Histone deacetylases (HDACs) play important roles in glial cell development and in disease states within multiple regions of the central nervous system. However, little is known about HDAC expression or function within the optic nerve. As a first step in understanding the role of HDACs in optic nerve, this study examines the spatio-temporal expression patterns of methylated histone 3 (K9), acetylated histone 3 (K18), and HDACs 1–6 and 8–11 in the developing murine optic nerve head.Using RT-qPCR, western blot and immunofluorescence, three stages were analyzed: embryonic day 16 (E16), when astrocyte precursors are found in the optic stalk, postnatal day 5 (P5), when immature astrocytes and oligodendrocytes are found throughout the optic nerve, and P30, when optic nerve astrocytes and oligodendrocytes are mature. Acetylated and methylated histone H3 immunoreactivity was co-localized in the nuclei of most SOX2 positive glia within the optic nerve head and adjacent optic nerve at all developmental stages. HDACs 1–11 were expressed in the optic nerve glial cells at all three stages of optic nerve development in the mouse, but showed temporal differences in overall levels and subcellular localization. HDACs 1 and 2 were predominantly nuclear throughout optic nerve development and glial cell maturation. HDACs 3, 5, 6, 8, and 11 were predominantly cytoplasmic, but showed nuclear localization in at least one stage of optic nerve development. HDACs 4, 9 and10 were predominantly cytoplasmic, with little to no nuclear expression at any time during the developmental stages examined.Our results showing that HDACs 1, 2, 3, 5, 6, 8, and 11 were each localized to the nuclei of SOX2 positive glia at some stages of optic nerve development and maturation and extend previous reports of HDAC expression in the aging optic nerve. These HDACs are candidates for further research to understand how chromatin remodeling through acetylation, deacetylation and methylation contributes to glial development as well as their injury response. Astrocytes, once thought to be relatively inert cells, have been shown to play a multitude of roles in the developing, mature, and injured or diseased central nervous system,2. For iin vitro, but there are conflicting reports in the literature as to whether this also occurs in vivo dissolved in 1XPBS at 1:800 dilution for 1 hr in dark at room temperature. Secondary antibodies were removed by washing the slides in 1XPBS (2 × 10 min). Sections used to label NKX2.2, the marker used for oligodendrocytes, were treated with biotinylated secondary antibody  for 1 hr and then washed with 1XPBS (2X10 min). Incubation with Streptavidin 549  was then performed for 1 hr. Sections were mounted with anti-fade mounting medium containing DAPI . In some experiments, antibodies were preabsorbed with 32 μg/ml of the peptide or protein immunogen that was used to generate the antibodies for 1 hr prior to incubation with sections and immunofluorescence performed as usual. Additional sections were incubated with 0.25 μg/ml of IgG in blocking solution in place of the primary antibody and immunofluorescence performed as usual. Negative controls peptides corresponding to each HDAC are shown in Table  × 10 minConfocal images of sections co labeled with SOX2 and PAX2, SOX2 and NKX2.2 were taken using FV1000 Olympus Fluoview microscope. Cells showing positive label in the optic nerve were counted over an area of 200 μm at all three stages  using retina on both sides as a reference. Cells were counted in every third section of through the optic nerve and three different nerves were analyzed for each timepoint.in silico PCR on the Santa Cruz Genome Browser [http://genome.ucsc.edu/], build mm10[Relative Expression Software Tool-Multiple Condition Solver  using the three reference genes for normalization[Optic nerves from the exit of the optic nerve up to, but not including the optic chiasm, were quickly isolated from mice following euthanasia and preserved in RNA stabilizing agent  at 4°C until ready for total RNA extraction. Total RNA was isolated using affinity columns  with DNAse treatment. Quality analysis and quantification of total RNA was done using Nanodrop 2000c (ThermoScientific). For each RNA sample, cDNA was synthesized using oligo-dT primers as per manufacturer’s instructions . Briefly, cDNA was reverse transcribed by incubating 1μg of RNA template with nuclease-free water, 1X buffer reaction mix and reverse transcriptase at 25°C for 5 min, 42°C for 20 min, and 85°C for 5 min. cDNA was stored at -20°C until used for PCR reactions. Primer pairs  and 1 mM PMSF  on ice. Tissue lysates were centrifuged at 14000 rpm for 10 min at 4°C and supernatant was collected. Total protein concentration was determined using BCA protein Assay reagent (ThermoScientific). Proteins were resolved using SDS-PAGE (4-20% gradient gel), transferred to PVDF membranes, and blocked using 5% milk or protein-free tween 20 buffer  and immunoblotted using specific HDAC antibodies. The concentrations of antibodies employed are specified in Table The authors have no competing interests to declare in relation to this manuscript.Experimental conception and design: TLB-A. Performance of experiments: ST, SD and MS. Analysis of results: TLB-A, ST and DCO. Manuscript preparation: TLB-A, DCO, ST, SD, and MS. All authors read and approved the final manuscript.HDAC2 localization pattern in developing murine optic nerve. (A) Low magnification confocal micrograph showing the P30 optic nerve (ON), optic nerve head and retina (R) double-labeled for SOX2  and HDAC2 (red). Optic nerve sections at E16 (B-E), P5 (F-I) and P30 (J-M) were triple-labeled with DAPI (blue), SOX2 (green) and HDAC2 (red).  Overlays of SOX2 and DAPI to aid in orientation of the image. Subsequent panels did not include DAPI to better show the co-localization of the SOX2 and HDAC2 labels.  HDAC2;  double-label of SOX2 and HDAC2. Arrows indicate nuclei that were co-labeled with SOX2 and HDAC2. Composite images in E, I and M show z stacks of optic nerve at high magnification; horizontal yellow lines correspond the x axis plane and vertical yellow lines corresponds to the y axis. The x,z axis is shown at the bottom of each panel, while the y,z axis is shown to the right of the panel . Negative controls at each stage showed a lack of immunoreactivity following preabsorption of antibodies with the peptide immunogen (N-P). Abbreviations: R; retina, ON; optic nerve. Scale bars = 50 μm. Scale bar in B applies to B-D, F-H, and J-L, scale bar in E applies to E, I and M.Click here for fileHDAC5 localization pattern in developing murine optic nerve. (A) Low magnification confocal micrograph showing the P30 optic nerve (ON), optic nerve head and retina (R) double-labeled for SOX2  and HDAC5 (red). Optic nerve sections at E16 (B-E), P5 (F-I) and P30 (J-M) were triple-labeled with DAPI (blue), SOX2 (green) and HDAC5 (red).  Overlays of SOX2 and DAPI to aid in orientation of the image. Subsequent panels did not include DAPI to better show the co-localization of the SOX2 and HDAC5 labels.  HDAC5;  double-label of SOX2 and HDAC5. Arrows indicate nuclei that were co-labeled with SOX2 and HDAC5. Composite images in E, I and M show z stacks of optic nerve at high magnification; horizontal yellow lines correspond the x axis plane and vertical yellow lines corresponds to the y axis. The x,z axis is shown at the bottom of each panel, while the y,z axis is shown to the right of the panel. Negative controls at each stage showed a lack of immunoreactivity following preabsorption of antibodies with the peptide immunogen (N-P). Abbreviations: R; retina, ON; optic nerve. Scale bars = 50 μm. Scale bar in B applies to B-D, F-H, and J-L, scale bar in E applies to E, I and M.Click here for file. (A) Low magnification confocal micrograph showing the P30 optic nerve (ON), optic nerve head and retina (R) double-labeled for SOX2  and HDAC6 (red). Optic nerve sections at E16 (B-E), P5 (F-I) and P30 (J-M) were triple-labeled with DAPI (blue), SOX2 (green) and HDAC6 (red).  Overlays of SOX2 and DAPI to aid in orientation of the image. Subsequent panels did not include DAPI to better show the co-localization of the SOX2 and HDAC6 labels.  HDAC6;  double-label of SOX2 and HDAC6. Arrows indicate nuclei that were co-labeled with SOX2 and HDAC6. Composite images in E, I and M show z stacks of optic nerve at high magnification; horizontal yellow lines correspond the x axis plane and vertical yellow lines corresponds to the y axis. The x,z axis is shown at the bottom of each panel, while the y,z axis is shown to the right of the panel . Negative controls at each stage showed a lack of immunoreactivity following preabsorption of antibodies with the peptide immunogen (N-P). Abbreviations: R; retina, ON; optic nerve. Scale bars = 50 μm. Scale bar in B applies to B-D, F-H, and J-L, scale bar in E applies to E, I and M.HDAC6 localization pattern in developing murine optic nerveClick here for fileHDAC8 localization pattern in developing murine optic nerve. (A) Low magnification confocal micrograph showing the P30 optic nerve (ON), optic nerve head and retina (R) double-labeled for SOX2  and HDAC8 (red). Optic nerve sections at E16 (B-E), P5 (F-I) and P30 (J-M) were triple-labeled with DAPI (blue), SOX2 (green) and HDAC8 (red).  Overlays of SOX2 and DAPI to aid in orientation of the image. Subsequent panels did not include DAPI to better show the co-localization of the SOX2 and HDAC8 labels.  HDAC8;  double-label of SOX2 and HDAC8. Arrows indicate nuclei that were co-labeled with SOX2 and HDAC8. Composite images in E, I and M show z stacks of optic nerve at high magnification; horizontal yellow lines correspond the x axis plane and vertical yellow lines corresponds to the y axis. The x,z axis is shown at the bottom of each panel, while the y,z axis is shown to the right of the panel . Negative controls at each stage showed a lack of immunoreactivity following preabsorption of antibodies with the peptide immunogen (N-P). Abbreviations: R; retina, ON; optic nerve. Scale bars = 50 μm. Scale bar in B applies to B-D, F-H, and J-L, scale bar in E applies to E, I and M.Click here for file. (A) Low magnification confocal micrograph showing the P30 optic nerve (ON), optic nerve head and retina (R) double-labeled for SOX2  and HDAC9 (red). Optic nerve sections at E16 (B-E), P5 (F-I) and P30 (J-M) were triple-labeled with DAPI (blue), SOX2 (green) and HDAC9 (red).  Overlays of SOX2 and DAPI to aid in orientation of the image. Subsequent panels did not include DAPI to better show the co-localization of the SOX2 and HDAC9 labels.  HDAC9;  double-label of SOX2 and HDAC9. Composite images in E, I and M show z stacks of optic nerve at high magnification; horizontal yellow lines correspond the x axis plane and vertical yellow lines corresponds to the y axis. The x,z axis is shown at the bottom of each panel, while the y,z axis is shown to the right of the panel . Negative controls at each stage showed a lack of immunoreactivity following preabsorption of antibodies with the peptide immunogen (N-P). Abbreviations: R; retina, ON; optic nerve. Scale bars = 50 μm. Scale bar in B applies to B-D, F-H, and J-L, scale bar in E applies to E, I and M.HDAC9 localization pattern in developing murine optic nerveClick here for file. (A) Low magnification confocal micrograph showing the P30 optic nerve (ON), optic nerve head and retina (R) double-labeled for SOX2  and HDAC10 (red). Optic nerve sections at E16 (B-E), P5 (F-I) and P30 (J-M) were triple-labeled with DAPI (blue), SOX2 (green) and HDAC10 (red).  Overlays of SOX2 and DAPI to aid in orientation of the image. Subsequent panels did not include DAPI to better show the co-localization of the SOX2 and HDAC10 labels.  HDAC10;  double-label of SOX2 and HDAC10. Composite images in E, I and M show z stacks of optic nerve at high magnification; horizontal yellow lines correspond the x axis plane and vertical yellow lines corresponds to the y axis. The x,z axis is shown at the bottom of each panel, while the y,z axis is shown to the right of the panel . Negative controls at each stage showed a lack of immunoreactivity following preabsorption of antibodies with the peptide immunogen (N-P). Abbreviations: R; retina, ON; optic nerve. Scale bars = 50 μm. Scale bar in B applies to B-D, F-H, and J-L, scale bar in E applies to E, I and M.HDAC10 localization pattern in developing murine optic nerveClick here for file. (A) Low magnification confocal micrograph showing the P30 optic nerve (ON), optic nerve head and retina (R) double-labeled for SOX2  and HDAC11 (red). Optic nerve sections at E16 (B-E), P5 (F-I) and P30 (J-M) were triple-labeled with DAPI (blue), SOX2 (green) and HDAC11 (red).  Overlays of SOX2 and DAPI to aid in orientation of the image. Subsequent panels did not include DAPI to better show the co-localization of the SOX2 and HDAC11 labels.  HDAC11;  double-label of SOX2 and HDAC11. Arrows indicate nuclei that were co-labeled with SOX2 and HDAC11. Composite images in E, I and M show z stacks of optic nerve at high magnification; horizontal yellow lines correspond the x axis plane and vertical yellow lines corresponds to the y axis. The x,z axis is shown at the bottom of each panel, while the y,z axis is shown to the right of the panel . Negative controls at each stage showed a lack of immunoreactivity following preabsorption of antibodies with the peptide immunogen (N-P). Abbreviations: R; retina, ON; optic nerve. Scale bars = 50 μm. Scale bar in B applies to B-D, F-H, and J-L, scale bar in E applies to E, I and M.HDAC11 localization pattern in developing murine optic nerveClick here for fileCrossing point analysis.Click here for file


Curcumin has anti-inflammatory and antioxidative properties. The objective of this study was to investigate the therapeutic effects of curcumin on functional disturbances, oxidative stress, and leukocyte infiltration induced by renal ischemia/reperfusion (I/R).-1, i.p. after the ischemic period. The 72-h reperfusion groups also included Sham-72h, I/R-72h, I/R treated with curcumin at single dose of 20 mg kg-1, i.p., and I/R group which received three doses of curcumin at 20 mg kg-1, i.p., consecutively. Renal functional injury was assessed by measuring serum creatinine and urea-nitrogen concentrations. Oxidative stress was evaluated by assessment tissue malondialdehyde (MDA) and the ferric reducing/antioxidant power (FRAP) levels. Moreover, renal tissue leukocyte infiltration was measured by histopathology examination. Animals were randomly divided into 9 groups. The groups with 24-h reperfusion consisted of sham-24h, I/R-24h, and three I/R groups treated with curcumin at 10, 20, or 30 mg kgIschemia/reperfusion resulted in a significant increase in serum concentration of creatinine, urea-nitrogen, tissue MDA level, and leukocytes infiltration as well as reduced FRAP level. Treatment with curcumin in 24-h reperfusion groups could only lead to a significant change in the levels of MDA and FRAP. However, in 72-h reperfusion groups, curcumin was able to correct all functional disturbances, oxidative stress, and leukocytes infiltration with more effectiveness in groups that received three doses of curcumin.The administration of curcumin during 72-h reperfusion following 30 minutes of ischemia can decrease renal oxidative stress and leukocytes infiltration as well as improve kidney function. However, during first 24-h reperfusion, curcumin only decreased oxidative stress. Curcuma longa (turmeric) is an herbaceous plant that its rhizomatous parts have been widely used as food coloring and flavoring substance. Curcuma contains various compounds such as carbohydrates, proteins, fats, fiber, and about 3-5% of curcuminoids. Curcuminoids include curcumin (70%), demethoxycurcumin (17%), bisdemethoxycurcumin (3%), and 10% cyclocurcumin . All animals in this experiment were kept under controlled condition of temperature (22-24 °C), 12 hours light/dark cycles, and were provided with standard rat food and water ad libitum. Ischemia and reperfusion induction After anesthesia with ether  for pathological examination. At the end of experiment, the animals were sacrificed by deep anesthesia  were counted by a pathologist and the average was calculated as the number in a cubic millimeter . To determine leukocytes infiltration in the kidney, sections stained with H & E were prepared and cortical regions, outer medulla, and inner medulla were separately studied using a light microscope. Number of leukocytes in 20 microscopic fields  and also FRAP based on Ohkawa (1979)Statistical analysis Results were shown as mean ± SEM. The one-way ANOVA followed by Duncan's post hoc test was used for the intergroup comparison of all measured parameters, and LSD test was used to determine the exact p value. All data were analyzed by SPSS-18 statistical software and p<0.05 was considered as significant level.The effect of curcumin on renal functional disturbances Applying a half-hour bilateral renal ischemia and 24 hours reperfusion increased serum creatinine concentration (p<0.001) in the I/R group compared to the sham animals . HoweverSerum creatinine concentration in I/R-72h group was also considerably higher than that in sham-72h group (p<0.001). Administration of curcumin at a single dose or three doses of 20 mg/kg of body weight over a period of 72-hours of reperfusion (I/R+C20(1)-72h and I/R+C20(3)-72h groups) could significantly reduce serum creatinine compared to the I/R-72h group with a higher reduction in the group that received three doses of curcumin (p<0.01 vs. p<0.001). There was no significant difference in serum creatinine concentration between I/R+C20(1)-72h and I/R+C20(3)-72h groups and the sham group .As The serum concentration of urea-nitrogen in I/R-72h was significantly higher than the sham-72h group (p<0.001). Although a single dose of curcumin could significantly lower urea-nitrogen concentration in I/R+C20(1)-72h group as compared to I/R-72h group (p<0.05), it was still higher than that the related sham animals (p<0.01). Three doses of curcumin during 72 hours of reperfusion resulted in a significant decrease in serum urea-nitrogen concentration in the I/R+C20(3)-72h group compared to I/R-72h (p<0.01). Moreover, there was no significant difference between I/R+C20(3)-72h group in comparison with the related sham group .Effect of curcumin on oxidative stress Application of curcumin in single and three doses of 20 mg/kg body weight during the 72-hour period of reperfusion could reduce MDA level in the I/R+C20(1)-72h and I/R+C20(3)-72h groups compared to the I/R-72h group (p<0.05 vs. p<0.001). So that there was no significant difference in tissue MDA level between I/R+C20(3)-72h group and its related sham group .Our results also showed that I/R led to severe kidney tissue FRAP reduction (p<0.001) in I/R-24h group compared to its value in the sham-24h group . AlthougThe effect of curcumin on leukocyte infiltrationImages that are related to leukocyte infiltration in the renal interstitium are shown in The leukocyte infiltration in the I/R-72h group was also significantly higher than the corresponding sham group (p<0.001). Following curcumin administration, leukocyte infiltration decreased by 8.5 times in the I/R+C20(1)-72h group (p<0.01) in comparison to the I/R-72h group. However, in the I/R+C20(3)-72h group, its amount reached that of the sham group. The results of present study showed that intraperitoneal administration of curcumin during 72-h reperfusion after bilateral renal ischemia could partially improve functional disturbances, oxidative stress, and leukocyte infiltration in the kidneys. Moreover, it demonstrated that effect of three successive doses were more than a single dose. However, curcumin treatment in the I/R- 24h group could only improve renal oxidative stress. Serum creatinine and urea-nitrogen concentrations in I/R group highly increased, possibly because of a sharp decline in the glomerular filtration rate (GFR). Several studies have demonstrated that renal I/R by disturbing the balance between the production of vasoconstrictors such as adenosine and endothelin and vasodilators including nitric oxide and prostaglandins increase the renal vasoconstriction , and eicosanoids causes damage to the endothelial and tubular cells. In addition, leukocytes produce and secrete the myeloperoxidase enzyme which catalyzes H2O2 to hypochlorous acid (HOCl-) which is a more active oxidant  enzyme expression in renal epithelial cells through Nrf2/ARE pathway Therefore, it can be concluded that curcumin possibly reduces leukocyte infiltration and functional disturbances in the rat kidney via supporting the kidney against oxidative stress.


P. falciparum preerythrocytic vaccine candidates, a library of circumsporozoite (CS) T and B cell epitopes displayed on the woodchuck hepatitis virus core antigen (WHcAg) VLP platform was produced. To test the protective efficacy of the WHcAg-CS VLPs, hybrid CS P. berghei/P. falciparum (Pb/Pf) sporozoites were used to challenge immunized mice. VLPs carrying 1 or 2 different CS repeat B cell epitopes and 3 VLPs carrying different CS non-repeat B cell epitopes elicited high levels of anti-insert antibodies (Abs). Whereas, VLPs carrying CS repeat B cell epitopes conferred 98% protection of the liver against a 10,000 Pb/Pf sporozoite challenge, VLPs carrying the CS non-repeat B cell eptiopes were minimally-to-non-protective. One-to-three CS-specific CD4/CD8 T cell sites were also fused to VLPs, which primed CS-specific as well as WHcAg-specific T cells. However, a VLP carrying only the 3 T cell domains failed to protect against a sporozoite challenge, indicating a requirement for anti-CS repeat Abs. A VLP carrying 2 CS repeat B cell epitopes and 3 CS T cell sites in alum adjuvant elicited high titer anti-CS Abs (endpoint dilution titer >1x106) and provided 80–100% protection against blood stage malaria. Using a similar strategy, VLPs were constructed carrying P. vivax CS repeat B cell epitopes (WHc-Pv-78), which elicited high levels of anti-CS Abs and conferred 99% protection of the liver against a 10,000 Pb/Pv sporozoite challenge and elicited sterile immunity to blood stage infection. These results indicate that immunization with epitope-focused VLPs carrying selected B and T cell epitopes from the P. falciparum and P. vivax CS proteins can elicit sterile immunity against blood stage malaria. Hybrid WHcAg-CS VLPs could provide the basis for a bivalent P. falciparum/P. vivax malaria vaccine.In order to design  P. falciparum, P. vivax, P. ovale and P. malariae. Of these, P. falciparum (Pf) is the most lethal. The vast majority of deaths occur among young children in Africa. P. vivax is the most prevalent species outside of sub-Saharan Africa and responsible for approximately 50% of all malaria cases worldwide  for 30 min. Samples were then washed and green-fluorescent sporozoites were visualized using a Nikon Eclipse 90i fluorescent microscope. IFAs using air-dried sporozoites were performed as previously described .5) were cultured with varying concentrations of WHcAg, CS or synthetic peptides derived from WHcAg or CS protein. For cytokine assays, culture supernatants were harvested at 48 h for IL-2 determination and at 96 h for interferon-gamma (IFNγ) determination by ELISA. IFNγ production was measured by a two-site ELISA using mAb 170 and a polyclonal goat anti-mouse IFNγ .Spleen cells from groups of 3 each of (B10xB10.s) F1 mice were harvested and pooled 4–6 weeks after immunization with the various WHcAg hybrid VLPs. Spleen cells , synthesized to comprise the P. falciparum CSP C-terminal region. Thus, the CSP gene in the resulting plasmid, pIC-CSPPfCT, consists of the P. berghei N-terminal and repeat regions (base pairs 1 to 786) and the remainder of the P. falciparum CSP (base pairs 787 to 1026). We then excised the hybrid CSP gene from pIC-CSPfCT as a KpnI-XhoI fragment and inserted it into the transfection plasmid, pR-CSPfCT. KpnI and SacI were used to release the inserted fragment from pR-CSPfCT prior to transfection of WT P. berghei (ANKA strain) parasites, as previously described  that weV12.PF3.1. In thispathogen . This ispathogen ; incorpopathogen –43. Typipathogen , whereaspathogen ,42 contapathogen .In the absence of head-to-head comparative studies to date, the WHc(C61S)-Mal-78-3T candidate embodies a number of unique characteristics that may be advantageous in comparison to other CS-vaccine candidates. The enhanced immunogenicity and protective efficacy of WHc(C61S)-Mal-78-3T suggests that the suboptimal performance of a preerythrocytic vaccine candidate is not likely due to the selection of the CS repeat region as a target or to a paucity of B cell epitopes, but rather to insufficient production of protective Abs. For example, the RTS,S vaccine shares similar CS-specific B and T cell epitopes with WHc(C61S)-Mal-78-3T but the carrier moieties are markedly different. WHc(C61S)-Mal-78-3T efficiently self-assembles into hybrid VLPs, which are stable even at 65°C, whereas the HBsAg-based RTS,S requires the addition of excess native HBsAg particles. Compared to the HBsAg, hepadnavirus nucleocapsids are inherently more immunogenic in mice and humans during natural infection or after immunization –48, are P. vivax CS repeat B cell epitopes demonstrates the power and flexibility of the WHcAg VLP combinatorial technology, especially when combined with the hybrid Pb/Pv sporozoite technology [P. vivax immunogen capable of eliciting sterile immunity to blood stage infection in this hybrid Pb/Pv challenge model after active immunization. It is notable that passive transfer of 400 μg of the P. vivax CS-specific Mab 2F2 was not able to confer sterile immunitiy against a 5 min exposure to the bites of 4 Pb/Pv-infected mosquitoes [P. vivax CS-targeted immunogens such as WHc-Pv-78. The CS repeat epitopes and variants from other P. vivax strains can also be inserted onto WHcAg-CS hybrid VLPs. Further development of P. falciparum and P. vivax-specific WHcAg-CS hybrid VLPs would allow their use either separately or combined in a bivalent malaria vaccine, depending on the regional malaria threat.The immunogenicity and protective efficacy of WHcAg hybrid VLPs carrying chnology  used forsquitoes . The faiS1 FigA) Scheme representing the strategy used for replacing the CSP gene of P. berghei (ANKA) with the P. falciparum (3D7) C-terminal region. The annealing sites of the primers used to verify recombination by PCR are indicated below. Restriction sites shown are K—KpnI; Se-SexAI; P-PacI; Xh—XhoI; S—SacI. (B) The resulting amino acid sequence of the hybrid CS protein. The sequence derived from the P. falciparum (3D7) sequence is depicted in red text.((TIF)Click here for additional data file.S2 FigA) Amino acid sequences of each epitope engineered onto the WHc VLPs (see B) Amino acid sequences of the synthetic peptides used for antibody titer determination by ELISA.((TIF)Click here for additional data file.S3 FigMice were immunized with rCS and sera either unadsorbed or adsorbed with NANP/NVDP-containing VLPs  were incubated with 10,000 sporozoites prior to challenge. NMS, normal mouse sera.(TIF)Click here for additional data file.S4 FigGroups of 3 mice were immunized (2 doses: 20 μg and 10 μg in IFAd) with the indicated WHcAg hybrid VLPs. Secondary antisera were pooled and serially diluted and analyzed by ELISA for binding to solid-phase WHcAg, NANP and NVDP. End-point titers of pooled sera are shown.(TIF)Click here for additional data file.S5 FigP. berghei UTC, TH.3R and CS.T3 T cell domains with the P. falciparum T cell domains incorporated into the WHc-Mal-78-3T VLP. The percentage represents homologies between the two sequences.Alignment of the (TIF)Click here for additional data file.S1 TableMean endpoint dilution titers from 9–10 mice in each group are shown.(PDF)Click here for additional data file.S2 TableMean endpoint dilution Ab titers shown. 1°, primary; 2°, secondary antisera. IFA assay used Pb/Pv dry sporozoites.(PDF)Click here for additional data file.


Fasciola hepatica in some areas has increased considerably and the availability of a vaccine to protect livestock from infection would represent a major advance in tools available for controlling this disease. To date, most vaccine-target discovery research on this parasite has concentrated on proteomic and transcriptomic approaches whereas little work has been carried out on glycosylation. As the F. hepatica tegument (Teg) may contain glycans potentially relevant to vaccine development and the Newly Excysted Juvenile (NEJ) is the first lifecycle stage in contact with the definitive host, our work has focused on assessing the glycosylation of the NEJTeg and identifying the NEJTeg glycoprotein repertoire. After in vitro excystation, NEJ were fixed and NEJTeg was extracted. Matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of released N-glycans revealed that oligomannose and core-fucosylated truncated N-glycans were the most dominant glycan types. By lectin binding studies these glycans were identified mainly on the NEJ surface, together with the oral and ventral suckers. NEJTeg glycoproteins were affinity purified after targeted biotinylation of the glycans and identified using liquid chromatography and tandem mass spectrometry (LC-MS/MS). From the total set of proteins previously identified in NEJTeg, eighteen were also detected in the glycosylated fraction, including the F. hepatica Cathepsin B3 (FhCB3) and two of the Cathepsin L3 (FhCL3) proteins, among others. To confirm glycosylation of cathepsins, analysis at the glycopeptide level by LC-ESI-ion-trap-MS/MS with collision-induced dissociation (CID) and electron-transfer dissociation (ETD) was carried out. We established that cathepsin B1 (FhCB1) on position N80, and FhCL3  on position N153, carry unusual paucimannosidic Man2GlcNAc2 glycans. To our knowledge, this is the first description of F. hepatica NEJ glycosylation and the first report of N-glycosylation of F. hepatica cathepsins. The significance of these findings for immunological studies and vaccine development is discussed.Recently, the prevalence of  Fasciola hepatica is a parasite responsible for the zoonotic disease fasciolosis, prevalence of which has increased in recent years because of the emergence of triclabendazole-resistant strains as well as changing climatic conditions. A number of F. hepatica protein antigens are used for assessing the immune response of the definitive host for the development of recombinant vaccines but no such vaccine has been commercialised yet. Glycans that may contribute to the antigenic, immunological and protective properties in F. hepatica have not been characterised. Using a panel of plant lectins with defined sugar binding specificities alongside mass spectrometric analysis, we found that high mannose and oligomannose N-glycans are the most abundant in the surface of the juvenile fluke and identified eighteen proteins likely to be glycosylated. Additionally, we found that the proteases cathepsin B1 and L3 contain a glycosylation site occupied by an unusually short Man2GlcNAc2N-glycan. This work is the starting point for understanding how F. hepatica glycans interact with the definitive host at the initiation of infection. Additionally, it provides useful information for including glycans in the design of new vaccine candidates. Fasciola hepatica, commonly known as the liver fluke, is widely distributed across five continents and is responsible for fasciolosis in livestock and humans. It has a large economic impact on the livestock industry, causing production losses in terms of reduced milk yield, liver condemnation and problems with animal fertility among others −  (deoxyhexose (F)) and m/z 1582.75 [M−H]− (H3N4F1) seen in N-glycans  and isolation of the proteins (A-UB-NEJTeg) and glycoproteins (A-BB-NEJTeg) was confirmed by comparing the protein profile between fractions using silver staining and the streptavidin blot .F. hepatica molecules identified in this fraction including the asparaginyl endopeptidase legumain-1, the antioxidants GST and PRX and heat shock protein 70 (HSP70). It is noteworthy also to highlight the identification of the proteases FhCB3 and FhCL3 in the glycosylated fraction. Two isoforms of FhCL3 were identified in this extract. All the glycoproteins except GST and Histone H4 were predicted to contain potential N- glycosylation sites. GST and Histone H4 were predicted not to contain N-glycosylated sites, suggesting that they are O-glycosylated.Proteomic MS analysis identified a total of 20 proteins in B-NEJTeg and A-BB-NEJTeg preparations whose molecular weights correlated with protein bands found by silver staining. A total of eighteen proteins were identified in the A-BB-NEJTeg (glycosylated) fraction . There wCathepsins were the most abundant proteins found in the glycosylated fraction; due to their pivotal role in parasite survival and since cathepsins from the same family have been employed as vaccine candidates, we investigated the total NEJTeg material focusing on evaluating cathepsin-specific glycosylation at the glycopeptide level.N-glycosylation site in the amino acid sequence of two of the FhCL3 members (BN1106_s3008B000074 and BN1106_s10139B000014), at the positions N151 and N255, respectively. FhCB1, FhCB2 and the partial novel FhCB were predicted to contain two N-glycosylation sites at positions N145 and N156 for partial novel FhCB, N80 and N315 for FhCB1 and N345 and N356 for FhCB2. FhCB3 was predicted to have four N-glycosylation sites at positions N180, N223, N336 and N453. However, one of the FhCL3 (BN1106_s4187B000061) and the partial FhCB (BN1106_s8462B000006) were predicted not to be N-glycosylated. When 0 missed cleavages were allowed, tryptic peptide masses containing potential N-glycosylation vary from 617.337 to 2658.15 kDa.After selecting and excising bands found at the predicted molecular weight of the cathepsins , trypticN-glycosylation sites were identified, we wanted to determine whether these glycosylation sites were occupied by glycans and also the composition of the glycans involved.Once the cathepsin members and the relevant tryptic peptides with potential m/z 829.50 [M+3H]3+ in the 20.1 min elution was detected in band 2, giving rise to oxonium glycan fragment ions at m/z 366.09 [M+H]+ (H1N1) and m/z 528.21 [M+H]+ (H2N1), which indicated the presence of the terminal di-mannosylated HexNAc element. Further inspection of the CID-MS/MS spectrum of the parent ion m/z 829.50 [M+3H]3+ indicated the presence of a glycan with the composition H2N2 linked to a peptide of 1755 Da (3+ fragment ions were observed for this peptide carrying N2 (m/z 721.28) or a H1N2 trisaccharide (m/z 775.31) structure of an Asn-linked glycan. Furthermore, a [M+2H]2+ fragment ion at m/z 980.40 (N1-peptide) was fully in line with the presence of an N-glycan.All tryptic glycopeptides from the four bands were applied to a RP-nano-LC column coupled to an ion-trap MS/MS system and fragmented in the auto-MS/MS mode. A parent-ion of  1755 Da . [M+3H]3m/z 878.70 [M+3H]3+ in the 19.9 min elution indicated the presence of another sort of glycan with the composition F1H2N2 linked to a peptide with the same mass as the previous peptide described (3+ fragment ions were observed for this peptide carrying a Fuc attached to the N2 (m/z 770.75) or a F1H1N2 tetrasaccharide (m/z 824.64) structure of an Asn-linked glycan. In addition, a series of [M+2H]2+ fragment ions at m/z 1243.62 (H2N2-peptide), 1235.44 (F1H1N2-peptide), 1162.51 (H1N2-peptide), 1154.60 (F1N2-peptide), 1081.49 (N2-peptide), 1052.96 (F1N1-peptide) and 979.94 (N1-peptide) were fully in line with the presence of the fucosylated version of the dimannosyl core N-glycan.The CID-MS/MS spectrum of another parent ion escribed . [M+3H]3N-glycans matched the mass of one of the tryptic peptides of FhCB1 (BN1106_s6570B000050). To obtain information on the glycopeptide sequence and identify the cathepsin that contained the N-glycan, the ETD-MS/MS spectra of the same parent ions m/z 829.83 [M+3H]3+ and 878.7 [M+3H]3+ were recorded in which peptide cleavages were predominantly observed while leaving the glycosidic linkages intact [m/z 829.83 [M+3H]3+ Y sequence, including the mass increment of 732 Da accountable to the glycan H2N2. This sequence is the FhCB1 tryptic peptide (no missed cleavage) Y79NVSENDLPESFDAR93, containing the N-glycosylation consensus sequence NVS. On the other hand, when the ETD-MS/MS spectrum of the parent ion m/z 878.7 [M+3H]3+ was recorded, the VSENDLPESFDAR sequence containing the glycan F1N2H2 could not be directly read. Taken together, the MS/MS data indicated that FhCB1 is modified at the Asn in position N80 with a Manα1-3/6Manα1-4GlcNAcβ1-4GlcNAcβ1-Asn N-glycan. Nevertheless, despite the strong evidence of the presence of a monofucosylated version of the same N-glycan attached to the FhCB1 in CID-MS analysis, its confirmation remained inconclusive by ETD-MS.The mass of the peptide containing the N2H2 and F1N2H2 s intact . For the[M+3H]3+ , the c’-N-glycans attached to a peptide of 1741.2 kDa were detected in the chromatograms of the parent-ions of m/z 824.70 [M+3H]3+ in the 19.9 min elution and m/z 873.70 [M+3H]3+ in the 19.8 min elution respectively (m/z 825.20 [M+3H]3+ showed the VSDNDLPESFDAR sequence from the c2-c15 ion series and the DFSEPLDNDSVN(glycan)Y sequence from the z2-z15 ion series, including the mass increment of 732 Da of the glycan H2N2 . The result showed a 100% coverage and identity hit with FhProCB2 (gi|27526823) which contains the N-glycosylation consensus sequence NVS in position N70. Following identification of further cathepsin B class proteins for F. hepatica and the comparative analysis of those cathepsin sequences publically available, the FhProCB2 (gi|27526823) is consistent with the FhCB1 sequence QTLFSEQQVLDCTR and VLQQESFLTQN(glycan)QF respectively, taking into consideration the mass increment of the H2N2 glycan. This sequence is the FhCL3 tryptic peptide (no missed cleavage) F151QNQTLFSEQQVLDCTR167, containing the N-glycosylation consensus sequence NQT. No definitive identification of its fucosylated version could be achieved in the ETD-MS/MS analysis.As demonstrated for FhCB1, FhCL3 also showed firm evidence of the same glycosylation pattern. The fragmented ions confirming the glycan structures are described in N-glycopeptide species were found in the CID-MS/MS spectra whose parent ions were registered in the elution times from 2.5 to 29.3 min  DC-specific ICAM-3-grabbing non-integrin (DC-SIGN) [N-glycans in the NEJTeg are likely to interact with DCs and macrophages via Man-specific CLRs at the early stages of infection, impairing the DC function and augmenting the M2a population in the peritoneal cavity, as seen with adult F. hepatica homogenate or ES fractions [The lack of sialic acid and the wide presence and distribution of high mannose iverrini  Schistosoma spp. ,55, Haemontortus ,57 and Ta solium . These gDC-SIGN) , suppresractions –25, contractions .N-acetylglucosaminidase and β-galactosidase treated N-glycans did not show evidence of terminal β-Gal- or GalNAc- on hybrid or complex glycans in the MS spectra but did show positive recognition of terminal Gal/GalNAc- residues on the parasite surface. This may reflect a low abundance and potential masking by high mannose N-glycans which are very abundant in NEJTeg. As seen in lectin blots, not many glycoproteins contain these residues. Alternatively, lectin fluorescence staining may also indicate the presence of glycolipids as described in adult F. hepatica [O-glycosylation pattern of NEJTeg. Similar PNA recognition has been described in adult F. hepatica [O. viverrini where 80% of the O-glycan profile is based on the T-antigen structure [In our study, lectin affinity techniques in some cases revealed different results than those using other glycomics techniques with respect to identification of terminal carbohydrates. For instance, β-hepatica ,30, whichepatica ,31 and itructure . Tn antitructure . AlthougF. hepatica, O-glycan motifs may also play a role in intestinal epithelial invasion by this parasite, as has been shown also in studies of protozoal infections [Taking into account the presence of these glycans in the oral and ventral suckers of the parasite, we suggest that NEJ-derived glycoproteins decorated with terminal Man, Fuc and Gal/GalNAc are in close contact with host mucins and intestinal enterocytes. Based on PNA positive recognition at the parasite surface and around the suckers in fections –63.vice versa. Due to the size of the NEJ and the relatively large number of parasites needed to obtain enough material, it is very difficult to achieve a complete separation of NEJTeg and NEJSom. It is known that some proteins are present in both F. hepatica Teg and Som. For example, the protein PRX as well as paramyosin are found in both Teg and Som of adult F. hepatica [Differences in NEJTeg and NEJSom glycosylation were confirmed with four lectins. However, we also observed similar protein profiles in silver stained gels and in most of the lectin blots. It is possible that some tegumental material remained in the somatic preparation or hepatica –66, althF. hepatica PRX and GST, which have well known antioxidant and immunomodulatory properties [F. hepatica, it was not predicted to be potentially N-glycosylated, suggesting that it could be O-glycosylated. The facts that (1) the F. hepatica NEJTeg O-glycan profile could not be confirmed in our previous analysis and (2) the O-glycan consensus is not as specific as that for N-glycans limited our attempts to analyse potential O-glycosylation sites. It is reasonable to assume that mucin-type core-1 O-glycan would be the most likely to be attached to the GST due to the positive lectin binding of proteins at molecular weights of GST. Further studies will be required to assess whether O-glycan structures are present and involved in the detoxification and of immunoregulatory activities of these molecules.This is the first report that suggests that operties –70, are F. hepatica NEJ-specific cathepsin cysteine proteases. These molecules, which play an important role in tissue degradation, in parasite penetration in the host gut and in parasite feeding [N-glycan. Although there is some evidence of the presence of Fuc on the chitobiose core, our analysis was inconclusive due to restrictions on material. However, the presence of this glycan is shown in the PNG-F released glycan pool of NEJTeg. The confirmation of N-glycosylation of FhCL3 in our study differs from previous findings [F. hepatica genome encoding FhCL3 [N-glycan detected, which is lower than 1 kDa, it is very difficult to measure differences in protein migration in SDS-PAGE gels before and after deglycosylation. Therefore, this is not an accurate approach to verify protein glycosylation when the glycans present are small. The use of MS for glycan analysis is more sensitive and suitable for these purposes. The absence of N-glycosylation sites in other trematode CL-like proteases [Trypanosoma brucei Cathepsin B [To our knowledge, this is the first report describing the specific glycosylation of the  feeding ,71 are gfindings ,72. Fiveng FhCL3 , meaningng FhCL3 ,72. Becaroteases , as wellhepsin B  and humahepsin B  has beenN- and/or O- glycosylation on these vaccine candidate molecules. There is only one report in the literature where glycosylation of FhCL3 was taken into account for measuring vaccine efficacy. This account involved rats vaccinated with recombinant FhCL3 expressed by Saccharomyces cerevisiae and recombinant baculovirus [N-glycans by yeasts [N-glycan structure identified for the native FhCL3 in the present work. This could have an effect on the final protein folding and/or the differences in the antigenic epitopes that could exist between the native protein and recombinant versions, explaining lack of efficacy of recombinant vaccine candidates. It is possible that the presence of this unusual N-glycan attached to the FhCL3 peptide backbone is required for proper processing and presentation. Correct glycosylation is an important issue for the production of recombinant antigens of F. hepatica and other helminths [Crucially, GST, PRX and FhCL3 have been proposed and used as potential vaccine candidates. When these molecules were used as vaccines their maximum efficacy was observed when antigens were used in their native, rather than recombinant, versions ,15. As tulovirus . The fory yeasts  and mammy yeasts  differs elminths . FurtherF. hepatica NEJTeg and NEJSom, with differences in glycosylation between the two fractions. We also have performed a glycan characterisation of the NEJTeg extract showing that high mannose and oligomannose N-glycans are the most dominant glycan structures. Finally, this work has shown that a set of important immunomodulatory proteins from F. hepatica NEJ, including some of the cathepsin family clade, are glycosylated. Knowledge of the specific structures of the glycans decorating these target proteins is particularly valuable for enhancing our understanding of immunoevasion and parasite migration as well as optimising the efficacy of future vaccines.In conclusion, we have demonstrated the presence of carbohydrates in the S1 FigBoth NEJTeg (1) and NEJSom (2) preparations were SDS-PAGE fractioned, transferred to nitrocellulose membranes and incubated with the biotinylated-labelled lectins UEA, SJA, VVL, STL, LEL, DSL, SNA, MAL-II and RCA. An additional incubation with IRDye-labelled streptavidin was used to detect positive lectin binding at different molecular weights (MW). Glycoproteins were revealed by infrared imaging.(TIF)Click here for additional data file.S2 FigN-glycans were acquired by MALDI-TOF-MS. Unidentified peaks are represented with an asterisk (*).N-glycans released from NEJTeg using PNG-A were labelled with fluorophore 2-aminobenzoic acid (2-AA). MS spectra of AA-labelled (TIF)Click here for additional data file.S3 FigNEJTeg (1) was employed for glycoprotein biotinylation. Biotinylated-NEJTeg (B-NEJTeg) (2) was used for biotin-avidin affinity chromatography, separating avidin-unbound-NEJTeg (A-UB-NEJTeg (3) and the avidin-bound-NEJTeg (A-BB-NEJTeg) (4) fractions. (A) SDS-PAGE and silver staining was used to detect the protein profiles of the four preparations. (B) FITC labelled-streptavidin-incubated membrane was used to confirm correct glycoprotein biotinylation of B-NEJTeg and correct biotin-avidin affinity chromatography separation.(TIF)Click here for additional data file.S1 FileN-glycosylation site are highlighted in yellow. Consensus N-glycosylation is underlined.Multiple sequence alignment of FhCB (BN1106_s5100B000033), FhCB1 (BN1106_s6570B000050), FhCB2 (BN1106_s4482B000044) and FhCB3 (BN1106_s6570B000051) and ProCB2 (gi|27526823) were performed using Clustal Omega. Sequences are ordered according to aligned. Differences in the region containing the (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Bacillus subtilis (Ehrenburg) Cohn ATCC 49760, deposited as Bacillus globigii, is the source strain for the restriction enzymes BglI and BglII. Its complete sequence and full methylome were determined using single-molecule real-time (SMRT) sequencing. Bacillus globigii was originally provided by Gary A. Wilson, University of Rochester, NY, and is now housed in the ATCC (as ATCC 49760). It is the original source of the type II restriction enzymes (REases) BglI and BglII, first isolated in 1976 and sold commercially since 1978   systems, respectively . The SMRTPortal software called 98.8% of the BglI sites as modified and 97.7% of the BglII sites as modified, at an average coverage of 90× per motif (single strand of the DNA), indicating effectively complete modification for both R-M systems. The genes responsible for these modifications were identified and described previously by cloning the R-M systems (B. globigii genome sequence we determined, locus tag A1D11_20185 contains the M.BglI modification gene, and locus tag A1D11_20190 contains the BglI endonuclease gene of the BglI R-M system, while locus tag A1D11_20255 contains the M.BglII modification gene, locus tag A1D11_20260 contains the BglII endonuclease gene, and locus tag A1D11_20265 contains the C.BglII control gene of the BglII R-M system. The complete systems can be seen schematically on the REBASE database website (B. globigii protein sequences to all known DNA methyltransferase protein sequences. A putative type IV methyl-directed restriction enzyme similar to 5mC-specific type IV R-M enzymes, such as EcoK Mrr or McrBC, is present in locus tag A1D11_00130.ce 1978 13. BglI r as ATCC 9760. It  systems , 6. In tCP014840.This complete genome sequence has been deposited in DDBJ/ENA/GenBank under the accession no.


Moreover, the immune responses induced by EVLP-G protect all vaccinated mice from lethal challenge with RABV. These results suggest that EVLP-G has the potential to be developed as a novel vaccine candidate for the prevention and control of animal rabies.Rabies remains an important public health threat in most developing countries. To develop a more effective and safe vaccine against rabies, we have constructed a chimeric rabies virus-like particle (VLP), which containing glycoprotein (G) and matrix protein (M) of rabies virus (RABV) Evelyn-Rokitnicki-Abelseth (ERA) strain, and membrane-anchored granulocyte-macrophage colony-stimulating factor (GM-CSF), and it was named of EVLP-G. The immunogenicity and protective efficacy of EVLP-G against RABV were evaluated by intramuscular administration in a mouse model. The EVLP-G was successfully produced in insect cells by coinfection with three recombinant baculoviruses expressing G, M, and GM-CSF, respectively. The membrane-anchored GM-CSF possesses a strong adjuvant activity. More B cells and dendritic cells (DCs) were recruited and/or activated in inguinal lymph nodes in mice immunized with EVLP-G. EVLP-G was found to induce a significantly increased RABV-specific virus-neutralizing antibody and elicit a larger and broader antibody subclass responses compared with the standard rabies VLP . The EVLP-G also elicited significantly more IFN-γ- or IL-4-secreting CD4 Rabies is one of the oldest zoonotic diseases that occurs worldwide and afflicts nearly all mammalian hosts. Once the infected, the host manifests clinical symptoms of rabies, the result is almost invariably lethal . AccordiVirus-like particles (VLPs) formed by one or several viral structural proteins are stable, non-replicative, non-infective, and highly immunogenic and have, therefore, become a novel safe option as a vaccine candidate . VLPs caThe stimulation of antigen-presenting cells (APCs), especially dendritic cells (DCs), which are the most efficient of APCs, is considered as the key linkage between innate and adaptive immune responses against viral infection ,13. The In our earlier studies, we observed that glycoprotein (G) and matrix (M) protein of rabies virus (RABV) can generate standard rabies VLP (sRVLP) by self-assembly in insect cells . In this3, a RABV street strain, was isolated from a pig that died of rabies in the Hunan Province of China in 2006 and stored in our laboratory [Sf9 cells  were maintained in suspension with SF900II serum-free medium  at 27 °C in cell culture flasks at a speed of 120 rpm. HuNPBboratory . The RABE. coli (Life technologies) for transposition into a bacmid. Then, a Cellfection® II Reagent (Life technologies) was used according to the manufacturer’s instructions to generate the rBV rpFBD-2GMCSF. The rBVs rpFBD-2COG and rpFBD-2COM expressing G and M protein, respectively, were generated as reported previously [E. coli to obtain positive recombinant bacmids. Then, the bacmids were transfected into Sf9 cells to produce two rBVs rpFBD-2COG and rpFBD-2COM.We constructed a recombinant plasmid encoding a membrane-anchored GM-CSF gene consisting of a signal peptide (SP) from honeybee mellitin, full-length murine GM-CSF, and the transmembrane (TM) and cytoplasmic tail (CT) regions of G. All primers used for this study are listed in eviously . BrieflySf9 cells were infected with rpFBD-2COG, rpFBD-2COM or rpFBD-2GMCSF. At 48 h postinfection, cells were fixed with ice-cold 70% alcohol for 30 min at room temperature (RT) and blocked with 2% bovine serum albumin (BSA) for 60 min at RT. The cells were incubated with mouse anti-rabies G antibody , rabbit serum against RABV M or mouse anti-GM-CSF antibody  for 90 min at 37 °C. Finally, the cells were stained with Alexa Fluor 488-conjugated goat anti-mouse or goat anti-rabbit IgG  for 50 min at 37 °C and analyzed under a fluorescence microscope.g for 30 min to remove cells and then pelleted by ultracentrifugation at 30,000 × g for 60 min at 4 °C. The pellets were resuspended in PBS and purified through a 20%–40%–60% discontinuous sucrose gradient at 25,000 × g for 90 min at 4 °C. The EVLP-G band obtained between 40% and 60% density range was collected, washed, and resuspended overnight in PBS.To produce EVLP-G, which is essentially cRVLPs containing GM-CSF, Sf9 cells were coinfected with rBV expressing G, M and GM-CSF at multiplicities of infection of 3, 2, and 3, respectively, and incubated at 27 °C for 5 days. Culture supernatants were harvested and centrifuged at 2000 × For Western blot analysis, EVLP-G and control sample (cell culture supernatant) were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis under denaturing conditions, transferred onto a nitrocellulose membrane  and then probed with rabbit serum against M, mouse anti-rabies G or mouse anti-GM-CSF antibodies at a dilution of 1:200 overnight at 4 °C. The sample was then incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit secondary antibody at a dilution of 1:4000  for 60 min at 37 °C.For electron microscopy, EVLP-G was applied onto a carbon-coated formvar grid, which was immediately stained with 1% phosphotungstic acid and then observed by a transmission electron microscope. For immunoelectron microscopy, after binding EVLP-G to formvar-coated grids, which were sequentially incubated with mouse anti-rabies G or mouse anti-GM-CSF antibodies for 90 min at RT and gold-labeled goat anti-mouse IgG antibody  for 60 min at RT. Finally, the grids were stained with 1% phosphotungstic acid and examined under an electron microscope.50) of HuNPB3 in the muscle of the forelimb. The mice were observed for 21 days, any mice that developed clinical signs of rabies during the observation period were humanely euthanized by cervical dislocation under isofluorane anesthesia.Female BALB/c mice aged 6-8 weeks were purchased from Changchun Institute of Biological Products Co., Ltd, China. Mice were randomly divided into 3 groups and individually immunized twice with 10 μg/mouse EVLP , EVLP-G, or PBS by the i.m. route at two week intervals. At 4 weeks post the final immunization, mice were challenged i.m. with 100 × 50% intramuscular mouse lethal dose  were measured using fluorescent antibody virus neutralization (FAVN) . The ser6 cells/mL) were prepared in complete RPMI 1640 medium  with 10% fetal bovine serum . The cells were stimulated with inactivated ERA  at final concentration of 10 μg/mL and cultured for 24 h at 37 °C. The splenocytes producing IFN-γ or IL-4 were quantified by ELISpot assay  according to the manufacturer’s instructions. Spot-forming cells (SFCs) were enumerated using an automated ELISpot reader .The spleens were collected from mice at 2 weeks after the second immunization and single splenocyte suspensions  at 37 °C for 6 h. After surface staining with anti-mouse CD4 and CD8 antibodies (BD Biosciences) for 30 min at 4 °C, the cells were permeabilized with Cytofix/Cytoperm (BD Biosciences) for 30 min at 4 °C and stained with anti-mouse IFN-γ and IL-4 antibodies (BD Biosciences) for 30 min at 4 °C. These stained cells were analyzed by a flow cytometer.At 2 weeks post-vaccination, isolated splenocytes at 1 × 106 cells/mL) were prepared in PBS containing 2% FBS and stained with anti-mouse CD19, CD40, CD11C, CD80, CD86, MHC I, and MHC II antibodies (BD Biosciences) for 30 min at 4 °C  [The inguinal lymph nodes were collected at 3, 6, and 9 days after primary immunization. Single cell suspensions (1 × 10for DCs) ,30. AfteAll animal studies were conducted with prior approval from the Animal Welfare and Ethics Committee of the Veterinary Institute at the Academy of Military Medical Sciences (permit number SCXK-2012-017). The environment and housing facilities satisfied the National Standards of Laboratory Animal-Requirements of Environment and Housing Facilities (GB 14925-2001) of China. Experiments involving the use of the RABV street strain were approved by the Military Veterinary Research Institute of the Academy of Military Medical Sciences and conducted in a biosafety level 3 laboratory.As depicted in EVLP-G particles were generated by coinfection with three rBVs expressing G, M, and GM-CSF, respectively. Western blotting analysis demonstrated that EVLP-G consists of G, M, and GM-CSF contents D. In addTo evaluate the immunogenicity of EVLP-G in a mouse model by using the vaccination schedule as described in Materials and Methods. To further investigate whether the pattern of IgG subclasses induced by EVLP-G containing GM-CSF could be changed or/and broadened, the specific serum IgG and subclass responses were determined by ELISA. As shown in After confirming that EVLP-G successfully induced enhanced VNA response in mice, we next evaluated the antigen-specific IFN-γ and IL-4 activities in splenocytes by ELISpot assays. As shown in + and CD8+ T cells. As shown in + T cell response compared with PBS treatment, but the percentage of CD4+IFN-γ+ or CD4+IL-4+ cells was significantly higher in the EVLP-G group than that in the EVLP group. As shown in + T cells secreting IFN-γ or IL-4 induced by EVLP-G was significantly more than the EVLP or PBS induction. The difference between EVLP and PBS groups was not statistically significant. These data demonstrated that mice immunized with EVLP-G containing GM-CSF elicited a notably enhanced IFN-γ- or IL-4-secreting CD4+ and CD8+ T cell response.To further characterize the T cell’s response induced by rabies VLPs, we evaluated the ability of EVLP or EVLP-G to induce IFN-γ- or IL-4-secreting CD4+CD40+ cells) in lymph nodes than PBS alone. However, the percentage of CD19+CD40+ cells in mice vaccinated with EVLP-G was significantly higher than that induced by EVLP at six and nine days after immunization. As shown in +CD86+ and CD11c+CD80+ cells) were detected at all time points in lymph nodes from mice vaccinated with EVLP-G compared with mice immunized with EVLP or mice treated with PBS alone. The expression of MHC I and MHC II on the surface of DCs is shown in To investigate whether membrane-anchored GM-CSF incorporated into cRVLP can function as an adjuvant and induce more B cells and DCs, lymph node cells were analyzed by flow cytometry. As shown in 50 RABV street strain  in the inguinal lymph nodes of EVLP-G-immunized mice. In combination with CD28, the co-stimulatory markers CD86 and CD80 expressed on mature DCs deliver a signal to the T cells, which is often referred to as the second signal and is essential for the induction of effector Th cells [The recognition of antigenic peptides displayed by MHC molecules on APCs initiates the adaptive immune response. As critical specialized APCs, DCs are positioned throughout the body to detect environmental and pathogenic threats from a large variety of microorganisms . DCs plaTh cells . EVLP-G Th cells .+ T and CD8+ T cells, which mediate antigen-specific cellular immune responses. Flow cytometry analysis for ICS showed that EVLP-G containing GM-CSF functions as an adjuvant and activates significantly more IFN-γ- or IL-4-secreting CD4+ T cells. IFN-γ secreted by Th1 cells activates macrophages, thereby increasing the killing of phagocytosed microbes. On the other hand, IL-4 produced by Th2 cells drives the maturation of B cells into plasma cells, resulting in antibody production, isotype-switching and affinity maturation [+ T cells. The CD8+ T cells further differentiate into CTLs to kill the infected cells [+ T cells responses. However, further knowledge of detailed mechanisms including the expression and regulation of relevant cytokine needs further research.Based in the type of co-receptor expressed on their surface, T lymphocytes have been divided into CD4turation . The resed cells . These r+ T and CD8+ T cell response. Both arms of the immune response mentioned above resulted in complete protection when the mice were challenged with a high dose of lethal RABV street strain. Taken together, the increased immunogenicity, stronger immune response, and improved protection against RABV infection underscored the potential for EVLP-G to be developed as a novel vaccine candidate for the prevention and control of animal rabies.The most important characteristic of a good vaccine candidate, especially in a non-infectious and non-replicative form, is its immunogenic potential. In this study, the immunogenicity of EVLP-G containing membrane-anchored GM-CSF was found to be much higher compared with sRVLP. The significantly higher RABV-specific VNA titers was detected in EVLP-G-immunized mice. Moreover, EVLP-G also induced a stronger CD4


HLA-G influence the magnitude of the protein by modulating HLA-G mRNA stability. Soluble HLA-G has been associated to 3’UTR +2960 Ins/Ins and +3035 C/T (lower levels) and +3187 G/G (high levels) genotypes. HLA-G 3’UTR SNPs have never been explored in CRC outcome. The purpose of this study was to investigate if common HLA-G 3’UTR polymorphisms have an impact on DFS and OS of 253 stage II-III CRC patients, after primary surgery and ADJ-CT based on FL. The 3’UTR was sequenced and SNPs were analyzed for their association with survival by Kaplan-Meier and multivariate Cox models; results underwent internal validation using a resampling method . In a multivariate analysis, we estimated an association with improved DFS in Ins allele (Ins/Del +Ins/Ins) carriers  and in patients with +3035 C/T genotype . The +3187 G/G mutated carriers (G/G vs A/A+A/G) were associated to a worst prognosis in both DFS  and OS . Our study shows a prognostic and independent role of 3 HLA-G 3’UTR SNPs, +2960 14-bp INDEL, +3035 C>T, and +3187 A>G.An important hallmark of CRC is the evasion of immune surveillance. HLA-G is a negative regulator of host’s immune response. Overexpression of HLA-G protein in primary tumour CRC tissues has already been associated to worse prognosis; however a definition of the role of immunogenetic host background is still lacking. Germline polymorphisms in the 3’UTR region of  Colorectal cancer (CRC) is still a clinical burden being the third most common cancer in the United States  and the HLA-G gene codifies for a tolerogenic molecule with well recognized immune-inhibitory properties on both innate and adaptive immune responses ) years, median age was 62.5 (range 24–82) years at onset. Most tumors (181/253 = 72%) presented stage III at the time of onset and were preferentially located at the left colon portion (191/253 = 76%). At diagnosis, stage II patients (N = 72) had pT3N0M0 (94%) of whom 6% had pT4aN0MO stage; most of them had >12 lymph nodes excised (72%), while 16% of patients had <12 lymph nodes excised. In 12% of cases the number of the analysed lymph nodes was not reported, although there was confirmation of no nodal involvement (N0). Mean follow-up time was 44.4 months for DFS (95% CI 42.1–46.7) and 74.8 months for OS (95% CI 71.9–77.8). Median follow-up time was 56.3 (range 1.2–186.3) months for DFS and 62.8 (range 4.6–186.3) months for OS. Five-year DFS was 65%  and 5-ye= 0.629) . A signi= 0.050) . MoreoveHLA-G 3’UTR segment was analyzed by direct sequencing. We detected 9 common SNPs at HLA-G 3’UTR: +2960 14-bp INDEL (rs371194629), +3003 T>C (rs1707), +3010 C>G (rs1710), +3027 C>A (rs17179101), +3035 C>T (rs17179108), +3142 G>C (rs1063320), +3187 A>G (rs9380142), +3196 C>G (rs1610696) and +3227 C>A (rs1233331). The germinal allele frequencies for HLA-G 3’UTR polymorphisms detected in this set of CRC patients were reported in 2 = 0.98) was found between +3010 C>G and +3142 G>C polymorphisms , +3035 C>T , and +3187 A>G (log-rank P = 0.019) were found (vs Del/Del) model  model  in the analysis . Statistre found . The polHLA-G 3’UTR SNPs and related reconstructed most abundant haplotypes  and DFS of CRC patients by means of multivariate Cox models  as well Ins allele carriers of (low-HLA-G secretor) according to the dominant model . Forty-two percent of patients with the Del/Del genotype (N = 78) had a recurrence (33/78), while a lower incidence (28%) was observed in patients with Ins/Del (N = 115) and Ins/Ins (N = 60) genotypes . The 21% of heterozygous (C/T) patients for +3035 C>T SNP had a relapse (10/47) with respect to the 35% found in the wild type +3035 C/C combination (72/205). CRC patients carrying the +3187 G/G mutated genotype (high-HLA-G secretor) were associated with an increased risk or relapse . A detectable relapse was observed in the 60% (9/15) of CRC patients with the +3187 G/G mutated genotype (N = 15), in 33% (31/93) of heterozygous A/G (N = 93), and in 29% (42/145) of wild-type A/A (N = 145) patients.A borderline statistically significant association with reduced risk of disease recurrence was determined in patients carrying the +3035 C/T (low-HLA-G secretor) genotype  dose  (P = 0.013).UTR-1 haplotype, containing the +3187= 0.014) . The HR P = 0.036), and in the corresponding recessive model  and consistently in the recessive model . Mortality rate was higher (47%) in homozygous mutated +3187 GG (7/15) carriers than in the heterozygous +3187 A/G  and in the wild type +3187 A/A  carriers (P = 0.021) (The +3187 A>G SNP was the only one to be associated with OS (log-rank carriers . Multiva= 0.021) . The obsThe relation between types of polymorphisms and DFS or OS risk, was further examined stratifying by stage (II and III) (not shown). Although some differences in the hazard ratios were detected across strata, the observed associations were still confirmed. Moreover, these associations were compatible with the effect of random variation since heterogeneity tests were not significant.Emerging data demonstrates a key role of genes involved in immune response checkpoints and their associations with the CRC clinical outcomes . In ordeHLAG polimorphisms (in particular the +2960 14-bp INDEL) and cancer risk [HLA-G 3’UTR regulatory SNPs in DFS and OS after adjuvant treatment of CRC. Our results emphasize the role of the host’s immunogenetic background in the CRC prognosis as well as report the molecular characterization of the 3’UTR region at the germinal level in subjects affected by colorectal cancer [The potential clinical relevance of HLA-G in cancer as a negative regulator due to its direct or indirect tolerogenic properties to avoid immune cells response, was previously highlighted in several studies ,24. Howecer risk ,34, but l cancer .HLA-G 3’UTR polymorphisms were investigated and after multivariate survival analyses using Cox’s regression models we found that +2960 14-bp INDEL, +3035 C>T and +3187 A>G SNPs had a significant and independent prognostic role with high internal validity by bootstrap modeling. HLAG 3’UTR SNPs previously reported to be associated with a reduced protein production such as +2960 14-bp INDEL and +3035 C>T were linked to a better prognosis, whereas the +3187 A>G SNP (increased HLAG production), was associated to worse DFS and OS.The common HLA-G mRNA expression and higher levels of the soluble HLA-G, whereas the Ins/Ins genotype displays a lower production of mRNA and soluble or membrane bound molecules [Ins allele in the heterozygous Ins/Del patients and in (Ins/Del+Ins/Ins) agreement with the dominant model . CRC patients with the Del/Del genotype showed an increased relapse rate and reduced 5-year DFS %.This study shows that the +2960 14-bp INDEL SNP, already described as a disease risk-marker, is also a prognostic marker for DFS in CRC patients treated with standard ADJ-CT. Intriguingly, our results on survival for +2960 14-bp INDEL SNP are in agreement with those observed in a non-solid tumour  and alsoolecules . At the Ins allele even if not statistically significant probably due to the small sample size. In reference to SNP +3035 C>T (rs17179108), it has been reported, in a recent published study [P = 0.045) statistically significant association in the multivariate Cox’s model. The +3035 C/T genotype was associated to prolonged 5-year DFS (77%) and a lower (21%) recurrence incidence (10/47) with respect to the wild type +3035 C/C combination that presented a decreased 5-year DFS (63%) and a 35% of relapse (72/205). CRC carriers for +3035 C/C had a decreased 5-year DFS % and an increased relapse incidence. It should be pointed out that the +3035 C/T heterozygous genotype, detected in 47 patients, was always associated to the Ins allele, in heterozygous Ins/Del (N = 27) and in homozygous Ins/Ins (N = 20) patients. Even if a protective HR in DFS resulted in the combined analysis of +3035 C/T and +2960 14-bp INDEL polymorphisms, no firm conclusion about a multiplicative or additive effect of the 2 SNPs cannot be inferred from this study. Regarding the +3187 A>G polymorphism (rs9380142), the +3187A allele has been associated to decreased HLA-G expression and the presence of +3187 G/G genotype to significantly increased soluble levels of HLA-G [HLA-G SNP was reported. We found an association between +3187 G/G carriers , and according to the recessive (G/G vs A/A+A/G) model  with reduced DFS. Similarly, the +3187 G/G carriers were associated to a reduced OS , also in the recessive model . These results may highlight that the modulation of the clinical outcome in CRC patients harbouring the +3187 A>G change is due to the contribution of the G allele in double dose. CRC patients carriers for +3187 G/G mutated genotype (N = 15) had increased recurrence rate and a reduced 5-year DFS %. Carriers of +3187 G/G mutated combination were also associated to a reduced 5-year OS percentage and increased mortality rate. The opposite prognostic associations found in CRC patients for SNPs +2960 14-bp INDEL and +3187 A>G, are corroborated by the evidence that these two polymorphisms are not in LD (r2 = 0.27). Furthermore, presence of +3187G allele is always associated to the Del allele, which are represented in the reconstructed UTR-1 haplotype. The latter, when present in double dose (UTR-1/UTR-1), has been associated to an unfavourable prognosis such as +3187 G/G genotype.Concerning OS, we found a similar trend of improved prognosis for ed study , that suof HLA-G . To dateHLA-G 3’UTR variants to test whether haplotypes are more predictive than single variants.The variation sites described in the 3’UTR are mainly arranged in haplotypes (known as UTR-1 to UTR-44) with the UTR-1 and UTR-2 as the most frequent in the worldwide population . TherefoDelCTGCGCCGCGTCGCG) haplotype carrying the 14-bp Del, +3003T, +3010G, +3027C, +3035C, +3142C, +3187G and +3196C alleles, has been considered as a high expressing haplotype. In particular, individuals with the +3187 G/G genotype and thus the UTR-1/UTR-1 combination in double dose, exhibit significant higher levels of the soluble HLA-G [HLA-G 3’UTR haplotype associated with prognosis in CRC patients in multivariate regression Cox’s analysis. We found an association of reduced DFS  and OS  in CRC patients carrying the UTR-1 haplotype in double (Hom) dose. Estimations found are not surprising since patients homozygous for UTR-1 haplotype (N = 15) are the same carriers for +3187 G/G change and share the same survival pattern.UTR-1  were associated with a favourable DFS with a reduced risk of relapse (protective prognostic role). CRC patients carriers for the +3187 G/G genotype and UTR-1/UTR-1 haplotype (higher HLA-G producer) were associated with an unfavourable prognosis in both DFS and OS. Furthermore, patients carrying the +3035 C/T genotype (lower HLA-G producer) and therefore the Ins allele (in heterozygous or homozygous state) were associated with an improved prognosis though with a borderline significant association.Moreover, we observed a strong LD . In summet al [HLA-G 3’UTR polymorphisms in the outcome of metastatic CRC patients. These results could provide new insights to better stratify patients and also for combination therapy between CT and monoclonal antibodies (i.e. cetuximab and bevacizumab) currently adopted in clinical practice.Finally, this study supports associations between the non-metastatic colorectal cancer outcome after CT treatment and polymorphisms of a gene involved in immune tumour escape. Our preliminary findings share a functional rationale considering that patients with higher levels of HLA-G would be more immunosuppressed and have a worse clinical prognosis as postulated by Rizzo et al . The effet al . Due to HLA-G 3’UTR SNPs should be analyzed for their prognostic role in an independent CRC cohort treated with surgical resection and without any chemotherapy. Moreover, our data should be explored considering also the contribution of validated prognostic and predictive biomarkers for CRC. Another limit is the lack of biological samples such as tumour or plasma samples to correlate genetic data with tissue and/or soluble levels of HLA-G to sustain functional hypothesis about the regulatory role of these SNPs, even if previous published studies support consistent data [HLA-G 3’UTR region in prognosis of colorectal cancer provide the basis for implementation of personalized cancer treatments. Identification of high risk patients with well-recognized prognostic, predictive and novel immune-related genomic biomarkers may represent a new frontier in the management of CRC patients. In conclusion, +3035 C>T and in particular, +2960 14-bp INDEL and +3187 A>G polymorphisms in the regulatory 3’UTR of the HLA-G gene, have emerged as novel prognostic biomarkers in determining survival outcome in colorectal cancer. Therefore, our exploratory findings should be verified in independent larger CRC cohorts as well as further relevant functional studies are required.Some limitations of this work should be highlighted. Despite an internal validation  confirming the results, ent data –32,51. NS1 Fig2) between variants is illustrated with dark shading. The r2 values (x100) for the marker pairs are listed in the corresponding boxes.LD plot generated by Haploview shows correlations between all pairs of variants with MAF >2%. High pairwise LD (r(TIF)Click here for additional data file.S2 Fig(A) Disease free survival curve for the UTR-1 haplotype in heterozygous (Het) and homozygous (Hom) CRC patients. (B) Overall survival curve for the UTR-1 haplotype in heterozygous (Het) and homozygous (Hom) CRC patients.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.


Scientific Reports5: Articles number: 9302; 10.1038/srep09302 published online: 03202015; updated: 01062016This Article contains errors in Figure 3C. The image depicting the 293T cell line co-infected with SDPR siRNA is a duplicate of the Control image of the SH-SY5Y cell line. In addition, the image depicting the 293T cell line co-infected with SDPR siRNA + miR-206 inhibitor is a duplicate of the image depicting the SH-SY5Y cell line co-infected with miR-206-inhibitor. The correct Figure 3C appears below as Figure 1.


S. aureus were grown in a defined media containing isotope labels [1-13C]glycine and L-[ε-15N]lysin, L-[1-13C]lysine and D-[15N]alanine, or D-[1-13C]alanine and [15N]glycine, to selectively 13C-15N pair label peptidoglycan bridge-link, stem-link, and cross-link, respectively. 13C{15N} and 15N{13C} rotational-echo double resonance NMR measurements determined that cyclic lipopeptide-treated S. aureus exhibited thinning of the cell wall, accumulation of Park’s nucleotide, inhibition of glycine utilization for purine biosynthesis, reduction of ester-linked D-Ala in teichoic acids, and reduction of peptidoglycan cross-linking. Whole cell NMR analysis also revealed that S. aureus, in presence of amphomycin and MX-2401, maintained the incorporation of D-Ala during peptidoglycan biosynthesis while the incorporation of D-Ala into teichoic acids was inhibited. These effects are consistent with amphomycin’s dual inhibition of both peptidoglycan and wall teichoic acid biosyntheses in S. aureus.Amphomycin and MX-2401 are cyclic lipopeptides exhibiting bactericidal activities against Gram-positive pathogens. Amphomycin and MX-2401 share structural similarities with daptomycin, but unlike daptomycin they do not target bacterial membrane. In this study, we investigate  Daptomy+ leakage is thought to be the killing mechanism of daptomycin. In contrast, friulimicin B2, amphomycin, and MBX-240in vitro assays, have shown that friulimicin B2 and MX-240155–P). The structure of amphomycin bound to C55–P is unknown, but the x-ray crystal structure of tsushimycin suggests that a binding cleft for the phosphate of C55–P may be formed at the dimer interface between the cyclic peptide core structuresDespite their structural similarities, daptomycin and amphomycin-class of antibiotics exhibit different modes of action. Daptomycin undergoes a large conformational change upon calcium-ion binding to form a 10 to 16-mer pore structurein vivo mode of action of amphomycin and MX-2401 in intact whole cells of S. aureus pair-labeled with [1-13C]Gly and L-[ε-15N]Lys, L-[1-13C]Lys and D-[15N]Ala, and D-[1-13C]Ala and [15N]Gly using solid-state NMR. Cyclic lipopeptides were added to S. aureus at mid-exponential growth phase at sub-MIC, then the changes of peptidoglycan (PG) and wall teichoic acid (WTA) compositions were determined by 13C{15N} and 15N{13C} rotational-echo double resonance (REDOR) NMRIn this study, we investigate S. aureus  as lysyl-ε-amine can be observed as a peak with the chemical shift of 10 ppm due to its open bridge-link. Amphomycin-treated S. aureus show the reduction of lysyl-ε-amide at 95 ppm and increase in lysyl-ε-amine at 10 ppm. The reduction of 95 ppm corresponds to the loss of PG through cell wall thinning, and the increased lysyl-ε-amine at 10 ppm corresponds to the accumulation of Park’s nucleotide.Growing ge-links . 15N-CPM treated , red and13C{15N} REDOR spectra of whole cells of S. aureus with and without amphomycin treatment after 1.6 ms of dipolar evolution are shown in S0 spectra (bottom) are normalized to natural abundance peaks from 10 to 30 ppm range. The 170-ppm glycyl peak intensities in S0 spectra (bottom) are directly proportional to the total amount of [1-13C]Gly found in S. aureus. The reduced glycyl-carbonyl carbon in the S0 of amphomycin-treated S. aureus indicates the thinning of cell wall due to inhibition of PG biosynthesis. Covalently bonded 13C-15N spin pairs unique to the PG bridge-link are selected in ΔS spectra after 1.6 ms dipolar evolution. Reduced ΔS 170-ppm intensity in amphomycin-treated S. aureus is the result of increased incorporation of endogenous unlabeled L-Lys into PG-stem structure from biosynthesis of cytoplasmic PG precursors. The 149-ppm peak in the S0 is due to the metabolism of [1-13C]Gly into purine biosynthesis. The reduction of 149-ppm peak in amphomycin-treated S. aureus indicates decreased amount of [1-13C]Gly entering purine biosynthesis pathway due to the heavy demand on [1-13C]Gly by PG biosynthesis pathway.S. aureus was grown in defined media with D,L-[1-13C]Ala and [15N]Gly to selectively label 13C-15N pair of the PG cross-link in order to determine the effect of amphomycin on transpeptidation step of PG biosynthesis (13C{15N} REDOR spectra of whole cells of S. aureus grown in presence (40 μg/mL) and absence of amphomycin with dipolar evolution of 1.6 ms are shown in 0 spectra are normalized to natural abundance peaks from 10 to 30 ppm range. The S0 spectrum of amphomycin-treated S. aureus shows reduction of D,L-[1-13C]Ala incorporation into PG. In ΔS spectra, 175-ppm peak intensity is proportional to the total amount of covalently bonded 15N-13C spin pairs found in S. aureus. Although the intensity of ΔS 175-ppm peak includes contributions from [15N]Gly-L-[1-13C]Ala sequences found in proteins, the primary contribution to the intensity comes from cross-linked PG. Alanine racemase inhibitor alaphosphin has been used to ensure selective labeling of PG with D-[1-13C]Ala and to remove protein contribution to the ΔS 175-ppm intensity821222324252627S. aureus showed reduction in ΔS 175-ppm peak intensity, which is directly proportional to the total number of PG cross-links. This reduced PG cross-linking in amphomycin-treated S. aureus is apparent in the enlarged carbonyl carbon region of the S0 overlaid with the dephased (S) spectra  and WTA.The lineshape of SS. aureus were grown in defined media containing L-[1-13C]Lys and D-[15N]Ala  from PG stem-links are observed at 95 ppm. The ΔS 95-ppm peak intensity is directly proportional to the total number of PG stem-links in S. aureus. The absence of change in ΔS 95-ppm intensity for S. aureus grown in the presence of antibiotics indicated that amphomycin and MX-2401 did not inhibit the incorporation of D-[15N]Ala into PG-stem structure. In contrast, a large reduction of 10-ppm peak intensity in S0 spectra of amphomycin and MX-2401 treated S. aureus which corresponds to ester-linked D-[15N]Ala found in WTA and LTA was observed. Thus, amphomycin and MX-2401 at sub-MIC concentrations significantly impair the incorporation of ester-linked D-[15N]Ala into teichoic acids in S. aureus.To determine the degree of amphomycin inhibiting the incorporation of ester-linked D-Ala into teichoic acids, [15N]Ala . The grot 10 ppm . 15N{13Cphomycin  and MX-2phomycin  are showin vivo mode of action of amphomycin and MX-2401 by measuring the changes in cell wall composition of intact whole-cells of S. aureus using solid-state NMR. It was found that amphomycin-treated S. aureus exhibit cell wall thinning  to inhibit the formation of lipids I, II, and III. Lipids I and II are the membrane transporters for PG biosynthesis, and lipid III is for WTA biosynthesis.We investigated thinning  and 3b cthinning  and 3d, 55-P is a central lipid transporter shared by PG and WTA biosyntheses, we investigated the comparative effects of amphomycin and MX-2401 on PG and WTA biosyntheses. Accurate biochemical quantification of PG and WTA are difficult, especially for WTA analysis15N]Ala incorporation into PG and WTA in intact whole cells. We were able to clearly quantify the amount of D-[15N]Ala incorporated into PG by measuring the amount of PG stem-links using 13C{15N} REDOR  direct inhibition of WTA biosynthesis, and/or ii) inhibition of D-Ala incorporation by targeting of D-alanylation pathway specific to WTA biosynthesis. Although solid-state NMR measurements cannot differentiate between these two mechanisms, it is clear that amphomycin and MX-2401 had a direct and consequential effect on wall teichoic acid composition. Surprisingly, amphomycin and MX-2401 had no visible effect on the incorporation of D-[15N]Ala into PG stem-link , which can result in mislocalization of penicillin-binding protein 4 (PBP4). PBP4 is the transpeptidase responsible for high levels of PG cross-linking observed in S. aureus. WTA has been shown to be essential in the recruitment of PBP4 to the septum during the cell division. In WTA deficient tagO-deletion mutant of S. aureus, PBP4 is shown to be dispersed over the cell surface resulting in reduced PG cross-linking3S. aureus. Similar mechanism of action can be envisioned for amphomycin, but the binding of amphomycin to PG remains yet to be demonstrated.Whole cell analysis by solid-state NMR indicates that . aureus  presumab. aureus . This reS. aureus (ATCC 6538P) grown overnight in 5 ml of trypticase soy broth in 37 °C at 250 rpm was added  to two one-liter flasks, each containing 250 mL of S. aureus Standard Medium (SASM) supplemented with calcium chloride to final concentration of 50 μg/mL333413C-15N isotope pair labeled  to SASM. WTA was 15N-isotope labeled  of 0.7, amphomycin or MX-2401 was added to final drug concentrations of 20 and 40 μg/mL. Cells were harvested after 60 minutes of further growth (OD660 of 0.9) by centrifugation at 8,000 g for 10 min at 4 °C in Sorvall GS-3 rotor. The cells were washed twice with 50 mL of ice-cold 40 mM triethanolamine buffer, pH 7.0, and resuspended in 10 mL of water and lyophilized.At mid-exponential growth of 13C{15N} and 15N{13C} REDOR NMR. REDOR is the solid-state NMR method that recouples heteronuclear dipolar interactions under magic-angle spinning0 spectrum), and the other in the presence of the coupling . In the S0 spectrum, dipolar dephasing is refocused over a single rotor period due to the spatial averaging by the motion of rotor under magic-angle spinning. In the S spectrum, spin part of the dipolar interaction is manipulated by the application of rotor-synchronized dephasing π-pulses that prevent full refocusing. Dipolar evolution over a rotor period in the S spectrum results in reduced peak intensity for dipolar coupled spin pairs. The difference in signal intensity  for the observed spin (13C or 15N) in two parts of the REDOR experiment is directly related to the heteronuclear dipolar coupling from which the corresponding distance to the dephasing spin is determined. The difference spectra are typically collected as a function of N rotor periods to map out the dipolar evolution.Cell wall compositions were analyzed by 1H, 75 MHz for 13C, and 30 MHz for 15N) provided by 89-mm bore Oxford  superconducting solenoids. The four-frequency transmission-line probe used in the 7.0-T spectrometer had a 14-mm long, 9-mm inner-diameter sample coil, while that used in the 4.7-T spectrometer had a 17-mm long, 8.6-mm inner-diameter sample coil. Both probes were equipped with Chemagnetics/Varian magic-angle spinning ceramic stator, and samples were spun at room temperature at 5 kHz (maintained within ±2 Hz). Radio-frequency pulses were produced by 1-kW Kalmus, ENI, and American Microwave Technology power amplifiers, each under active control; π-pulse lengths were 10 μs for both 13C and 15N. Proton-carbon and proton-nitrogen matched cross-polarization transfers were at 50 kHz for 2 ms. Proton dipolar decoupling during signal acquisition was 105 kHz. Standard XY-8 phase cycling0) eliminated long-term drifts in the performance of the spectrometer. The normalized REDOR difference (ΔS/S0) is a direct measure of dipolar coupling, and this quantity was calculated using modified Bessel function expressions given by Mueller et al.1/2 pair.REDOR experiments were performed on whole cells at 7.0 T  supplemented with 20 mM Ca2+. Cyclic lipopeptide was added to the suspension to final drug concentrations of 0, 0.5, 1, 2, 5, 10, 50, and 100 μg/mL and the cells incubated for 20 min at 37 °C. After the incubation, bacteria were pelleted and removed from the suspension by centrifuging. The amount of leaked ATP in the supernatant was quantified by directly adding 100 μL of CellTiter-Glo® 2.0 reagent  to equal volume of supernatant. After 10 minutes of equilibration, luminescence given off by the mixture was measured using Fluoroskan Ascent FL Luminometer (Thermo Scientific) with the integration time of 200 ms.ATP-leakage assay was performed on overnight cultures of How to cite this article: Singh, M. et al. Solid-state NMR characterization of amphomycin effects on peptidoglycan and wall teichoic acid biosyntheses in Staphylococcus aureus. Sci. Rep. 6, 31757; doi: 10.1038/srep31757 (2016).


Interleukin-7 (IL-7) is a non-hematopoietic cell-derived cytokine with a central role in the adaptive immune system. It promotes lymphocyte development in the thymus and maintains survival of naive and memory T cell homeostasis in the periphery. Moreover, it is important for the organogenesis of lymph nodes (LN) and for the maintenance of activated T cells recruited into the secondary lymphoid organs (SLOs). The immune capacity of cancer patients is suppressed that is characterized by lower T cell counts, less effector immune cells infiltration, higher levels of exhausted effector cells and higher levels of immunosuppressive cytokines, such as transforming growth factor β (TGF-β). Recombinant human IL-7 (rhIL-7) is an ideal solution for the immune reconstitution of lymphopenia patients by promoting peripheral T cell expansion. Furthermore, it can antagonize the immunosuppressive network. In animal models, IL-7 has been proven to prolong the survival of tumor-bearing hosts. In this review, we will focus on the mechanism of action and applications of IL-7 in cancer immunotherapy and the potential restrictions for its usage. Interleukin-7 (IL-7) is a cytokine essential for the adaptive immune system. T lymphopoiesis in the thymus has been shown to be highly IL-7 dependent in mice . In addiCancer is a common disease worldwide and causes thousands of deaths annually. One important reason for tumor progression lies in that tumors can suppress the normal immunological surveillance function of the immune system of the organism to avoid detection . Cancer in vivo and antagonize the immunosuppressive network. Thus, we focused on the application of IL-7 as an immunotherapy for cancer treatment and the action mechanism of IL-7. We also discuss limitations for the use of IL-7.In addition to killing cancer cells by chemotherapy and radiotherapy, immunotherapy has emerged as an attractive area of interest in recent years, which utilizes the immune system of patients themselves. IL-7 is an ideal target used to enhance the function of the immune system. It can reconstitute the immune system ,6, improIL-7 is mainly produced by non-hematopoietic cells including keratinocytes in the skin, fibroblastic stromal cells in the bone marrow ,9, epithThe receptor of IL-7 is a heterodimer that consists of two chains: IL-7Rα (CD127), which is shared with thymic stromal lymphopoietin (TSLP), and the common γ chain (CD132) for IL-2, IL-4, IL-9, IL-15 and IL-21. The γ chain is expressed on all hematopoietic cell types, while IL-7Rα is mainly expressed by lymphocytes, including common T/B lymphoid precursors, developing T and B cells, naïve T cells and memory T cells . Innate kip1 to induce the expression of cyclin D1 for cell cycle progression [There are two main signaling pathways responsible for the function of IL-7: Jak-Stat and PI3K-Akt . By actigression . Moreovegression ,24.+ T cell pool. This pool is maintained in a dynamic balance. Antigen-specific effector T cells fulfill their mission and subsequently die. Then expansion of new T cells supplements this pool. IL-7 retains this balance in three ways: thymopoiesis, homeostasis proliferation and life-support.The immune system protecting the organism from cancer relies on the size of its T lymphocyte pool, especially the CD8−/−, IL-7Rα−/−, γc−/− or Jak3−/− suffer from severe lymphopenia, particularly for T and NK cells. A human mutation in IL-7Rα results in a syndrome of severe combined immunodeficiency (SCID) with an absence of T cells [i.e., double-negative (DN) stages (DN1–4), immature single positive (ISP), double-positive (DP) and single positive (SP) CD4+ or CD8+ T cells. Early thymocyte progenitors do not express IL-7Rα. As T cell lymphopoiesis progresses, DN2 progenitors have the highest IL-7Rα expression while DN4 cells express the lowest levels of IL-7α. IL-7 activates the transcription factor NFATc1 in DN thymocytes by phosphorylating Tyr371 in the regulatory region of NFATc1, a pathway that is critical for the survival and development of DN thymocytes [T lymphocytes are mainly generated in the thymus where IL-7 is necessary for their development. Mice deficient in IL-7 T cells . During  T cells . The devymocytes . Furtherymocytes . IL-7 siymocytes .in vivo. Stromal cells produce IL-7 at relatively constant amounts and independently of external stimuli. Regulation of the function of IL-7 relies on IL-7Rα levels [IL-7 plays a critical role in peripheral T cell homeostasis . There aα levels . In addiα levels . IL-7Rα + T lymphopenia, high dose chemotherapy and auto-immune diseases, patients often have increased circulating levels of IL-7 and a strong inverse correlation between the levels of IL-7 and the number of CD4+ T cells. Higher levels of IL-7 induce T cell homeostasis proliferation outside of the thymus. After the recovery of CD4+ T cell populations, IL-7 returns to homeostatic levels due to naive CD4+ T cells expression of IL-7R, which consumes IL-7 to maintain their survival [+ T cells compared with CD4+ T cells. In addition, CD8+ T cells survive better and proliferate faster than CD4+ T cells. Furthermore, IL-7 signaling on IL-7Rα+ DCs downregulates the expression of MHC class II on DCs, which is critical for the expansion of CD4+ T cells [+ T cell homeostasis, IL-7 signaling must be intermittent due to continuous IL-7 signaling results in increased pSTAT5, which can activate IFN-γ gene expression [In human lymphopenia diseases, such as HIV infection, idiopathic CD4survival . In lympsurvival . However T cells . During pression  and cellpression . The intpression . The TCRpression .+CD31−) in SLOs secrete chemokines CCL19 and CCL21, to attract T cells and DCs and orchestrate a complex network of collagens and extracellular matrix to facilitate the migration of these cells [+CD31+), which not only convey signaling in the lymph further upstream, but it also induces peripheral tolerance [Adaptive immune responses against tumors are generated in secondary lymphoid organs (SLOs), particularly in LN draining of pathological sites. They provide suitable microenvironments for the activation and expansion of antigen-specific lymphocytes. In these collaborative environments, lymphocytes move along stromal networks to scan the surfaces of antigen-presenting cells (APCs) for cognate antigens. The interactions between stromal and hematopoietic cells in SLOs are critical for the function of immune cells . There ase cells ,37,38. Solerance ,39,40. L+ T cells [+ T cells [Cytokines have been applied in cancer immunotherapy for many years, of which IL-2 was approved by the U.S. Food and Drug Administration (FDA) for the treatment of patients with metastatic melanoma. But it required high-dose of IL-2 to achieve significant clinical response which resulted in dose-related fever, serious biochemical abnormalities in the liver and kidney, and capillary leak . IL-2 st T cells . But it  T cells . It also T cells .in vitro with bryostatin and ionomycin, that were infused back into the body of the animals, has greater regression of established melanoma and 4T1 mammary carcinomas in vivo if the cells were cultured in IL-7 and IL-15 compared with IL-2 alone [+ cells for subcutaneous prostate cancer challenge [+ and CD8+ T cells increased significantly and the number of Treg reduced. TCR repertoire diversity increased after IL-7 therapy. But the anti-tumor activity of IL-7 was not well evaluated [Investigators aim to utilize IL-7 to enhance the efficacy of tumor regression. A set of preclinical trials has proven that IL-7 can prolong the survival of tumor-bearing hosts in several tumor models. In murine mammary carcinoma, the administration of IL-7 and IL-15 after radiofrequency thermal ablation (RFA) resulted in a relapse-free survival and showed inhibition of metastatic nodules in their lungs . Adoptiv-2 alone ,47. Inte-2 alone . IL-7 prhallenge , while thallenge . In the valuated ,52,53.+ and CD8+ T lymphocytes in patients with refractory malignancy [Cancer patients often suffer from immunosuppression with low T cell counts . This silignancy .in vivo. As an adjuvant, IL-7 enhances long-term tumor antigen-specific CTLs responses in both quantity and quality after immunization with recombinant lentivector [+ T cells is well known in promoting inflammation and pathology in animal models. In addition to CTLs, the number of infiltrated Th17 cells also increases with IL-7 adjuvant [+ T cells. Next, it represses Casitas B-lineage lymphoma-b (Cbl-b), a negative regulator of T cell activation, to maintain CD8+ T cells survival [+ T cells can continue to proliferate even under conditions where the antigen and other cytokines are limited. Simultaneously, increased CTLs infiltration produces more IFN-γ in mouse melanoma, colon and breast cancer models [+ central memory populations and promote the production of IL-7Rα+ long-living memory stem T cells (TSCM), which can self-renew and demonstrates the plasticity to differentiate into effectors [+ T cells [The IL-7 treatment can improve the function of immune cells tivector . First, adjuvant , which isurvival . These Cr models ,61,62 anr models . Memory ffectors . The ant T cells .+ T cells exhibit a phenotype known as exhaustion, with high PD-1 expression. IL-7 can diminish PD-1 expression on activated CD8+ T cells such that they are revitalized [+ T cells. IL-7 can abrogate this inhibitory effect via an induction of the expression of SMAD ubiquitylation regulatory factor 2 (SMURF2) [Several reports have shown that IL-7 is able to antagonize the immunosuppressive network . This neitalized . Some tu(SMURF2) . Treg ac(SMURF2)  and decr(SMURF2) . Intrale(SMURF2) . HoweverIL-7 signaling is well known to occur in either the initiation or maintenance of some lymphocyte-derived tumors, such as T-cell acute lymphoblastic leukemia ,68. IL-7There is no data demonstrating the exact cell source of IL-7 detected in tumor sample. Was it obtained from tumor cells themselves or from stromal cells in the tumor tissue? Moreover, the source of increased IL-7 in serum was not defined to-date. It is unclear whether IL-7 may signal tumors themselves to accelerate the progression of cancer. There has not been good data to demonstrate that IL-7 receptor expressed on solid tumors is functional or that IL-7 contributes to tumor growth . As a reModulation of the IL-7 axis is an ideal tool for repairing damaged immune systems in cancer patients, particularly those who received chemotherapy . IL-7 ca