The 1.95 Å crystal structure of the specificity subunit from Thermoanaerobacter tengcongensis (TTE-HsdS) shows an unreported open form inter-domain orientation of this subunit. Based on the crystal structure of TTE-HsdS and the closed state model of M.EcoKI-M2S1, we constructed a potential open state model of type I methyltransferase. Mutational studies indicated that two α-helices (aa30-59 and aa466-495) of the TTE-HsdM subunit are important inter-subunit interaction sites in the TTE-M2S1 complex. DNA binding assays also highlighted the importance of the C-terminal region of TTE-HsdM for DNA binding by the TTE-M2S1 complex. On the basis of structural analysis, biochemical experiments and previous studies, we propose a dynamic opening and closing mechanism for type I methyltransferase.Type I DNA methyltransferases contain one specificity subunit (HsdS) and two modification subunits (HsdM). The electron microscopy model of M.EcoKI-M An open state model of the M2S1 complex is needed in order to understand the structure of the complex and to model the dynamic opening and closing of the complex. Three dimensional structures of the HsdS subunit with an open form domain-orientation are therefore needed.The orientation of the TRDs and the CRs are quite different between the two published structures for the HsdS subunit Mja-HsdS . The dif2S1 reveals that the N terminal domains of the two HsdM subunits contact each other, while the C terminal domain of the HsdM subunits contact the HsdS subunit The EM model of M.EcoKI-MThermoanaerobacter tengcongensis in an open form conformation at 1.95 Å resolution. Based on structural comparisons and modelling, we propose a hemi-open state model for the M2S1 complex. Also, mutational studies were used to reveal the inter-subunit interaction sites of type I methyltransferases from T. tengcongensis (TTE-M2S1). Based on the structural and mutational evidence presented here, we have supposed a dynamic “opening and closing ” way of the M2S1 complex.We report here the crystal structure of HsdS from tte-hsdS and tte-hsdM gene were amplified by PCR from T. tengcongensis genomic DNA tte-hsdS (ORF: TTE1545) and tte-hsdM (ORF: TTE1547) were cloned into the pET-DUET co-expression vector at cloning sites 1  and 2 (without tag) respectively. Based on this co-expression vector of “wild type TTE-HsdS/wild type TTE-HsdM”, we also constructed several co-expression vectors of “wild type TTE-HsdS/mutant TTE-HsdM”. Details of these co-expression vectors are summarized in tte-hsdS into the pHAT-2 expression vector.The Escherichia coli cells. The cells were grown in LB media supplemented with 100 mg/mL ampicillin until they reached log phase growth (OD600 = 0.6). The expression of TTE-HsdS was induced by stimulation with IPTG (0.4 mM) at 28°C for 10 h. Cells were harvested and resuspended in buffer A  and then lysed by sonication. The lysate was clarified by centrifugation and purified by passage through a nickel-affinity column. A further purification step was then performed using size exclusion chromatography on a Superdex 200 column (Amersham). The purified protein was concentrated to 15 mg/mL for crystallization in buffer B . A number of TTE-HsdS/TTE-HsdM complexes were expressed and purified using the same protocol.All vectors were transformed into BL21 (DE3) 4)2SO4). Crystals suitable for X-ray diffraction studies were obtained after 5 days growth. Hg derivatives were obtained using the same protocol as in our previous work 2SO4 for 2 min before data collection and were flash-frozen in liquid nitrogen. Native crystal data were collected on a beamline NW12A . Derivative data were collected on a Rigaku FR-E X-ray generator with a Rigaku R-AXIS IV++ image plate detector. Data were integrated and scaled with HKL2000 Crystals of recombinant TTE-HsdS were grown at 20°C using the hanging-drop, vapor-diffusion method. Drops consisted of 2 µL of protein solution and 2 µL of mother liquor . The samples were subjected to agarose gel electrophoresis after 1 h incubation at 20°C.A non-radioactive electrophoretic mobility shift assay (EMSA) method was used to inspect the DNA binding properties of wild type and mutant TTE-MThe crystal structure of TTE-HsdS was determined to 1.95 Å resolution by the single wavelength anomalous diffraction method using a mercury derivative Table 2.α atoms. A 2-stranded antiparallel β-sheet was found at the beginning and end of each TRD . The core structure of each TRD consisted of three α-helices and two β-sheets with four and three short strands respectively  . A three amino acid loop (Gln375–Glu377) is inserted in the CR2 α-helice. And there is a fifty degree bend in CR2. The angle and distance between TRD1 and TRD2 indicated the open-form domain-orientation of TTE-HsdS . When only the TRDs were superimposed, the following rmsd values were obtained: 3.7 Å for 146 equivalent Cα atoms (TRD1s of TTE-HsdS and Mja-HsdS), 3.9 Å for 117 equivalent Cα atoms (TRD1s of TTE-HsdS and Mge-HsdS) , 2.1 Å for 124 equivalent Cα atoms (TRD2s of TTE-HsdS and Mja-HsdS) and 2.9 Å for 123 equivalent Cα atoms (TRD2s of TTE-HsdS and Mge-HsdS) . Although both TRD1 and TRD2 have similar folds in the three HsdS subunits, the overall domain orientation is quite different. Hence, significant domain motion could happen within the HsdS subunit. However, the intrasubunit conformational changes are not well understood. By superimposing the TRD2s of the three HsdS subunits, differences between the TTE-HsdS and the other two HsdS subunits could be described in three ways. Firstly, significant bending and twisting of the CRs occurs within TTE-HsdS, giving rotations of 23.7° and 33.8° when compared to Mja-HsdS and Mge-HsdS . Comparisons among the above structures revealed that the TTE-HsdS subunit is in a relatively open conformation. The proposed HsdS-HsdM interaction sites are located in the connection region of CRs and TRDs and in a helix-loop region in TRDs 2S1 complex is able to undergo conformational changes.Superposition of the overall structure of TTE-HsdS and two other HsdS subunits (Mja-HsdS and Mge-HsdS) using the DALI PAIRWISE COMPARISON SERVER gave an rmsd of 8.1 Å for 360 structurally equivalent C2S1 complexes were purified by co-expression of TTE-HsdS and TTE-HsdM in E.coli. Expression of TTE-HsdM alone was insoluble. The molecular weight of the protein complex was determined by analytical ultracentrifugation to be 165 kDa, indicating that the protein complex consists of two HsdM subunits (MW: 58.5 kDa) and one HsdS subunit (MW: 46.5 kDa) . The open form conformation structure of TTE-HsdS and the closed state model of M.EcoKI-M2S1 complex  . As expected, the TTE-M2S1 model revealed a completely different open state conformation than the M.EcoKI-M2S1 model. In addition, the open state TTE-M2S1 model clearly showed that the clamp-like closed state complex converts to the open state by movement of the N-terminal domains of the HsdM subunits. By using the same protocol, crystal structures of Mja-HsdS and Mge-HsdS can not result in a reasonable open form of the M2S1 complex .Stable M2S1 EM model shows that the HsdM subunit C-terminal region contacts the HsdS subunit while the N-terminal regions of the HsdM subunits contact each other. A series of mutation assays were designed in order to confirm these proposed contact regions and identify the specific interaction sites. Firstly, we constructed four co-expression vectors consisting of wild type TTE-HsdS and different TTE-HsdM C-terminal deleted mutants  ((Type I methyltransferase will remain in the closed state when no DNA is entering or leaving the complex _SMΔc40) Table 1.2S1 complex.Four co-expression vectors were also constructed of wild type TTE-HsdS with different TTE-HsdM N-terminal deleted mutants  Table 2.T. tengcongensis Type I methyltransferase M2S1 complex. Results of our EMSA assay showed that the mixture of linear vector DNA and wild type TTE-M2S1 was less mobile than free DNA, an effect that was more obvious as the concentration of protein complex was increased . TTE-MΔn102S1 and TTE-MΔn302S1 had similar linear DNA binding affinities as wild type TTE-M2S1 , which is consistent with the results of random point mutagenesis studies in EcoKI In order to identify the TTE-HsdS DNA binding sites, TRD1 and TRD2 were entered into the DALI server 2S1 model, the minimum distance between the N-terminal domains of the two HsdM subunits is about 10.3 Å, which is close to but not sufficient to allow the passage of DNA. Therefore, the TTE-M2S1 model might reflect an intermediate state which is near to the fully open state. Obviously, this open state model is not very sophisticated for lacking of direct experimental evidence, but it implicates a reasonable picture of the flexible clamp-like enzyme.Compared to the EcoKI-HsdS computational model, significant bending and twisting of the CRs in TTE-HsdS crystal structure enlarges the angle and distance between the TRDs and also shows a small range of rotation between the TRDs. Changes of domain orientation in the HsdS subunit are accompanied by movement of the HsdM subunits that interact with the CRs and TRDs. Interactions of N-terminal domains of the HsdM subunits are also lost. We assume that this series of conformational changes reveals the structural basis mediating the conversion between closed and open states. In our TTE-M2S1 complex. However, the N-terminal domains of the HsdM subunits move apart to open the clamp-like complex when DNA is entering or leaving the complex. Target DNA might act as a bridge connecting the N-terminal domains of the HsdM subunits, thereby stabilizing the complex. Therefore, target DNA could facilitate the conversion of the complex from closed to open state.Our mutational experiments indicated that α-helices formed by residues 30–59 and 466–495 of the HsdM subunits are important sites for HsdM-HsdM and HsdM-HsdS interactions, respectively. Damage to either region will disrupt the corresponding interaction and affect assembly of the M2S1 and TTE-M2S1 models represent the potential closed and open states of type I methyltransferase respectively  are shown in stick model.(TIF)Click here for additional data file.Figure S2Superimposition of HsdS Structures. (A) Stereo view of the overall superimposition of the TTE-HsdS (blue), Mja-HsdS (yellow) and Mge-HsdS (red) structures. (B) Stereo view of superimposition of TRD1s. (C) Stereo view of superposition of TRD2s.(TIF)Click here for additional data file.Figure S3Superimposition of CRs. Superimposition of CRs of the TTE-HsdS (blue), Mja-HsdS (yellow) and Mge-HsdS (red) structures.(TIF)Click here for additional data file.Figure S4Analytical ultracentrifugation analysis. The molecular weight of the protein complex was determined to be 165 kD, indicating that the protein complex consists of two TTE-HsdM subunits (MW:58.5 kD) and one TTE-HsdS subunit (MW:46.5 kD).(TIF)Click here for additional data file.Figure S5The modeling procedure of TTE-M2S1. EcoKI-HsdS (red) and TTE-HsdS (blue) are shown in cartoon model. HsdM subunits are shown in surface model.(TIF)Click here for additional data file.Figure S62S1 complex.The potential states of M The potential states of M2S1 complex based on the conformations of Mge-HsdS (A), Mja-HsdS (B), EcoKI EM model (C) and TTE-HsdS (D). HsdS subunits and HsdM subunits are shown in cartoon model and surface model respectively.(TIF)Click here for additional data file.


Lolium rigidum population recurrently selected with low (below recommended label) doses of the herbicide diclofop-methyl. We report the variability in herbicide resistance levels observed in F1 families and the segregation of resistance observed in F2 and back-cross (BC) families. The selected herbicide resistance phenotypic trait(s) appear to be under complex polygenic control. The estimation of the effective minimum number of genes (NE), depending on the herbicide dose used, reveals at least three resistance genes had been enriched. A joint scaling test indicates that an additive-dominance model best explains gene interactions in parental, F1, F2 and BC families. The Mendelian study of six F2 and two BC segregating families confirmed involvement of more than one resistance gene. Cross-pollinated L. rigidum under selection at low herbicide dose can rapidly evolve polygenic broad-spectrum herbicide resistance by quantitative accumulation of additive genes of small effect. This can be minimized by using herbicides at the recommended dose which causes high mortality acting outside the normal range of phenotypic variation for herbicide susceptibility.The interaction between environment and genetic traits under selection is the basis of evolution. In this study, we have investigated the genetic basis of herbicide resistance in a highly characterized initially herbicide-susceptible  S-transferases can metabolize certain herbicides and evolved resistance can be due to enhanced rates of herbicide metabolism endowed by these enzymes  and Lolium rigidum (Gaud.), enhanced rates of herbicide metabolism have been shown  families and discuss the management of low-dose-selected polygenic herbicide resistance.Here, we are concerned with herbicide resistance evolution from selection at low herbicide doses. Our studies conducted with L. rigidum parental population VLR1 (hereinafter referred to as S) was exposed to three cycles of recurrent selection with low (below the recommended label dose) doses of the ACCase-inhibiting herbicide diclofop-methyl (selecting agent) as described in (Neve and Powles −1) applied to plants at the first, second and third cycles of recurrent selection, respectively. Plant survival at 0.1, 0.5 and 2.0 dose was 36%, 33% and 44%, respectively.The well-characterized herbicide-susceptible –1) to confirm resistance, and six randomly chosen resistant R plants were each pair-crossed to one plant of the susceptible original unselected S parental to produce a total of 6 F1 pair crosses. Seed progeny was collected from both parental plants generating a total of 12 F1 families comprising six F1 maternal R (numbered from 1 to 6 and hereinafter referred to as ♀ R F1) and six reciprocal F1 maternal S (same numeration and hereinafter referred to as ♂ R F1).R plants as described above were treated with a single herbicide dose  was treated at a different diclofop-methyl dose: 188 (low dose L), 375  or 1500 (high dose H) g diclofop-methyl ha−1. Each clone was assessed for survival 28 days after herbicide treatment. F1 families 5 and 6 were chosen to generate F2 families because, based on survival of cloned plants, there were at least two surviving plants at each L, M and H treatment.Twenty plants from each of the six ♀ R F2 families  were generated by pair-crossing two cloned plants within the same F1 family that survived the specific L, M or H diclofop-methyl dose. Type L F2 families were generated by pair-crossing cloned plants from F1 families that were only able to survive 188 g diclofop-methyl ha−1 (cloned counterparts were killed at 375 and 1500 g ha−1). Type M F2 families were generated by pair-crossing cloned plants surviving 188 and 375 g diclofop-methyl ha−1 but killed at the highest dose (1500 g ha−1). Type H F2 families were generated by pair-crossing cloned plants that were able to survive the high dose of 1500 g diclofop-methyl ha−1.Three types of F1 families were treated with 188 g diclofop-methyl ha−1 at the two-leaf stage to eliminate susceptible individuals. The mean plant survival ratio in F1 families was 75%, significantly different from survival in the S , but not significantly different to survival observed in the R plants . This suggested the phenotypic herbicide resistance trait(s) were endowed by dominant gene(s). As suggested by Tabashnik .Plants from ♀ R Fabashnik , F1 survNE) for resistance to diclofop-methyl in parental, F1 and F2 families was estimated as suggested by Lande (The effective (minimum) number of genes  of parental populations (R and S) and σ2 is the difference in variance for the same phenotypic trait between F2 and F1 families.μ1, F2 and BC families were tested for conformity to an additive-dominance model. The quantities A and C and their variances were calculated as described by Mather's equations  endowing diclofop-methyl resistance were assessed by a scaling test as described in Mather and Jinks . Observe1 families , six F2 families  and two ♀ BC families (no. 5 and 6) were tested in final diclofop-methyl dose–response studies conducted at different times but under identical glasshouse conditions. Seeds were germinated on 0.6% (w/v) agar. Seedlings were transplanted and grown in 18-cm diameter pots containing a potting mix  and maintained in glasshouse conditions. Plants at the two-leaf stage were treated with 0, 46, 188, 375 or 1500 g diclofop-methyl ha−1 (recommended label dose 375 g ha−1). In each experiment, there were three replicates per treatment with a minimum of 37 plants treated at each herbicide dose depending on seed availability and germination. Plant survival and aboveground dry biomass were evaluated 21 days after herbicide treatment.The resistant parental R, the unselected susceptible parent S and four FAs described in Busi et al. , maternad is the upper limit, b is the slope of the curve, x is the herbicide dose, and e is the dose producing a 50% reduction in response. Regression assumptions were held under square root data transformation (i.e. Box-Cox transformation lambda λ = 0.5)  or susceptible (dead) plants compared with the expected survival/mortality Ho ratios  multiplied by the observed survival (%) in R, F1 and S at that specific dose  test was used to compare the observed plant survival with the expected calculated values according to one-, two- or three-resistance gene segregation models , 0.75 F1:0.25S (2 genes) or 0.875 F1:0.125S (3 genes) , 375 (medium) or 1500 (high) g diclofop-methyl haabashnik  and Presabashnik , the sego ratios . The expfamilies . Thus, tn models . P-value3 genes) . The sig1 and F2 families was also treated with a discriminating (label) dose of the ACCase-inhibiting herbicides sethoxydim (186 g ha−1) or diclofop-methyl (375 g ha−1) or the very dissimilar ALS-inhibiting herbicides, sulfometuron (30 g ha−1) or chlorsulfuron (30 g ha−1). Survivors of chlorsulfuron and diclofop-methyl were treated with 2 kg malathion ha−1 followed eight hours later by another treatment with the same herbicide at the same dose. In L. rigidum, malathion can overcome chlorsulfuron resistance by inhibiting cytochrome P450 enzymatic activity  for resistance to both herbicides would be equally inhibited by the P450 inhibitor malathion. Sethoxydim and sulfometuron were used to identify the presence of resistant individuals carrying mutations in the ACCase or ALS gene, respectively  indicated that, at the population level, at least three resistance genes were present in the original parental herbicide-susceptible (S) L. rigidum population. Most probably, all three genes were not all initially stacked within S individuals; however, over three cycles of recurrent low-dose diclofop-methyl selection, these three resistance genes became accumulated within surviving individuals through cross-pollination. Once the three resistance genes were present within individuals, then they collectively endowed relatively high-level diclofop-methyl resistance. Pooled data obtained from parental families (R and S) and respective F1, F2 and BC crosses at the three diclofop-methyl doses tested were assessed by a joint scaling test. Student's t-test revealed no significant deviation from zero of quantities A and C calculated in the scaling test  (1 families was lower than the respective R parent (P < 0.01).As expected, the herbicide dose–response study confirmed the R parent as diclofop-methyl resistant and the original parental S population as susceptible . The com > 0.48) A,B. Over1 cloned plants were confirmed to be diclofop-methyl resistant and cloned S confirmed as susceptible  between plant response at treatments with L versus H dose (188 vs 1500 g diclofop-methyl ha−1). The other F1 families were not studied further because either this difference in herbicide response was not significant (P = 0.79) or there were no survivors at the H dose . Similarly, the chi-square test revealed that a one-gene model best fitted the data obtained with families L and M. For these two families, any other genetic model involving a greater number of genes did not fit the data . Consistently, the one-gene model poorly fitted the segregation data in the F2 no. 5 family H (P < 0.02), whereas two-gene and three-gene models were found to be appropriate with no significant statistical deviations (P > 0.13) . The observed survival in family L was significantly lower than expected by the one-gene model at doses 375 and 1500 g diclofop-methyl ha−1 (P < 0.01). Family H exhibited the highest resistance level  (P > 0.08)  . F2 no. sistance D. Familyhyl ha−1 . Family hyl ha−1 . Two- an= 0.001) D. Either > 0.08) .1 no. 5 lines (P = 0.40), whereas BC no. 6 exhibited a significantly lower resistance level than F1s no. 6 (P < 0.001)  E,F. The family M . It is e1 and F2 (H type) displayed diclofop-methyl resistance and a variable degree of cross-resistance to the dissimilar ALS-inhibiting herbicide chlorsulfuron  by a subsequent diclofop-methyl plus malathion treatment  of different mode of action. Here, parental R, Fsulfuron A. It is sulfuron  and it hreatment B. This i−1) resulted in 100% mortality in R, S, F1 and F2 lines and likewise sethoxydim (186 g ha−1) killed all the plants treated as they do not have a resistance-endowing mutation in the ALS or ACCase gene, respectively  dose of the herbicide diclofop-methyl evolved a high level of phenotypic resistance  endowing herbicide resistance  of quantitative genes  in the R, F1 and F2 lines. Thus, we hypothesize that these are P450 genes also because diclofop-methyl and chlorsulfuron are known to be P450-metabolized in wheat  that minimize herbicide damage and thus endow plant survival. On the contrary, use of high (recommended) herbicide doses may eliminate those weakly endowing resistance gene traits. Herbicide resistance evolution in weed species is a global challenge in modern agro-ecosystems and increasingly evolutionary concerns are raised for the sustainability of world food production in a changing climate (Neve et al. Data for this submission available as supplementary material hosted by Wiley Online.


S. aureus. Variable attenuation of activity was observed in the presence of Ca2+ and Mg2+. Introduction of D-amino acids abrogated the need for a disulfide bridge for exhibiting activity. Confocal images of carboxyfluorescein (CF) labeled peptides indicated initial localization on the membrane and subsequent translocation into the cell. Analogs corresponding to cationic rich segments of human defensins substituted with L- and D-arginine, could be attractive candidates for development as future therapeutic drugs.We have examined the antimicrobial activity of C-terminal analogs of human β-defensins HBD-1and-3 wherein lysines have been selectively replaced by L- and D-arginines and L-isoleucine substituted with its D-enantiomer. The analogs exhibited antibacterial and antifungal activities. Physiological concentration of NaCl did not attenuate the activity of the peptides against Gram-negative bacteria considerably, while some attenuation of activity was observed against  Mammalian defensins are cationic peptides with three disulfide bridges -3. They in vitro, HBD-1 is relatively less potent against the Gram-positive bacterium Streptococcusaureus . Peptides were cleaved from the resin using triﬂuoroacetic acid containing thioanisole, meta-cresol and ethanedithiol . Formation of disulﬁde bonds was accomplished by air oxidation in 20% (v/v) aqueous dimethyl sulfoxide  at a conLabeling of peptides with carboxyﬂuorescein (CF) at the free amino group of the N-terminal amino acid was carried out by treating 10 mg of resin-bound peptide with 0.8 ml of dimethylformamide containing CF and activating agents as described earlier . The depE. coli (MG 1655), S. aureus (ATCC 8530), and P. aeruginosa (NCTC 6751). The antibacterial activity of the peptides was examined in sterile 96 well plates at a ﬁnal volume of 100 µl as follows: Bacteria were grown in nutrient broth (Bacto Difco nutrient broth) to mid-log phase and diluted to 106 colony forming units (cfu)/ml in 10 mM sodium phosphate buffer (pH 7.4). Bacteria were incubated with different concentration of peptides for 2 h at 37°C and suitably diluted aliquots were spread on nutrient agar plates. After the plates were incubated at 37°C for 18 h, colonies formed were counted. Lethal concentration (LC) is the concentration of the peptides at which no viable colonies were formed. Cell survival is expressed as a percentage of the control. Percentage killing was calculated as: [(colonies from control cells- colonies from treated cells)/colonies from control cells x 100]. The LC determined was average of three independent experiments done in duplicate. In control experiments, cells were incubated with only buffer.Bacterial strains used were Activity of the peptide analogs were also tested in the presence of 1mM DTT. The disulfide bridges were broken by incubation with DTT at 37°C for 1h. Antimicrobial assay was performed with reduced peptide in the presence of 1 mM DTT. In all the experiments, untreated peptide and cells in the presence of 1mM DTT were used as controls.2+ and Mg2+ , were included in the buffer to determine their effect on activity at lethal concentration (LC) of the peptides. In control experiments, cells were incubated with only buffer. Data are presented as mean ± standard deviation computed for three independent replicates.To determine the effect of salt on antibacterial activity, different concentrations of NaCl was included in the incubation buffer at their LC. Different concentrations of divalent cations CaC. albicans aerobically in yeast extract-peptone-dextrose (YEPD) medium at 30°C. After 20 h, 0.5 ml from this suspension was subcultured for 2 h in 20 ml of YEPD broth to obtain a mid-log-phase culture. Cells were harvested by centrifugation, washed with 10 mM phosphate buffer (PB), pH 7.4, and resuspended in the same buffer, and the concentration was adjusted to 106 cells/ml. Aliquots of diluted cells were incubated with peptides in 100 µl volume at 30°C for 2 h. Cell suspensions were diluted and spread on YEPD agar plates and the plates were incubated for 24 h at 30°C. Colonies were counted, and the concentrations of the peptides at which no viable colonies were formed were taken as the Minimum fungicidal concentration (MFC). Cell survival was expressed as a percentage of the control. Percentage killing was calculated as [(colonies from control cells- colonies from treated cells)/colonies from control cells x 100].Minimum fungicidal concentrations (MFC) of the peptides were determined by growing The average of results from three independent experiments done with duplicate samples was taken for the calculation of MFC. In control experiments, cells were incubated with only buffer.Spectra were recorded in 10 mM phosphate buffer (pH 7.4), TFE, and 10 mM SDS micelles on a JASCO J-715 automatic recording spectropolarimeter at 25°C using a quartz cell of 1 mm path length. Each spectrum (185-250 nm) was an average of six scans. Data are represented as mean residue ellipticities.E. coli and C. albicans with CF-peptides and FM4-64 and propidium iodide (PI), respectively. E. coli and C. albicans were grown overnight in nutrient broth and yeast extract-peptone-dextrose medium, respectively, washed with 10 mM phosphate buffer (pH-7.4) and the concentration were adjusted to 1 x 107 cells/ml in 10 mM phosphate buffer. These cells were treated with sub-lethal concentration of peptide for different time intervals. Peptides were incubated for 5 min, 10 min, 20 min and 30 min at 37°C or 30°C for E. coli and C. albicans respectively, to capture different stages of peptide entry, localization on the membrane and killing. After addition of peptide, the live cells were examined with a Zeiss LSM 510 META confocal microscope. Optical sectioning was done at 1 airy unit by using the 488- and 543-nm-wavelength laser lines with a 63 water lens objective. Emission data were collected using 500- to 530-nm band-pass and 565- to 615-nm band-pass ﬁlters for CF and PI, respectively, in the multitrack mode. Z-sections were acquired at 0.35-µm intervals and projected using the LSM-FCS software version 3.2. The bright-ﬁeld images were obtained simultaneously using the transmitted-light detector. Three independent experiments were done in case of bacteria and fungi to record different stages of peptide entry into the cell. Images shown are representative of the different events  that could be captured from these independent experiments.Localization of CF-labeled peptides was examined by treating The sequences of HBD-1and-3 and the peptides investigated in the present study are shown in C. albicans. The activity of HBD-1 analogs, HC-1(R) and [D]HC-1(R) are comparable except against C. albicans, where HC-1(R) is more active. The analog of HBD-3 shows comparable activity against bacteria and C. albicans. [D]HC-(3) is 2-fold more active against bacteria as compared to HC-1(R) and [D]HC-1(R). Loss in antimicrobial activity was observed for HC-1(R) when the disulfide bridge was broken by DTT similar to Phd-1-3. The activities of the analogs with D-amino acids, [D]HC-1(R) and [D]HC-3(R), were not lost when the disulfide bridge was broken by DTT. All the analogs were more active than Phd-1 and -3.The antimicrobial activity of the peptides is summarized in S. aureus was attenuated to a greater extent. The effect of Ca2+ and Mg2+ on antibacterial activity is shown in 2+, the activity of HC-1(R) and [D]HC-1(R) is not attenuated against Gram-negative bacteria, whereas, loss in activity is observed for [D]HC-3(R) that is further attenuated at 5 mM Ca2+. At 5 mM Ca2+, loss in activity is more pronounced for HC-1(R) and [D]HC-3(R) against P. aeruginosa as compared to [D]HC-1(R). Loss in activity is observed for HC-1(R) against S. aureus but not for [D]HC-1(R) at 1 mM Ca2+. However, loss in activity for all the peptides is observed at 5 mM Ca2+. Loss in activity is evident for all the peptides in the presence of Mg2+against Gram-negative bacteria but not S. aureus. Loss in activity at 5 mM Mg2+ against P. aeruginosa is more for [D]HC-1(R) and [D]HC-3(R) as compared to HC-1(R).The antibacterial activity of the peptides in the presence of varying concentration of NaCl is shown in Secondary structure of the peptides was examined in buffer, SDS micelles and TFE . In buffE. coli and C. albicans was examined using CF-labeled peptides by confocal microscopy. Antimicrobial activity of the CF-labeled peptides was comparable to that of the unlabeled peptides. CF labeled HC-1(R) and [D]-HC-1(R) interacted with E. coli . After entry, it is likely that the mechanism of killing is similar.In the present study, confocal images clearly showed that the peptides did not cause gross membrane disruption and membrane destabilization. The images indicate that HC-1(R) and [D]-HC-1(R) were localized on the membranes initially and subsequently translocated across the membrane. Localization of [D]-HC-3(R) on the membrane was not discernible. The peptide appears to rapidly enter bacterial cells. The mode of entry of HC-1(R) and [D]-HC-1(R) into Electrostatic charge-based mechanisms rather than formation of bilayer-spanning pores have been proposed for β-defensins -49. The C. albicans, which indicates that the peptides, though are conformationally flexible, have the ability to interact with membranes with varying lipid composition.Components of bacterial membranes appear to play an important role in discriminating interactions with different defensins. These components include lipopolysaccharides ,53, teicIn summary, our study indicates that substituting R for K and introducing D-amino acids results in peptides whose antibacterial activity is attenuated only marginally at high NaCl concentrations. When D-amino acids are introduced, even the linearized peptides are active unlike their counterparts with L-amino acids. Selective introduction of D-amino acids would result in increased structural flexibility which appears to result in improved antimicrobial activity. While it remains to be established that similar engineering of full length defensins would also result in improved antimicrobial activity, short peptides with broad spectrum antimicrobial activity can be engineered from defensins, as described in this study. Such peptides could be attractive candidates for the development of therapeutic agents.


Helicobacter pylori.Many bacteria can deliver pathogenic proteins (effectors) through type IV secretion systems (T4SSs) to eukaryotic cytoplasm, causing host diseases. The inherent property, such as sequence diversity and global scattering throughout the whole genome, makes it a big challenge to effectively identify the full set of T4SS effectors. Therefore, an effective inter-species T4SS effector prediction tool is urgently needed to help discover new effectors in a variety of bacterial species, especially those with few known effectors, e.g., H. pylori, an important human pathogen associated with gastritis, ulcer and cancer. In total, 24 new highly possible H. pylori T4S effector genes were computationally identified.In this research, we first manually annotated a full list of validated T4SS effectors from different bacteria and then carefully compared their C-terminal sequential and position-specific amino acid compositions, possible motifs and structural features. Based on the observed features, we set up several models to automatically recognize T4SS effectors. Three of the models performed strikingly better than the others and T4SEpre_Joint had the best performance, which could distinguish the T4SS effectors from non-effectors with a 5-fold cross-validation sensitivity of 89% at a specificity of 97%, based on the training datasets. An inter-species cross prediction showed that T4SEpre_Joint could recall most known effectors from a variety of species. The inter-species prediction tool package, T4SEpre, was further used to predict new T4SS effectors from We conclude that T4SEpre, as an effective inter-species T4SS effector prediction software package, will help find new pathogenic T4SS effectors efficiently in a variety of pathogenic bacteria. Helicobacter Cag-T4SS in human gastric ulcer and cancer, Legionella Dot/lcm-T4SS in Legionnaires’ disease, and Bartonella VirB/VirD4 in cat scratch disease [ADEKNPQ]E’, ‘E[AEGKMNQR][DEKNPQ]E’, and ‘S[GIKLMNQRST][PQRS]S’, which were significantly enriched in T4S sequences .Besides sequence-based Aac preference in T4S signal peptides, the position-specific Aac profiles were also compared between T4S and non-T4S sequences. As shown in Additional file To further evaluate whether the observed amino acid preference (or depletion) is statistically significant, we set up a binomial distribution model for each amino acid at each position of T4S and non-T4S C-terminal 50 positions. At positions of T4S C-termini, the 20 amino acid species did not show equal preference. Some amino acids were enriched while some others depleted significantly Figure C. The moThe primary peptide sequence determines its secondary structure (Sse) and solvent accessibility (Acc), which may be associated with the specificity of signal recognition. Therefore, we compared the Sse and Acc composition in each C-terminal position of T4S effectors with those of the non-T4S proteins. As expected, T4S effectors showed a position-specific Sse preference pattern apparently different from that of the non-T4S proteins in the C-terminal region, especially at the C-terminal 40 positions  and YP_094180.1 had an extremely similar 3D structure, with a mirror symmetry for the C-terminal end parts , motifs, Sse and Acc profiles can be used for distinguishing T4S proteins. Support Vector Machine (SVM) based machine learning models were therefore trained with different features and/or their combination, and comparison was performed on their classification power. SVM was adopted since it often generates high classification accuracy and especially high specificity -28,31. AAs shown in Table Inclusion of secondary structure and solvent accessibility improved the distinguishing power of both sequence-based models and position-specific Bayesian models. The model based on sequential joint features of Aac, Sse and Acc outperformed any other pure sequential features-based model Table . Most stWe also tested the influence of different signal sequence length on model performance. Among the models based on C-terminal 25aa, 30aa, 40aa, 50aa and 100aa , C100 models apparently outperformed the others (data not shown). Since the models based on combined SPB Aac and sequential Aac features (T4SEpre_psAac), BPB Aac features (T4SEpre_bpbAac) and position-specific joint features of Aac, Sse and Acc (T4SEpre_Joint) showed the best performance on classification of T4S and non-T4S sequences, the rest parts of the research will only use these three models based on C-terminal 100aa signals. To further confirm the classification performance of these three models, we changed the size of negative dataset  could still correctly recognize most of the known Legionella effectors . One genus, Ochrobactrum, was an apparent exception: the models based on the effectors of other genera could at best recall 2/5 of the known effectors  for possible T4S effectors.ffectors ,20, amonT4SEpre_Joint, T4SEpre_bpbAac and T4SEpre_spAac identified 58, 78 and 37 T4S effectors respectively , the percentages of positive T4S proteins predicted in S. tyhimurium  were lower than in H. pylori . Furthermore, the prediction results of the three software tools were combined to increase prediction specificity, as performed in H. pylori. We found only 13 proteins were predicted by both T4SEpre_Joint and at least one other software tool . Similar to the distribution of T3S signals among different bacteria, it is not surprising to find T4S signal containing proteins in strains without protein-transporting T4SSs such as S. typhimurium LT2, though the number of positive proteins could be much smaller [Salmonella.As a control, we also made a whole-genome T4S effector prediction from  smaller ,29,30. T smaller . The funL. pneumophila, but very few new effectors are being identified for other important species, e.g., H. pylori. These factors prompted us to develop an inter-species T4S effector prediction method.Bacteria encode diverse protein secretion or translocation systems to effectively interact with host cells. Type III and type IV secretion systems play especially important roles in gram-negative bacteria ,9,33,34.In this study, we focused on sequence and structure-derived features. Through sequence-based single-, bi-, tri-residue Aac and motif analysis, we found distinct composition preference in C-terminal sequences of T4S effectors relative to control proteins. Glutamic acid and serine were most strikingly preferred by T4S effector sequences Figure . In the de novo structure prediction, it is not feasible to predict the structure of all T4S effectors with high precision. However, it is still interesting to observe the structure basis of specific type IV secretion recognition.We also tried to observe the different secondary structure and solvent accessibility determined by the different Aac features between T4S and control proteins. The T4S effectors had much more flexible and exposed C-terminal regions than the control proteins . Remarkably, the T4SEpre_Joint model trained on the sequences of the other species  could still correctly recall ~ 81% of the known Legionella effectors  and sequence-based Aac features, considerably outperformed the others in terms of sensitivity, specificity, accuracy, AUC and MCC . It should be pointed out that, even with the highest cross-validation specificity 97%, ~86 false positives would be predicted from a genome encoding 2850 non-effector proteins. The sensitivity of T4SEpre_Joint is 89% at the specificity of 97%, so about 134 effectors can be correctly predicted assuming there are 150 effector proteins in the same genome. Therefore, in a genome encoding 3000 total proteins and 150 (5%) T4S effectors, T4SEpre_Joint will predict 220 candidates, 61% (134/220) among which are true positives. In order to further increase the specificity, we suggested the following two strategies as we adopted in H. pylori effector prediction: (1) combining all the three tools and looking for the effectors predicted by both T4SEpre_Joint and at least one other software tool, and (2) increasing the prediction threshold value to 0.5 or higher. From our observations, the true positives are more often predicted by combining multiple models, and with higher prediction scores. Therefore, both the strategies should decrease the ratio of false positives in the prediction results.An ideal computational model could predict all the true positive effectors (highest sensitivity) without any false positive effector (highest specificity). However, it is infeasible to develop such a perfect model. In practice, we have to make a balance between sensitivity and specificity to cope with different situations. For example, in bacteria with many known effectors such as S. typhimurium LT2, Additional file S. typhimurium LT2, the number of total proteins with T4S signals in bacteria without protein-transporting T4SSs should be much smaller than strains with functional protein-transporting T4SSs.The T4S proteins were also predicted from bacteria without known protein-transporting T4SSs . The ratio between the similarity score of pairwise sequences and self similarity score was calculated. Conserved paralogs or orthologs were identified when a pair of sequences had an above-stated similarity score ratio higher than 0.30. For each orthologous or paralogous cluster, only one representative was selected as the training sequence. This homology-filtering procedure reduced the number of T4S peptides to 347. The non-redundant peptides constitute the positive training dataset. Non-T4S proteins were randomly selected from the same strains where the positive training sequences were originated, followed by removal of the known T4S effectors and their homologs. The C-terminal 100-aa peptide fragment was also extracted from each non-T4S protein, and the same homology-filtering procedure was performed. Finally, for each strain, the ratio of non-T4S: T4S peptides was set as 2:1, and the GC content for encoding nucleotides was generally maintained equal or similar between the two types of sequences (T4S 40% vs. Non-T4S 41%) [Experimentally validated T4S effectors were retrieved from literature and their putative orthologs were extracted from genome annotation files. In total, we analyzed 1913 effectors from 10 genera, including T4S 41%) . The 347To observe whether the size of negative dataset influence the classifying prediction performance, another independent negative dataset was prepared  or non-T4S (negative) sequences, with a larger absolute value for a more striking advantage. The bi-residue (bAac) and tri-residue (tAac) compositions were calculated with a similar procedure. Putative and conserved motifs were screened with MEME [Sequence-based Aac was calculated for each T4S or non-T4S sequence. Each of the 20 amino acid species was counted for its occurrence within the C-terminal 100, 50 and 30 positions . An Aac frequency vector was obtained for each sequence, and the vectors for all sequences composed a frequency matrix. The composition of each amino acid species was compared between T4S and non-T4S sequences with Student’s two-tail rrection . The sigith MEME , followeS = s1, s2, s3,…, sn denote a peptide sequence in which s represents amino acid while 1, 2,… or i represents position and n represents sequence length. For m sequences, the position-specific occurrence of a certain amino acid A is described as: p(Ai) = f (Ai)/mi, in which f (Ai) denotes the frequency of amino acid A at position i. For each position, the p(Ai) of different amino acids form a position set, and for a sequence S with n amino acids, n values (extracted from each position set) comprise a composition vector. A binomial distribution Bi was modeled for each amino acid species at each position, where paa was set as p(Ai) of negative dataset or 1/20  for different comparison purpose. A Bonferroni-corrected binomial test was performed based on the distribution model to find out the significantly preferred or un-favored amino acids at corresponding position of T4S sequences. The significance level was also set as p < 0.05.The position-specific Aac features were extracted as follows. Let vector SCRATCH was used to predict the secondary structure  and solvent accessibility  . Tertiarhttp://cran.r-project.org), with radial basis kernel function. The parameters for SVM were optimized using grid search based on 10-fold cross-validation.Sequence-based Aac features were directly represented by the frequency of each amino acid species (‘Seq_Aac’) or each bi-residue (‘Seq_bAac’). The combination of all the ‘Seq_Aac’ and ‘Seq_bAac’ features or those significantly preferred/depleted in T4S peptides led to the features of model ‘Seq_Aac, bAac’ or ‘Seq_Sig’, respectively. The sequence-based joint Aac, Sse and Acc features were extracted with the strategy described in Yang et al., . PositioA), Specificity (Sp), Sensitivity (Sn), Receiver Operating Characteristic (ROC) curve, the area under ROC curve (AUC) and Matthews Correlation Coefficient (MCC) were utilized to assess the predictive performance. In the following formula, A denotes the percentage of both positive instances (T4S) and negative instances (non-T4S) correctly predicted. Sn (true positive rate) and Sp (true negative rate), respectively, represent the percentage of positive instances (T4S) and the percentage of negative instances (non-T4S) correctly predicted. An ROC curve is a plot of Sn versus (1 - Sp) and is generated by shifting the decision threshold. AUC gives a measure of classifier performance. MCC takes into account true and false positives and false negatives and is generally regarded as a balanced measure which can be used even if the classes are of very different sizes.The model performance was evaluated and compared with a five-fold cross-validation and Leave-One genus-Out strategy . Accuracwhere, and denote the number of true positives, true negatives, false positives and false negatives, respectively.H. pylori genome (NC_000915) and S. typhimurium LT2 (NC_003197) DNA sequences downloaded from the NCBI Genome database. The sequences were screened for possible T4S effectors with three independent models in the T4SEpre package . The default cutoff SVM scores (≥ 0.5) were adopted for all the three models. The standalone T4SEpre package could be freely downloaded from the web site: http://biocomputer.bio.cuhk.edu.hk/softwares/T4SEpre/.The proteins were deduced from the The authors declare that they have no competing interests.YW and SLL conceived and designed the project; YW and XW annotated T4S effectors and control proteins; YW, XW and HB analyzed the data; YW developed the models and wrote the software; YW and SLL wrote the manuscript. All authors read and approved the final manuscript.Logarithm of Aac ratios between T4S and non-T4S C-terminal sequences. The different amino acids were listed along the horizontal axis while the length of bars represented the logarithm of composition ratio of the corresponding amino acid. Three lengths of T4S and non-T4S C-terminal sequences were analyzed, with C100, C50 and C30 representing C-terminal 100-aa, 50-aa and 30-aa peptides, respectively.Click here for fileSignificantly biased sequential bi-Aac.Click here for fileMotifs in signal sequences of T4S effectors.Click here for file(A) and (B) represent T4S proteins and control proteins, respectively.Position-specific Aac profiles of T4S and control proteins for C-terminal 100 positions. The horizontal axis indicates the C-terminal position number. Click here for fileSignificantly biased position-specific Aac.Click here for file(A) and (B) represent the Sse of T4S proteins and control proteins, respectively. (C) and (D) represent the Acc of T4S proteins and control proteins, respectively.Position-specific Sse and Acc profiles of T4S and control proteins for C-terminal 100 positions. The horizontal axis indicates the C-terminal position number. Click here for file(A)Legionella VipE; (B)Legionella YP_094180.1; (C)Legionella YP_094076.1; (D)Coxiella YP_001597263.1; (E)Legionella YP_094096.1; (F)Legionella YP_094157.1.3D structure of C-terminal 100aa peptides of T4S effectors. Click here for file(A) The structure cluster formed by all the six T4S effectors with high prediction accuracy ; (B) Structure alignment between Legionella VipE and YP_094180.1; (C) Structure alignment between Legionella YP_094076.1 and Coxiella YP_001597263.1; (D) Structure alignment among Legionella VipE, YP_094180.1, YP_094076.1 and Coxiella YP_001597263.1.Structural similarity among C-termini of T4S effectors. Click here for fileOptimized parameters for different SVM models classifying T4S effectors and control proteins.Click here for file6-fold negative dataset.Click here for filePerformance of models classifying T4S effectors and non-effectors .Click here for filePerformance of models classifying T4S effectors and non-effectors .Click here for fileT4SEpre package.Click here for fileH. pylori.T4SEs predicted from Click here for fileS. typhimurium LT2.T4SEs predicted from Click here for fileTraining datasets.Click here for file


Prion diseases or transmissible spongiform encephalopathies are a rare group of fatal neurodegenerative illnesses in humans and animals caused by misfolding of prion protein (PrP). Prion protein is a cell-surface glycosylphosphatidylinositol (GPI)-anchored glycoprotein expressed mostly in the central and peripheral nervous system, and this membrane-bound protein can be cleaved from the cell membranes by phosphoinositide phospholipase C. Numerous studies have investigated GPI-free recombinant PrP, but the role of GPI on misfolding of PrP is not well known. In this study, we synthesized a GPI analog that was covalently linking to a PrP S230C mutant, resulting in S230C-GPI. The structural changes in S230C-GPI upon binding to lipid vesicles composed of mixtures of the zwitterionic lipid (POPC) and the anionic lipid (POPG) were analyzed by circular dichroism spectroscopy, and the amyloid aggregation of S230C-GPI in the liberation from phospholipid vesicles was monitored by proteinase K-digestion assay. Our results indicate that S230C-GPI in the liberation of lipid vesicles has high tendency to misfold into amyloid fibrils, while the membrane-bound S230C-GPI proteins are highly stable and rarely convert into amyloid forms. In addition, the role of cholesterol in S230C-GPI was studied. The effect of GPI, cholesterol and phospholipid vesicles on misfolding of PrP is further discussed. C) into the β-stranded scrapie isoform of PrPC (PrPSc) is characteristic of TSEs. PrPSc potentially aggregates into amyloid fibrils having protease resistance.Prion diseases or transmissible spongiform encephalopathies (TSEs) are a rare group of fatal neurodegenerative illnesses in humans and animals. Some TSEs include Creutzfeldt–Jakob disease (CJD), Kuru, Gerstmann–Sträussler–Scheinker (GSS) syndrome, and fatal familial insomnia (FFI). Conversion of the native, predominantly α-helical conformation of prion protein -anchored glycoprotein, expressed mostly in the central and peripheral nervous system  is the concentration of the quencher. A purely static quenching process would exhibit a good linear relationship. According the results illustrated in 0/F) − 1 vs. GPI and (F0/F) − 1 vs. cholesterol indicates the static quenching process in S230C and MoPrP, respectively. The binding event can be further analyzed. The number of ligand binding sites (n) can be determined from where FKa is the binding constant for ligand–MoPrP interaction. Linear regression analyzed in C-terminus. In the same way, the binding site of cholesterol is calculated to be 0.9, very close to one, as determined in where C-terminus. Thus, mild modification caused by attachment of GPI analog at the C-terminus was expected. Interestingly, this structural modification enhances rather than weakens the α-helical conformation.After confirmation of interaction of S230C with GPI and MoPrP with cholesterol, the protein structure resulting from attachment of the ligands was determined by circular dichroism (CD) spectroscopy. As compared in A noteworthy structural effect of GPI and cholesterol is the stability of α-helical structure. The structural stability of the S230C mutant, S230C-GPI and MoPrP-cholesterol were determined by heat-induced denaturation monitored at 222 nm as shown in C to lipid membrane via a GPI anchor induces intermolecular β-sheet structure [218/θ208 . Similar to PrPSc, prion fibrils are resistant to PK digestion at the C-terminal regions. PK-resistant bands with molecular weights 8, 10, and 12 kDa have been clearly identified in amyloid fibrils converted from recombinant MoPrP [One special feature of PrPnt MoPrP . In FiguC-terminal region rather than the N-terminus as illustrated in N-terminal binding of PrP to lipid vesicles as illustrated in C-terminal region would become highly accessible for PK digestion resulting strong PK resistant band. In the case of C-terminal binding of PrP to lipid vesicles, if proteins aggregate as shown in Sc in a cell-free system, where PrP is associated with raft membranes until the GPI anchor is cleaved [In contrast to other PK-resistant patterns, abundant S230C-GPI/SUV monomer rather than PK-resistant fragments were observed. This phenomenon indicates that S230C-GPI proteins are highly stable when inserted into lipid vesicles and barely convert to amyloid forms. This result is different from the extensive survey on the lipid-induced amyloid formation of prion in which the PK-resistant fragments in the mixture of POPC and anionic lipid was reported . The inc cleaved . Our res cleaved .GPI-lipid anchoring is an important post-translational modification in eukaryotes. GPI-anchored proteins are functionally diverse. They are involved in functions including signal transduction, cell–cell interaction, cell adhesion, and host defense . Among tC-terminus in the native condition [Clustering of GPI-anchored proteins on the membrane microdomain of the cell surface is cholesterol-dependent ,30. Cellondition  and at tondition . It is kondition , and a h1 and 2 were mixed in acetic acid and heated to 170 °C for 6 h. Compound 3 was formed after returning the solution to the room temperature [sn-glycero-3-phosphate (compound 4) was dissolved in pyridine and warmed to 35 °C and added with 2,4,6-triisopropylbenzenesulfonyl chloride. Reaction of compounds 3 and 4 at 35 °C for 4.5 h yielded compound 5 [Briefly, compounds perature ,39. Subsmpound 5 . At the Experimentally, the mixture of MoPrP and GPI analog at ratio of 1:3 was incubated in a cold room overnight. The S230C-bound, and free GPI analog, were separated by HPLC equipped with a reverse-phase C4 column (Waters) and eluted with gradient of water and acetonitrile. The formation of a covalent linkage between MoPrP S230C and GPI analog was confirmed by 16% Tricine SDS-PAGE .® XL site-directed mutagenesis kit  with primers:S230C single point mutation was introduced into MoPrP 23-230 gene using QuickChangeTGTTAAAAGGGCGAGCTCAAGGC-3′Forward: 5′-GAAGAACATTATTC-5′Reverse: 3′-GGATAATGCTGCCCTCTTCTS230C plasmid obtained from PCR was confirmed by DNA agarose gel and the sequence of S230C plasmid was further confirmed by sequence analysis.Escherichia coli BL21 star™  cells respectively, in 50 mL LB medium overnight. In large-scale culture, cells were transferred to TB medium and grew until optical density at 600 nm reached 0.6. Subsequent addition of 1 mM isopropyl β-d-1-thiogalactopyranoside induced expression of proteins in inclusion body. The proteins were purified by immobilized metal affinity chromatography and reversed-phase C4-HPLC as previously described procedure [The plasmids pET101 encoding MoPrP 23-230 (WT) and S230C were transformed into competent rocedure ,42. The sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-1′-rac-1-glycerol (POPG) both purchased from Avanti Polar Lipids were preliminarily dissolved in chloroform. Mixture of POPC and POPG at the molar ratio of 3:1 was dried in a test tube. Subsequently, the lipids were dissolved with distilled water and sonicated at 40 °C for 40 min. The sonicated solution was quickly frozen with liquid nitrogen for 20 min, and then heated to 40 °C for 20 min. This freezing and heating cycle was repeated 5 times. Finally, the lipids were sonicated for 20 min followed by centrifugation at 12,000 rpm in eppendorf tubes. The desired SUV was populated in the supernatant. The radius of SUV was about 10 nm as determined by dynamic light scattering .In the preparation of SUV, 1-palmitoyl-2-oleoyl-GPI and cholesterol solutions were titrated into 10 μM MoPrP S230C and WT, respectively, in 50 mM MES buffer (pH 6.0). The spectra of tryptophan residues in the proteins upon each titration were collected by Hitachi F-4500 fluorometer with excitation wavelength at 280 nm. The fluorescence intensity at 350 nm was recorded for further analysis.CD spectra of MoPrP samples were recorded with a Jasco J-815 spectrometer. For measurements in the far-UV region, a quartz cell with a path length of 0.1 cm was used in nitrogen atmosphere. For protein samples, the concentration was kept constant at 10 μM in 20 mM MES (pH 6.0). An accumulation of five scans, with a scan speed of 50 nm per minute, was performed at 20 °C. The heat-induced denaturation of proteins was conducted with heating protein solutions at the rate of 1 °C/min, and the ellipticity at 222 nm was collected every 0.5 °C.2O for further experiments.For fibril conversion, proteins were added into 50 mM MES (pH 6) in the absence or in the presence of 2 M GdnHCl. The samples were incubated at 37 °C, as described in previous studies on protein fibrillation ,18. When2O twice. The samples were air-dried overnight before imaging. The TEM images were collected by a Joel JEM-2100 TEM. The analysis of fibril length was carried out on WICF ImageJ software .The fibril samples were stained with 2% tungsten phosphoric acid onto carbon-coated 200-mesh copper grids. The samples were adsorbed onto the copper grids for 30 s and subsequently washed with PBS and HThe amyloid fibrils (10 μM) were dialyzed and treated with PK (0.2 μM) at 37 °C for 1 h in 100 mM Tris–HCl buffer (pH 7.2). This PK-digestion was stopped by addition of urea to a final concentration of 2.25 M. Samples were heated at 95 °C for 10 min and analyzed by 12.5% SDS-PAGE followed by analysis with silver staining. In addition to the silver staining, aliquots of PK-treated samples were analyzed with mass spectroscopy for precise molecular weight.C into disease-related amyloid form, PrPSc. Enhancement of linking PrP on the membrane domain can be a direction to develop approaches to prevent and to alleviate TSE.GPI-anchoring on lipid vesicles keeps PrP in native structure and with normal function. Liberation of PrP from membrane surface potentially converts normal PrP


Paramyxoviridae family. It is a common cause of respiratory tract infections in children, adults, and immunocompromised patients, for which no specific treatment or vaccine is available. Recent investigations in our lab identified hMPV glycoprotein G as an important virulence factor, as a recombinant virus lacking the G protein (rhMPV-ΔG) exhibited enhanced production of important immune and antiviral mediators, such as cytokines, chemokines and type I interferon (IFN) in airway epithelial cells, and expression of G protein alone inhibits cellular signaling dependent on retinoic induced gene (RIG)-I, a RNA helicase with a fundamental role in initiating hMPV-induced cellular responses. In this study, we have further investigated the mechanism underlying the inhibitory role of hMPV G protein on RIG-I-dependent signaling. We found that the interaction of hMPV G with RIG-I occurs primarily through the CARD domains of RIG-I N-terminus, preventing RIG-I association with the adaptor protein MAVS , recruitment of RIG-I to mitochondria, as well as the interaction between mitochondria and mitochondria-associated membrane (MAM) component of the endoplasmic reticulum (ER), which contains STINGS, an important part of the viral-induced RIG-I/MAVS signaling pathway, leading in the end to the inhibition of cytokine, chemokine and type I IFN expression. Mutagenesis analysis showed that hMPV G protein cytoplasmic domain played a major role in the observed inhibitory activity, and recombinant viruses expressing a G protein with amino acid substitution in position 2 and 3 recapitulated most of the phenotype observed with rhMPV-ΔG mutant upon infection of airway epithelial cells.Human metapneumovirus (hMPV) is a recently identified RNA virus belonging to the  Paramyxoviridae and is closely related to the avian metapneumovirus (AMPV) subgroup C in vivo, as a recombinant hMPV in which the G protein was deleted (rhMPV-ΔG) exhibited reduced replication in the upper and lower respiratory tract of Syrian hamsters and African green monkeys in vivo.Human metapneumovirus (hMPV) was isolated for the first time in 2001 in the Netherlands, and has been quickly recognized as a leading cause of respiratory tract infections in infants, elderly and immunocompromised patients worldwide Toll-like receptor (TLR)- and RNA helicase-mediated signaling plays a critical role in host innate immunity against viral infection We have recently demonstrated that the hMPV G protein is an important virulence factor, as it inhibits host innate immune responses LLC-MK2, A549 and 293 cells  and BSR T7/5 cells (baby hamster kidney cells that constitutively express the T7 RNA polymerase) were maintained as described Recombinant viruses derived from hMPVCAN-83 were propagated in LLC-MK2 cells at 35°C in the absence of serum and in the presence of 1 µg/ml of trypsin , and were sucrose purified, as previously described To investigate the role of rhMPV-G site mutants in modulating cellular signaling, A549 or U4A cell monolayers were infected with rhMPV, WT or G mutants, at a MOI of 2. Uninfected cells in serum-free media supplemented with trypsin, containing similar amount of sucrose as infected cells, were defined as mock-infected and used as a negative control.gaattcATGACAATACAAAAAACCTCATC -3′, and reverse: 5′- TctcgagTCACGTAGAATCGAGACCGAGGAGAGGGTTAGGGATAGGCTTACCGTTTTGCATTGTGCTTACAGATGCCTG-3′. Italicized letters indicate restriction enzyme site. Underlined letters indicate V5 sequence. The cloned V5-tagged C-termini was first cloned into the TOPO cloning vector, and then cut by EcoRI and XhoI and subcloned into pCAGGS vector.The C-terminus of hMPV G protein was cloned using the full length G in pCAGGS expression plasmid as template Site mutants of G protein N-terminus were generated by replacing Glu 2, Val 3, Lys 4, Val 5, Glu 6, Asp 7, IIe 8 or Arg 9 with Ala using a multiple-site mutagenesis kit  and the full-length G in pCAGGS vector as template. All plasmid constructs were verified by sequencing performed by the protein chemistry core laboratory at UTMB. Information regarding the primers used to generate these mutants is available upon request.GCTGTCAAAGTAGAGAACATTCGAGCAATAGAC-3′5′-GGGACAAGTAGTTATG. The primer to change Val 3 to Ala is GCGAAAGTAGAGAACATTCGAGCAATAGAC-3′5′- GGGACAAGTAGTTATGGAG. Italicized letters encode Ala. Bold letters indicate mutated residues either for Ala coding or silent mutation for cloning screening. The mutated fragment II was then cloned back into pBSKSII using NheI and KpnI sites as described Construction of G mutant cDNA was performed using a similar approach we used to generate rhMPV-ΔG To investigate the effect of deletions or site mutations of hMPV G protein on RIG-I dependent gene transcription, logarithmically growing A549 cells were co-transfected in triplicate in 24 well plates with the a plasmid containing the N-terminus of RIG-I , pCAGGS containing full length, truncated or mutated G protein or the control vector, and a luciferase reporter plasmid containing the IFN-β promoter, using FuGene 6 . Cells were harvested 30 h post-transfection to independently measure luciferase and β-galactosidase reporter activity, as previously described Mitochondria were isolated using the Qproteome Mitochondria Isolation Kit from Qiagen , according to manufacturer's instructions. Purity of mitochondria was assessed by the absence of β-actin, a marker of cytoplasmic proteins. Isolated mitochondria were resuspended in SDS sample buffer for Western blot analysis.Logarithmically growing 293 cells in 6-well plate were cotransfected with pcDNA6 containing C-terminus or N-terminus Flag-tagged RIG-I domain  and either pCAGGS encoding V5-tagged G protein or the empty vector, used as a negative control. Cells were harvested 30 h post-transfection and immunoprecipitation was carried out using immunoprecipitation Kit from Roche  and an antibody against either V5 or Flag or an isotype antibody control, as previously described To investigate the molecular mechanisms underlying the disruption of RIG-I-mediated signaling, logarithmically growing A549 cells in 6-well plates were co-transfected with a fixed amount of plasmids encoding Flag-tagged RIG-I and EGFP-tagged MAVS , or their control vectors, and increasing concentrations of V5-tagged G expression plasmid. Cells were harvested 30 h after transfection and immunoprecipitation was carried out using anti-Flag antibody to pull down RIG-I, followed by Western blot using anti-EGFP or anti-V5 antibody to detect associated MAVS or hMPV G protein, respectively.3VO4, 1 mM NaF, 1% Triton X-100 and 1 μg/ml of aprotinin, leupeptin and pepstatin). After incubation on ice for 10 min, the lysates were collected and detergent insoluble materials were removed by centrifugation at 4°C at 14,000 g. After normalizing for protein content, using Bio-Rad, Hercules, CA. Nuclear extracts or total cell lysates were fractionated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% milk in TBS-Tween and incubated with the proper primary and secondary antibodies according to manufacturer's instruction.Nuclear extracts of uninfected and infected cells were prepared using hypotonic/nonionic detergent lysis, according to Schaffner protocol IFN-β, IL-8 and RANTES levels in cell supernatants were quantified by ELISA .P value of less than 0.05 was considered significant. Mean ± standard error (SE) is shown.Statistical significance was analyzed using analysis of variance (ANOVA). The RIG-I/MAVS pathway plays an essential role in initiating cellular signals leading to the activation of transcription factors and subsequent induction of inflammatory/immune and antiviral mediators in response to viral infections To determine whether hMPV G protein could bind to RIG-I CARD domains, leading to the disruption of RIG-I/MAVS association, 293 cells were transfected with expression plasmids encoding V5-tagged G and Flag-tagged N-terminus of RIG-I . Vectors expressing V5 or Flag only were used as negative controls. Cells were lysed followed by immunoprecipitation using anti-V5 antibody and immunoprecipitated complex was subjected to Western blot analysis using anti-Flag antibody. The results showed that hMPV G protein interacts with RIG-I-N , an inteTo investigate whether the interaction between hMPV G and RIG-I could prevent its association with MAVS, 293 cells were transfected with Flag-tagged RIG-I, EGFP-tagged MAVS, and increasing concentrations of V5-tagged G. Vectors expressing Flag or EGFP or V5 only were used as negative controls. G/RIG-I/MAVS complex was immunoprecipitated using anti-Flag antibody and the immunoprecipitated complex was subjected to Western blot analysis using anti-EGFP and anti-V5 antibodies. The results showed that RIG-I interacted with MAVS in the absence of G protein expression , lane 4.To confirm that G protein affects RIG-I-MAVS interaction in airway epithelial cells, we investigated RIG-I recruitment to mitochondria in response to rhMPV-WT or -ΔG infection. Uninfected and infected A549 cells were harvested at various times p.i. to prepare mitochondrial fraction. We found that rhMPV-ΔG-infected cells had more RIG-I recruited to mitochondria than WT-infected cells at all times p.i. There was also a slight reduction in abundance of MAVS at 15 and 24 h p.i., with no change in levels of the mitochondrial protein SDHA , used as loading control , suggestTo investigate the effect of hMPV G on RIG-I/MAVS interaction in the absence of IFN signaling, we used U4A cells, which lack JAK1 expression, a molecule necessary for type I, II and III IFN signaling To determine whether lack of RIG-I recruitment leads to disruption of viral-induced mitochondrial signalosome, we investigated ER-MAM recruitment to mitochondria. Calnexin is a marker ER-MAM, as well as STING, an adaptor molecule that interacts with RIG-I/MAVS and facilitates viral-induced signaling through this pathway G protein is a type II mucin-like glycosylated transmembrane protein, with the N-terminus facing intracellular space and the C terminus oriented externally To confirm the role of Glu 2 and Val 3 G residues in cellular signaling inhibition, we generated recombinant rhMPV harboring site mutations of G protein, where Glu 2 or Val 3 was replaced with Ala. We named these mutants rhMPV-E2A and rhMPV-V3A, respectively, and we tested the effect of these two mutations on hMPV-induced cellular signaling. A549 cells were infected with rhMPV-E2A, V3A or WT, and cell supernatants were harvested at various times p.i to measure IL-8, RANTES and IFN-β by ELISA. As shown in We and others have shown that transcription factors belonging the NF-κB and IRF families control pro-inflammatory and antiviral gene expression in response to paramyxoviruses To investigate changes in IRF- and NF-κB activation, A549 cells were infected with rhMPV-WT or G site mutants and harvested at different times p.i. to prepare nuclear extracts. Nuclear abundance of IRF-3, p65 and p50 , including TLRs and RIG-I-like receptors (RLRs), and activate a cascade of cellular responses, leading to antiviral and immune gene expression. Innate cellular signaling in response to viral infection is cell-type dependent and we have recently shown that, in the context of hMVP infection, TLR-4 and RIG-I play a major role in viral-induced signaling in immune cells and airway epithelial cells, respectively in vitro mutagenesis, focusing on the N-terminal domain of G, as it faces the intracellular compartment, therefore being more likely to interact with the RIG-I/MAVS pathway. Our results show that indeed the N-terminal domain and in particular the Glu 2 and Val 3 aa residues are involved in the inhibition of the RIG-I pathway, as individual mutations of these two aa impairs the ability of G protein to interfere with the RIG-I/MAVS interaction, using an overexpression system, and with the recruitment of RIG-I and ER-MAM to mitochondria/MAVS, resulting in enhanced activation of NF-κB and IRF-3, and their downstream target genes.In order to dissect the functional domains of hMPV G protein, to better understand the mechanism leading to the inhibition of immune and antiviral gene expression, we performed in vivo, as a potential vaccine candidate. On the other hand, in a rodent model of infection rhMPV-ΔG induces two and a half log2 lower neutralizing antibody titers compared to infection with rhMPV-WT. Furthermore, there was also detectable viral replication in the upper airways of animals immunized with rhMPV-ΔG, but not with rhMPV-WT, following viral challenge with naïve hMPV, suggesting that G protein may play an important role in inducing protective immune responses in vivo, using a mouse model of infection Although hMPV is a leading cause of both upper and lower respiratory tract infections in infants, elderly and immunocompromised patients worldwide, no treatment or vaccine is currently available Figure S1Effect of G protein deletion on innate immune response in U4A cells. U4A cells were infected with rhMPV-WT or rhMPV-ΔG, and harvested at 6, 15 and 24 h p.i. to measure secretion of IL-8 (A) and RANTES (B) by ELISA. Cell lysates were subjected to Western blot to compare the viral protein expression in response to WT and ΔG infection (C). Data shown are representative of two independent experiments. **, P<0.01 relative to rhMPV-WT.(TIFF)Click here for additional data file.Figure S2Expression levels of hMPV G protein full length, extracellular domain or mutants. A549 cells were transfected with a plasmid containing either full length G or G extracellular domain (Gc) (A) or G mutants carrying Ala mutation of individual amino acids, as indicated (B), or the corresponding empty vector. Cells were harvested 30 h post-transfection to prepare total cell lysates, after collection of cell supernatants. Expression of full length G and Ala site mutants was detected in total cell lysates, while hMPV cG expression was detected in cell supernatants by Western blot assays using an anti-V5 antibody. Membranes were stripped and reprobed with anti-β-actin, as control for comparable loading of samples. NS indicates a non-specific band. Data shown are representative of two independent experiments.(TIFF)Click here for additional data file.Figure S3Amino acid residues Glu 2 and Val 3 of hMPV G are responsible for inhibiting the interaction between RIG-I and MAVS. 293 cells were transfected with a fixed amount of plasmids encoding Flag-tagged RIG-I and EGFP-tagged MAVS, and a plasmid expressing either V5-tagged G, E2 G mutant, V3 G mutants, N or their control vector (CV). Total cell lysates were immunoprecipitated with anti-Flag antibody to pull down RIG-I, followed by Western blot using anti-EGFP antibody to detect associated MAVS. Total cell lysates were subjected to Western blot to determine levels of hMPV G and site mutants, RIG-I-N and MAVS expression. Data are representative of two independent experiments.(TIFF)Click here for additional data file.


Out of the examined markers of angiogenesis and lymphangiogenesis only the pronounced expression of VEGF-C was found to correlate with patient poor clinical outcome (p = 0.009). The positive correlation between AT-1R and VEGF-A and VEGF-D could point to stimulatory action of Ang II on their expression what might result in augmented lymph- and angiogenesis in IDC.Recent studies point to the involvement of angiotensin II (Ang II) receptor type 1 (AT-1R) on processes of metastasing, stimulation of invasiveness and angiogenesis in tumours. In this study, the correlation between intensity of AT-1R expression and expression of lymph- and angiogenesis markers in invasive ductal breast cancers (IDC) was examined. Immunohistochemical studies (IHC) were performed on archival material of 102 IDC cases. Only 28 (27.5%) cases manifested low AT-1R expression while 74 (72.5%) cases demonstrated a moderate or pronounced AT-1R expression. Expression intensity of AT-1R was found to correlate with expressions of VEGF-A ( Breast cancer is the most frequent female malignancy all over the world. In 2008 its incidence was appraised at 1.38 million new cases  with increasing frequency of its manifestation [via two types of receptors, AT-1R and AT-2R, which are coupled to G proteins [i.a., for induction of apoptosis, is less common and expressed mainly in fetal cells. In an adult healthy organism, it is expressed in cells of vascular endothelium, uterine muscle, ovaries, some cerebral structures and in selected structures of skin, kidneys, heart [Angiotensin II (Ang II) represents an important element of renin-angiotensin system (RAS). It is a multifunctional octapeptide, formed by excision of two amino acids (histidine and leucine) from carbonyl terminus of its precursor, angiotensin I. The reaction is executed by angiotensin converting enzyme (ACE), secreted mainly by the endothelium of pulmonary blood vessels . Ang II proteins . Ang II proteins –7. Expreproteins . On the s, heart , 10.Several earlier studies pointed to a significant function of Ang II on several aspects of neoplastic disease and compounds suppressing action of Ang II were suggested to have an anti-neoplastic significance . Overexpi.a. due to changes in activity and accessibility of Ang II in the pathway linked to AT-1R [In mammary gland, presence of both receptor types was demonstrated . AT-1R ito AT-1R , 19. It to AT-1R . No suchAng II is also engaged in the process of angiogenesis, of key importance from the point of view of neoplastic progression , 22. In In this study, we undertook an attempt to define the significance of AT-1R expression, with particular attention paid to its correlation with selected markers of angiogenesis  and lymphangiogenesis  in IDC.The studies were conducted on archival paraffin blocks of IDC subjected to radical surgery in 1999–2002 in the Lower Silesia Centre of Oncology in Wroclaw. Clinical and pathological data were obtained from hospital archives. A group of 102 patients, aged between 30 and 83 years (mean of 56 years) was retrospectively analyzed. Duration of observation ranged between 1 and 125 months (mean of 61 months). The clinical and pathological data are summarized in Table Immunohistochemical reactions were performed on paraffin sections using DAKO Autostainer Link48  in case of the following antibodies in respective concentrations: mouse anti-AT-1R , mouse anti-CD31 , mouse anti-D2-40 , mouse anti-VEGF-A  and mouse anti-Ki-67 . In order to deparaffinize, rehydrate and unmask the antigens the sections were boiled in Target Retrieval Solution buffer  using Pre-Treament Link Platform (Dako) and, subsequently, cooled in a rinsing buffer (TBS). EnVision™ FLEX (Dako) was used to visualize the antigens. Only in the case of VEGF-A IHC reaction, incubation with primary antibodies was extended from 20 min at RT to 18 hours at the temperature of 4°C and reaction was amplified using the EnVision™ FLEX + Mouse LINKER system (Dako).2O2. Subsequently, primary antibodies to ER  or PR  and incubated for 1 h at RT. The subsequent reaction stages followed manufacturer’s instructions for the LSAB + System-HRP visualization system (Dako). 3,3′-diaminobenzidine (DAB) was used as a chromogen. In order to visualize expression of HER2, the HercepTest™ kit (Dako) was used, according to the procedure recommended by the producer.Reactions with ER-specific antibodies (detecting estrogen receptors), PR-specific antibodies (detecting progesteron receptors) and with HER-2-specific antibodies were conducted according to the earlier described procedure . The sec2O2. The sections were incubated with primary antibody overnight at the temperature of 4°C  or for 30 min at room temperature (Lyve-1). Then the antigens were visualized using the EnVision™ Detection Systems Peroxidase/DAB, Rabbit/Mouse (Dako).In order to examine the expression of VEGF-C, VEGF-D and Lyve-1, mouse antibodies anti-VEGF-C (2 μg/ml), anti-VEGF-D (2 μg/ml) and anti-Lyve-1 (1 μg/ml) were used . The sections were deparaffinized in xylene, rehydrated and boiled in a citrate buffer, pH 6  or pH 9 (for VEGF-D). Subsequently, activity of endogenous peroxidase was blocked by 5 min incubation in 3% HAll slides were counterstained with Mayer’s hematoxylin (Dako). Subsequently, the preparations were mounted in SUB-X Mounting Medium (Dako).In all cases hematoxylin-eosin (H + E) staining of the preparations was performed in order to determine grade of tumour (G) and to evaluate the extent of necrosis in the tumours.+, Lyve-1+ and D2-40+) Chalkley point array  was used. Under magnification of 200x, the vessels were scored in three intratumoural and peritumoural areas of potentially the highest density (hot spots). All the preparations were examined using OLYMPUS BX-41 light microscope .The intensity of IHC reactions for AT-1R, VEGF-A, VEGF-C and VEGF-D was estimated using the semi-quantitative immunoreactive (IRS) scale of Remmele and Stegner . Determip < 0.05.The results were subjected to statistical analysis using Prism 5.0 . Relationships between expression of studied markers and clinicopathological factors were examined using the Spearman rank correlation test, the Kruskal-Wallis test and the exact Fisher’s test. Kaplan-Meier’s survival curves for the patients survival were analysed using the test of Mantel Cox. The differences were regarded significant at p = 0.85).In most cases  cytoplasmic expression of AT-1R was noted. Among them, 28 (27.5%) cases demonstrated low expression of AT-1R (IRS 0–4) Fig. , while 7r = 0.26, p = 0.008) and VEGF-D expression  on the other  manifested a cytoplasmic reaction in tumour cells. A pronounced expression of VEGF-A was noted in 52 (51%), of VEGF-C in 56 (54.9%), and of VEGF-D in 48 (47.1%) studied cases. A moderately positive correlation was documented between expression of AT-1R on one hand and expression of VEGF-A , presence of lymph nodes metastases (pN) (p = 0.0005) and high intensity of VEGF-C expression (p = 0.009) , although a positive correlation has been noted between AT-1R expression and intensities of VEGF-A and VEGF-D expression. Earlier studies of Herr et al. performed on clinical material of breast cancer and on two cellular models of the tumour suggested that Ang II might stimulate production of proangiogenic factors only in cases of triple negative breast cancers . Many coOur studies have demonstrated no relationship between intensity of AT-1R expression and some clinicopathological parameters, i.e. grade of malignancy, primary tumour size, presence of lymph node metastases, menopausal status or expression of ER, PR and HER2 receptors. In addition, statistical analysis has documented no effects of AT-1R expression intensity on survival of the patients.ACE gene, which is linked to higher levels of the circulating ACE isoform in blood of these patients [Although our results have documented no relationships between AT-1R expression intensity and the listed clinicopathological parameters, in our opinion studies on the role of RAS in development of breast cancer should be continued, taking into account all proteins composing the system. Aside from our results, such a conclusion stems also from recent reports suggesting significant effects of an imbalance between activities of angiotensin-converting enzymes (ACE and ACE2) in the development of breast cancer . This hypatients , 45. In


However, the recovery of peripheral blood B cells and CD4+ and CD8+ T cells was not significantly affected by SB and/or G-CSF treatment. These results suggest that the treatment with SB and/or G-CSF can reduce IR-induced BM injury probably in part via promoting HSC and HPC regeneration.The acute and residual (or long-term) bone marrow (BM) injury induced by ionizing radiation (IR) is a major clinic concern for patients receiving conventional radiotherapy and victims accidentally exposed to a moderate-to-high dose of IR. In this study, we investigated the effects of the treatment with the p38 inhibitor SB203580 (SB) and/or granulocyte colony-stimulating factor (G-CSF) on the hematoimmune damage induced by IR in a mouse model. Specifically, C57BL/6 mice were exposed to a sublethal dose (6 Gy) of total body irradiation (TBI) and then treated with vehicle, G-CSF, SB, and G-CSF plus SB. G-CSF (1 µg/mouse) was administrated to mice by intraperitoneal (ip) injection twice a day for six successive days; SB (15 mg/kg) by ip injection every other day for 10 days. It was found that the treatment with SB and/or G-CSF significantly enhanced the recovery of various peripheral blood cell counts and the number of BM mononuclear cells 10 and 30 days after the mice were exposed to TBI compared with vehicle treatment. Moreover, SB and/or G-CSF treatment also increased the clonogenic function of BM hematopoietic progenitor cells (HPCs) and the frequency of BM lineage Hematoimmune injury is one of the most important side effects of radiotherapy. Some patients receiving radiotherapy might develop both acute and long-term myelosuppression ex vivo HSC expansion and attenuate hematopoietic cell senescence induced by IR The p38 mitogen-activated protein kinase (p38) pathway can be activated in response to a variety of extracelluar stimuli, particularly to cellular stress such as osmotic shock, hypoxia, and IR Male C57BL/6 mice were purchased from Vital River  and housed in the certified animal facility at the Institute of Radiation Medicine of the Chinese Academy of Medical Sciences (CAMS). All mice were used at approximately 8–12 weeks of age. All experimental procedures were performed with the approval of the Animal Use Committee at the Institute of Radiation Medicine of CAMS.137Cs source housed in an Exposure Instrument Cammacell-40  at a dose-rate of 0.78 Gy per minute. After irradiation, animals were returned to the animal facility for daily observation.Mice were exposed to 6 Gy TBI from a SB 203580 , a specific p38 inhibitor, was dissolved in a saline solution containing 30% DMSO. G-CSF  was diluted to 5 µg/ml with a saline solution containing 30% DMSO. For SB treatment, mice were given SB at 15 mg/kg via intraperitoneal injection (ip) 24 h after irradiation, and then thereafter every other day for a total of 5 injections. For G-CSF treatment, mice were administered with G-CSF at a dose of 1 µg/mouse by ip injection at 2 h and 6 h on day 1 after irradiation, and then twice per day for 5 days. For the combined therapies, mice were given both SB and G-CSF as described above. As a control, mice were irradiated and then treated with vehicle  in a similar manner as that described for SB and/or G-CSF treatment 3EDTA. BM cells were flushed from mouse femurs with Hank’s balanced salt solution (HBSS) after mice were euthanized by CO2 suffocation followed by cervical dislocation. The numbers of various blood cells and BMMNCs was counted using a pocH-100i hemocytometer  and expressed as 109/L and ×106/femur, respectively.Blood samples were obtained from the orbital sinus using a micro-pipette coated with the anticoagulant KThe spleen and thymus were collected and weighted after mice were euthanized. The spleen and thymus coefficients were calculated by dividing individual organ weight (mg) with their body weight (g).3) per femur.The CFC assay was performed by culturing BM cells in MethoCult GF M3534 methylcellulose medium  according to the manufacturer’s instruction. The colonies of CFU-granulocyte macrophage (CFU-GM) with more than 30 cells were scored under an invert microscope after 7 days of culture. The results were expressed as the numbers of CFU-GM , the remaining cells in blood were washed twice with phosphate-buffered saline containing 1% bovine serum albumin (PBS-BSA) (pH 7.4) and re-suspended in 100 µl PBS-BSA solution. They were stained with phycoerythrin (PE)-conjugated anti-CD8, fluorescein isothiocyanate (FITC)-conjugated anti-CD4 antibodies, PE-cy5-conjugated anti-B220  for 15 min at room temperature in the dark. The samples were analyzed by a Beckman Coulter flow cytometer  using the Expo32 analysis software.BMMNCs were incubated with biotin-conjugated rat antibodies specific for murine CD5, Mac-1, CD45R/B220, Ter-119, and Gr-1 for 15 min at room temperature. After washed by PBS twice, the cells were stained with APC-Cy7-conjugated Streptavidin, FITC-conjugated anti-CD34, PE-conjugated anti-Flt3, PE-Cy7-conjugated anti-Sca1, and Alexa Fluor 700-conjugated anti-c-kit antibodies  and analyzed by flow cytometry. The numbers of a particular cell population were calculated using the following formula: cell numbers/femur = BMMNCs/femur×percentage of positive cells in BMMNCs.t-test or one-way analysis of variance (ANOVA). p value of less than 0.05 was considered significant.The results were expressed as the mean ± standard deviations (SD). The data were analyzed by Student’s Hematopoietic cells are highly sensitive to IR and their injury is the primary cause of death after exposure to a dose of TBI in the range of 4–10Gy To determine whether SB and/or G-CSF facilitate hematopoietic recovery after TBI via stimulating BM hematopoiesis, we analyzed BMMNC counts 10 and 30 days post 6Gy TBI. As demonstrated in To determine whether SB and/or G-CSF can increase BM hematopoiesis after 6Gy TBI by stimulating HSC/HPC proliferation, we examined the effects of SB and/or G-CSF on BM CFU-GM. As shown in To determine whether SB and/or G-CSF can promote immune cell recovery after 6Gy TBI, we first examined the effects of SB and/or G-CSF on immune organ coefficients but found no significant effects on the organ coefficients at 10d post 6Gy TBI . However+ T cell, CD8+ T cell, and B cell counts in blood. As shown in + T cells in peripheral blood from irradiated mice received SB treatment were slightly higher than those from vehicle and G-CSF treated mice 30 days after TBI. As shown in + cells in peripheral blood from irradiated mice receiving SB treatment were significantly higher than those from vehicle treated mice. As shown in Next, we examined the effects of SB and/or G-CSF on CD4−Sca-1+c-kit+ cells (LSK cells), long-term hematopoietic stem cells , short term hematopoietic stem cells  in BM 30 days after 6Gy TBI .and/or G-CSF on p38 activation in lineage negative BM hematopoietic cells . The labeled mature lymphoid and myeloid cells were depleted twice by incubation the cells with goat anti-rat IgG paramagnetic beads  at a bead:cell ratio of 4∶1 and were removed with a magnetic field. Lin− cells (2–3×106/ml in RPMI1640 medium contained 10% FBS) were incubated with vehicle, SB (5 µM), G-CSF (10 ng/ml), or both at 37°C, 5% CO2, and 100% humidity for 30 min prior to exposure to 4 Gy IR in vitro. Two h after IR, the cells were fixed and permeablized with BD CytoFix/Cytoperm solutions , and then stained with a rabbit anti-phosphorylated p38 (p-p38) antibody  according to the manufacturers’ instructions. p-p38 staining was detected by a flow cytometer after staining with FITC–conjugated goat anti-rabbit IgG . (A-E) Representative flow cytometric analyses of p-p38 in Lin− cells with different treatments; (F) The merged flow cytometric analysis graph of A-E; and (G) The percentages of p-p38 positive cells under various treatment conditions are presented as mean± SD (n = 3). a, p<0.05, vs. Ctr; b, p<0.05, vs. V; c, p<0.05, vs. CSF; d, p<0.05, vs. SB.(TIF)Click here for additional data file.Figure S2Effects of SB and/or G-CSF treatment on various lymphocytes in blood. Mice were treated with ip injection of vehicle (V), SB 203580 (SB), G-CSF (CSF), or both (C+S) after exposure to 6Gy TBI as described in the Methods. A group of sham-irradiated control mice was included as a control (Ctr). Blood were collected after the mice were euthanized 30 days after 6Gy TBI and analyzed by flow cytometry. The data are expressed as mean± SEM (n = 5) of percentage of CD4+ T cells (A), CD8+ T cells (B), and B cells (C). a, p<0.05, vs. Ctr; b, p<0.05, vs. V; c, p<0.05, vs. CSF; d, p<0.05, vs. SB.(TIF)Click here for additional data file.


In searching for phosphorylation sites that could account for suppression of GC-B activity by S1P (sphingosine-1-phosphate), mutations were introduced into several candidate serine/threonine and tyrosine residues. Although no novel phosphorylation sites that influenced the suppression of GC-B were identified, experiments revealed that mutations in Tyr808 markedly enhanced GC-B activity. CNP-stimulated activities of the Y808F and Y808A mutants were greater than 30-fold and 70-fold higher, respectively, than that of WT (wild-type) GC-B. The Y808E and Y808S mutants were constitutively active, expressing 270-fold higher activity without CNP stimulation than WT GC-B. Those mutations also influenced the sensitivity of GC-B to a variety of inhibitors, including S1P, Na3VO4 and PMA. Y808A, Y808E and Y808S mutations markedly weakened S1P- and Na3VO4-dependent suppression of GC-B activity, whereas Y808E and Y808S mutations rather elevated cGMP production. Tyr808 is conserved in all membrane-bound GCs and located in the niche domain showing sequence similarity to a partial fragment of the HNOBA (haem nitric oxide binding associated) domain, which is found in soluble GC and in bacterial haem-binding kinases. This finding provides new insight into the activation mechanism of GCs.In this study, Tyr The membrane-bound GC (guanylate cyclase) family consists of five (human) and seven (rodent) members . Among t2+ to stabilize GC activity orthophosphate for 4 h. HeLa cells expressing Myc-GC-B were homogenized on ice with RIPA buffer , and the homogenates were centrifuged for 10 min at 21000 g at 4°C. The resulting supernatants were transferred to a tube containing mouse anti-myc antibody (5 μg/ml) and mixed for 4 h at 4°C. Protein G-Sepharose was then added to each tube and further mixed for 2 h at 4°C. The Protein-G-Sepharose-anti-myc antibody complex was washed five times with RIPA buffer, and the final pellet was mixed with 2× SDS/PAGE sample buffer (Bio-Rad) containing 5% (v/v) 2-mercaptoethanol. Samples were boiled for 3 min, aliquots of the homogenate were separated by SDS/PAGE (6% polyacrylamide gel), and then blotted on to a nitrocellulose membrane. Phosphorylated WT Myc-GC-B and Y808E bands were detected by autoradiography, and WT Myc-GC-B and Y808E proteins were detected using anti-myc antibody and the ECL system (GE).Myc-GC-B or Y808E mutant cDNA was transfected into HeLa cells grown in 6-well plates, and the cells were cultured in the growth medium overnight. After 24 h culture in DMEM containing 0.5% FBS, cells were labelled with serum- and phosphate-free DMEM containing 0.1 mCi/ml [http://www.cbs.dtu.dk/services/NetPhos/), and selected residues having scores greater than 0.800 for mutation. Based on this search, I identified 14 potential serine/threonine phosphorylation sites and two potential tyrosine phosphorylation sites which had not previously been examined . To further analyse the significance of Tyr808 for GC-B activity, I substituted this residue with amino acids having different chemical characteristics. The effect of residue volume was examined by substituting Tyr808 with smaller  or larger (tryptophan) residues , I found that cells expressing the Y808F mutant produced more than 30-fold higher levels of cGMP than cells expressing WT GC-B upon stimulation with 0.1 μM CNP B, despitectively ). The im WT GC-B . Upon st WT GC-B G. These 808 mutations affected the suppression of GC activity by well-characterized GC-B inhibitors, such as S1P, PMA and Na3VO4. As demonstrated above, S1P-dependent suppression of GC-B activity was unaffected by the Y808F mutation, nor was it affected by the Y808W mutation  in the presence of Mn ligands . To testractions . Consist523 and Ser526 to alanine or glutamine greatly reduced GC-B activity [P< 0.01) of 32Pi incorporation level into WT GC-B  into all six potential phosphorylation sites in the juxtamembrane portion of both WT GC-B (WT GC-B-6D) and the Y808E mutant (Y808E-6D), and measured cGMP production in cells expressing these constructs, in the absence or presence of CNP B. Consis808 plays a critical role in the regulation of the enzymatic activity of GC-B. Mutations of Tyr808 resulted in a wide range of effects on cyclase activity, depending on the nature of the substituting residue  or charged (glutamic acid) amino acid, probably because these mutations alter the orientation of GC domain to KHD, allowing GTP to access the catalytic site with less steric hindrance  domain, which is found in soluble GCs and bacterial haem-binding protein kinases [808 is located within the helical linker region. Mutations of Tyr808 may alter the dimerization state of GC-B and thereby control its catalytic activity, which fits our hypothesis shown in Based on NCBI  Conserved Domain Database , Tyr808  kinases ,34 (Figu808-equivalent tyrosine residue with alanine or phenylalanine, and of obtaining constitutively active cyclases by replacing that residue with serine or glutamic acid. Interestingly, mutations in Glu837, Arg838 and Thr839 of GC-E (shown in bold italics in 808 in GC-B, have been linked to dominant cone–rod dystrophy [All membrane-bound GCs, as well as adenylyl cyclases 2 and 4, have tyrosine at the position corresponding to residue 808 of GC-B B. Therefystrophy –38. Thes808 or its neighbouring residues. Current models suggest that membrane-bound GCs are present in an auto-inhibited conformation in unstimulated cells, and that binding of natriuretic peptides to their extracellular domains releases this inhibitory constraint. Mechanisms that underlie GC auto-inhibition have remained elusive, primarily because three-dimensional structures of the intracellular domains of membrane-bound GCs have not yet been solved. My results suggest that Tyr808 contributes to the auto-inhibition of GC-B, and raises the possibility that binding of CNP to the receptor induces a conformational change that displaces this residue from its inhibitory orientation.Due to its location between the KHD and the GC domain, scant attention has been paid to the potential biochemical significance of Tyr