%, quite normal blood glucose levels. The case with c.127T>A mutation treated with insulin for 8 years has physical development delay, poor blood glucose control with HbA1C of 11.4%.3 cases (one girl and two boys) onset at 126.6 ±56.7 days of age with gestation age of 38.0 ± 1.4 weeks, birth weight of 2850 ± 494.9 g. All of them admitted with the feature of diabetic ketoacidosis with pH of 6.94 ±0.16; HCOIt is important to perform screening gene mutation for patients with diabetes diagnosed before 6 months of age to control blood glucose and follow up the patients.


Several sirtuins that exhibit weak deacetylase activity have recently been shown to possess more efficient activity for the removal other acyl lysine modifications, such as succinyl lysine and palmitoyl lysine. Here, we demonstrate that even the well-known deacetylase SIRT2 possesses efficient activity for the removal of long-chain fatty acyl groups. The catalytic efficiency (cat/Kmk) for the removal of a myristoyl group is slightly higher than that for the removal of an acetyl group. The crystal structure of SIRT2 in complex with a thiomyristoyl peptide reveals that SIRT2 possesses a large hydrophobic pocket that can accommodate the myristoyl group. Comparison of the SIRT2 acyl pocket to those of SIRT1, SIRT3, and SIRT6 reveals that the acyl pockets of SIRT1-3 are highly similar, and to a lesser degree, similar to that of SIRT6. The efficient in vitro demyristoylase activity of SIRT2 suggests that this activity may be physiologically relevant and warrants future investigative studies.Sirtuins are a class of enzymes originally identified as nicotinamide adenine dinucleotide (NAD)-dependent protein lysine deacetylases. Among the seven mammalian sirtuins, SIRT1-7, only SIRT1-3 possess efficient deacetylase activity  A malaria parasite SIR2, PfSIR2A, was also shown to possess a similar activity toward long-chain fatty acyl groups15catk of demyristoylation is lower than that of deacetylation.Previous studies demonstrating the defatty-acylase activity of SIRT1-3 only used one peptide sequence15kcat for deacetylation (0.275 s−1) was 15-fold higher than that for demyristoylation (0.018 s−1), the Km for deacetylation (19.00 μM) was approximately 80-fold higher than that for demyristoylation (0.24 μM). Therefore, kcat/Km for demyristoylation  was approximately 5-fold higher than that for deacetylation .To quantitatively compare the activity of SIRT2 toward acetyl and long-chain fatty acyl peptides, we performed kinetics studies using acetyl and myristoyl H3K9 peptides, respectively. As shown in To further understand the defatty-acylase activity of SIRT2, we sought to obtain an X-ray crystal structure of SIRT2 in complex with a myristoyl peptide substrate or analog. Upon screening of crystallization conditions, we obtained crystals of SIRT2 in complex with a thiomyristoyl peptide, referred to as BHJH-TM1 . BHJH-TMAs observed in the crystal structures of apo SIRT2 and other sirtuins, SIRT2/BHJH-TM1 consists of two distinct domains: a Rossmann fold domain and a zinc-binding domain. The BHJH-TM1 peptide inserts into a large groove between the two domains .Archaeoglobus fulgidus SIR2The BHJH-TM1 peptide (PKK(TMy)TG) consists of five amino acid residues  with a tmK value of SIRT2 toward the myristoyl lysine peptide  indicates that the zinc-binding domain moves approximately 10 Å toward the groove, closing the binding site in the SIRT2/BHJH-TM1 structure . A similin vitro. As observed in SIRT2, SIRT6 also possesses a large hydrophobic pocket that accommodates the long-chain fatty acyl groupsWe have previously reported that SIRT6 prefers to hydrolyze long-chain fatty acyl groups from protein lysine residues Comparison of the overall structures of SIRT2/BHJH-TM1 with SIRT3 (PDB ID: 3GLT)in vitro. The catalytic efficiency of SIRT2 for the hydrolysis of myristoyl lysine is slightly higher than that for deacetylation. This increased catalytic efficiency originates primarily from the decreased mK values. In contrast, the catk value for demyristoylation is lower than that for deacetylation. The crystal structure of SIRT2 in complex with a thiomyristoyl peptide, BHJH-TM1, reveals a larger hydrophobic pocket that is responsible for binding the long-chain fatty acyl groups. The hydrophobic acyl pocket of SIRT2 resembles that of SIRT6, which is a sirtuin that has been previously demonstrated to possess efficient defatty-acylase activity, although the two acyl pockets differ in certain aspects. In addition, the hydrophobic acyl pocket of SIRT2 is highly similar to that of SIRT1 and SIRT3, thus providing a reasonable model to account for the ability of SIRT1 and SIRT3 to remove long-chain fatty acyl groups. Moreover, SIRT1, SIRT2 and SIRT3 can remove lipoic acid based on assays utilizing [32P]NAD+We have demonstrated that SIRT2 is an efficient demyristoylase in vitro, belongs to class II. The desuccinylase/demalonylase/deglutarylase SIRT5 belongs to class III. SIRT6  and SIRT7 both belong to be class IV. Our kinetic and structural studies on SIRT2 suggest that the defatty-acylase activity of SIRT2 (and likely that of SIRT1 and SIRT3) is equally efficient, if not more efficient, than the deacetylase activity. Thus, class I sirtuins have likely evolved to recognize both short and long-chain fatty acyl groups. Class IV sirtuins, particularly SIRT6, exhibit a preference for long-chain fatty acyl groups, at least in vitro. However, several studies suggest that the deacetylase activity is important in vivo. Class III sirtuins preferentially recognize negatively charged acyl groups. Human SIRT5 is specific for negatively charged acyl groups, such as malonyl, succinyl, and glutaryl groups. However, certain class III members, such as Escherichia coli CobB, may be multifunctional and can remove different acyl groups (such as succinyl and acetyl) with similar efficiencySirtuins are classified into four different classes based on sequence similarityOur kinetic and structural analyses of SIRT2 suggest that the defatty-acylase activity may be physiologically relevant. A recent study shows that SIRT2 can regulate protein lysine fatty-acylation in cells26273233Acetyl and myristoyl peptide substrates used in the SIRT2 assay were synthesized as previously reported. The thiomyriostyl peptide BHJH-TM1 was synthesized as a mechanism-based inhibitors for SIRT1-3 and SIRT6 as recently reportedSirt2 gene from Homo.Sapiens was cloned into a pET28b(+) (Novagen) vector containing an N-terminal hexahistidine and sumo tag. SIRT2 was overexpressed in E. coli BL21 (DE3) grown in Luria-Bertani (LB) media. The cells were grown at 37°C to an OD600 of ~0.6 and induced with 0.2 mM isopropyl 1-β-D-galactopyranoside (IPTG) for 18–24 h at 18°C. Cell pellets from 2 L of culture were lysed by sonication on ice in 20 mM Tris, 200 mM NaCl, pH7.5. The resulting supernatants were loaded onto a Ni-NTA column and the column was washed with 10 column volumes of buffer containing 20 mM Tris, 200 mM NaCl, and 20 mM imidazole, pH 7.5. The bound SIRT2 was eluted with 20 mM Tris, 200 mM NaCl, and 250 mM imidazole, pH 7.5. The eluted protein fractions were cleavage by Ulp1 overnight, then loaded onto a Ni-NTA column and collected the flow out solution. The solution were incubated with 10 mM Myr Peptide (PKK(Tmy)TG) and 2 mM dithiothreitol for 1 h at 4°C and then further purified in 20 mM Tris, 50 mM NaCl, pH 7.5 buffer, using a Superdex G200 column . The peak corresponding to SIRT2 was pooled, concentrated to 10 mg/mL and stored at −80°C.The The activity of SIRT2 was assessed by high-performance liquid chromatography (HPLC). Purified SIRT2 was incubated in 60 μL of reaction buffer  with 32 μM H3K9, H2BK12, or H4K16 acyl peptides, respectively, at 37°C for indicated time. The reaction was quenched with 60 μL of 200 mM HCl and 320 mM acetic acid in methanol and spun down at 18,000 g for 10 min to remove the precipitated SIRT2. The supernatant was analyzed by HPLC on a Kinetex XB-C18 column .For kinetics, 0.2 μM SIRT2 was incubated in 60 μL of reaction buffer  with H3K9 acetyl or myristoyl peptides at varied concentrations at 37°C for 2 min (H3K9 acetyl) or 3 min (H3K9 myristoyl). Peptide concentration used for H3K9 acetyl was 1, 2, 4, 8, 16, 32, 64, 128 μM. Peptide concentration used for H3K9 myristoyl was 0.25, 0.5, 1, 2, 4, 8, 16, 32 μM. The reactions were quenched and analyzed as described above. The product and substrate peaks were quantified by integrating their absorbance area at 280 nm, which were converted to initial rates. The initial rates were then plotted against the peptide concentrations and fitted using the Kaleidagraph program.Crystals of SIRT2/BHJH-TM1 were grown using the vapor diffusion hanging drop method. A solution containing 10 mg/mL of SIRT2 in 20 mM Tris, 50 mM NaCl, pH 7.5 was preincubated on ice with 10 mM BHJH-TM1 for about 1 h. Hanging drops were formed by mixing 1.5 μL of protein solution and 1.5 μL of well solution containing 25% (v/v) PEG 3350, 0.1 M Hepes buffer, pH 7.5. Rod shape crystals grew in about 6 days to their maximum size of 0.2−0.3 mm × 0.1−0.2 mm.X-ray diffraction data for SIRT2/BHJH-TM1 were collected at Beamline A1 at the Cornell High Energy Synchrotron Source (CHESS) using an ADSC Quantum 210 CCD detector. The data collection temperature was 100 K. A total of 180° of data was collected with an oscillation range of 1° per frame and an exposure time of 5 s per frame. X-ray diffraction data were indexed, integrated, scaled, and merged using the program HKL2000CCP4REFMAC5 as part of CCP4The structure of SIRT2/BHJH-TM1 was determined by molecular replacement using PhaserAtomic coordinates and structure factors for the reported crystal structure have been deposited with the Protein Data Bank under accession code 4R8M.


The 2.4x increased SNR, that is predicted by theory at 7T compared to 3T, should allow detection of smaller changes in metabolite concentrations or measurement of changes in smaller patient groups, which will further our understanding of cardiac energetics. This study represents the first cardiac 31P-MRS patient data acquired at a 7 Tesla field strength.Phosphorous magnetic resonance spectroscopy . All patients were NYHA Class I (40%) or Class II (60%), and the mean LV ejection fraction was 38%.10 dilated cardiomyopathy patients were enrolled. All underwent As expected, the change in field strength from 3T to 7T resulted in an increased mean SNR from 7 ± 1.4 to 18 ± 9.3 for PCr (p 0.005 paired t-test) and for γATP an increase from 4.1 ±1.1 to 12.0 ± 3.4 (p 0.0002).The ATP line width increased from 8.9 ±1.7Hz at 3T to 34.1 ± 14.5 at 7T (p 0.0008) a 281% increase, compared to a previously reported increase of 131%. This suggests that more careful B0 shimming may be required in patients than in healthy volunteers.The PCr/ATP ratio  was consistent between the two field strengths; with as expected no statistically significant difference, as demonstrated in figure The Cramer-Ráo Lower Bounds (CRLB) are shown in figure 31 P-MRS has been previously shown to be feasible in healthy volunteers and now in patients, which in future should allow more accurate quantification and understanding of myocardial energetics.Compared to a 2.8x SNR increase from 3T to 7T previously reported in normal volunteers, we observed a 2.6x increase in this population of patients. The PCr/ATP ratio is not significantly different between the field strengths, showing there is no significant new bias at 7T. 7T Dr Victoria Stoll is funded by the British Heart Foundation FS/12/14/29354. The research was supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society [Grant Number 098436/Z/12/Z] and by the National Institute for Health Research (NIHR) Oxford Biomedical Research Centre based at The Oxford University Hospitals Trust at the University of Oxford.


Compared to bare Bi2WO6 and Ag2S nanoparticles, the as-formed Bi2WO6/Ag2S heterostructures exhibit enhanced photocatalytic activity for the degradation of rhodamine B (Rh B) under visible-light irradiation. This kind of enhancement in the photocatalytic activity is considered to be the synergistic effects of both the effective electron-hole separation and expansion of the light-absorption range. The pH of the solution is of vital importance to the photocatalytic activity of the as-formed Bi2WO6/Ag2S heterostructures. Under low pH value, the photosensitization process is suppressed, while under higher pH value, the photosensitization process is favored. The mechanism of the photocatalytic process was proposed by the active-species-trapping experiments, indicating that the photogenerated holes (h+) play a crucial role in the degradation of Rh B under visible light. The enhanced photocatalytic performance of this heterostructure makes it a promising material for the treatment of dye-containing wastewater.In this report, a novel photocatalyst based on BiThe online version of this article (doi:10.1186/s11671-016-1319-7) contains supplementary material, which is available to authorized users. Therefore, many efforts have been devoted for the preparation of Bi2WO6-based photocatalysts, such as the solid-state method [2WO6 can only respond to visible light with the wavelength shorter than 450 nm [2WO6, which prevents it from further large-scale applications [2WO6, two main methods are employed. The first one is based on the element doping of Bi2WO6  [2WO6 and other kind of materials, such as g-C3N4 [With the widespread application of visible-light-driven photocatalysts, Bioperties –3. As the method , microwae method , ultrasoe method . Howevern 450 nm , which aications . To broaodoping) –14. The s g-C3N4 , C60 [16s g-C3N4 , metals s g-C3N4 , 19, and2WO6 with other semiconductors has been proved to be an effective method for the preparation of the photocatalysts with enhanced photocatalytic performances. On the one hand, the coupling of Bi2WO6 with other semiconductors will broaden the light-absorption range via the formation of intermediate energy levels. On the other hand, the recombination rate of photo-induced charge carriers will be decreased because of the charge transfer on the interfaces of heterostructures. As a result, a variety of heterostructures based on Bi2WO6 have been successfully prepared, which exhibit enhanced photocatalytic activities under visible light. For instance, Yang and coworkers reported the preparation of a BiOCl-Bi2WO6 heterojunction with a chemically bonded interface. The decomposition rate constant for rhodamine B is about 2 times faster than that for pure BiOCl (0.029 min−1) and 1.5 times faster than that for Bi2WO6 (0.041 min−1) [2S3/Bi2WO6 composite photocatalyst using hydrothermal method. The apparent rate constant is calculated to be 0.0062 min−1 for the Bi2S3/Bi2WO6 composite, which is 6.2 times higher than the corresponding value of bare Bi2WO6 (0.001 min−1) [2WO6/α-Fe2O3 [2WO6/TiO2 [2WO6/BiOBr [2WO6/BiIO4 [2WO6/BiVO4 [2WO6. However, developing new heterostructures based on Bi2WO6 is still a big challenge for the chemists, especially by a simple and economic method.The combination of Bi1 min−1) . Zhang a1 min−1) . Other k/α-Fe2O3 , Bi2WO6/WO6/TiO2 , Bi2WO6/O6/BiOBr , Bi2WO6/O6/BiIO4 , and Bi2O6/BiVO4  have als2S has been widely used in various fields such as photoconductors, photovoltaic cells, IR detectors [2S can absorb light with the wavelength lower than 1000 nm, which covers the whole visible-light region. Meanwhile, the conduction band (CB) and valence band (VB) position of Ag2S is higher than the corresponding values of Bi2WO6, which can form the type-II heterostructures when coupling with Bi2WO6. These two fascinating characteristics make the Bi2WO6/Ag2S heterostructure a good candidate for the photodegradation of organic dyes. However, few reports are concerned on the fabrication and photocatalytic activity of Bi2WO6/Ag2S heterostructure. In this report, the Bi2WO6/Ag2S heterostructures were successfully prepared by a surface functionalization method using 3-mercaptopropionic acid (MPA) as the surface-functionalizing agent. The as-formed Bi2WO6/Ag2S heterostructures exhibit enhanced photocatalytic activity as compared to bare Bi2WO6 and Ag2S. Accordingly, a rational model is proposed to illustrate the key roles of Ag2S in the photocatalytic process and the corresponding photocatalytic mechanism of the as-formed heterostructure is also proposed.As a semiconductor with narrow bandgap (1.0 eV), Agetectors , 27, phoetectors , and lumetectors . BecauseAll the reagents are commercially available and used without further treatments.2WO6 were synthesized by a hydrothermal method as we have previously reported [3)3·5H2O and 1 mmol Na2WO4·2H2O were added to 22.5 mL of deionized water under magnetic stirring, respectively. Then, the two solutions were mixed and stirred for another 30 min. The resulting white suspension was then transferred into a 50-mL Teflon-lined autoclave and heated at 200 °C for 12 h. After cooling to room temperature naturally, the precipitates were collected by centrifugation, washed with deionized water and ethanol, and then dried at 60 °C for 6 h in vacuum.The flower-like Bireported . In a ty2WO6/Ag2S heterostructures were prepared by a surface functionalization route which employs MPA as the surface-functionalizing agent [2WO6 was dispersed in 40 mL of deionized water to form a slurry under magnetic stirring. Then, 20 μL of MPA was added into the above suspension, followed by vigorous stirring for 4 h to ensure the complete surface functionalization of Bi2WO6. In the next step, 0.05 g of AgNO3 was added to the above reaction mixture and the suspension was stirred for another 2 h at room temperature. At last, 0.03 g of Na2S·9H2O was added dropwise to the abovementioned system. The resulting suspensions were stirred at room temperature for another 1 h. The molar ratio between elements Ag and S was 2:1. Finally, the product was separated by centrifugation, washed with ethanol and water for several times, and dried under vacuum at 60 °C to obtain the Bi2WO6/Ag2S heterostructures. The resulting powder was collected for further characterization.Bing agent . In a ty2S nanoparticles were also synthesized by a simple precipitation method. In a typical process, 1.0 g of AgNO3 was dissolved in 40 mL of deionized water to form a transparent solution. Then, 0.71 g of Na2S·9H2O was added to the above solution and stirred for another 1 h at room temperature. The resulting products were separated by centrifugation, washed with deionized water and absolute alcohol for 3 times, and then dried at 60 °C for 12 h in vacuum.For comparison purpose, Agλ = 1.5416 Å). The scanning rate of 0.05°s−1 was applied to record the patterns in the 2θ range of 10°–70°. The scanning electron microscope (SEM) characterizations were performed on the S-4800 (Hitachi) field emission scanning electron microscope (FESEM) equipped with a GENESIS4000 energy-dispersive X-ray spectroscope. The transmission electron microscope (TEM) analyses were performed using a Hitachi H-7650 transmission microscope at an accelerating voltage of 100 kV, and the high-resolution transmission electron microscopy (HRTEM) images were obtained on a JEOL-2010 TEM at an acceleration voltage of 200 kV. The Brunauer-Emmett-Teller (BET) tests were determined via a Quantachrome autosorb IQ-C nitrogen-adsorption apparatus. All the as-prepared samples were degassed at 150 °C for 4 h prior to nitrogen-adsorption measurements. X-ray photoelectron spectroscopy (XPS) analysis was performed on Thermo ESCALAB 250 system with a monochromatic Al Kα source with 1486.6 eV of energy and 150 W of power. The light-absorption properties were measured using a UV-vis diffuse reflectance spectrophotometer (DRS)  by using BaSO4 as the background at room temperature and were converted from reflection to absorbance by the Kubelka-Munk method. The photoluminescence (PL) spectra of the samples are recorded with the F-7000 FL spectrophotometer.X-ray diffraction (XRD) patterns were monitored by a Philips X’Pert Pro Super diffractometer using Cu Kα radiation (−5 mol L−1). Temperature of the beaker containing the dispersion of Rh B and the samples was maintained at 20 °C by using circulating water during the whole process. Prior to irradiation, the mixture was magnetically stirred in the dark for 30 min to ensure the adsorption/desorption equilibrium between the photocatalyst and Rh B. Then, light was turned on, and optical power is maintained at 25.8 mW cm2. At given time intervals, 6 mL of the suspension was taken out and centrifuged at 9000 rpm to remove the residual photocatalyst powders for analysis. The clear solution was analyzed through UV-vis spectrophotometer (Agilent Cary 5000E) by recording the variations of the absorption band at 553 nm.The photocatalytic activities of the photocatalysts were evaluated by the degradation of rhodamine B (Rh B) using a 500-W Xe lamp with a 400-nm cutoff filter. The working distance from the Xe lamp to the beaker is 20 cm. In this process, 80 mg of the photocatalyst was added into 80 mL Rh B solution (102·−), ammonium oxalate (AO) (a quencher of h+), AgNO3 (a quencher of e−), and t-BuOH (a quencher of ·OH) were employed. And the amount of scavenger was fixed to be 10 mM except BQ, which was 1 mM. The whole process was similar to the photocatalytic experiments mentioned above.For the detection of active species generated in the photocatalytic reaction, active-species-trapping experiments were carried out. In this process, various kinds of scavengers such as benzoquinone (BQ)  electrochemical working station with a standard three-electrode configuration under visible light provided by a 500-W Xe lamp. The as-prepared photoelectrode, Pt wire, and Ag/AgCl electrode were used as the working electrode, counter electrode, and reference electrode, respectively. During the electrochemical test, 0.1 M phosphate-buffered saline (pH = 7.4) was used as the electrolyte, and the optical power density on the ITO electrode was determined to be 70 mW cm2.For the preparation of the photoelectrode, the ITO (Indium tin oxide) glass was firstly cut into 3 cm (length) × 0.7 cm (width) slices, washed with water and 1 M NaOH solution several times before use. In the next step, 10 mg of the photocatalysts was dispersed in 1 mL of absolute ethanol by ultrasonication. Then, 10 μL of the resulting dispersion was drop-casted onto the ITO slice by a pipette and the area was fixed to be 0.7 cm2WO6 with cell constants of a = 0.5456 nm, b = 0.5434 nm, and c = 1.64 nm, which is consistent with the previous reports  [2WO6/Ag2S heterostructure is also shown in Fig. 2WO6, no obvious change can be observed. The absence of the peaks corresponding to Ag2S may result from the low content and poor crystallinity of Ag2S nanoparticles.The phase purity of the as-prepared samples was investigated by the X-ray diffraction (XRD) method, and the corresponding result is shown in Fig. 73-1126) . The XRD2S in sample Bi2WO6/Ag2S, the as-prepared heterostructures were further investigated using X-ray photoelectron spectroscopy (XPS). The binding energies in the spectrum were calibrated using that of C 1s (284.62 eV). Figure 2WO6/Ag2S heterostructure, in which elements Bi, W, O, S, and Ag can be clearly detected. The peaks centering at 164.73 and 159.43 eV  was detected in the XPS spectrum, indicating that Ag mainly exists in the form of Ag2S. The atomic ratio of Bi:W:Ag:S is determined to be 2:1:1.19:1, which is much larger than the theoretical value (2:1:0.2:0.1). This phenomenon clearly indicates that the as-formed Ag2S are mainly dispersed on the surfaces of Bi2WO6 because the XPS is a surface analysis technique indeed.To verify the existence as well as the chemical states of Ag eV Fig.  can be d eV Fig.  can be a reports , 34. The2WO6/Ag2S heterostructures were further investigated by SEM, TEM, and HRTEM. Figure 2WO6/Ag2S heterostructures, which clearly indicates that the sample is composed of flower-like microstructures with diameter of about 2.0 μm. This result agrees well with the microstructure of bare Bi2WO6 in our previous report [2WO6/Ag2S heterostructure, suggesting the existence of small nanoparticles on the surfaces of these nanoplates. These nanoparticles, as investigated by the element mapping method, are determined to be Ag2S nanoparticles , indicating the existence of Ag2S nanoparticles. The typical lattice spacing, being determined to be 0.316 nm, is consistent with the “1 1 3” planes of Bi2WO6.The structure and morphology of the as-prepared Bis report . Figure 2WO6 and Bi2WO6/Ag2S were determined by nitrogen-adsorption BET method. The specific surface areas  [2 g−1, respectively, which are also a little smaller than the surface areas of CNTs (59.2 m2 g−1).In general, the BET surface area of the photocatalyst can greatly affect its photocatalytic performance, and a high BET surface area is usually beneficial for the improvement of photocatalytic activity , 37. The, Br, I) . In this2WO6, Ag2S and Bi2WO6/Ag2S heterostructures. Flower-like Bi2WO6 exhibits photo-absorption from UV light to visible light, and the absorption edge locates at ~450 nm. After surface modification with Ag2S, an obvious red shift in the absorption edge can be observed. The absorption edge of the Bi2WO6/Ag2S extends to ~550 nm because of the coupling of Ag2S nanoparticles with Bi2WO6 nanoplates. The optical bandgaps of a semiconductor can be calculated using the following equation [α is the absorption coefficient, h is the Planck constant, ν is the frequency of vibration, A is a constant, and Eg is the bandgap of the semiconductor. Among them, n is determined by the optical transition type of a semiconductor. For direct transition, n = 1/2, while for the indirect transition, n = 2 [n is determined to be 1/2 for the as-formed heterostructures. The bandgap energies (Eg) of Bi2WO6 and Ag2S are estimated to be 3.08 and 1.60 eV, respectively. The bandgap of the as-prepared Bi2WO6/Ag2S heterostructures is estimated to be 2.68 eV, which lies between the bandgaps of Bi2WO6 and Ag2S.Figure equation :Fig. 4an, n = 2 . Accordi2WO6/Ag2S heterostructure, values of the relative conduction band (CB) and valence band (VB) position for Bi2WO6, Ag2S, and Bi2WO6/Ag2S are calculated according to the following equations [To further explore the bandgap configuration of the as-prepared Biquations , 41:\docEg, ECB, EVB, and X represent the bandgap energy, conduction band potential, valence band potential, and the electronegativity (the geometric average of the absolute electronegativity of the constituent atoms) of the semiconductor, respectively. E0 is the scale factor relating to the reference electrode redox level to the absolute vacuum scale . The calculated CB and VB potential of Bi2WO6, Ag2S, and Bi2WO6/Ag2S is summarized in Table ECB (−0.27 eV) and EVB (1.33 eV) of Ag2S are lower than the corresponding ECB (0.36 eV) and EVB (3.44 eV) values of Bi2WO6, corresponding to the type-II bandgap alignment when forming the heterostructure. This kind of bandgap alignment will favor for the separation of electrons and holes during the photocatalytic process. Therefore, the Ag2S-modified Bi2WO6 could be excited to produce more electron-hole pairs under the same visible-light illumination, which would result in higher photocatalytic activity.andmentclass2pt{minim2WO6/Ag2S, photodegradation experiments were carried out using Rh B as the target pollutant under visible-light irradiation (λ > 400 nm). For comparison purpose, the blank experiments  and the photocatalytic activities of bare Bi2WO6 and Ag2S nanoparticles were also investigated. The photocatalytic efficiencies of the samples mentioned above are shown in Fig. 2WO6 and Ag2S are employed, about 68 and 12 % of the Rh B molecules can be decomposed after irradiation for 1.5 h. Although Ag2S can absorb light in the whole visible-light region, the photocatalytic activity of Ag2S is still lower than Bi2WO6. The poor photocatalytic activity of Ag2S nanoparticles may result from its poor crystallinity, which has been confirmed from the corresponding XRD pattern  versus irradiation time are shown in Fig. k for Bi2WO6/Ag2S, Bi2WO6, and Ag2S nanoparticles are determined to be 0.02079, 0.01318, and 0.00139 min−1, respectively. The corresponding values for all the samples are summarized in Table 2WO6/Ag2S heterostructure is 1.57 and 14.95 times that of Bi2WO6 and Ag2S nanoparticles, respectively. The enhanced photocatalytic activity of the as-formed Bi2WO6/Ag2S heterostructure can be attributed to the effective electron-hole separation at the interfaces of the two semiconductors.The kinetic behavior of the degradation process can be described using a pseudo-first-order kinetic model . Therefoentclass1pt{minima2WO6/Ag2S heterostructures exhibit higher transient photocurrent density than bare Bi2WO6 and Ag2S nanoparticles  using diluted nitrate acid or sodium hydroxide, while keeping other conditions constant. According to the previous reports, the influence of pH mainly takes effect in two aspects. On the one hand, the pH of the solution will influence the adsorption of dye molecules via changing the surface charges of the photocatalysts [−1 when pH = 2, while the corresponding value decreases to 0.018 min−1 when the pH of the solution increases to 8.To study the influence of pH on the photocatalytic activity of Biatalysts . The varatalysts , 53. On atalysts . To be s2WO6/Ag2S heterostructure was investigated, and the corresponding result is shown as the inset of Fig. 2WO6/Ag2S heterostructure was investigated. The result clearly indicates that the point of zero charge is less than 2 , holes (h+), electron (e−), and hydroxyl radicals (·OH), respectively [2WO6/Ag2S was nearly unchanged when t-BuOH (quencher of ·OH) was added, indicating that hydroxyl radicals are not the driving force for the degradation of Rh B. Oppositely, the photodegradation of Rh B was greatly suppressed by the addition of AO, indicating that the photogenerated holes (h+) play vital roles in the photodegradation of Rh B over Bi2WO6/Ag2S heterostructures under the visible-light irradiation. Upon the addition of AgNO3 and BQ, the photodegradation efficiencies will also decrease, indicating that both electron (e−) and superoxide radical anions (O2·−) are present in the reaction system. Based on the experimental results, the photodegradation process of Rh B in the presence of Bi2WO6/Ag2S heterostructure can be described as follows:e−CB and h+VB represent the electrons in the conduction band and holes in the valence band, respectively.To investigate mechanism of the photocatalytic process, the trapping experiments were conducted to detect the active species during the photodegradation process of Rh B. The experiments involve the utilization of benzoquinone (BQ), ammonium oxalate (AO), AgNOectively . Before 2WO6/Ag2S heterostructure are presented in Scheme 2WO6 and Ag2S can be excited. Driven by the built-in electric field, the photogenerated electrons and holes can be separated at the interfaces of the as-formed Bi2WO6/Ag2S heterostructures. Therefore, the electrons and holes can be efficiently separated, offering enough holes for the degradation of dye molecules. As a result, the photocatalytic activity of the as-formed heterostructures was greatly enhanced.Accordingly, the schematic diagrams for the photocatalytic process in the presence of Bi2WO6/Ag2S heterostructures was prepared by a facile surface functionalization method. The as-prepared Bi2WO6/Ag2S heterostructure displays enhanced photocatalytic activity for the degradation of Rh B under visible-light irradiation compared to its individual components. The decomposition rate for Rh B in the presence of Bi2WO6/Ag2S is about 1.6 times higher than the corresponding value when Bi2WO6 is used as the photocatalyst. The PL and photocurrent measurement were applied to verify the effective separation of electron-hole pairs, which indicate that the enhanced separation of the charge carriers is the main reason for the enhanced photocatalytic activity of the heterostructure. The pH of the solution exerts tremendous influence on the photocatalytic activity of the as-formed heterostructures. According to the active-species-trapping experiments, the photogenerated holes (h+) are determined to be the main reactive species for Bi2WO6/Ag2S in this photocatalytic process.In this paper, a novel photocatalyst based on Bi