Enterococcus faecalis strainscan be assigned to one of four serotypes (CPS-A to CPS-D). The structural basisfor enterococcal serotypes, however, is still incompletely understood. Here wedemonstrate that antibodies raised against lipoteichoic acid (LTA) from a CPS-Astrain are opsonic to both CPS-A and CPS-B strains. LTA-specific antibodies alsobind to LTA of CPS-C and CPS-D strains, but fail to opsonize them. From CPS-Cand CPS-D strains resistant to opsonization by anti-LTA, we purified a noveldiheteroglycan with a repeating unit of→6)-β-Galf-(1→3)-β-D-Glcp- and type 5 (CPS-D). Rabbitantiserum raised against purified diheteroglycan opsonized CPS-C and CPS-Dstrains and passive protection with diheteroglycan-specific antiserum reducedbacterial counts by 1.4 – 3.4 logs in mice infected with E.faecalis strains of the CPS-C and CPS-D serotype.Diheteroglycan-specific opsonic antibodies were absorbed by whole bacterialcells of E. faecalis FA2-2 (CPS-C) but not by its isogenicacapsular cpsI-mutant and on native PAGE purifieddiheteroglycan co-migrated with the gene product of thecps-locus, suggesting that it is synthesized by this locus. Insummary, two polysaccharide antigens, LTA and a novel diheteroglycan, aretargets of opsonic antibodies against typeable E. faecalisstrains. These cell-wall associated polymers are promising candidates for activeand passive vaccination and add to our armamentarium to fight this importantnosocomial pathogen.In a typing system based on opsonic antibodies against carbohydrate antigens ofthe cell envelope, 60% of  E. faecalis strain12030 bind to the group antigen LTA E.faecalis strains were typeable and assigned to four serotypes,designated CPS-A to CPS-D E.faecalis: a rhamnopolysaccharide  and a diheteroglycan. The rhamnopolysaccharide was firstdescribed by Elliott et al. as the type antigen of Streptococcusfaecalis in 1960 E. faecalisand named it diheterglycan The classification system for streptococci was developed by Rebecca Lancefield at thebeginning of the 20th century E.faecalis. Hancock, Gilmore and Thurlow E. faecalis synthesized by the cps locus. Thiscapsule mediates resistance to killing by serum and neutrophils or macrophages,augments bacterial persistence in vivo, and impedes C3b deposition on the bacterialsurface Enterococcus is currently ranked third among Gram-positive pathogens to causehospital-associated infections in the US E.faecalis and investigated their role as antigens in the CPS-serotypingsystem by Hufnagel and colleagues. Using highly purified polysaccharides, we wereable to show that opsonic antibodies are directed against only two of theseantigens: In acapsular strains, LTA is the major opsonic epitope and in encapsulatedstrains opsonic antibodies bind to a novel diheteroglycan, the putative capsularpolysaccharide of E. faecalis in CPS-C and CPS-D strains.In the current study, we have revisited the cell wall carbohydrates of E. faecalis strains belonging to theCPS-A serotype are opsonized by antibodies specific to a teichoic-acid likepolysaccharide, which was later shown to be structurally identical to LTA E. faecalis strains, however, belong to serotypesCPS B – D and are not opsonized by this antiserum E.faecalis strains, we vaccinated a rabbit with LTA from E.faecalis 12030 (CPS-A), which was extracted and purified usingnon-degrading conditions. In a western blot analysis with whole cell lysates ofthe vaccine strain this antiserum was monospecific to LTA (data not shown). Inthe opsonophagocytic killing assay, anti-LTA antibodies mediated killing notonly of CPS-A strains, but also of E. faecalis strains thatbelong to the CPS-B serotype. In contrast, CPS-C and CPS-D strains were notopsonized , CPS-B (12107), CPS-C (FA2-2) and CPS-D(type 5) strains, suggesting that antigenic variability of LTA is irrelevant forthe lack opsonic activity of LTA antiserum against CPS-C and CPS-D strains and CPS-D  strains by enzymaticdigestion of peptidoglycan and separated the extracted material bygel-permeation chromatography. Carbohydrate eluting at void volume consisted ofLTA as determined by 1H NMR analysis. A large second peak elutingaround a Kav of 0.45 was further purified by anion-exchangechromatography. The majority of the material eluted from Q-Sepharose around 300mM NaCl and contained rhamnose, glucose, galactose,N-acetylglucosamine and N-acetylgalactosamine,as determined by sugar analysis. The compositional analysis of thispolysaccharide was consistent with the previously described rhamnopolysaccharide1H-NMR spectroscopy revealed that this material contained a noveldiheteroglycan and impurities of lipoteichoic acid, which were removed by afinal purification step using gel-permeation chromatography on a Toyopearlcolumn HW-40S. This final preparation contained <3% protein and<1% phosphorus and was used for consecutive chemical and biologicalanalysis.Since CPS-C and CPD-D strains are not killed by anti-LTA antibodies, wehypothesized that LTA in these strains is masked by a polysaccharide capsule, anassumption also supported by agglutination experiments by Thurlow and coworkers1H NMRspectrum of the diheteroglycan isolated from E. faecalis type 2, and at δ4.491 , which were assigned to H-1 ofβ-Galf and β-Glcp, respectively.In addition, a broad signal at δ 5.379 was identified, which was assigned toproton H-5 of β-Galf due to the substitution of positionC-5 by an O-acetyl group (δ 2.164). Furthermore, thedoublet at δ 1.366 was recognized as methyl group belonging to lactic acid(LA) residue Compositional analysis identified the presence of D-Glc. The 2CPS-C,  showed t1H and 13C chemical shifts of the capsularpolysaccharides were established from 1H,1H correlationand total correlation as well as 1H,13C heteronuclearmultiple quantum coherence NMR experiments. Low-field shifted signals of carbonatoms demonstrated substitutions at C-6 and C-3 of β-Galf and substitution at C-3 ofβ-Glcp (δ 82.58) The chemical shifts are summarizedin All interresidual NOE contacts were observedbetween H-1 A/H-3 B (δ 5.297/3.622), as well asbetween H-1 B/H-6a A (δ 4.491/3.897), and H-1B/H-6b A (δ 4.491/4.101). Additional weak NOEcontacts were found between H-5 A and LA methyl group(δ 5.379/1.336), and H-5 A and O-acetyl methylgroup (δ 5.379/2.164). The HMBC data confirmed the sequence of theconstituents assigned from ROESY data. The followinginterresidual proton-carbon correlations were observed: H-1A/C-3 B (δ 5.297/82.58), C-1A/H-3 B (δ 109.20/3.622), C-1 B/H-6aA (δ 103.44/3.897), as well as H-3 A/C-2LA (δ 3.747/77.88), and C-3 A/H-2LA (δ 84.85/3.966). The chemical structure of the isolatedpolysaccharide is shown on the The sequence of the residues in the repeating unit of the capsularpolysaccharides was established by rotating frame nuclear Overhauser effect(NOE) spectroscopy ROESY,  and hete1H NMR spectroscopy of diheteroglycans isolated from E.faecalis FA2-2 (CPS-C) and type 5 (CPS-D) revealed a polysaccharidethat they only differed from the E. faecalis type 2 (CPS-C)diheteroglycan by their lack of O-acetylation ofβ-Galf  and type5 (CPS-D) that opsonize CPS-C or CPS-D strains E. faecalis type 2 and type 5  and88% of opsonic killing of E. faecalis type 5 (CPS-D) bythe respective antiserum indicating that the majority of opsonic antibodiesraised by whole cell vaccination are directed against this antigen. To furtherexplore the potential of diheteroglycan as candidate for an enterococcalvaccine, we immunized a rabbit with purified antigen from E.faecalis type 2 (CPS-C). Western blot analysis of this antiserumwith whole cells lysates of the same strain as antigen confirmed the presence ofantibodies against the high molecular weight band of diheteroglycan. Inaddition, we detected antibodies against a second, broad band that migratedaround 20–30 kDa, suggestive of antibodies against LTA (data not shown).Diheteroglycan – like many other bacterial polysaccharide antigens –was overall poorly immunogenic and induced only moderate levels of specificantibodies as quantified by ELISA . At higher serum dilutions, opsonickilling activity of ≥50% was observed only for three out of fiveheterologous E faecalis strains, indicating overall moderatetiters of opsonic antibodies induced by vaccination with diheteroglycan  with anacapsular cpsI mutant in E. faecalis FA2-2 andthe isogenic wild type strain (CPS-C) E.faecalis but not with the acapsular mutant abolished opsonickilling of the E. faecalis FA2-2  48 h and 24 h before and 4 h after i.v. challenge. A total of threeE. faecalis strains were evaluated , type 5 [CPS-D]).Anti-LTA was chosen as control because natural LTA-specific antibodies werepresent in the diheteroglycan antiserum and because identical vaccinationprotocols including complete Freunds adjuvant were used for production of bothantisera. Forty-eight hours after infection, bacterial counts from the blood,kidneys and liver were enumerated. At this time point, mice of both immunizationgroups had cleared the bacteremia, but – depending on the E.faecalis challenge strain - bacterial counts in the kidney andliver were reduced 1.4 to 3.4 logs in mice immunized withdiheteroglycan-specific antibodies compared to mice that had received rabbitanti-LTA  and CPS-Bstrains . The discrepancy of ourcurrent and previous study may be related to the different antigen preparations usedfor production of rabbit antisera. For a previous investigation we utilized apartially deacylated and dealanylated LTA molecule that may have lost antigenicproperties during the purification process cps-locus: CPS-A and CPS-B strains both contain only the firsttwo cps-genes (cpsA and cpsB) butlack remaining the genes of this locus (cpsC –cpsK) which are essential for capsule production Our group has demonstrated previously that the teichoic acid-like cell envelopepolysaccharide that is the target of opsonic antibodies in CPS-A strains isstructurally identical to LTA E. faecalis, we nextpurified cell envelope polysaccharides from the CPS-C strain E.faecalis type 2 and identified a novel diheteroglycan.In contrast to acapsular CPS-A and CPS-B strains, our and Thurlow's dataindicate that LTA of CPS-C and CPS-D strains is not available on the bacterialsurface to bind specific antibodies for opsonization via the classical pathway,probably because a polysaccharide capsule masks this antigen E. faecalis strains(FA2-2 [CPS-C] and type 5 [CPS-D]) contained a polysaccharidewith an identical repeating unit of →6)-β-Galf-(1→3)- β-D-Glcp-. They also can be part of antigenic epitopes of bacterialpolysaccharides recognized by opsonic antibodies The isolated diheteroglyan represented only a small proportion of carbohydratesreleased by enzymatic digestion of peptidoglycan. Most of the material obtained bythis mode of extraction was a rhamnopolysaccharide with similar composition asdescribed previously E.faecaliscps locus and was identified as a 130 kDa glycan. Thecomposition of the material isolated from strain FA2-2 (CPS-C) was determined to beglucose, galactose, glycerol and phosphate at a ratio of 4∶1∶1∶2cps locus - are identical. Ourstudies demonstrate that opsonic antibodies directed against diheteroglycan bind tocapsule-bearing CPS-C strains, but not to its isogenic acapsular mutant. Also, bothantigens co-migrate in native PAGE and contain glucose and galactose, albeit atdifferent ratios. We can only speculate about the reason for this discrepancy inmonosaccharide composition, but impurities in the capsular polysaccharide describedby Hancock may explain the compositional differences. As mentioned above, thediheteroglycan partially co-elutes during the chromatography process with LTA andonly an additional purification step following ion exchange chromatography yieldedpure material in our study. Hence, impurities of LTA could explain why Hancock andGilmore found more glucose, glycerol and phosphate in their preparations of capsularpolysaccharide. Of note, high-pH anion-exchangechromatography–pulsed-amperometric detection employed for compositionalanalysis in Hancock's study is not suitable to detect LTA in mixtures ofcomplex carbohydrates Pazur et al. described a diheteroglycan of very similar composition in E.faecalis infection, we passively immunized mice with eitheranti-diheteroglycan or anti-LTA rabbit serum and challenged them with E.faecalis CPS-C and CPS-D strains intravenously. Bacterial counts were1.4–3.4 logs lower in kidneys and livers of mice immunized withanti-diheteroglycan for the three strains tested. The variable protective efficacyagainst the tested E. faecalis strains is somewhat surprising, butmay be explained by differences of in vivo expression or the degree of O-acetylationof diheteroglycan between strains. In broader terms, this level of reduction ofbacterial load by passive protection is comparable if not superior to vaccineefficacy achieved for various vaccine antigens evaluated in StaphylococcusaureusStreptococcus pneumoniae or group-B streptococci, which arevirulent pathogens in humans and mice, mice clear enterococcal bacteremia orperitonitis spontaneously unless a very high inoculum(>108–109 bacteria per mouse) is given. Hence, inour animal model we may have overwhelmed adaptive immunity with bacterial loads muchhigher than encountered during human infection, a problem that also riddles vaccineresearch for other nosocomial pathogens of low virulence .Nevertheless, we expect that conjugation of the diheteroglycan capsule to a proteincarrier will enhance immunogenicity of the vaccine and elicit better protection invivo. Active and passive vaccination strategies with a diheteroglycan conjugatevaccine could potentially be employed in patients that are at high risk of invasiveenterococcal infections such as patients hematologic malignancies, neutropenia, orliver-transplant recipients To test if capsule-specific antibodies can protect against E. faecalis. Active vaccination with LTA anddiheteroglycan or a passive immunotherapy approach using recombinant humanmonoclonal antibodies could therefore target non-encapsulated and capsule-expressingstrains and may provide protection against a majority of E.faecalis strains.In summary, the results presented in this study provide evidence that twocarbohydrates of the cell envelope are targets of opsonic antibodies in typeablestrain of Bacterial strains and the respective serotype according to Hufnagel et al. arespecified in E. faecalistype 2 and type 5 have been described previously E. faecalis 12030 bybutanol extraction and hydrophobic interaction chromatography Rabbit antisera against whole bacterial cells of E. faecalis was isolated by methods similar tothose described previously E. faecalis strains type2, FA2-2 and type 5 were cultivated as described above and harvested bycentrifugation. The bacteria were washed in PBS and cell walls were digested byaddition of mutanolysin and lysozyme  at 37°C for 18 h.Insoluble material was removed by centrifugation, and the supernatant wastreated with nucleases  at 37°C for 4h followed by addition of proteinase K  at56°C for 18 h. The supernatant was precipitated by the addition of ethanol, collected by centrifugation, dialyzed againstdeionized H2O, and lyophilized. For size exclusion chromatography,the material was redissolved in 0.01 M ammonium carbonate buffer (pH 8.0) andapplied to a column (1.6×90 cm) of Sephacryl S-400 . Fractions eluting at around a Kav of 0.45 werecombined, dialyzed and lyophilized. The material was resuspended in 20 mMNaHCO3, pH 8.4 and applied to an anion-exchange column . Bound antigen was eluted from the column by a lineargradient of 0–1 M NaCl and fractions were assayed for hexose content bythe Dubois assay and for phosphorus using the Lowry method Diheteroglycan of LTA was prepared by butanol extraction and hydrophobic interaction chromatographyas described previously Protein and phosphorus content of purified diheteroglycan as quantified usingstandard assays E. faecalis FA2-2 orHG101 by treatment with mutanolysin, lysozyme and nuclease and proteinase Ktreatment as described above. Next, 25% ethanol was added andprecipitated material was discarded. More ethanol was added to a finalconcentration of 75% for the precipitation of carbohydrates. Purifieddiheteroglycan and cell wall extracts were analyzed by electrophoresisthrough 3% polyacrylamide (33∶1) in Tris-boratebuffer , and detected usingStains-Allaccording to the method of Hancock et al. Cell wall carbohydrates were released from 2H2O,lyophilized, and redissolved in 99.99% 2H2O. Allone-dimensional and two-dimensional spectra were recorded at 27°C with aBruker DRX Avance 600 MHz spectrometer as described previously H2.225; δC 31.45).Samples were exchanged three times with 99.90% p-nitrophenyl phosphate was used as asubstrate (Sigma). After 60 min of incubation at 37°C, the absorbance wasmeasured at 405 nm.ELISA experiments were performed by standard methods as described previouslyE. faecalis strains weregrown to logarithmic phase (OD 600 nm 0.4) in TSB and diluted in RPMI plus15% heat-inactivated fetal calf serum. Baby rabbit serum  absorbed with the target bacterial strainserved as a source of complement. White blood cells (WBC) were isolated fromhealthy volunteers by sedimentation with heparin-dextrane 6white blood cells, 2.5×106 CFU of E. faecalis,0.00125–2.5% rabbit antiserum , and 1.7% complement in a total volume of 400 µl wereincubated in tubes and rotated end over end at 37°C for 90 min. Negativecontrols included tubes from which leukocytes, complement or serum were omitted.The opsonic activity of the serum was calculated as follows: {1 - (CFU immuneserum at 90 min/CFU of control without WBC at 90 min)} x 100. For studies oninhibition of opsonophagocytic killing, rabbit antiserum was incubated at aconcentration of 1∶800  with various concentrationsof inhibitor for 60 min at 4°C. After incubation, the opsonophagocytickilling assay was continued with the absorbed antiserum as described above.Inhibition assays were performed at serum dilutions yielding ∼70 –80% killing of the inoculum without the addition of the inhibitor . The percentage of inhibition of opsonophagocytickilling was compared to controls without inhibitor.An opsonophagocytic killing assay was used with modifications as previouslydescribed 1 CFU.To test protective efficacy of rabbit immune serum, we employed a modified mousebacteremia model developed previously in our laboratory All animal experiments were performed in compliance with the German animalprotection law (TierSchG). The mice were housed and handled in accordance withgood animal practice as defined by FELASA and the national animal welfare bodyGV-SOLAS. The animal welfare committees of the University of Freiburg(Regierungspräsidium Freiburg Az 35/9185.81/G-07/15) approved all animalexperiments. The institutional review board of the University of Freiburgapproved the study protocol and written informed consent was obtained from allstudy participants.Statistical significance for two-way comparisons was determined by an unpaired ttest. Analysis of variance (ANOVA) for multigroup comparisons was used onlog-transformed data, and the Tukey's multiple-comparison test was used forposthoc analysis for pairwise comparisons. Statistical results were calculatedusing the Prism 3 software package.Table S11H and 13C NMR data of the capsular polysaccharide fromE. faecalis strain type 2. Spectra were recorded of asolution in 2H2O at 600 MHz and 27°C relative tointernal acetone .(DOC)Click here for additional data file.Table S21H and 13C NMR chemical shifts [δ] ofdiheteroglycan isolated from of E. faecalis type 5. Spectrawere recorded of a solution in 2H2O at 600 MHz and27°C relative to internal acetone .(DOC)Click here for additional data file.Figure S1E.faecalis type 2. Lane 1 protein molecular mass marker, lane 2Coomassie stain, lane 3 PAS stain.SDS PAGE electrophoresis of purified diheteroglycan from (TIFF)Click here for additional data file.Figure S2E.faecalis type 2. The interresidualconnectivities are underlined.Section of the HMBC spectrum of diheteroglycan isolated from (PDF)Click here for additional data file.Figure S31H NMR spectrum of diheteroglycan isolated from:AE. faecalis type 5, BE. faecalis FA2-2. The letters refer tothe carbohydrate residues as shown in chemical structure Click here for additional data file.Figure S4E.faecalis type 5. The spectrum was recorded at 600 MHz and27°C. The interresidual NOE contacts areunderlined.Section of the ROESY spectrum of diheteroglycan isolated from (PDF)Click here for additional data file.Figure S5E. faecalis type 2 and type 5. Microtiter plates werecoated with the respective polysaccharide (1 µg/well) and incubatedwith serum dilutions of immune rabbit serum against the homolgous strain asindicated in the graph.ELISA of rabbit antiserum raised against purified diheteroglycan from(TIFF)Click here for additional data file.


Helicobacter pylori clarithromycin-resistant strains. Clarithromycin (CLR)-resistant strains were obtained under the exposure of H. pylori strain 26695 on agar plates with low clarithromycin concentrations. The genome data provide insights into the genomic changes of H. pylori under selection by clarithromycin in vitro.We report the complete genome sequences of two  Helicobacter pylori is a spiral Gram-negative bacterium that infects more than half of the world’s population and is currently accepted to play a causative role in the pathogenesis of chronic gastritis, peptic ulcer diseases, gastric cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma  obtained in vitro. The genomes of these strains provide insights into the genomic changes of H. pylori under CLR selection.In the present report, we announce two genome sequences of CLR-resistant in vitro (0.03 and 0.06 µg/ml CLR agar plates), and whole-genome sequencing was performed using 90-base paired-end reads on the Illumina HiSeq 2000 genome sequencer . The whole-genome sequences of the two strains were reconstructed by mapping the short-read sequences on the genome of H. pylori 26695 (GenBank accession no. NC_000915) using the CLC Genomics Workbench version 4.0 . The lengths of the 26695-1CL and 26695-1CH genomes are 1,667,239 bp and 1,667,302 bp, with coverage depths of 840× and 1,257×, respectively. The G+C content of both strains is 38.87%.Two CLR-resistant strains were obtained under exposure to low concentrations of CLR hp0190, hp0471, and hp1048, respectively) were present in both strains. Two mutations were found only in the H. pylori 26695-1CL strain, without mutations in the 23S rRNA gene, and four mutations were found only in the H. pylori 26695-1CH strain, including the A2143G mutation in the 23S rRNA gene.In the two CLR-resistant strains, 5 and 7 mutations (including the well-known mutations in the 23S rRNA gene) were found. Three mutations (C712T, T743A, and G160A in the genes H. pylori strains 26695-1CL and 26695-1CH were deposited at GenBank under the accession no. AP013356 and AP013355, respectively.The genome sequences of clarithromycin-resistant


To prospectively evaluate the impact of 3.0 T Cardiac MR imaging using dual-source parallel radiofrequency (RF) transmission with patient-adaptive B1 shimming compared with single-source RF transmission in the RF homogeneity, image contrast and image quality.The study was approved by the local institutional review board, and all subjects provided written informed consent. Fourteen healthy volunteers were examined at 3.0 T MR, with both the conventional single-source and the new dual-source RF transmission. B1 calibrations (RF shimming) of the heart region were performed to acquire a percent of the prescribed flip angle (FA) of B1 maps, which were used for quantitative assessment of RF homogeneity. Contrast ratios (CRs) between ventricular blood pool and septum were calculated on balanced-turbo field echo (B-TFE) cine images. The off-resonance artifacts of cine images were blindly assessed by two radiologists according to a 4-point grading-scale.P = 0.0094). Dual-source RF shimming significantly increased the CRs (P<0.05) and reduced the off-resonance artifacts of B-TFE cine images (P<0.05). Inter-observer agreement for the off-resonance artifacts of B-TFE cine images was good to excellent (k >0.65).A significantly lower mean coefficients of variance of the achieved FA with dual-source revealed better RF homogeneity compared to single-source (Dual-source parallel RF transmission significantly improves the RF homogeneity, increases image contrast and reduces image artifacts of cardiac B-TFE images compared to single-source mode. This may be of value in reducing the observer-dependence of cardiac MR images and enhancing diagnostic confidence for clinical practice using CMR at 3.0 T. In recent years, higher-field strength MR systems (≥3.0 T) have been more and more used in both clinical diagnosis and scientific researches by using the balanced steady-state free precession (b-SSFP) imaging, such as cardiac function, cardiac flow analysis and the other imaging techniques Multi-channel parallel RF transmission system has been proposed as a means for improving B1 homogeneity Due to its susceptibility to B1 inhomogeneity in the cardiac balanced-turbo field echo (B-TFE) cine sequence The study protocol was conducted with the approval of the local institutional review board of Shandong Medical Imaging Research Institute, Shandong University. Informed written consent was obtained from all participating volunteers before their enrollment in the study.Fourteen healthy volunteers without history of cardiovascular disease  were consecutively enrolled in a prospective study. Inclusion criteria for participation in this study were ability to offer written informed consent and age no less than 18 years. Exclusion criteria were contraindications to MR imaging .Cardiac MR imaging were performed on a 3.0 T MR scanner  equipped with flexible dual-source parallel RF transmission system . Images were acquired once with Multi-Tx switched on , and once with Multi-Tx switched off (single-source). Two 16-channels phased-array coils integrated were used for signal reception. All MR imaging acquisitions were ECG gated.B1 Map imaging: For each subject, a cardiac cine imaging was performed after the B1 calibration scanning. The B1 calibration image of the short-axis plane across the heart was acquired utilizing a saturation-recovery, dual-flip angle method with a segmented echo-planar imaging read out B-TFE imaging: A cardiac cine acquisition of short-axis plane perpendicular to the inter-ventricular septum with breath-holding was performed with parameters: 6 slices from the ventricular base to the apex; slice thickness, 8 mm without slice gap; FOV, 320×320 mm; matrix, 200×256; number of heart phases, 30; TR was chosen as short as the system allowed with maximal readout bandwidth ; echo time (TE) was set to half of TR. In order to compare the effect in MR images acquisition between the dual-source and single-source, with a special focus on the revised TR or FA in the dual-source transmit system, four groups of parameters were designed to compare with each other. Group M0 was with single-source RF excitation, and with typical parameters: shortest TR/TE, 3.4/1.7 ms; FA, 45°. Groups M1, M2 and M3 were utilizing dual-source excitation with local B1 shimming. The parameters of group M1 were the same as group M0; FA increased from 45° to the maximum allowable FA 58° in group M2 with TR/TE kept to 3.4/1.7 ms; TR/TE values reduced to the shortest TR/TE 2.8/1.4 ms in group M3 with FA kept to 45°. Each volunteer was imaged according to the above 4 groups of parameters described. The CMR imaging parameters were summarized in RF homogeneity: The homogeneity of the RF field in the local volume was calculated from all pixels inside the ROI of the B1 map with and without dual-source excitation  respectively. The mean signal intensity was obtained automatically. Each data was measured 3 times by the same reader, and the average of results was used finally. CR between blood pool and interventricular septum was calculated as follows bp = signal intensity of blood pool and SIis = SI of interventricular septum). CRs of 4 groups of cardiac cine images (M0–M3) were compared with each other.Image quality: Image quality evaluation for cardiac cine imaging included image contrast (CRs) and the occurrence of off-resonance artifacts. In each subject, CRs between blood pool and interventricular septum consisted of left ventricular (LV)-to-septum contrast and RV-to-septum contrast. A ROI covering an area of approximately 120 mmThe occurrence of off-resonance artifacts was assessed by two experienced radiologists independently on a 4-point grading-scale: 4–No off-resonance artifacts, excellent image quality; 3–Mild off-resonance artifacts, diagnostic but not perfect; 2–Moderate off-resonance artifacts, diagnostic on most segments but not all; 1–Serious off-resonance artifacts, not diagnostic.P<0.05 was considered to be significant.Data analysis was performed using SPSS 17.0 . A paired t-test was performed to compare RF homogeneity data with and without dual-source excitation. The calculated CRs of multiple groups were compared with one-way analysis of variance (ANOVA), followed by SNK (Student-Newman-keuls) test. The artifacts data were analyzed with non-parametric Wilcoxon signed rank test. Inter-observer agreement was evaluated with Cohen's kappa test. The strength of inter-observer agreement was defined as follows: 0.0–0.2, poor agreement; 0.21–0.4, fair agreement, 0.41–0.6, moderate agreement; 0.61–0.80, good agreement; and 0.81–1.0, excellent agreement. Statistical tests were two-tailed, and All the fourteen healthy volunteers  underwent CMR imaging successfully. The mean body mass index of the included volunteers was 25.7±3.9. All subjects showed regular heart rate and rhythm .P = 0.0152). The mean CV of dual-source mode and single-source mode were 0.066±0.022 and 0.103±0.05 respectively, with statistically significant difference (P = 0.0094). The quantitative analysis of the B1 maps was summarized in A representative image demonstrating the benefit of local volume RF shimming using the dual-source RF excitation was shown in P1<0.001, P2<0.001, P3 = 0.001 for LV-CRs, and P1<0.001, P2<0.001, P3<0.001 for RV-CRs, respectively). Within the dual-source mode, the CR of group M2 was the largest with significant difference , while there was no significant difference between group M1 and M3 (P = 0.874 for LV and P = 0.689 for RV). Comparisons of LV and RV CRs between the four groups were summarized in The CRs of group M1, M2 and M3 were all significantly higher than that of group M0 both in LV-to-septum and RV-to-septum . The artifacts scores of group M3 were the highest among the three groups with dual-source mode . The artifacts scores of the four groups were summarized in The artifacts scores of group M1, M2 and M3 were higher than that of group M0, while there was only statistical significance between group M3 and group M0  improved the homogeneity of RF field, led to more uniform distribution of FA across the heart region and increased CRs of the blood-to-myocardium; 2) reduced the off-resonance artifacts of the heart and its surrounding tissues by using the shortest TR available with the use of dual-source mode.Initial studies have reported several approaches on improving B1 field uniformity, such as post-processing methods or using dielectric cushions Traditional single-source RF excitation, using a single transmit source with a 90° phase shifter, the single RF signal is transported to the two ports of a quadrature RF coil. Dual-source parallel RF excitation, two completely independent RF sources are delivered to independent ports of the system quadrature body coil by using two independent RF transmit channels with software control P = 0.0152). Dual-source parallel RF excitation with patient-adaptive B1 shimming in the heart region would enable us to achieve a relatively higher FA, which was closer to the prescribed FA for cardiac imaging (85.38% with volume RF shimming vs 76.74% without). The mean CV of the percentage of the achieved FA decreased from 0.103±0.05 with single-source to 0.066±0.022 with dual-source patient-adaptive RF shimming (P = 0.0094), which revealed a more uniform RF field distribution. Therefore, the use of dual-source RF excitation is effective in getting a relatively higher, more uniform and consistent FA in the heart region, which will be conducive to improving the quality of cardiac MR imaging.The use of dual-source parallel RF excitation allows for calibration of the RF pulse (the shape of RF waveforms) and thus enables local B1 shimming, which can be clearly illustrated on B1 maps In addition, the more uniform B1 field and subsequently homogeneous FA distribution result in an optimized SAR distribution within the imaging volume, thereby reducing local SAR peaks. This makes it possible for high-field strength MR at 3.0 T to lower the TR  B-TFE sequences could yield higher SNR and contrast between myocardium and blood than spoiled gradient-echo sequences k >0.65), preferably dual-source RF shimming. The gains in imaging quality of B-TFE with dual-source RF transmission are conducive to reduce the observer-dependence on cardiac MR images post-processing For 3.0 T MR, B1 inhomogeneity also induces B-TFE amplified off-resonance artifacts, decreasing image quality and limiting its clinical use in cardiac imaging In conclusion, dual-source parallel RF transmission with patient-adaptive RF shimming can significantly improve B1 field homogeneity and increase contrast of cardiac B-TFE cine images. An improved B1 homogeneity with a shortest TR can also help to reduce image artifacts substantially and accelerate imaging speed. These gains in acquisition with the dual-source parallel RF transmission have shown us a wide application prospects in clinical CMR at 3.0 T.


Burkholderia cenocepacia J2315 bceN gene, encoding a protein with GDP-D-mannose 4,6-dehydratase enzyme activity (E.C.4.2.1.47). Data presented indicate that the protein is active when in the tetrameric form, catalyzing the conversion of GDP-D-mannose into GDP-4-keto-6-deoxy-D-mannose. This sugar nucleotide is the intermediary necessary for the biosynthesis of GDP-D-rhamnose, one of the sugar residues of cepacian, the major exopolysaccharide produced by environmental and human, animal and plant pathogenic isolates of the Burkholderia cepacia complex species. Vmax and Km values of 1.5±0.2 µmol.min−1.mg−1 and 1024±123 µM, respectively, were obtained from the kinetic characterization of the B. cenocepacia J2315 BceN protein by NMR spectroscopy, at 25°C and in the presence of 1 mol MgCl2 per mol of protein. The enzyme activity was strongly inhibited by the substrate, with an estimated Ki of 2913±350 µM. The lack of a functional bceN gene in a mutant derived from B. cepacia IST408 slightly reduced cepacian production. However, in the B. multivorans ATCC17616 with bceN as the single gene in its genome with predicted GMD activity, a bceN mutant did not produce cepacian, indicating that this gene product is required for cepacian biosynthesis.This work reports the biochemical and functional analysis of the  Paramecium bursaria chlorella virus 1 where D-rhamnose is believed to be a component of the major viral capsid glycoprotein, the occurrence of this rare deoxysugar seems to be restricted to bacteria Burkholderia cepacia complex (Bcc), a group of 17 closely related species that can cause infections in several hosts, including the human host The rare 6-deoxysugar D-rhamnose is often a component of bacterial cell surface glycans, such as lipopolysaccharides (LPS), extracellular polysaccharides (EPS), and other glycoconjugate-containing bacterial cell components Cepacian has been pointed out as contributing to the overall pathogenicity of Bcc bacteria. For example, cepacian interferes with human neutrophils phagocytosis by facilitating the bacterial persistence in a mice model of infection, inhibits the production of oxygen reaction species (ROS) by neutrophils, and scavenges ROS, playing a role in the survival of cepacian-producing strains in different environments Besides D-rhamnose, cepacian also contains residues of D-glucose, D-mannose, D-galactose, and D-glucuronic acid as sugar components of its heptasaccharide repeat unit B. cepacia IST408 B. cepacia IST408 and B. multivorans ATCC17616 showed that both strains produced EPSs with FTIR spectra identical to that of cepacian Cepacian was previously identified as the sole EPS produced by the clinical isolate bceI and bceIIBurkholderia genus sequenced so far, except of B. mallei strains bceII gene cluster comprises several genes putatively involved in the synthesis of cepacian, including the bceM and bceN genes, presumably encoding proteins with putative GDP-4-keto-6-deoxy-D-mannose reductase (RMD) and GDP-D-mannose-4,6-dehydratase (GMD) activities, respectively Previous work allowed the identification of genes involved in cepacian biosynthesis in two clusters, named bceN gene product with predicted GMD activity, from the highly epidemic clinical isolate B. cenocepacia J2315, a member of the ET12 lineage that caused several fatalities among CF patients from both sides of the Atlantic , suggesting that the cofactor is tightly bound to the enzyme.In the present work, we report the cloning, purification, and kinetic characterization of the B. cepacia IST408 and B. multivorans ATCC17616 confirmed the requirement of this enzyme activity for its synthesis.The comparison of cepacian production ability of wild-type and mutant strains derived from the cepacian producers Escherichia coli strains were maintained on Lennox broth  agar plates, supplemented with 100 µg/ml trimethoprim, 150 µg/ml ampicillin, or 50 µg/ml kanamycin, when appropriate. Unless otherwise stated, liquid cultures were carried out in LB liquid medium supplemented with the appropriate antibiotics with orbital agitation (250 rpm), at 30°C for Bcc strains or 37°C for E. coli strains. Bacterial growth was assessed by measuring the cultures optical density at 640 nm (OD640).Bacterial strains and plasmids used in this study are described in E. coli transformation were carried out using standard procedures B. cenocepacia J2315 bceN gene (BCAM1004) was performed using the oligonucleotides UP-GMD (GCTAGCATGAGCCAAA CTC-3′5′-TT) and LW-GMD (AAGCTTGAACGTGTCATG-3′5′-AAA) containing, respectively, the NheI and HindIII restriction sites (underlined) at their 5′ ends. For the amplification of the bceN gene and the neighbour region, oligonucleotides UP-GMDmut (GGTACCGTCGGAGAAATC-3′5′-AAA) and LW-GMDmut (AAGCTTC GATTCGTTCTG-3′5′-TTT) containing at their 5′ end, respectively, the KpnI and HindIII restriction sites (underlined). These primers were designed based on the genome sequence of B. cenocepacia J2315, available at the Sanger Institute Homepage (http://www.sanger.ac.uk/Projects/ B_cenocepacia).Genomic DNA was extracted from exponentially-growing liquid cultures of Bcc strains using the High Pure PCR Template Preparation Kit (Roche). Plasmid isolation and purification, DNA amplification, restriction and ligation were carried out, respectively, using kits from Macherey-Nagel, and enzymes from Finnzymes and Fermentas. Agarose gel electrophoresis and B. cenocepacia J2315 and other Burkholderia strains were carried out using the Integrated Microbial Genomes system Nucleotide and amino acid sequences were analysed using bioinformatic tools resident at the National Center for Biotechnology Information (NCBI) or the ExPASy-Prosite websites. Searches for homology within the genome sequences of B. cenocepacia J2315 bceN gene sequence (BCAM1004) lacking the stop codon was amplified using B. cenocepacia J2315 genomic DNA as template and primers UP-GMD and LW-GMD. The 1061 bp amplicon was digested with NheI and HindIII and was subsequently cloned into the NheI/HindIII cloning sites of pET23a+, yielding plasmid pJFR4. pJFR4 allows the control of protein expression by the T7 promoter, expressing the BceN protein with a 6×His-tag at its C-terminus. Plasmid pJFR4 was sequenced to confirm the correct insertion of the bceN gene, and transformed into E. coli BL21 (DE3). Overexpression of the C-terminally 6×His-tagged BceN protein was performed by growing E. coli BL21 (pJFR4) in 100 ml of LB liquid medium (supplemented with 150 µg/ml ampicillin) at 37°C and with orbital agitation (250 rpm). When the culture OD640 reached 0.6, isopropylthiogalactoside (IPTG) was added  and incubation was prolonged for 2 hours. Bacteria were harvested by centrifugation at 7000× g for 5 min at 4°C, and ressuspended in 8 ml of start buffer  containing 20 mM imidazole. Cell suspensions were kept at −80°C until further processing. Aliquots of these cell suspensions were processed and protein overproduction was assessed by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). To confirm the overproduction of the 6×His-tagged BceN protein, the samples were transferred onto a nitrocellulose membrane. The membrane was incubated with a monoclonal anti-polyhistidine antibody conjugated with peroxidase (Sigma-Aldrich) diluted 1∶2000, followed by the addition of the peroxidase substrate 3,3′,5,5′ tetramethylbenzidine .The g for 1 hour at 4°C. The 6×His-tagged BceN protein was purified by affinity chromatography using a Hi-Trap chelating column , following the suppliers instructions. Purified fractions were analysed by SDS-PAGE followed by staining with Coomassie brilliant blue R-250. The eluted fractions containing the purified 6×His-tagged protein were dialysed overnight at 4°C in a 10 kDa cutoff Slide-A-Lyzer Dialysis Cassette (Pierce) against 25 mM sodium phosphate buffer (pH 7.4) supplemented with 50 mM NaCl and 20% (v/v) glycerol. Protein concentration was estimated by the method of Bradford Cell suspensions prepared as described above were lysed by ultrasonic vibration with a Branson sonifier 250 (Branson). Cell debris were removed by centrifugation at 12,500× Native protein gel electrophoresis of the purified 6×His-tagged BceN was performed based on previously described methods B. cenocepacia J2315 6×His-tagged BceN protein to convert GDP-D-mannose into GDP-4-keto-6-deoxy-D-mannose was assessed using GDP-D-mannose as substrate, and analysing the reaction products by Nuclear Magnetic Resonance (NMR) spectroscopy, based on the methods described by King et al.+ 400 MHz (1H) spectrometer equipped with a 5 mm BBO probe. The reacting mixture , was placed in a 5 mm NMR tube (Norrel 100 MHz) with concentrations of GDP-D-mannose (Sigma) ranging from 1 to 5 mM, in the absence or presence of 5 mM NADP+ (Sigma), and MgCl2 (1 mol MgCl2 per 1 mol of purified 6×His-tagged BceN protein). Experiments were conducted at 25°C. The reaction was started by the addition of 90 µg of the purified 6×His-tagged BceN protein to the reaction buffer, and the proton spectrum was recorded at fixed periods of time. Spectral data was collected over time and treated with TopSpin software v2.1. Resonance of the acetone methyl group was used as internal reference for all spectra . Different concentrations of MgCl2  and NADP+ , and different reaction temperatures (25°C and 37°C) were tested for the reaction conditions optimization. Reaction products were characterized by 1D (1H and 13C) and 2D (HSQC) NMR experiments. Protein stability was only achieved when using 20% (v/v) glycerol in all experiments, which interfered with the substrate and product peaks in the range of 3.5 to 3.9 ppm. Therefore, kinetic measurements were based on the proton signals corresponding to the anomeric protons in GDP-D-mannose and the two reaction products as described by King and coworkers 3δH = 2.225 ppm) and plotted versus time o was obtained from the slope of the exponential part of the curve obtained with the increase of products concentration, calculated using the equation [P]t = [S] (1-SNAt), where [P]t is the concentration of products on time t, [S] is the initial substrate concentration, and SNAt is the substrate normalized integral signal area at time t. NMR experimental data was further analyzed using Graphpad Prism v6.The ability of the 2 at the ratio of 1 mol MgCl2 per 1 mol protein, 25 mM sodium phosphate buffer, 50 mM NaCl, 20% (v/v) glycerol, 90% H2O/10% D2O, pH 7.4]; b) addition of 10 mM NADPH, after incubation of the reaction mixture for 30 minutes at 25°C.The hypothesis that the 6×His-tagged BceN protein can catalyse both the GDP-D-mannose 4,6-dehydratation and the subsequent reduction reaction to produce GDP-D-rhamnose was investigated based on the two following NMR assays: a) addition of 10 mM NADPH to the reaction mixture , which takes substrate inhibition into account. The values obtained for Km and Vmax were 1024±123 µM and 1.5±0.2 µmol min−1mg−1, respectively, corresponding to a kcat value of 1.0 s−1 and catK/mK value of 9.77×102 M−1 s−1. A iK value for GDP-D-mannose of 2913±350 µM was estimated, a value almost 3-fold that estimated for mK, suggesting that the substrate binds with high affinity to the active site of the enzyme. The kinetic parameters obtained for the B. cenocepacia J2315 6×His-tagged BceN protein, as well as available kinetic parameters for the GMD enzymes of P. aeruginosa, H. pylori, E. coli, and the fungi Mortierella alpina are summarized in A decrease in the reaction rate was observed for substrate concentrations higher than 2 mM . These rB. cenocepacia J2315 6×His-tagged BceN protein is a monofunctional enzyme, at least under the assay conditions used in this work.Some bacterial GMDs are bifunctional, being able to catalyze the 4-keto reduction of GDP-4-keto-6-deoxy-D-mannose B. cepacia JFR1 with the bceN gene insertionally inactivated was constructed from B. cepacia IST408, previously described as a high EPS producer B. cenocepacia J2315 was previously reported as a cepacian non-producer due to a frameshift mutation in the bceB gene bceN mutant in the EPS producer B. multivorans ATCC 17616, a strategy based on the interference of an antisense RNA targeting the bceN mRNA was employed to construct a conditional mutant. This approach has been used successfully in Staphylococcus aureus and Mycobacteria to analyse specific gene essentiality, and more recently in B. cenocepacia J2315 bceN gene in the antisense orientation was inserted downstream of the pBAD promoter, resulting in the antisense plasmid pJRF6. This plasmid was introduced into B. multivorans ATCC 17616 cells by triparental conjugation, yielding B. multivorans (pJRF6), which, upon induction with arabinose, expresses the antisense transcript of bceN, effectively blocking the translation of the mRNAs corresponding to bceN (results not shown).Since GDP-D-rhamnose is one of the sugar nucleotides required for the synthesis of the extracellular polysaccharide cepacian, the mutant strain B. cepacia IST408, the mutant B. cepacia JFR1, B. multivorans ATCC17616 and the B. multivorans (pJFR6) strain in SARA liquid medium (data not shown). The ability of the wild-type strain B. cepacia IST408, the mutant strain B. cepacia JFR1, B. multivorans ATCC17616 and B. multivorans (pJRF6) to produce EPS was compared in SARA liquid medium at 30°C and 37°C with orbital agitation. Although the B. cepacia JRF1 mutant was still able to produce EPS in SARA liquid medium, a significant reduced yield was observed when compared to the IST408 wild-type strain after 48 hours of growth . Therefore, we have conducted homology searches for putative GMD encoding genes within publicly available genome sequences of strains of Bcc. One, two or three putative GMD-encoding genes were found in sequenced strains of the Burkholderia genus  induced with arabinose. Induction of the antisense bceN mRNA in B. multivorans (pJFR6) strain completely abolished the EPS production  encoding protein(s) with GMD activity, thus compensating the inactivation of the bceN gene.Similar growth curves were observed for the wild-type strain f growth . Since Gia genus  panel A.oduction . Taken tbceN gene from the opportunistic pathogens B. cenocepacia J2315 and B. cepacia IST408, which are 97% identical. This last strain is a clinical isolate previously reported as a high producer of cepacian B. cenocepacia J2315 does not produce cepacian due to a frameshift mutation on the bceB gene In this work we report the cloning, purification, biochemical and functional characterization of the bceN gene is part of the bceII cluster of genes found in all the genomes of Burkholderia strains sequenced so far The P. aeruginosa GMD H. pylori HP0044 E. coli GMD BceN homologue proteins are involved in the synthesis of the rare deoxysugar nucleotide GDP-D-rhamnose, as is the case of the B. cenocepacia J2315 BceN protein enzyme activity was investigated based on NMR analysis. This technique was used previously by others to identify the products of the reaction catalyzed by the GMD and RMD enzymes of P. aeruginosaB. cenocepacia J2315 BceN protein indicate that this protein is monofunctional. This is not the case of the P. aeruginosa protein with GMD activity, which is bifunctional and can catalyze the two reactions necessary to convert GDP-D-mannose into GDP-D-rhamnose P. aeruginosa GMD protein, the GMD proteins from H. pylori and E. coli were both found to catalyse only the 4,6 dehydratase reaction, thus yielding only the intermediate GDP-4-keto-6-deoxy-D-mannose B. cenocepacia J2315, the conversion of the intermediate into the final product GDP-D-rhamnose is most probably performed by the product of the bceM gene, previously predicted to encode a GDP-6-deoxy-D-lyxo-4-hexulose reductase. bceM is also located in bceII cluster, downstream bceNbceM gene has not yet been functionally characterized.The B. cenocepacia BceN was found to be a tetramer, like the GMD proteins from P. aeruginosa and Helicobacter pyloriK. pneumoniae, E. coli, and the Human GMDs The active form of the + or NADP+ did not affect the rate of the reaction catalysed by B. cenocepacia J2315. Since the dehydration step requires an electron acceptor E. coli GMD and P. aeruginosa GMD The addition of exogenous NADP. aeruginosa GMD, the H. pylori HP0044 and the E. coli GMD The 6×His-tagged BceN exhibited a non-Michaelis-Menten kinetics, with a typical substrate inhibition model and a cooperative binding to the substrate. The kinetic parameters for 6×His-tagged BceN are comparable with those of other GMD enzymes previously described, such as the P. aeruginosa alginate pathway, the step catalysed by the GDP-mannose dehydrogenase is essential for the control of the biosynthetic pathway B. cenocepacia isolated from a CF patient with chronic infection were the bceF and bceG genes, encoding a tyrosine autokinase and a glycosyltransferase, respectively. This is in good agreement with previous work, in which a bceF mutant from B. cepacia IST408 was not able to produce EPS, exhibiting a reduced virulence in a X-CGD mice model when compared with the wild-type strain In the bceN gene, results from this work reinforce previous observations that the control of cepacian biosynthesis does not occur at the sugar nucleotide level, when additional genes encoding similar functions are encoded within the genomes, as are the BceA, BceC and BceN bceN in the B. multivorans ATCC17616, mutation in genes encoding protein functions at the sugar nucleotide level can abolish cepacian biosynthesis.Besides the functional analysis of the Figure S1Burkholderia strains encode 1 to 3 proteins with GMD activity. (A) Genomic context of the bceN gene orthologues (represented as solid black arrows) in sequenced genomes of strains of the Burkholderia genus. (B) Unrooted phylogenetic tree for the B. cepacia IST408 BceN and the putative BceN orthologues from B. lata sp. 383 (B383), B. cenocepacia J2315 (BcenJ), B. cenocepacia AU1054, B. cenocepacia HI2424 and B. cenocepacia MC0-3 (Bcen), B. cenocepacia PC184 (BcenP), B. ambifaria AMMD (Bamb), B. dolosa AUO158 (Bdol), B. vietnamiensis G4 (Bviet), B. thailandensis E264 (Bthai), B. multivorans ATCC17616 (Bmul), B. mallei ATCC23344  and B. pseudomallei K96243 (Bpseud). The phylogenetic tree was constructed based on the alignment of amino acid sequences with CLUSTAL X2 using the neighbor-joining method with a minimum of 100 bootstraps. Clade A includes putative GMDs located in gene clusters similar to bce-II of chromosome 2 that is involved in Cepacian biosynthesis Burkholderia putative GMD homologies located on chromosome 1, and the surrounding genes, arranged in clusters most probably involved in LPS biosynthesis. The B. vietnamiensis G4 BceN ortologue is not included in clade A or B, and is the only GMD homologue from a Burkholderia strain encoded on chromosome 3. Genes: a, b – ABC transporters; c, j, m, o – glycosiltransferases; d – type II mannose-6-phosphate isomerase; e – transposase; f – GDP-D-mannose 4,6-desydratase; g, k – NAD-dependent epimerases/dehydratases; h – methyltransferase FkbM; i – hypothetical protein; l – methyltransferase type 11; n – wcbA; p – wcbC; q – bexA; r – bexB; s – wcbD; t – transferase hexapeptide repeat containing protein; u – polysaccharide biosynthesis protein; v – exopolysacharide transport protein; x – tyrosine phosphatase; w – polysaccharide export protein; y – sugar transferase.(DOC)Click here for additional data file.Table S1Bacterial strains and plasmids used in this work.(DOC)Click here for additional data file.Table S213C and 1H NMR data for GDP-D-mannose (A), GDP-4-keto-6-deoxy-D-mannose (B), and the gem-diol form of compound B (C). Experiment with 5 mM GDP-D-mannose, 14 hours of incubation with 90 µg BceN protein and 10 mM MgCl2 in H2O 90%/D2O 10% buffered saline solution. Resonances were referenced to an internal acetone standard at δH = 2.225 p.p.m and δC = 30.89 p.p.m. For compounds A, B and C, the coupling constants (JHH) for the anomeric proton were 2.08 Hz, 1.56 Hz and 1.56 Hz, respectively. * Low resolution or signals obscured by glycerol peaks.(DOC)Click here for additional data file.


Trypanosoma cruzi exhibit a poorly functional T cell compartment, characterized by monofunctional (IFN-γ-only secreting) parasite-specific T cells and increased levels of terminally differentiated T cells. It is possible that persistent infection and/or sustained exposure to parasites antigens may lead to a progressive loss of function of the immune T cells.Adults with chronic T. cruzi-specific T cell responses were evaluated in T. cruzi-infected children and compared with long-term T. cruzi-infected adults with no evidence of heart failure. The phenotype of CD4+ T cells was also assessed in T. cruzi-infected children and uninfected controls. Simultaneous secretion of IFN-γ and IL-2 measured by ELISPOT assays in response to T. cruzi antigens was prevalent among T. cruzi-infected children. Flow cytometric analysis of co-expression profiles of CD4+ T cells with the ability to produce IFN-γ, TNF-α, or to express the co-stimulatory molecule CD154 in response to T. cruzi showed polyfunctional T cell responses in most T. cruzi-infected children. Monofunctional T cell responses and an absence of CD4+TNF-α+-secreting T cells were observed in T. cruzi-infected adults. A relatively high degree of activation and differentiation of CD4+ T cells was evident in T. cruzi-infected children.To test this hypothesis, the quality and magnitude of T. cruzi infection promotes eventual exhaustion of immune system, which might contribute to disease progression in long-term infected subjects.Our observations are compatible with our initial hypothesis that persistent  Trypanosoma cruzi-specific T cells inversely correlates with the severity of cardiac disease in the chronic phase of the infection, suggesting that the immune system becomes exhausted overtime. To test this hypothesis, the quality and magnitude of T. cruzi-specific T cell responses were measured in T. cruzi-infected children – who are presumed to have shorter-term infections - and compared with long-term T. cruzi-infected adults. The activation status of total T cells in T. cruzi-infected children was also evaluated. T. cruzi-infected children exhibit a more robust, and more highly functional parasite specific T cell responses compared to T. cruzi-infected adults. In spite of a more functional immune profile, T. cruzi-infected children have a heightened state of immune activation. These observations are compatible with the initial hypothesis that T cell responses specific for T. cruzi become exhausted overtime. The impairment in T cell responses might contribute to disease progression in long-term infected subjects.Chagas disease is a neglected tropical disease affecting approximately 10 million people in the world. As a consequence of migration flows, the disease has also become established in non-endemic countries. Previous studies have demonstrated that  Trypanosoma cruzi. As a consequence of migration flows, the disease has been also become established in non-endemic countries Chagas disease, a neglected tropical disease affecting approximately 10 million people from south of the United States to Mexico and Central and South America T. cruzi frequently results in the development of cardiomyopathy, generally many years after the initial infection. Three factors are likely associated with the development of severe disease: parasite burden; the effectiveness of the host immune response in controlling parasites in specific tissues, and the effectiveness of the host immune response in limiting peripheral damage T. cruzi-infections exhibit a prevailing profile of parasite-specific Interferon (IFN)-γ only secreting T cells T. cruzi-specific T cells were also found to inversely correlate with the severity of chronic Chagas disease T. cruzi-infected children are likely to have shorter-term infections than most adults, the overall CD8 T cell compartment in children in the early phase of chronic T. cruzi infection exhibits decreased levels of naïve T cells and increased levels of terminally differentiated antigen-experienced T cells T. cruzi-infected children have a mixed T cell profile with the production of IFN-γ and IL-4 T. cruzi specific T cells to co-express multiple functions has not been performed.Although, T. cruzi infection, we have assessed the quantitative and qualitative attributes of T. cruzi-specific T cell responses in early stages of Chagas disease in children in comparison to T. cruzi-infected adults. The degree of activation, differentiation and antigen exposure of total CD4+ T cells was also evaluated in T. cruzi-infected children. The primary finding is that T. cruzi-infected children have a higher frequency of polyfunctional and more robust T cell responses specific for T. cruzi compared to T. cruzi-infected adults, and a heightened state of immune activation of CD4+ T cells.In order to examine the progression of immune exhaustion in chronic This protocol was approved by the Institutional Review Boards of the Hospital Interzonal General de Agudos Eva Perón, and the Centro Nacional de Genética, Buenos Aires, Argentina. Informed written consent was obtained from adult subjects and the parents of all children included in this study.T. cruzi infection was determined by indirect immunofluorescence, haemagglutination and ELISA assays T. cruzi infection. Age- and sex-matched children with negative serological findings were recruited as uninfected controls. T. cruzi-infected subjects were classified according to a modified version of the Kuschnir grading system Five to 16-year old children and 29 to 63 year-old adults were enrolled at the Instituto Nacional de Parasitología Dr. Mario Fatala Chaben and at the Hospital Interzonal General de Agudos Eva Perón, . Children and adults with any impaired health condition such as severe nephropathy, liver disease, severe neuropathy, severe anaemia or homeopathy were excluded from the study. Data on the number, sex, age, clinical and epidemiologic features of the subjects included in this study are summarized in g for 15 min for sera separation.Approximately 10 mL of blood were drawn by venipuncture into heparinized tubes . PBMC were isolated by density gradient centrifugation on Ficoll-Hypaque (Amersham) and were cryopreserved for later analysis. Additional 2 mL of blood were allowed to coagulate at 37°C and centrifuged at 1000 T. cruzi amastigotes was obtained by freeze/thaw cycles followed by sonication as previously reported Protein lysate from trans-sialidase gene family of T. cruzi and peptides derived from Influenza (Flu) virus with high binding-affinity for the common class I HLA-supertypes A01, A02 and A03 were synthesized at the University of Georgia Molecular Genetics Instrumentation Facility .HLA-A01, A02, A03, A24 and B44-supertype binding epitopes encoded by the T. cruzi antigen-responsive IFN-γ- and IL-2-secreting T cells was determined by ex vivo ELISPOT using commercial kits (BD Biosciences), as described elsewhere 5 cells/well, and stimulated with T. cruzi lysate (10 µg/mL) or with peptide pools from the trans-sialidase protein family . For controls, PBMCs were incubated with either 20 ng/mL phorbol 12-myristate 13-acetate  plus 500 ng/mL ionomycin (Sigma), Flu virus , tetanus toxoid (4 I.U./ml) or media alone (unstimulated control) for 16–20 hr at 37°C and 5% CO2. Spot forming cells (SFC) were automatically enumerated using ImmunoSpot analyzer (CTL). The mean number of spots in triplicate wells was obtained for each condition, and the number of specific IFN-γ and IL-2-secreting T cells was calculated by subtracting the value of the wells containing media alone from the antigen-stimulated spot count. Responses were considered significant if a minimum of 10 spots/4×105 PBMC were present per well, and additionally, this number was at least twice the value of wells with media alone The number of T. cruzi lysate in the presence of anti-CD28 and anti-CD49d antibodies , for 16–20 h at 37°C. Ten µg/ml brefeldin A (Sigma) were added for the last 5 h of incubation, as previously described For polyfunctional analysis, 200 µL heparinized blood, half diluted in RPMI, were incubated with T. cruzi lysate were considered positive, for any particular subset, if the frequency of cytokine/CD154-positive T cells was threefold higher than the frequency in medium alone and above 0.07% of total CD4+ T cells, since the limit of detection was set at 0.01%.Typically, 500,000 lymphocytes were acquired on a FACScalibur (Becton Dickinson Immunocytometry Systems) and analyzed using FlowJo software  Lymphocytes were identified based on their scatter patterns and CD4 expression for the combination of IFN-γ, TNF-α and CD154; and based on scatter patterns as well as CD3 and CD4 expression for the combination of IFN-γ and TNF-α or IFN-γ and CD154. Boolean combination gating was then performed to calculate the frequencies of expression profiles corresponding to the seven different combinations of functions by using the FlowJo software. After subtracting the background values, the proportions of the different subsets were expressed as percentages of total cytokine or CD154-positive cells. Responses to the + T cells, 50 µL of whole blood were incubated with different combinations of CD4 (PerCP), CD45RA (FITC or APC), CD27 (PE), CD28 (PE), CD127 (PE), HLA-DR (FITC) and KLRG1 (APC) monoclonal antibodies (BD Biosciences), followed by red cell lysis and white cell fixation in FACS Lysing Solution (Pharmingen). Typically, 500,000 lymphocytes were acquired on the FACScalibur (Becton Dickinson Immunocytometry Systems) and analyzed using FlowJo software For phenotypic analysis of total CD4T. cruzi-infected subjects included in this study were summarized using range and median and compared with healthy participants using Kruskal-Wallis test with Dunn correction and Fisher's exact tests for numerical and categorical variables, respectively. Comparisons of the frequencies of responders in the different categories of ELISPOT responses among T. cruzi-infected children and between T. cruzi-infected children and the uninfected group were evaluated by use of the chi 2 test and Fisher's exact test. To compare the number of spots,as well as the magnitude of CD4+ T cells responses among triple, double or single functional profiles, the Kruskal-Wallis nonparametric analysis with Dunn correction was used. The percentages of responding CD4+ T cells for each cytokine within and among the children and adult groups were compared by the Kruskal-Wallis nonparametric analysis with Dunn correction and Mann-Whitney U test, respectively. T cell phenotypes in T. cruzi-infected children and uninfected controls were compared applying the Mann-Whitney U-test. Differences were considered to be statistically significant at P<0.05.Demographic and clinical characteristics of T. cruzi-infected and uninfected children enrolled in the study were all born from mothers with positive serology for T. cruzi infection and, at the time of the study all children were living in Buenos Aires, where T. cruzi infection is not endemic. T. cruzi-infected and uninfected groups of children included individuals born in areas endemic for T. cruzi infection and individuals born in non-endemic areas. Seropositive children who had not lived in endemic areas are presumed to have been infected congenitally. Only one child out of the fifty T. cruzi-infected children showed a right bundle branch block that is among the electrocardiographic alterations related to Chagas disease. Abnormal findings in the electrocardiography not related to Chagas disease, including sinusal tachycardia, incomplete right bundle branch block and premature atrial contraction T. cruzi-infected children born in areas endemic for T. cruzi infection than those born in non-endemic areas  T. cruzi–derived recombinant proteins We have previously reported an inverse correlation between the frequency of IFN-γ-producing T cells responsive to T. cruzi-antigens, with secretion of IFN-γ and IL-2 in nearly 60% of T. cruzi-infected children and, approximately 18% with IFN-γ-only responses. IL-2-only responses were not observed in T. cruzi-infected children  - a marker of costimulatory potential - was evaluated in 19 T. cruzi-infected children. For comparison, this functional profile was also assessed in 10 T. cruzi-infected adults without evidence of cardiac involvement or with mild cardiac disease. Examining IFN-γ, TNF-α and CD154+ individually, CD154 was the most highly expressed in T. cruzi-infected children and there was a trend for higher proportions of cells expressing one or more of these three markers in the in the T. cruzi-infected children relative to the adults   . CD4+ T children . Polyfunantigens . In cont subsets , with a unctions . In adulfunction .+ followed by monofunctional IFN+ T cells and double expressing IFN-γ+CD154+ T cells showed the highest magnitude of CD4+ T cell responses to T. cruzi antigens in T. cruzi-infected children , a marker of the number of TCR-triggering events −CD27−CD28− and CD45RA−CCR7−CD62L−) and terminally differentiated effector T cells (CD45RA+CCR−CD62L−) are increased in T. cruzi-infected children compared with uninfected controls , trans-sialidase-derived CD8+ target peptides showed a stronger and broader response compared with our previous findings in adults  T. cruzi-infected children with adults exhibiting severe cardiomyopathy who mostly showed negative T cell responses against T. cruzi antigens + T cell numbers with the ability to produce IFN-γ and IL-2 The analysis of IFN-γ ELISPOT responses to the lysate and to T. cruzi-infected children displaying both IFN+ and IL-2+ ELISPOT responses compared with those with single IFN-γ-producing T cells. It is likely that the maintenance of polyfunctional T cell responses provides more efficient control of T. cruzi infection and limits peripheral tissue damage. Indeed, most T. cruzi-infected children have not developed cardiac disease and previous studies from our group have shown higher frequencies of IFN-γ-producing T cells and higher magnitude of these responses in subjects with less severe forms of the disease compared with patients with severe cardiomypathy T. cruzi infection showed higher electrocardiographic abnormalities not related to Chagas disease compared to children born in non-endemic areas. This is likely due to co-morbidities and poorer socioeconomic conditions in rural areas. In this respect, we have shown that socioeconomic conditions had a significant influence on the progression of chronic Chagas disease which was independent of antiparasitic treatment and clinic characteristics T. cruzi-specific T cell responses were not different between seropositive children born in endemic areas and those born in non-endemic areas, who are presumed to have been infected congenitally.Multifunctional T cells are optimized for effector function, exhibiting a higher secretion of IFN-γ on a per-cell basis, more efficient killing, and IL-2-mediated expansion of T cells in an autocrine or paracrine manner T. cruzi infection, parasite persistence for decades, might drive a process of immune exhaustion. A broader range (relative to adults) of T. cruzi-specific immune phenotypes are present in T. cruzi-infected children – from “adult-like” monofunctional CD4+ T cell subsets to T cells exhibiting up to 3 functional properties. Thus, some T. cruzi-infected children display a T cell profile similar to that of adult patients. This is not unexpected since some children will likely have infections of >10 years. The increased frequencies of total effector/effector memory and activated CD4+ T cells in T. cruzi-infected children observed herein suggest a heightened state of immune activation as previously suggested in other studies + T cells shown herein, previous studies by our group in a similar patient population of children demonstrated a significant decrease in the levels of naïve CD8+ T cells compared to uninfected children +CD4+ T cells but lower frequencies of CD4+HLA-DR+ T cells along with unaltered levels of CD4+CD28+ T cells T. cruzi infection also show increased levels of activated, late differentiated memory and effector T cells, while naïve T cells are diminished as disease becomes more severe In Children have not only shorter term infections but are also younger. Aging of the immune system in elderly subjects , known as immunosenescence T. cruzi-infected subjects are younger than 60 years, in average, and there were not differences in the magnitude of IFN-γ ELISPOT to irrelevant antigens like toxoid tetanus and Flu among uninfected children, uninfected adults T. cruzi-infected adults,. Of note, in previous studies, we did not found any association between the magnitude of T cell responses specific for T. cruzi and age in a cohort of chronically T. cruzi-infected subjects in an age range of between 22 and 70 years T. cruzi-antigens in T. cruzi-infected adults display high expression of the inhibitory receptor CTLA-4 which is a feature of exhausted T cells T. cruzi-infected adults was confined to those specific for T. cruzi. The phenotype of the overall T cell compartment with an expansion of end-differentiated effector T cells and loss of naïve T cells in T. cruzi-infected children and adults compared with age-matched controls suggest that chronic T. cruzi infection might accelerate immune aging along with the induction of T cell exhaustion.T. cruzi infections T.cruzi T cell response prior to treatment.The quality of the immune response might also be a factor in the higher cure rates following drug treatment observed in children relative to adults Figure S1T. cruzi+ T cell profile in children and adults with Chagas disease. –specific CD4 Co-expression profiles of CD4+ T cells with the ability to produce IFN-γ, TNF-α, or to express the co-stimulatory molecule CD154 were measured by flow cytometry after 16–20 hs stimulation of whole blood with a T. cruzi lysate preparation. (A) Representative dot plots from one T. cruzi-infected child and one adult with chronic T. cruzi infection are shown. The numbers in the upper right quadrant indicate the percentage of CD4+IFN-γ+, CD4+TNF-α+ or CD4+ CD154+ T lymphocytes responsive to the lysate. (B) Representative dot blot of IFN-γ and TNF-α co-production by CD4+ T cells from the same patients showed in A. The numbers in the upper right quadrant indicate the percentage of CD4+IFN-γ+TNF-α+ T cells, while the numbers in the upper left represent the percentage of CD4+IFN-γ+TNF-α− T lymphocytes on total CD4+ T cells.(TIF)Click here for additional data file.Figure S2T. cruzi+ T cell responses in children and adults with Chagas disease.-specific CD4 PBMC were stimulated with a T. cruzi lysate preparation and stained for IFN-γ, TNF-α and CD154. Triple, double or single functional profiles were determined by FlowJo Boolean gating analysis in nineteen T. cruzi-infected children (A) and ten T. cruzi-infected adults (B). Data are presented as the percentage of responding CD4+ T cells in each subset. Boxes depicting median and 5th and 95th percentile values are shown. I, IFN-γ; T, TNF-α; D, CD154. (**) P<0.01 vs. ITD and TD among T. cruzi-infected children; (&) P<0.05 vs. IT and T among T. cruzi-infected children; (*) P<0.05 vs. ITD, IT, TD and T among T. cruzi-infected adults, by the Kruskal-Wallis test with Dunn correction.(TIF)Click here for additional data file.


Specific antiviral functions, however, have only been attributed to a few of them. + T-cells and PBMCs. 90K’s susceptibility to IFN-mediated stimulation of expression was cell type-specific. siRNA-mediated knockdown of 90K in TZM-bl cells and primary macrophages enhanced the incorporation of Env glycoproteins into progeny virions, boosted the particle infectivity of released HIV-1, and accelerated HIV-1 spread. Conversely, treatment of HIV-1 infected macrophages with IFN-α induced 90K expression and lowered the particle infectivity of HIV-1.Here, we show that 90K expression dose-dependently decreased the particle infectivity of HIV-1 progeny. The lower infectivity of released particles correlated with reduced virion incorporation of mature envelope glycoproteins gp120 and gp41. Further, proteolytic processing of the gp160 precursor and surface expression of gp120 in the producer cell were impaired in the presence of 90K expression. In contrast, expression of Gag, Nef and Vpu, and virus release were not grossly affected by 90K expression. 90K-imposed restriction occurred in the absence of direct interaction of 90K with HIV-1 Env or entrapment of Env in the ER. The cell-associated, but not the secreted species of 90K, mediated the antiviral effect. A truncated version of human 90K, solely consisting of the two intermediate domains, displayed a similar antiviral activity as the full-length wildtype 90K, indicating that the N-terminal SRCR-like domain and the C-terminal domain are dispensable for 90K’s antiviral activity. The murine homolog of 90K, CypCAP (Cyclophilin C-associated protein), neither modulated particle infectivity of HIV-1 nor lowered the virion incorporation of mature gp120, suggesting a species-specific mode of action. 90K was expressed at basal levels in TZM-bl cells and in primary macrophages, and at low levels in CD4Thus, 90K constitutes a novel antiviral factor that reduces the particle infectivity of HIV-1, involving interference with the maturation and incorporation of HIV-1 Env molecules into virions. Notable examples of anti-HIV-1 factors include members of the apolipoprotein editing complex (APOBEC) class of cytidine deaminases, which induce lethal hyper-mutations of the viral genome ,3, and S T-cells -6. Expre T-cells , and HIV [ex vivo and in vo[in vivo.90K/LGALS3BP, which encodes for a secretory glycoprotein of 585 amino acids, shares these properties. By virtue of an IFN-responsive element in the 90K promoter [in vivo following IFN-α administration to cancer patients [+ T-cell cultures [in vivo in HIV-1 infected individuals [in vivo.promoter , levels promoter -13. Indupromoter ,15 as wepatients ,16. Furtividuals -22, althN-glycosylated in the endoplasmic reticulum and Golgi complex [+ T-cell-mediated lysis activity and cytokine production [90K, originally identified as a tumor-associated antigen ,23,24, i complex . Secrete complex ,27,28. Ioduction . Furtheroduction  by bindide novo virus production and reduced incorporation of Env into progeny virions. The two central protein-binding domains of 90K were essential and sufficient for its antiviral infectivity. 90K protein expression was cell type-specific, and the degree of IFN-mediated upregulation of 90K at the protein level differed between macrophages and CD4+ T-cells. Depletion of endogenous 90K in HIV-1 susceptible cells, including TZM-bl cells and primary macrophages, boosted the particle infectivity of progeny HIV-1, ameliorated Env incorporation into nascent virions and accelerated the spread of HIV-1. Reduction of 90K expression in PBMCs likewise enhanced HIV-1 spread. Concordantly, IFN-α treatment of primary HIV-1 infected macrophages lowered the particle infectivity of viral progeny. These data suggest that 90K is an important contributor of the cellular antiviral defense against HIV-1 infection.The inducibility of 90K expression by IFNs and its upregulated expression in HIV-1 infected individuals prompted us to investigate whether 90K modulates HIV-1 replication. In this study, we establish 90K as an antiviral factor that is associated with the release of poorly infectious virions. This involved defective maturation of HIV-1 Env during In order to investigate whether 90K may affect HIV-1 replication, 293T cells were cotransfected with a proviral HIV-1 DNA and decreasing amounts of an expression plasmid for C-terminally myc-tagged 90K (90K-myc). Notably, transfection of 293T cells with a 90K-encoding plasmid resulted in levels of 90K expression that were clearly higher than those obtained following IFN stimulation -myc . NotablyNL4.3. Three days post infection, culture supernatants were analyzed for p24 capsid antigen release and particle infectivity. Importantly, 90K depletion barely affected HIV-1 p24 capsid release   domain, a BTB/POZ domain, an IVR domain, and a C-terminal part without known homologies to other protein domains . In ordeConclusively, a region within the two intermediate protein-binding domains of 90K, the BTB/POZ and the IVR domain, is required and sufficient for its ability to interfere with HIV-1 Env maturation and incorporation, whereas the scavenger receptor cysteine-rich domain and the C-terminal domain of 90K are dispensable.Mice encode a 90K homolog, Cyclophilin C-associated protein (CypCAP), which displays 69% homology on the amino acid level . Human 9We thus tested the ability of mouse CypCAP to reduce the particle infectivity of progeny HIV-1. Remarkably, the mouse homolog of 90K failed to act antivirally, whereas human 90K reduced the particle infectivity of HIV-1  and CD4s Figure C, despits Figure A, whereas Figure C. IFN-α,s Figure C. Both I+ T-cells, whereas the other T-cell lines scored negative. In macrophages, screening of macrophages from multiple donors by anti-90K ELISA confirmed that this cell type expresses higher 90K protein levels than T-cells.In order to verify the findings on 90K expression obtained by Western Blotting, we made use of a sensitive commercial anti-90K antigen ELISA for analysis of cell-associated 90K using lysates of T-cell lines and primary HIV-1 target cells. This assay revealed that 90K levels were close to the detection limit in Jurkat T-cells and CD4+ T-cells than macrophages  . IFN-α treatment potently reduced the levels of released HIV-1 infectivity  which is highly efficiently cleaved by furin and may be less sensitive to modest changes of furin efficacy. HIV-1 Env, in contrast, has been reported to be a notoriously poor furin substrate [Since HIV-1 Env and 90K share, by means of their N-terminal signal peptide, subcellular trafficking through the secretory pathway, it was tempting to speculate that a direct or indirect association of these two proteins is required for 90K’s restricting activity. HIV-1 gp160Env molecules require proteolytic cleavage by cellular furin and furin-like enzymes located in the ubstrate  and mightrans-Golgi compartment by a direct interaction, resulting in an abolished proteolytic cleavage and, as a consequence, in a reduced incorporation of mature gp120 into budding particles. This scenario, however, could not be substantiated by deglycosylation experiments and immunoprecipitation assays. These data, however, do not exclude an indirect association of 90K and HIV-1 Env within a multi-protein complex. Mutational analysis revealed that the two central protein-binding domains, BTB/POZ and IVR, are crucial for 90K’s antiviral activity, whereas the SRCR domain and the carboxyterminal domain of 90K were dispensable. Future work will consist to determine the exact mechanism of 90K-imposed inhibition.We next hypothesized that 90K may prevent access of the unprocessed gp160 precursor to the furin-positive cis-trans isomerase reactions and assist protein folding [Interestingly, the mouse homolog of 90K, CypCAP, failed to act antivirally. Cyclophilins catalyze petidyl-prolyl  folding . Whetherin vivo that may depend on its reported immunostimulatory functions [trans-infection of T-cells [In healthy individuals, 90K is present in multiple body compartments, including blood, semen, urine, tears, saliva, and breast milk ,27,28. Dunctions . In lineunctions . Also, eunctions . Further T-cells  at sitesin vivo, as well as possible viral evasion strategies that may circumvent 90K-imposed antiviral activity.A third criterion of many restriction factors is the existance of virally encoded antagonists against them, expressed either by HIV-1 or HIV-1 precursors. Work of our laboratory has provided preliminary evidence for the presence of an HIV-1 encoded antagonist of human 90K (unpublished observation), yet the specificity of the observed antagonism is not established and requires substantial experimental proof. Of note, low density of glycoprotein molecules on the virus surface is a specific property of HIV-1, which has been suggested to provide a selective advantage. Specifically, it has been hypothesized that this feature may enable efficient escape from antibody-mediated immune response . It is uThis study establishes 90K as an antiviral factor which impairs the infectivity of HIV-1 progeny, involving defective maturation and incorporation of Env glycoproteins. Further investigation and exploitation of its mode of action may provide a platform for the design of novel therapeutic antiviral approaches and widen our understanding of cell-mediated antiviral defense mechanisms.+ T-cells were isolated from Buffy Coat by negative selection with the RosetteSep Human CD4+ T Cells Enrichment Cocktail (StemCell Technologies). For the 90K expression studies, CD4+ T-cells were stimulated with IL-2/PHA for 2-3 days prior to IFN-α (Roche) or IFN-γ (Sigma-Aldrich) treatment. 293T cells were transfected by calcium phosphate DNA precipitation, SupT1 cells were transfected by electroporation.293T cells, TZM-bl cells and the T-cell lines SupT1, JTag, PM1, CEM, Jurkat were cultured as recommended by ATCC. Cultures of primary PBMCs, from random human donors were generated as previously reported . Taking NheI and BamHI. The truncated variants of 90K-myc all contained the authentic signal peptide and were generated by amplification of 90K with the following primers using HindIII and XbaI and insertion into pcDNA6: fwd GCAAGCTTGGATGACCCCTCCG and rev CGTCTAGAGGGGAGGAGGATGGCAAAGAGGCTTGC -myc); fwd CGAAGCTTATGACCCCTCCGAGGCTCTTCTGGGTGTGGCTGCTGGTTGCAGGAACCCAAGGCGTGAACGATGGTGACACCAGGAGCACCCACACCCTGGACCTC and rev CGTCTAGAAATCCGGGGCTTGTAGGTATCCT -myc); fwd CGAAGCTTATGACCCCTCCGAGGCTCTTCTGGGTGTGGCTGCTGGTTGCAGGAACCCAAGGCGTGAACGATGGTCTCCTCCCCCAGGACCCCTCGTTCCAGATG and rev CCTCTAGAGTCCACACCTGAGGAGTTGGTCAGG -myc). pcDNA6-mouse CypCAP-myc was generated by amplification of the mouse cDNA  using primers carrying HindIII and XbaI restriction sites (fwd GCAAGCTTATGGCTCTCCTG and rev GCTCTAGACACCATGTCAGTG) and ligation of the amplicon into pcDNA6/myc. Expression constructs for Glypican-3-HA and H7 Influenza virus HA (derived from A/chicken/Rostock/8/1934(H7N1)) were provided by Guido David and Wolfgang Garten, respectively. The expression plasmid pEbola GP was from Mark Goldsmith. The CD4 and the HIV-1NL4.3 Env expression plasmid was from Oliver Keppler. The Vpu-IRES GFP expression construct and the respective control vector have been described elsewhere [pcDNA6 and pcDNA6.90K-myc plasmids were provided by Ji Hee Lee, South Korea. pIRES2 90K-myc IRES GFP was generated by PCR amplification of 90K-myc and insertion into pIRES2-GFP (Clontech) using lsewhere .nef ORF [Infectious HIV-1 was generated by calcium phosphate DNA precipitation of 293T cells with plasmids encoding HIV-1 and harvest of cell-free virion-containing supernatant. Plasmids encoding NL4.3, LAI, YU-2 were obtained from Oliver Keppler, plasmids encoding transmitter founder viruses  were obtained from Frank Kirchhoff. The construct pBR.HIV-1 NL4.3-IRES GFP carries an IRES-EGFP cassette downstream of the nef ORF . The expThe infectivity of HIV-1 virions was assessed by applying a standardized TZM-bl based firefly luciferase assay or beta-galactosidase assay. Readouts were obtained luminometrically . For calculation of the particle infectivity, luminometric counts were divided by the content of p24.Supernatants were analyzed for p24 content (ng/ml) by applying a home-made anti-p24 antigen ELISA .Cells were stained with 7-AAD for detection of dead or unhealthy cells (BD PharMingen). For measuring metabolic activity, cell lysates were analyzed using the Cell Titer Glow assay (Promega). As a positive control for apoptosis induction or cell death, cells were treated UV-irradiated.The following siRNAs were used: control siRNA (Dharmacon), 90K-specific siRNA . TZM-bl cells and primary macrophages were transfected 2-3 times with siRNAs at the final concentration of 5-15 nM, using RNAiMax (Invitrogen). PBMCs were transfected twice by Amaxa nucleofection (Lonza) with final siRNA concentration of 2.5 μM. Knockdown efficiency was determined by quantitative RT-PCR of 90K mRNA and RNaseP mRNA and/or by anti-90K Western Blotting or anti-90K ELISA (eBioscience).RNaseP mRNA (Applied Biosystems) as internal reference. Each sample was analyzed in duplicates. Data analysis was performed using Applied Biosystems Step One Software v2.1.Total RNA extraction from untreated or IFN-treated cells was performed with the Qiagen RNeasy kit (Qiagen), followed by DNase treatment (Ambion) and cDNA synthesis . Quantification of relative 90K mRNA levels was performed with the Step One Plus Real Time PCR System (Applied Biosystems) using TaqMan PCR technology with the following oligonucleotide primers: 5′-GCTTCCTTCCTCTCTGCAATGA-3′ (forward primer), 5′-TCAGGTGAGTAGGGCGACATC-3′ (reverse primer), 5′-FAM-CTTCAACAACCGGCCAC-TAMRA-3′ (fluorescent probe). Relative 90K mRNA levels were determined using the ∆∆Ct method with human http://tech.knime.org/faq#q1_1) in combination with the KNIME Image Processing 1.0.3 extension. For segmentation, DAPI stained nuclei were used. The images were smoothed using a Gaussian Filter followed by a simple Otsu thresholding. Consecutively, cells were splitted using a standard cell clump splitter and additionally filtered by size. After identification of the cells, the intensity of each pixel was measured, the mean of intensity for each cell was calculated.Cells grown on coverslips were transfected with Lipofectamin2000 (Invitrogen) and fixed with 4% paraformaldehyde. They were permeabilized with 0.2% Saponin or 0.1% TritonX-100, blocked and stained with goat polyclonal anti-90K (R&D Systems), followed by anti-goat Alexa647, goat polyclonal anti-myc (novus), followed by anti-goat Alexa488, or Chessie8 Hybridoma supernatant, followed by anti-mouse Alexa647. Coverslips were mounted with Mounting medium containing DAPI (Vectashield) and analysed with a Zeiss 710 LSM confocal microscope. Images were recorded and processed with the ZEN (2010) software. Quantification of 90K expression was performed by using the open-source KNIME (Kontanz Information Miner) platform , mouse anti-myc (provided by Jens von Einem), goat polycolonal anti-myc , rabbit anti-MAPK (Santa Cruz), mouse anti-HIV-1 p24 (Exbio), rabbit anti-HIV-1 gp120 and mouse anti-HIV-1 gp41 , human 2G12 (NIH), mouse anti-HA (abcam), anti-Ebola GP (provided by Stephan Becker), anti H7-serum (provided by Wolfgang Garten), rabbit anti HIV-1 Vpu (provided by Ulrich Schubert), mouse anti-CD4 (abcam), anti-CD4-APC (BD).Cells were lysed with M-PER Mammalian Protein Extraction Reagent (Pierce), and virions were prepared from supernatants and ultracentrifugation through a 20% (w/v) sucrose cushion. Proteins were run on a 12.5% or 7.5% SDS-PAGE and transferred onto nitrocellulose. Blocked membranes were incubated with primary antibodies. Secondary antibodies conjugated to Alexa680/800 fluorescent dyes were used for detection by Odyssey Infrared Imaging System (LI-COR Biosciences) and quantification by Odyssey software.Cells were lysed with 0.5% NP40 lysis buffer and a small aliquot was used for control of protein expression (Input). Residual lysate was filled up to final volume of 500 μl with 0.5% NP40 and incubated with antibody for 1 hour at 4°C. 30 μl G-Sepharose Beads  were added and precipitated 90 minutes at 4°C. Beads were washed with 0.1% NP40, treated with Western Blot Sample Buffer and boiled for 5 minutes. Supernatants contained the precipitated proteins.Cell lysates were treated with PNGase (NEB BioLabs) and EndoH (NEB BioLabs) according to instructor’s protocol. Digestion was performed with 625 Units PNGase for 20 minutes or with 100 Units EndoH for 5 minutes at 37°C.A.U: Arbitrary units; HIV: Human immunodeficiency virus; IFN: Interferon; LGALS3BP: Lectin galactoside-binding soluble 3 binding protein; MFI: Mean fluorescence intensity; RLU: Relative light units.The authors declare that they have no competing interests.VL and CG conceptualized and designed the study; VL, KS, NS, CK, CMS, TW, DK, CG performed the study; TW and CD performed the immunofluorescence-based quantification of 90K expression; VL, KS, CK, CMS, CG analyzed data; VL and CG wrote the paper. JM and FK provided reagents. All the authors reviewed the manuscript and approved the final version.Heterologous 90K levels compared to endogenous 90K levels. (A) 293T cells were cotransfected with various amounts of pcDNA6.90K-myc , stimulated with 100 Units IFN-α, IFN-γ for 48 h, or left untreated. (B) Western Blot of cell lysates of 293T cells overexpressing various amounts of 90K-myc (A) and untreated TZM-bl cells. (C) Western Blot of cell lysates of 293T cells overexpressing various amounts of 90K-myc (A) and primary macrophages stimulated with 100 IFN-α, IFN-γ for 48 h, or left untreated. The numbers depict the relative 90K expression, measured by normalization of the 90K signal to the MAPK signal obtained by Infrared-imaging based quantification. The ratio obtained for the lowest antivirally active 90K plasmid dose , the ratio obtained for TZM-bl cell lysates (B) or the ratio obtained for untreated macrophages (C) was set to 1, respectively.Click here for file90K reduces the particle infectivity of multiple HIV-1 strains. (A) 293T cells were cotransfected with the indicated proviral plasmids and highest amount of pcDNA6.90K-myc or empty vector. Supernatants were analyzed for infectious HIV-1 using a luminometric TZM-based luciferase assay. Shown are the results of one representative experiment out of three-six. (B) Relative levels of particle infectivity, defined as HIV-1 infectivity per ng p24 capsid are depicted. (C) Sucrose cushion-purified virions were analyzed by immunoblotting. Percentages indicate the relative gp120 incorporation, as measured by Infrared imaging-based quantification of the amount of gp120 per p24. The signal intensity in absence of 90K expression was set to 100%. (D) Cell lysates were analyzed by immunoblotting using the indicated antibodies. Numbers indicate the efficiency of gp160 processing. * : p < 0.05; **: p < 0.02 (Student’s T-Test).Click here for file90K-myc expression is not associated with toxicity or reduced metabolic activity. (A) 293T cells were transfected with pcDNA6.90K-myc (1.3 μg), empty vector, or UV-irradiated and stained two days post transfection and one day post UV-irradiation with 7-AAD. Shown are representative dot plots of one experiment out of two. Numbers indicate percentage of 7-AAD-positive cells. (B) Quantification of 7-AAD FACS analysis. (C) Cells were lysed and analysed for metabolic activity by Cell Titer Glow assay. Shown are the RLU of triplicates obtained from one representative experiment out of two.Click here for file90K does not reduce the cell surface levels of CD4. (A) 293T cells were cotransfected with pcDNA.CD4 and pIRES2EGFP.90K-myc or empty vector, Cells were stained with APC-conjugated anti-CD4 and analyzed by flow cytometry. Shown are representative dot plots of one experiment out of three. (B) 293T cells were cotransfected with pcDNA.CD4 and pVpu-IRES GFP or empty vector and processed like in (A). (C) CD4 cell surface levels were calculated by comparing, within the same sample, CD4 levels on non-GFP-expressing cells (gate P2) with CD4 levels on cells with medium-high GFP expression levels (gate P3). CD4 levels on vector transfected cells were set to 100%. (D) An aliquot of the cells shown in (A) and (B) were lyzed and analyzed by Western Blotting using the indicated antibodies.Click here for filevice versa using ZEN2010 software. The data represent the arithmetic mean ± S.D. of 105 analyzed cells.90K and Env colocalize to a high extent. (A) 293T cells were cotransfected with pcDNA6.90K-myc and an HIV-1 Env expression plasmid, and stained for 90K-myc (green) and Env (red). Scale bar: 10 μm. (B) The classic colocalization coefficient was calculated for the colocalization of 90K protein with Env protein or Click here for fileNo evidence for a direct interaction of 90K and HIV-1 Env. (A-C) 293T cells were cotransfected with pcDNA6, pcDNA6.90K-myc, an HIV-1 Env expression plasmid, pcDNA.CD4 or a combination out of these. (A) 90K, CD4 and bound proteins were precipitated from cell lysates by an anti-90K or anti-CD4 antibody, respectively. (B) 90K, CD4 and bound proteins were precipitated from cell lysates by an anti-myc or anti-CD4 antibody, respectively. (C) Env and bound proteins were precipitated from cell lysates by an anti-gp120 antibody. For each experimental set up, an aliquot of whole cell lysate for expression control (Input) and the precipitated proteins were analyzed by Immunoblot with indicated antibodies.Click here for file90K does not retain Env in the ER. (A-B) 293T cells were cotransfected with pBR.HIV-1 IRES GFP and vector or pcDNA6.90K-myc. (A) Cell lysates were treated with PNGase. (B) Cell lysates were treated with EndoH. Deglycosylated and control proteins were analyzed by Western Blot. Numbers indicate the efficiency of gp41 processing, calculated as the signal ratio of gp41 relative to (gp41 + gp160), or the percentage of deglycosylated gp160 to the total gp160 signal.Click here for fileSpecies specificity of 90K-imposed antiviral activity. (A) 293T cells were cotransfected with pBR.HIV-1 NL4.3-IRES GFP and 1.3 μg of the indicated expression constructs or empty vector. Two days post transfection, supernatants were analyzed for particle infectivity, defined as infectivity per ng p24. Cell lysates were analyzed by immunoblotting. (B) Sucrose cushion-purified virions were analyzed by immunoblotting. Shown is the relative gp120 incorporation, as measured by Infrared imaging-based quantification of the amount of gp120 per p24. The signal intensity in absence of 90K expression was set to 100%. (C) Cell lysates were analyzed by immunoblotting. Shown is the percentage of gp120, as measured by Infrared imaging-based quantification of the amount of gp120 per (gp120 + gp160). Bar diagrams show the arithmetic means ± S.E.M. of 3-4 independent experiments. The Western Blot shown is representative for one of the experiments included in the calculation. **: p < 0.02; n.s. : > 0.05 (Student’s T-Test).Click here for file


Reproducible and accurate T2 measurements are required for the characterization of myocardial tissue, e.g. to distinguish healthy, scar and edematous myocardium. It is the objective of this study to investigate the impact of the fitting algorithm applied for the T2 quantification from data derived from a single-breathhold T2-prepared steady-state free precession (T2p-SSFP) imaging technique.In direct comparison to multi spin echo technique, superior performance of T2p-SSFP-based T2 quantification has been reported. In spite of availability of a variety of quantification algorithms, the use of mono-exponential model is still the standard for fitting the T2 decay curve.2 was used to describe the quality of the resulting fit. Significance of the results was tested by applying two-tailed paired Student’s t-test.3 different fit-models were investigated: a) numerical approach . Acquisition parameters: matrix 240x240, slice thickness 5mm, FOV 2302mm2, matrix 288x288, slice thickness 10mm, bandwidth 515.7Hz/pixel, acquisition window 102ms and scan duration of 12RR cycles. Image registration was performed to compensate for residual respiratory motion.The identified optimal parameter set was applied for in vivo measurements. T2-prepared experiments with 6 preparation times  were performed consecutively within a single breathhold Fig. . Data waThe phantom data Fig.  revealedThe phantom data showed superior performance of the offset model for T2 quantification as compared to the mono-exponential and numerical model for T2p-SSFP. The respective quantification of myocardial T2 values in vivo yielded a lower value as previously reported. This may indicate that applying simple mono-exponential decay model may overestimate resulting T2 value in myocardium.EU FP7 REBORNEProject Reference 241879.