These peptides elicited significant numbers of IFN-γ secreting cells, compared with other M nonapeptides and one irrelevant nonapeptide. Bioinformatics analysis showed that the former is an H-2Kd-restricted CTL epitope, and the latter is an H-2Dd-restricted CTL epitope. Multiple amino acid sequence alignment among different PRRSV M sequences submitted to GenBank indicated that these two CTL epitopes are strongly conserved, and they should therefore be considered for further research on the mechanisms of cellular immune responses to PRRSV.Twenty-seven nanopeptides derived from the matrix (M) protein of porcine reproductive and respiratory syndrome virus (PRRSV) were screened for their ability to elicit a recall interferon-γ (IFN-γ) response from the splenocytes of BALB/c mice following DNA vaccination and a booster vaccination with recombinant vaccinia virus rWR-PRRSV-M. We identified two peptides (amino acid residues K Characterization of field isolates suggested that PRRSV are genetically diverse, and this genetic variation increases the difficulty of developing effective vaccines. Based on significant sequence differencesPRRS viruses are grouped into two distinct genotypes, European isolate  and North American isolate VR-2332) PRRSV has332 PRRSVAt present, effective vaccination strategies for the prevention and control of PRRSV infection are not available. Vaccines based on inactivated PRRSV virus have been ineffective at inducing protective immune responses. Live-attenuated PRRSV vaccines can provide protection against this pathogen, but have been observed to revert to virulence , restric+CD8+ splenocytes of vaccinated mice. A multiple amino acid sequence alignment among different PRRSV M proteins indicates that these two peptides are strongly conserved across multiple PRRSV strains and therefore should be considered for further research.Unlike many other pathogens, there is limited knowledge of the specific PRRSV-derived peptides targeted by T-cells. In the current study, we report the identification of CTL epitopes from the PRRSV (Ch-1a strain) M protein in a mouse model. We screened peptides derived from the PRRSV M protein for their ability to induce interferon (IFN)-γ in splenocytes harvested from BALB/c mice following DNA vaccination and a booster vaccination with recombinant vaccinia virus expressing M protein. The screen identified two peptides that elicited IFN-γ production in CD32 incubator.The PRRSV Ch-1a strain, the vaccinia virus WR strain, and the Akabane virus OBE-1 strain were part of our laboratory collection. The former was propagated in Marc-145 cells, and the latter two were propagated in BHK-21 cells, respectively, and these two cell lines were cultured in DMEM (Invitrogen) supplemented with 10% of fetal calf serum  in a humidified 37°C, 5% COTotal RNA was extracted from the PRRSV CH-1a strain and the spleens of BALB/c mice. Full length cDNAs were amplified based on the complete open reading frames (ORFs) of M and ubiquitin (Ub) following reverse transcription (GenBank accession numbers AY032626 and AZ033428) using the M-F and M-R primers pair or the Ub-F and Ub-R primer pair (Table E.coli BL21 (DE3) and recombinant protein expression was induced with 0.5 mM IPTG. The samples were harvested by centrifugation and the pellets were resuspended with phosphate buffered saline . After being analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and Western blot with anti-PRRSV-M monoclonal antibody (our laboratory collection), the fusion-expressed truncated M protein was purified by GST tag according to the manufacturer's instructions . Finally, the concentration of the purified protein was determined using a Bradford kit (Bio-Rad) according to the manufacturer's instructions.An indirect enzyme-linked immunosorbent assay (iELISA) method was established to evaluate specific antibody against M protein. A truncated M protein 85-174aa), which was used as the coating antigen, was expressed and purified using a prokaryotic expression system. The truncated M gene cDNA was amplified from pMD-M using the truncated-M-F and truncated-M-R primers . The Ub and M genes were amplified from pMD-U and pMD-M with the primer pairs Ub-F and Fusion-M-Ub-R and Fusion-M-Ub-F and M-R, respectively Table . The fusIn vitro expression of the Ub-M was demonstrated by transient transfection of BHK-21 cells using FuGene 6 (Roche) according to the manufacturer's instructions. Ub-M protein expression was detected by indirect immunofluorescence analysis (IFA). An anti-PRRSV-M monoclonal antibody and a FITC-conjugated rabbit anti-mouse IgG secondary antibody (Sigma) were used for IFA detection of M protein. Fluorescence was observed using a fluorescence microscope (LEICA DM IRE2).The transferring vector pSC11 (our laboratory collection), which is composed of the early promoter P7.5 and late promoter P11 of vaccinia virus, and the LacZ and Amp genes controlled by the promoter P11 as the reporter genes, was used in this study. To construct the transferring vector pSC11-M, the complete M gene was amplified with the pSC11-M-F and pSC11-M-R primer pair (Table -143 cells cultured in MEM medium containing 25 μg/ml BrdU (Sigma). The viruses were collected after the appearance of cytopathic effect (CPE), and recombinant vaccinia virus was purified according to the expression of LacZ gene.Homologous recombination was performed by lipofectin-mediated co-transfection of the transferring plasmid pSC11-M and the WR strain vaccinia virus into 80% confluent TK2, 0.5% NP40, 20 μg/ml DNase I). Cell lysates were separated by SDS-PAGE and were subsequently transferred to a membrane for Western blot analysis using an anti-PRRSV-M monoclonal antibody, HRP-conjugated goat anti-mouse IgG secondary antibody (Bio-Rad), and DAB substrate. The positive recombinant virus was named rWR-PRRSV-M.Western blot analysis was performed as described previously . BrieflyThe expression of M was further confirmed by IFA. Briefly, BHK-21 cells were infected with either rWR-PRRSV-M or the vaccinia virus WR strain when the BHK-21 cells reached 70-80% confluence in a 24-well plate. M protein expression was evaluated by IFA 3 days post infection using the anti-PRRSV-M monoclonal antibody followed by a FITC-conjugated rabbit anti-mouse IgG secondary antibody (Sigma). Specific fluorescence was observed using a fluorescence microscope (LEICA DM IRE2).d/H2-Dd/H2-Ld 9-mer epitopes using the algorithms from the SYFPEITHI website [BALB/c [The sequences of M were screened for potential H2-K website , the HLA website  and PRED [BALB/c . The 27 All animal experiments were performed according to national and institutional guidelines. One hundred and ninety female BALB/c mice  were maintained in isolation cages at the Experimental Animal Center of Harbin Veterinary Research Institute . Mice were divided into three groups: the pVAX1-Ub-M vaccination group (n = 120), the pVAX1 control vaccination group (n = 35) and the PBS control vaccinated group (n = 35).The plasmid DNA used for immunization was purified using the EndoFree Mega plasmid preparation kit (Qiagen). The pVAX1-Ub-M and pVAX1 cohorts were intramuscularly (i.m.) vaccinated with 100 μg pVAX1-Ub-M or pVAX1 plasmid DNA in 100 μl PBS, respectively. The PBS control group received an i.m. injection of 100 μL PBS. Each group was vaccinated four times at 3-week intervals. To enhance the specific CTL responses to M protein, the mice received an intraperitoneal (i.p.) injection containing 0.1 MOI of rWR-PRRSV-M on day 7 following the fourth DNA vaccination. At the same time, five mice from the pVAX1 and PBS vaccination groups were also inoculated intraperitoneally with 0.1 MOI of rWR-PRRSV-M on day 7 following the fourth vaccination in order to compare the specific antibody raised against M protein of different experimental groups following vaccination with rWR-PRRSV-M. All procedures were conducted with the protocols approved by Experimental Animal Center of Harbin Veterinary Research Institute (HVRI) of the Chinese Academy of Agricultural Sciences (CAAS).2O2 for 10 min, after which the reaction was stopped by the addition of 1 M H2SO4. Finally, the optical density (OD) was read at 492 nm. An anti-PRRSV-M monoclonal antibody was used as a positive control. Serum containing antibodies against Akabane virus and the sera of control group mice served as negative controls.To investigate the specific antibody response, serum samples was obtained from vaccinated mice 7 days after each DNA vaccination and 3 days after boosting with rWR-PRRSV-M. An iELISA was performed according to methods described previously . The pur6 cells/well in 100 μL complete RPMI-1640 medium supplemented with 10% FCS. The cells were then mixed with 100 μl media containing a nonamer peptide derived from PRSSV M protein at 20 μg/ml, or phorbol-12-myristate-13-acetate (PMA 10 ng/mL) and ionomycin (500 ng/mL) as a positive control. Cells incubated in medium alone or with a peptide derived from the infectious bronchitis virus (IBV) H52 strain [Isolated splenocytes were added to U-bottomed 96-well plates (Corning Inc) at 102 strain  were use+CD8+ T cells producing IFN-γ on days 3, 5 and 12 after boosting with rWR-PRRSV-M was determined using flow cytometric analysis. ICS analysis was performed using a FACSCalibur flow cytometer (BD) according to the methods described previously [in vitro with M protein-derived peptides, as detailed in section 2.12. Ten thousand CD3+ T lymphocytes were acquired per sample and the number of IFN-γ-secretion CD3+CD8+ T cells were enumerated. The CD3+CD8+ lymphocytes that expressed IFN-γ following peptide stimulation were considered to be peptide-specific CTLs. Specific CTL responses were evaluated as the increase in the number of CD3+CD8+IFN-γ+ cells. Reagents used include PerCP-conjugated CD3e, PE-conjugated CD8a and FITC-conjugated IFN-γ, and BD Cytofix/Cytoperm™, all purchased from Becton, Dickinson & Co (BD).The number of CD3eviously . Twenty 2.13). Data are expressed as the number of IFN-γ-secreting cells per two hundred thousand splenocytes. Peptide-specific IFN-γ ELISPOT responses were considered to be positive if the response (minus media background) was ≥ 3-fold above the media response and ≥ 50 spot-forming cells (SFC)/2 × 105 splenocytes were registered.To further confirm the results of the ICS, the other sixty mice from the group vaccinated with pVAX1-Ub-M and thirty mice from the groups vaccinated with pVAX1 and PBS were tested by the IFN-γ ELISPOT assay at three different time points , and a probability value below 0.05 was considered significant.The iELISA results, the percentage of IFN-γ positive CD3in vitro, we expressed a truncated version of M that lacks 84 hydrophobic amino acids at the N-terminus. SDS-PAGE analysis showed that cells transformed with the pGEX-M expression vector produced a large amount of protein with a molecular mass of approximately 36 KDa, consistent with the expected molecular weight of the truncated M protein fused with a GST tag (data not shown). Western blot analysis using an anti-PRRSV-M antibody confirmed the expression and identity of the truncated M protein fused with a GST tag  is the principal mechanism for protein catabolism in the mammalian cytosol and nucleus. In order to enhance the Ub-mediated degradation efficiency of M protein, we expressed a Ub-M fusion protein in BHK-21 cells using the eukaryotic expression plasmid pVAX1-Ub-M, in which the Ub coding sequence was fused in-frame with the PRRSV M coding sequence. Following transient transfection of BHK-21 cells with the pVAX1-Ub-M plasmid, the Ub-M fusion protein was expressed and accumulated predominantly in the cytoplasm , whereas the ELISPOT assay assesses the number of IFN-γ+ cells among all splenocyte cell types (0.05%), and cannot definitively assign the production of IFN-γ to a particular cell type. Thus, the two assays use different denominators in calculating the frequency of IFN-γ+ production by splenocytes. Importantly, each method clearly identified K93FITSRCRL and F57GYMTFVHF as the only two peptides from a panel of 27 that elited significant IFN-γ production from the splenocytes of vaccinated mice.It is important to recognize that the ICS assay calculates the percentage of IFN-γ+ T cell responses during PRSSV infection and immuinization. Whether these two epitopes can bind the SLA class I molecules of pigs remains to be determined. In some cases, specific peptide epitopes are known to be recognized by cytotoxic T cells in different animal species. For instance, the core region of HCV contains an epitope that is recognized by cytotoxic T cells of both mice and humans [In this study we used BALB/c mice as a model system to identify CTL epitopes in the M protein of PRSSV to circumvent limitations derived from shortages of inbred pigs and a paucity of reagents to evaluate porcine immune responses. The identification of PRSSV CTL epitopes in BALB/c mice allow for the future generation of reagents, such as MHC class I: peptide staining reagents, that will enable the in-depth investigations of CD8d humans . Further93FITSRCRL" and "F57GYMTFVHF" from the M protein of PRSSV as H-2Kd and H-2Dd restricted CTL epitopes, respectively. In this study, we also developed a mouse model of PRRSV infection and this will undoubtedly contribute to our understanding of the cell-mediated immune responses to PRRSV.In conclusion, we identified peptides "KThe authors declare that they have no competing interests.DLW and GLL gave me the idea of this study. WJZ and YL participated in the design and conducted the majority of the experiments as well as drafted the manuscript. TGT helped revise the manuscript and participated in the first stage of the experiments. YB and QW participated in the prediction of CTL epitopes and analyzed the data. YHX, NHL and TY participated in the sequence alignment. ZGB provided the expression system of recombinant vaccinia virus, and GZT provided the PRRSV Ch-1a strain. All the authors read and approved the final manuscript.Western blotting results of the recombinant protein PRRSV-M expressed in E.coli. Fig.S1. Western blotting analysis of the recombinant protein PRRSV-M expressed in E.coli BL21 (DE3) cells transformed with the pGEX-M expression vector. SDS-PAGE analysis showed that cells transformed with the pGEX-M expression vector produced a large amount of protein with a molecular mass of approximately 36 KDa, consistent with the expected molecular weight of the truncated M protein fused with a GST tag (data not shown). And western blot analysis using an anti-PRRSV-M antibody confirmed the expression and identity of the truncated M protein fused with a GST tag (Lane 1), while there was no such signal at the corresponding position of the negative control sample (Lane 2). Lane M. prestained protein mass marker; Lane 1. recombinant PRRSV M protein; Lane 2. pGEX-6P-1.Click here for fileIFA results of eukaryotic expression plasmid pVAX1-Ub-M in transfected BHK-21 cells. Fig.S2. IFA result of BHK-21 cells transfected with pVAX1-U-M. BHK-21 cells were transfected with pVAX1-U-M or empty pVAX1 plasmids. After 36 hours, IFA was performed using an M protein-specific antibody. A. pVAX1-U-M; B. pVAX1. (magnifications are × 200).Click here for fileWestern blot results of recombinant vaccinia virus rWR-PRRSV-M in infected BHK-21 cell. Fig.S3. Western blot analysis of BHK-21 cell lysates following infection with rWR-PRRSV-M. BHK-21 cells were infected with rWR-PRRSV-M or WR strain vaccinia virus. After 72 hours, cell lysates were generated for Western blot using an M protein-specific antibody. The results showed that the recombinant vaccinia virus rWR-PRRSV-M drove expression of a complete M protein with the expected molecular weight (17 KDa) when transfected into BHK-21 cells. Lane M: prestained protein mass marker; Lane 1. Lysate from cells infected with rWR-PRRSV-M; Lane 2. Lysate from cells infected with WR strain vaccinia virus.Click here for fileIFA results of recombinant vaccinia virus rWR-PRRSV-M and vaccinia virus WR strain in infected BHK-21 cell. Fig.S4. IFA result of BHK-21 cells infected with rWR-PRRSV-M and vaccinia virus WR strain. BHK-21 cells were infected with (A) rWR-PRRSV-M or (B) vaccinia virus WR strain. After 72 hours, IFA was performed using an M protein-specific antibody. (magnifications are × 100).Click here for fileELISA antibody response in mice after immunization following DNA vaccination and a booster vaccination with recombinant vaccinia virus. Fig.S5. M protein-specific antibody responses in mice immunized with PBS, or pVAX1 or pVAX1-U-M DNA, and boosted with rWR-PRRSV-M. Serum samples was obtained from vaccinated mice 7 days after each DNA vaccination and 3 days after boosting with rWR-PRRSV-M, and were evaluated for reactivity to M-protein in an ELISA based on coating with the truncated M protein fused with a GST tag. And, the day 0 represents the day of the first DNA immunization. Statistically significant differences are indicated by "*" or "**" for p-values < 0.05 or < 0.01, respectively, as determined by ANOVA.Click here for file


Cyclic phosphatidic acid (cPA) is a structural analog of lysophosphatidic acid (LPA), but possesses different biological functions, such as the inhibition of autotaxin (ATX), an LPA-synthesizing enzyme. As LPA is a signaling molecule involved in nociception in the peripheral and central systems, cPA is expected to possess analgesic activity. We characterized the effects of cPA and 2-carba-cPA (2ccPA), a chemically stable cPA analog, on acute and chronic pain.(1) The systemic injection of 2ccPA significantly inhibited somato-cardiac and somato-somatic C-reflexes but not the corresponding A-reflexes in anesthetized rats. (2) 2ccPA reduced sensitivity measured as the paw withdrawal response to electrical stimulation applied to the hind paws of mice through the C-fiber, but not Aδ or Aβ. (3) In mice, pretreatment with 2ccPA dose-dependently inhibited the second phase of formalin-induced licking and biting responses. (4) In mice, pretreatment and repeated post-treatments with 2ccPA significantly attenuated thermal hyperalgesia and mechanical allodynia following partial ligation of the sciatic nerve. (5) In rats, repeated post-treatments with 2ccPA also significantly attenuated thermal hyperalgesia and mechanical allodynia following chronic sciatic nerve constriction.Our results suggest that cPA and its stable analog 2ccPA inhibit chronic and acute inflammation-induced C-fiber stimulation, and that the central effects of 2ccPA following repeated treatments attenuate neuropathic pain. Physarum polycephalum, in 1992 . TheData were expressed as mean ± S.E. The data were statistically analyzed by Student's t-test, Welch's test, Steel's test, Dunnett's test, Wilcoxon test, or Tukey's multiple comparison tests. A P value < 0.05 was considered statistically significant.All the experiments were performed with an approval of the Animal Care and Use Committee at the Tokyo Metropolitan Institute of Gerontology and Nagasaki University Animal Care Committee at the Nagasaki University Graduate School of Biomedical Sciences (reference number 0706130596).Single shock stimulation of A- and C-afferent fibers of the tibial nerve (at 10 V with 0.5 ms pulse duration) elicited 2 types of cardiac sympathetic reflex discharges as shown in Figure Intravenous injection of 0.1 mg/kg of 2ccPA depressed the C-reflex but not the A-reflex Figure . In mostFigure The hypothesis that cPA acts at the primary afferent nerve terminals was tested by recording spinal somato-somatic reflexes in 4 rats Figure . StimulaWe established the EPW test to distinguish responses mediated by different sensory fibers . The hinIn the formalin-induced nociception tests, ICR (CD1) mice were given an s.c. injection of formalin solution to the hind paw. As shown in Figure LPA is produced by ATX in the early phase after nerve injury ,14; therWe examined the effects of 2ccPA on established neuropathic pain in C57BL/6 mice. In the thermal withdrawal test, mice with sciatic nerve injury exhibited a decreased threshold at day 7. Under these conditions, a single 2ccPA dose  significantly increased nociceptive latency at 30 min on the day 7 after injury (considered as day 1), as shown in Figure In the paw-pressure test, repeated post-injection (i.t. or i.v.) of 2ccPA yielded similar effects on day 8 Figure .Similar studies to examine the analgesic effects of 2ccPA against established chronic pain were performed using a different chronic-pain model in rats. In the chronic constrictive sciatic nerve injury (CCI) model, the same experimental schedule was performed, as described above Figure . In the sn-2  injection in naïve mice. The thresholds were measured at 90 min (A) and on days 1 and 7 (B) after 2ccPA injection, using the thermal withdrawal test. All data represent the mean ± S.E. from 3-6 individual mice per group.Click here for fileTime variation of relative C-reflex level after oral administration of 2ccPA (16:1). Relative C-reflex levels were measured after oral administration of 2ccPA (16:1) at 1 mg/kg and plotted over time until 120 min. Each data point represents the average four independent measurements, and vertical bar represents S.E. **P < 0.01 and *P < 0.05; significantly different from time 0 by one-way ANOVA, Dunnett's multiple comparison test.Click here for fileEffects of i.v. 3ccPA on the somato-carcdiac C-reflex. Relative C-reflex levels of the somato-cardiac response were measured after i.v. injection of 3ccPA (18:1) at 100 μg/kg (n = 3). Vertical bar represents S.E. **P < 0.01; significantly different from the vehicle by Student's t-test.Click here for file


Pulmonary complications after cardiac surgery like ARDS are frequent and linked to high mortality . Pro-adrIn this prospective cohort study, 39 patients undergoing cardiac surgery using CPB were included. Blood was collected before surgery (T0), after induction of anesthesia (T1), after termination of CPB (T2), at ICU arrival (T3) and 3 hours (T4), 6 hours (T5) and 18 hours (T6) after arrival. Pro-ADM was measured with a sandwich immunoassay. Primary endpoints were length of ICU and hospital stay .P = 0.032) and the difference at T3 was a significant predictor of hospital-LOS (P = 0.001).An increase of arterial and venous pro-ADM plasma concentrations was observed after surgery. Immediately after termination of CPB the venous concentration was significantly lower than arterial pro-ADM concentration, but at T6 the venous concentration was significantly higher, indicating a switch from a negative to positive transpulmonary gradient Figure . The proWe found that the transpulmonary gradient of pro-ADM was a predictor for ICU-LOS and hospital-LOS at T3 and T5, respectively. Pro-ADM might be a promising marker for prediction on outcome of patients undergoing cardiac surgery on CPB. The transpulmonary shift of pro-ADM might be caused by an inflammatory response.


Streptomyces spp., can cause dramatic plant cell hypertrophy and seedling stunting. Thaxtomin A is the dominant form from Streptomyces scabies and has demonstrated herbicidal action. TxtE, a cytochrome P450 enzyme from Streptomyces scabies 87.22, catalyzes direct nitration of the indolyl moiety of L-tryptophan to L-4-nitrotryptophan using nitric oxide, dioxygen and NADPH. The crystal structure of TxtE was determined at 2.1 Å resolution and described in this work. A clearly defined substrate access channel is observed and can be classified as channel 2a, which is common in bacteria cytochrome P450 enzymes. A continuous hydrogen bond chain from the active site to the external solvent is observed. Compared with other cytochrome P450 enzymes, TxtE shows a unique proton transfer pathway which crosses the helix I distortion. Polar contacts of Arg59, Tyr89, Asn293, Thr296, and Glu394 with L-tryptophan are seen using molecular docking analysis, which are potentially important for substrate recognition and binding. After mutating Arg59, Asn293, Thr296 or Glu394 to leucine, the substrate binding ability of TxtE was lost or decreased significantly. Based on the docking and mutation results, a possible mechanism for substrate recognition and binding is proposed.Thaxtomins, a family of phytotoxins produced by  Cytochromes P450 superfamily (CYP) is a large and diverse group of enzymes containing a heme cofactor which participate in a variety of physiological roles including hormone and secondary metabolite biosynthesis, as well as xenobiotic metabolism,2. Most Streptomyces spp., can cause dramatic plant cell hypertrophy and seedling stunting[Streptomyces scabies which also has demonstrated herbicidal action. Until recently, six proteins were identified in the Thaxtomin A biosynthesis pathway: TxtA and TxtB are non-ribosomal peptide synthases[Thaxtomins, a family of phytotoxins produced by  stunting. Thaxtomsynthases,7; TxtC[synthases and TxtEsynthases are CYPssynthases; TxtR issynthases. TxtE casynthases, TxtE issynthases. Howeversynthases.In this paper, we report the crystal structure of TxtE in substrate-free form. TxtE shows an open conformation. A clearly defined substrate access channel and a potential proton transfer pathway were observed. According to docking and mutagenesis experiments, we propose a possible substrate recognition and binding mechanism.S. scabies 87.22 (406 amino acids) was subcloned into plasmid pET-28a, which produced an N-terminal His-tagged protein with a thrombin digestion site. The R59L (TxtE-R59L), Y89F (TxtE-Y89F), N293L (TxtE-N293L), T296L (TxtE-T296L) and E394L (TxtE-E394L) mutants were produced by applying mutagenic PCR to the pET28a-TxtE vector according to QuikChange protocol.The full-length TxtE DNA sequence from Escherichia coli Rosetta(DE3) plysS cells in LB broth medium for 12 hours at 20°C with 1 mM IPTG induction. The resulting cell pellet was re-suspended in buffer A . Cells were lysed by passing through a microfluidizer  twice, and then the lysate was centrifuged at 30,000 g for 30 minutes. The supernatant was loaded into a 5 mL Ni-IDA column  which had been equilibrated with buffer A, and then eluted by using buffer B . After dilution with buffer C , the sample was loaded into HiTrap Q FF and eluted by using a linear gradient. Finally, TxtE was loaded into a Superdex 75 16/60 column which was equilibrated with buffer D . The resulting protein peak was concentrated to 15 mg/mL by using a 10 kDa MWCO Amicon Ultra. Aliquots were snap frozen in liquid nitrogen and stored at −80°C until they were used for crystallization. TxtE-R59L, TxtE-Y89F, TxtE-N293L, TxtE-T296L and TxtE-E394L mutant proteins were expressed and purified using the same method as mentioned above.Wild-type TxtE protein was expressed in Before crystallization, fresh DTT and imidazole pH 6.5 were added to protein solution. The final concentrations were 2 mM and 200 mM, respectively. Crystals of TxtE were grown by the hanging-drop vapor diffusion method at 18°C. Protein solution was mixed with equal volumes of reservoir solution containing 20% MPD and 20% PEG6000. Crystals were cryo-protected with cryoprotectant before freezing in liquid nitrogen.X-ray diffraction data were collected at beamline BL17U1 of the Shanghai Synchrotron Radiation Facility. Data were processed using the HKL2000 software suite. The TxtL-tryptophan was docked into the structure of TxtE in the zwitterionic form using AutoDock 4.2. ImidazoPurified wild-type TxtE, TxtE-R59L, TxtE-T296L and TxtE-E394L were exchanged to buffer E (25 mM Tris-HCl pH 8.0) by ultrafiltration. Finally, protein was diluted to about 15 μM using buffer E. Spectra were recorded between 325 nm and 500 nm with or without L-tryptophan using a UV-vis spectrophotometer . The final concentration of L-tryptophan was 1 mM.Purified wild-type TxtE, TxtE-Y89F, and TxtE-N293L were exchanged to buffer E (25 mM Tris-HCl pH 8.0) by ultrafiltration. Finally, the protein was diluted to about 8 μM using buffer E. L-tryptophan was added to the sample cuvette until the protein was saturated. The added substrate solution did not exceed 2% of the total volume. The protein solution without L-tryptophan was served as control. This procedure was repeated three times for each sample. The average values of the difference in absorbance of each spectrum at 390 nm and 420 nm were plotted against the concentration of the substrate. The data were fitted to a one site binding model using Qtiplot.a = 46.3 Å, b = 52.6 Å, c = 88.2 Å, α = 77.8°, β = 82.2° and γ = 68.4°. Detailed crystallographic data statistics for TxtE are shown in The purified wild-type TxtE used in this study is catalytically active. L-4-nitrotryptophan could be separated and identified in the reaction products using LC-MS (1 and B′2) are separated by an inserted loop (residues 69 - 77). The helix B′1 of TxtE is well overlapped with helix B′ of CYP101A1. The helix B′2 locates at the similar position of the B-C loop of CYP101A1, and is parallel to the helix B′1 of TxtE. Among the reported CYP structures, it is unusual to find two B′ helices separated by a short loop. Two B′ helices together with B′2-C loop, F helix and G helix are located in the distal side of the heme, and are involved in substrate access and binding. In both molecules, the F-G loop (residues 175 - 183) is disordered. These residues may form a lid on the top surface of the substrate access channel  in TxtE  can be t channel . This chThe orientation of heme in TxtE is similar to that in other CYPs . The thiBecause we could not find crystallization conditions without imidazole, attempts to co-crystallize TxtE with L-tryptophan were unsuccessful. The program AutoDock was therefore used to explore potential L-tryptophan binding poses. Three lowest-energy docking solutions from 2,500,000 searching results were chosen . The cald) of TxtE-Y89F and TxtE-N293L were 120.63 ± 4.30 μM and 1.70 ± 0.28 mM, respectively, while the Kd of the recombinant wild-type TxtE used in this study was 43.59 ± 2.26 μM -OH intermediate, which is formed at the end of the reaction cycle, to regenerate the resting state of the enzyme. However, in many known CYP structures, the complete proton transfer pathway is not clear with the exception of CYP158A2 and P450In vitro, TxtE can accept electrons from spinach ferredoxin (Fd) and ferredoxin reductase (Fr). Usually, in P450 enzyme reactions, there are two electrons transferred from NAD(P)H through the Fd and FrFr pathway, which can be divided into two transfer processes. The first one happens between the CYP-substrate complex and Fd, and the second one happens between CYP-dioxygen-substrate complex and Fd. According to the theoretical and experimental results, in CYP101A1, Arg112 plays important roles in the first electron transfer[The heme proximal face, where the iron atom is closest to the enzyme surface, is the likely binding site for the electron transfer complex.  transfer. Compare1 and helix G which can be classified as channel 2a according the common nomenclature[2[2 helix only has one turn, and in P450epoK these two helices are nearly vertical. Although the overall folds of CYPs are strongly conserved, the precise position of various structure elements differs substantially, especially in the regions controlling substrate specificity. In the structure reported here, TxtE has the characteristic P450 fold. A clearly defined substrate access channel was observed between helix B′enclature. Channelenclature, CYP121[enclature and CalOclature[2 form twoclature[2 and P450clature[2. In CYP1CYP101A1 and a water molecule are required to form hydrogen bonds with dioxygen simultaneously for the cleavage of dioxygen. In TxtE, Thr250 and HOH309 may have the same roles in oxygen activation although the precise positions of HOH309 and Thr250 may be changed after dioxygen binding due to the rearrangement of water molecules. In CYP101A1, protons are also transferred through widened helix I, but the water chain only extend to Glu366CYP101A1. Compared with CYP101A1, the kink of the helix in TxtE is wider than that in CYP101A1. Therefore it is easier to form a complete water chain through this kink in the reaction process.In the reaction of CYPs, protons should reach the active site through a hydrogen bond chain involving water molecules. Unfortunately, few CYP structures show a clear proton transfer pathway from the active site to the external solvent except CYP158A2 and P450The molecular docking suggests that carboxyl group and amino group of L-tryptophan form salt bridges or hydrogen bonds to Arg59, Tyr89, Asn293, Thr296 and Glu394. The bond lengths (from proton donor to accepter) are shown in Figure S1The enzyme activity assay. The enzyme reaction products were analyzed by using LC-MS. The m/z of L-tryptophan is 205, and m/z of L-4-nitrotryptophan is 250. Ions at m/z = 250 were detected in the products which were catalyzed by using un-boiled TxtE, but could not be detected in the products which were catalyzed by using boiled TxtE.(PDF)Click here for additional data file.Materials and Methods S1(PDF)Click here for additional data file.


Pro have been described, including the Ala-specific editing domain (INS) of most bacterial prolyl-tRNA synthetases (ProRSs) and an autonomous single-domain INS homolog, YbaK, which clears Cys-tRNAPro in trans. In many species where ProRS lacks an INS domain, ProXp-ala, another single-domain INS-like protein, is responsible for editing Ala-tRNAPro. Although the amino acid specificity of these editing domains has been established, the role of tRNA sequence elements in substrate selection has not been investigated in detail. Critical recognition elements for aminoacylation by bacterial ProRS include acceptor stem elements G72/A73 and anticodon bases G35/G36. Here, we show that ProXp-ala and INS require these same acceptor stem and anticodon elements, respectively, whereas YbaK lacks inherent tRNA specificity. Thus, these three related domains use divergent approaches to recognize tRNAs and prevent mistranslation. Whereas some editing domains have borrowed aspects of tRNA recognition from the parent aminoacyl-tRNA synthetase, relaxed tRNA specificity leading to semi-promiscuous editing may offer advantages to cells.Errors in protein synthesis due to mispairing of amino acids with tRNAs jeopardize cell viability. Several checkpoints to prevent formation of Ala- and Cys-tRNA ARSs catalyze aminoacylation in a two-step reaction, wherein the cognate amino acid is first condensed with ATP to form an aminoacyl-adenylate intermediate followed by transfer of the amino acid to the 3′-adenosine of tRNA . Aminoactrans . The roltrans ,3,5.Pro  inserted between motifs 2 and 3 of the ProRS aminoacylation domain  to discriminate against Ala and Cys, which leads to the formation of Ala- and Cys-tRNAPro ,7. In mon domain A catalyzcylation . Howeverg domain A and thuin trans ,10.FiguE. coli ProRS and tRNAPro anticodon bases G35 and G36, in addition to acceptor stem bases G72 and A73 , C. crescentus ProXp-ala recognition is independent of the anticodon but shows a strong dependence on the same tRNAPro acceptor stem elements used for recognition by ProRS. Finally, E. coli YbaK appears to lack any tRNA specificity and is likely to rely exclusively on ternary complex formation with ProRS to specifically deacylate Cys-tRNAPro . Our data show that although evolutionarily related, these enzymes have evolved divergent mechanisms of tRNA recognition. Whereas INS does not possess intrinsic recognition of tRNA-tRNAPro . Overall14C]-Ala (151 mCi/mmol), [α-32P]ATP and [32S]-Cys (1075 Ci/mmol) were from PerkinElmer Life Sciences.All amino acids and chemicals were purchased from Sigma unless otherwise noted. -Ala in buffer A  for ∼2 h at room temperature. G1:C72/U70, C70U, ΔC1 and WT tRNAPro (8–10 µM) were first 3′-[32P]-labeled using tRNA nucleotidyltransferase as described  and 1–300 mM Ala in buffer A for ∼2 h at 25°C. Escherichia coli AlaRS and E. coli and human ProRS charging efficiencies were ∼70%.All escribed . Ala-tRNPro mutants and microhelixPro was carried out using biotinylated dinitro-flexizyme (dFx) (Thermo Scientific) following published conditions with minor modifications  to a final concentration of 25 mM and 5 mM, respectively. dFx-catalyzed aminoacylation was carried out for 2 h on ice. Reactions were quenched with 120 μl of 0.3 M NaOAc, pH 5 . Flexizyme aminoacylation efficiency ranged from 30 to 60%. Biotinylated dFx was removed by incubating the reaction mixture with 150 μl of streptavidin agarose resin (Novagen) for ∼15 min at room temperature followed by centrifugation in a table top centrifuge at 4°C for 2 min at 2000×g. The supernatant containing the aminoacylated tRNA was removed. WT and C70U tRNAPro and 3′-[32P]-labeled A73U and ΔC1 tRNAPro were aminoacylated using WT E. coli ProRS and [35S]-Cys or unlabeled Cys, respectively, while WT, G70U and G3:U70 tRNACys were aminoacylated by WT E. coli CysRS as previously described  was carried out for 6 h on ice with an efficiency of ∼15%. Following aminoacylation, aminoacyl-tRNAs (aa-tRNAs) were phenol-chloroform-extracted and ethanol precipitated. The aa-tRNA pellet was dissolved in diethylpyrocarbonate-treated water and stored at −80°C for use in deacylation assays.Aminoacylation of C1G, G1:C72, A73C, G72C, G1:C72/C73, G35C, G36A, G36C, G37A, G37C and G38C tRNAications . BrieflyE. coli ProRS or 1 µM ProXp-ala was carried out at 25°C according to published protocols , 5 mM MgCl2 and 0.1 mg/ml BSA]. Reactions containing ProXp-ala were performed in buffer C. Deacylation of 0.4–0.7 µM Cys-tRNAs by 0.2–0.7 µM E. coli YbaK was carried out at 37°C according to published protocols  in 200 mM NaOAc (pH 5.0) on ice. Aminoacyl-[32P]AMP and [32P]AMP products were separated on polyethyleneimine-cellulose TLC plates and analyzed as previously described (kobs. All data represent the average of three experiments with the standard deviation shown.Deacylation of Ala-tRNAs 0.75–1 µM) by 0.3 or 3 µM rotocols . ProRS a5–1 µM byescribed . FollowiE. coli ProRS contain an INS editing domain between conserved class II synthetase consensus motifs 2 and 3, some bacteria such as C. crescentus, lack this domain and instead possess a truncated mini-INS domain. Thermus thermophilus ProRS is an example of a ProRS that lacks INS but contains a noncatalytic C-terminal extension. Several free-standing INS domain homologs are also widely encoded in bacteria (E. coli and C. crescentus) and ProXp-ala (C. crescentus)  A.Proin vitro, and this interaction may facilitate correct hydrolysis of misacylated Cys-tRNAProin vivo  and a G72C variant at rates similar to WT tRNAPro. We next charged a 21-nt RNA that mimics the acceptor stem of E. coli tRNAPro  or the loop sequences alone (AC-loop) resulted in deacylation rates similar to those measured for WT E. coli tRNAPro   of a discriminator base variant of tRNAPro (A73C) was observed in the presence of ProXp-ala. The triple mutation G1:C72/A73C resulted in the strongest effect in ProXp-ala deacylation (556-fold decrease). In contrast, single point changes at positions C1 (ΔC1 and C1G) and G72 (G72C) only resulted in relatively minor (up to 13-fold) defects in editing. Thus, the discriminator base A73 and the C1:G72 base pair provide the major contribution to ProXp-ala specificity. This conclusion is reinforced by the low activity of ProXp-ala against human cytosolic Ala-tRNAPro, which naturally contains a G1:C72 base pair and a C73 as discriminator base  . Interesro)  depends on the anticodon base G34, which is also critical for aminoacylation (Phe by Streptococcus pneumoniae PheRS (E. coli isoleucyl-tRNA synthetase (IleRS) appears to use different combinations of nucleotides for aminoacylation and editing. Whereas A73, the anticodon triplet, and the reverse Hoogsteen U8:A14 base pair of tRNAIle are critical for aminoacylation, editing is only affected by changes in the D-loop and the anticodon triplet (Aquifex aeolicus leucyl-tRNA synthetase (LeuRS) requires A73, tertiary interactions that stabilize the core structure, as well as a specific orientation of the variable arm found in tRNALeu. Although not required for aminoacylation, LeuRS editing activity is mildly affected by elements in the tRNALeu anticodon arm  are not only important for aminoacylation but also are indispensible for ProRS post-transfer editing. In contrast, the activity of the ProRS editing domain does not depend on the acceptor stem sequence of tRNAPro, as shown by the efficient deacylation of E. coli tRNAPro acceptor stem variants, as well as human tRNAPro. This is in sharp contrast to the aminoacylation activity of E. coli ProRS, which seems to rely on two critical recognition elements (A73 and G72) in the acceptor stem. Human tRNAPro, which lacks these elements is not aminoacylated by the bacterial enzyme. Moreover, the lack of WT Ala-tRNAAla deacylation, and the robust deacylation of a chimeric tRNAAla variant containing a Pro-specific AC-loop, suggests that editing of Ala-tRNA by ProRS requires specific interaction between the ProRS ABD and the tRNAPro anticodon loop. The co-crystal structure of T. thermophilus ProRS in complex with tRNAPro shows that the three anticodon bases are splayed out and interact via specific hydrogen bonding to ProRS ABD residues , others, such as YbaK, are more promiscuous, using interactions with synthetases and other cellular factors to avoid correct aa-tRNA hydrolysis. Semi-promiscuous editing may offer advantages to cells, in providing a single factor to carry out multiple proofreading activities.YbaK, ProXp-ala and INS are all part of the INS superfamily of proteins that also includes three other putative editing factors, ProXp-x, ProXp-y and ProXp-z, whose functions have not yet been reported ,25. The National Institutes of Health [RO1 GM049928 to K.M-F.] and the Japan Society for the Promotion of Science Grants-in-Aid for Specially Promoted Research [21000005 to H.S.]. Funding for open access charge: Institutional support.Conflict of interest statement. None declared.


Adoptive transfer of antiviral T cells enhances immune reconstitution and decreases infectious complications after stem cell transplantation. Information on number and function of antiviral T cells in stem cell grafts is scarce. We investigated (1) immunomodulatory effects of G-CSF on antiviral T cells, (2) the influence of apheresis, and (3) the optimal time point to collect antiviral cells.CMV-, EBV- and ADV-specific T cells were enumerated in 170 G-CSF-mobilized stem cell and 24 non-mobilized platelet donors using 14 HLA-matched multimers. T-cell function was evaluated by IFN-γ ELISpot and granzyme B secretion. Immunophenotyping was performed by multicolor flow cytometry.in vitro expansion rate upon antigen restimulation. However, T-cell function was significantly impaired, as expressed by a mean reduction in secretion of IFN-γ  and granzyme B  as well as CD107a expression (27%). Clinical follow up data indicate that the first CMV-reactivation in patients and with it the need for T-cell transfer occurs while the donor is still under the influence of G-CSF.G-CSF treatment did not significantly influence frequency of antiviral T cells nor their To overcome these limitations, T-cell banking before mobilization or recruitment of third party donors might be an option to optimize T-cell production. Peripheral blood stem cells (PBSCs), which can be collected after granulocyte colony-stimulating factor (G-CSF) treatment, have been increasingly used for allogeneic stem cell transplantation in recent years Apart from GvHD, infectious complications of persistent viruses like cytomegalovirus (CMV), Epstein-Barr virus (EBV) and adenovirus (ADV) remain a problem in vivo and in vitro. To gain insight into the antiviral T-cell repertoire, we determined the frequencies of CMV-, EBV- and ADV-specific T cells in stem cell and HLA-typed platelet donors using overlapping peptide pools and multimer staining. Furthermore, we analyzed clinical data on recipients' and donors' CMV-serostatus and time of first CMV reactivation.To further clarify the effects of G-CSF on donor T cells, we addressed the questions of whether (1) multimer monitoring and detection of interferon-gamma (IFN-γ)-secreting cells by enzyme-linked immunospot (ELISpot)-based technique after short-term antigen stimulation are suitable tools to evaluate antiviral CTLs in various samples from stem cell donors, (2) G-CSF mobilization and apheresis influence antiviral T-cell levels, and (3) G-CSF treatment influences the functional activity of CTLs both Here, we analyze the influence of G-CSF stimulation on antiviral T-cells to obtain (1) better understanding of the kinetics of antiviral reconstitution, (2) determine the most effective subpopulation of antiviral T cells, and (3) select an optimal primary transplant or third-party donor for patients requiring antiviral T-cell therapy early after HSCT.Written informed consent was obtained from all donors and patients. Sample collection and analyses were part of an extended monitoring program conducted in the course of routine sampling for clinical follow-up. The study was approved by the Ethics Committee at the Hannover Medical School and is registered as #2906 for patients and as #1142-2011 for healthy donors. Sample material was obtained from 170 PBSC donors between February 2011 and December 2012. Each donor's HLA type, CMV and EBV serostatus were determined. ADV serology was not available. The following samples were collected: whole blood before G-CSF mobilization (WB), whole blood after G-CSF mobilization on the day of apheresis (WBM), blood from the apheresis tubing set (A) and an aliquot from the graft (G). All samples were processed within 48 h, as longer storage yielded no reliable results .G-CSF mobilization was performed by administering 10 µg/kg/day of G-CSF  for four consecutive days. Peripheral blood samples were collected via venipuncture into collection tubes containing EDTA as an anticoagulant . CD34All reagents/antigens used in this study are abbreviated as follows: multimer (m), peptide (p) or peptide pool (pp)-virus_viral protein_HLA restriction. For example, the multimer containing the HLA-A*02:01-restricted epitope NLVP of CMV protein pp65 is abbreviated “mCMV_pp65_A02”, and the EBNA1 peptide pool is abbreviated “ppEBV_EBNA1”. A nonsense peptide (mNeg) was used to determine unspecific background of multimer staining. A complete list of antigens/reagents is provided in Table S1 in 6 cells were used. Percentages of specific multimer staining were determined by subtracting the non-specific background value (mNeg). Flow cytometric analysis was performed on an FC500  as published previously 100 µl of blood  was stained with HLA-matched multimers (Table S1 in 7 cells per ml in 24-well plates (Sarstedt). G-CSF and/or antigens were added for the various read-out experiments detailed below.PBMCs were isolated from peripheral blood of platelet donors and from HSC donors before (WB) and on the day of donation . Cells were resuspended in T-cell culture medium (T-CM) consisting of RPMI1640  supplemented with 10% heat-inactivated human AB serum  plus 50 U/ml IL-2  and cultured overnight at a density of 1×10+ lymphocyte gate were analyzed, and the proportion of G-CSFR+/CD4+ and G-CSFR+/CD8+ cells was expressed as a percentage of all CD3+ lymphocytes analyzed. Upregulation of G-CSFR was three-fold on CD4+ T cells and 17-fold on CD8+ T cells after in vivo G-CSF treatment (data not shown).Flow cytometric analysis of G-CSF receptor (G-CSFR) expression was done on PBMCs  using peridinin chlorophyll (PercP)-conjugated anti-CD3, fluorescein isothiocyanate (FITC)-conjugated anti-CD4, allophycocyanin (APC)-conjugated anti-CD8 , and R-phycoerythrin (R-PE)-conjugated anti-G-CSFR monoclonal antibodies (mAbs) . Flow cytometric analyses were performed using a FACSCanto II flow cytometer (BD Biosciences) and FACSDiva software (V6.1.2). At least 30,000 events in the CD36 cells per well. Cells were incubated with 10 µg/ml Brefeldin A (Sigma-Aldrich) for 12 hours and stained with CD3-PerCP and IFN-γ-R-PE (Beckmann Coulter). Intracellular staining was performed using the IntraPrep Kit (Beckmann Coulter) according to the manufacturer's instructions. At least 30,000 CD3+ cells were acquired in the lymphocyte gate. The proportion of IFN-γ+/CD3+ cells was analyzed and expressed as a percentage of total CD3+ lymphocytes. The IFN-γ concentration in untreated PBMCs (controls) was used as the reference value (100%). At 10 ng/ml G-CSF a reduction of IFN-γ-producing cells to 39% compared to control cells was detected  7 cells/ml were stimulated in 24-well plates with 10 µg/ml of the respective single HLA-matched peptides (Table S1 in + cells). Supernatants were analyzed for granzyme B secretion by ELISA (eBioscience). The percentages of CD8+ naïve (N), central memory (CM), effector memory (EM) and terminally differentiated effector memory (TEMRA) T-cell subpopulations were analyzed by additional staining with anti-CD62L-APC-Cy7 and anti-CD45RA-PerCP-Cy5.5 mAbs (both BioLegend).Baseline frequencies of CMV- and EBV-specific T cells in freshly isolated PBMCs were assessed by multimer staining prior to peptide stimulation. 1×105 PBMCs  or 1.25×105 PBMCs (after 7-day stimulation) were plated in 100 µl T-CM/well and incubated overnight with 10 µg/ml CEF pool , 10 µg/µl peptide, 10 µg/ml peptide pool or without antigen (negative control). Spots were counted using an ImmunoScan Core Analyzer and the results analyzed using ImmunoSpot 5.0 Academic software . Means of tests were calculated and expressed as spot-forming units (sfu).Virus-specific IFN-γ-producing T lymphocytes were enumerated by IFN-γ ELISpot assay as described previously In order to determine the influence of G-CSF treatment on cytotoxic activity of antiviral T cells, we performed a granzyme B ELISpot assay as described previously. 6 peptide- or peptide-pool-stimulated PBMCs were restimulated with pCMV_pp65_A02, pEBV_BZLF1_B08, ppCMV_pp65 and ppEBV_BZLF1, respectively. Phycoerythrin-Cyanin 7 (PCy7)-conjugated anti-CD107a antibody  was added and cells were cultured at 37°C, 5% CO2. After one hour, Monensin  was added and cells were further incubated for 4 hours before staining with anti-CD3 mAb  and anti-CD8 mAb .Degranulation of antiviral T cells as a surrogate marker for cytotoxicity Statistical analyses were performed using the Mann-Whitney test or Kruskal-Wallis test followed by Dunn's multiple comparison on GraphPad Prism v5.02 software . Levels of significance were expressed as p-values .Healthy G-CSF-mobilized stem donors (n = 170) were included in this study. Serology for CMV was available for 139 donors . EBV serology was available for 121 donors . Determination of ADV serostatus was not possible, as no fast routine assays for detection of various strains are available. Intermediate analysis showed comparable assay results for mobilized samples  differed significantly between the different sample types , mCMV_pp65_B07 (2.46%) and mCMV_pp50_A01 (0.67%) were significantly different between CMV-seropositive and CMV-seronegative individuals  . A similSurprisingly, the mCMV_IE1-A02 gave false positive staining results in CMV-seronegative individuals . In ELISpot analysis, ppBZLF1 yielded the highest spot counts in 32 of 101 donors (53/156 samples).The current study results for dominance of CMV- and EBV-specific epitopes from pp65 and BZLF1, respectively, are in concordance with those from our recent study n vivo G-CSF–mobilized stem cell donors (n = 81) were compared for their IFN-γ production upon stimulation with CMV-, EBV- and ADV-overlapping peptide pools. Analysis of the total number of sfu showed that G-CSF treatment led to a significant reduction in spots for all antigens tested   (T cells from untreated platelet donors (n = 24) and from i5.8 sfu) . This covs. 0.8) .Secretion of IFN-γ by cells from the same donor before and after G-CSF treatment (n = 7) was additionally compared . Again, in vitro with 10 ng/ml G-CSF for one week were evaluated by multimer staining, IFN-γ ELISpot, granzyme B ELISpot, granzyme B ELISA and CD107a degranulation assay. The frequency of multimer-positive T cells obtained after 7 days of peptide stimulation did not differ significantly between G-CSF-treated and untreated cells for the single HLA-restricted peptides used , central memory (CM), effector memory (EM) or terminally differentiated effector memory (TEMRA) CD8+ T cells was observed after peptide-specific stimulation in the presence or absence of G-CSF . While the frequency of multimer+ T cells remained stable, the percentage of IFN-γ secreting cells decreased after G-CSF treatment for pCMVpp65_A02 (63.7%) and pCMVpp65_B35 (45.7%). For EBV, the percentage of IFN-γ producing cells decreased to 83.1% (pEBV_EBNA1_B35) and 50.1% (pEBV_BZLF1_B08).T-cell function was evaluated by IFN-γ ELISpot on day 7 ; shown iThe immunosuppressive effects of G-CSF treatment on the cytotoxic function of T cells were further assessed by detection of granzyme B secretion and expression of lysosome-associated membrane protein CD107a on the cell surface. Granzyme B was detected .in a target-independent ELISA,  as well Follow-up data for seropositive family donors and patients (3/5) showed reconstitution of CMV-CTLs after HSCT, as expected  and donors (D) and the time of first CMV reactivation in order to determine the demand for adoptive therapies. The proportion of high-risk patients with an R+D+ and 103 R+D− patients between June 1998 and June 2012. The mean day of CMV reactivation was day +39 and +40 in R+D+ and R+D− patients, respectively. PBSC transplanted from G-CSF treated donors may still be impaired in their function at this early time of CMV-reactivation Early CMV reactivation (<day +100) occurred in 172 RHematopoietic stem cell transplantation is used to cure many malignant, benign and genetic bone marrow disorders, solid tumors, immunodeficiencies, metabolic diseases and autoimmune disorders It is well known that despite to a one-log increase in the number of T cells transplanted with G-CSF–mobilized PBSCs compared to bone marrow grafts no higher rates of severe aGvHD are associated whereas the incidence of chronic GvHD increases in vivo G-CSF-mobilized T–cell populations suggesting that this cytokine has a direct effect on T cells.G-CSF influences T cells by various mechanisms: (A) It inhibits the secretion of type I cytokines on the single-cell level as well as by reducing the population of cytokine-secreting cells in vitro and in vivo G-CSF treatment, we were able to show that G-CSF mobilization does not affect the frequency of antiviral T lymphocytes, but strongly impairs the functionality by reducing the capability to secrete IFN-γ (40% to 75% reduction) and cytolytic molecules like granzyme B (32% reduction). The reduced cytolytic function as determined by the reduction of granzyme B release in a target cell dependent manner and the decrease of CD107a expression on cell surface of CD8+ T cells further confirmed the negative impact of G-CSF on T-cell functionality (in vitro G-CSF treated PBMCs were not conclusive (data not shown). In order to gain insight into the underlying mechanism in G-CSF-induced impairment of T-cell function, subpopulations of T cells should be investigated separately in a larger cohort.By evaluating the effector function of antiviral T cells after ionality . Prelimiet al.+ G-CSF-mobilized T cells were functionally similar to G-CSF-untreated CD154+ T cells, making this isolation strategy more suitable. However, this finding is based on the comparison of a small number of mobilized and non-mobilized donors and not on a comparison of the same donors before and during G-CSF treatment, as done in this study.Recently, Samuel et al. described the successful isolation of functional CMV-specific T cells from G-CSF-mobilized donors after short-term stimulation using CMVpp65 overlapping peptide pool in vivo. Furthermore, seropositive patients with grafts from seronegative donors were the group at highest risk of (recurrent) CMV reactivation While avoiding additional apheresis by using surplus material from mobilized donors might be beneficial for the donor, it remains unclear whether the G-CSF-treated T cells will achieve control of viral reactivation The duration and characteristics of functional impairment of T-cell functions after G-CSF treatment were investigated in several studies. Toh and colleagues Apart from considerations for adoptive T-cell transfer, the duration of G-CSF influence in each donor, in particular the effects on T-cell function after HSC donation should be monitored to determine when T cells recover. This might be essential for donor safety. There are reports showing that donors suffer from side effects like bone pain, headache and flu-like symptoms in vivo G-CSF treatment. This suggests that cells of G-CSF-treated donors might not be the best choice in cases where donor lymphocyte infusion or adoptive T-cell transfer is required for treatment of viral reactivation early after transplantation. Further studies are needed to determine whether the adoptive transfer of G-CSF-mobilized T cells is comparable to that of cells from non-mobilized donors or mobilized donors in whom the effects of G-CSF have worn off. In order to increase donor safety, the possibility of higher susceptibility to infections after G-CSF treatment should be investigated and the time needed for T-cell recovery should be determined.Our results demonstrate that antiviral T-cell function is impaired after File S1Supporting Information files.Figure S1 in File S1. Effects of sample storage time and temperature. Multimer analysis is usually performed on whole blood samples stored at room temperature within 24 h. As apheresis and graft processing took longer, some samples were analyzed later. Thus, we evaluated the influence of storage time and temperature on the reliability of the results. Nine patient samples were analyzed consecutively to detect changes in CD3, mNeg (non-specific background) and total percentages of mCMV_pp65_A02 and/or mCMV_pp65_B07 over time. Representative examples are shown. Storage time is plotted on the X-axis, and percentages of total CD3 (A), mCMV_pp65_A02 (B) and mCMV_pp65_B07 (C) on the Y-axis. Staining results for fresh samples (unstored) are depicted as a reference line , and those for samples stored at room temperature (RT) and 4°C as filled circles (black line) and white circles (dashed line), respectively. Changes in CD3 and specific multimer percentages over time were less pronounced at 4°C in all sample types. Background staining did not change significantly at 24 h or 48 h (data not shown). After 48 h of storage at 4°C, a mean of 93%  and 96%  of the frequency in the fresh sample (baseline) was obtained. After 72 h, the variation from baseline was more pronounced, even in samples stored at 4°C. For example, in the 5 samples analyzed with mCMV_pp65_A02, only 82% and 30% (mean) of the multimer-positive population detected in fresh material was detected after 72 h of storage at 4°C or room temperature, respectively (Table S2 in File S1). Therefore, donor samples analyzed after more than 48 h (n = 2) were excluded from further analysis. As storage at 4°C led to less deviation from baseline (percentage change), it is preferable. Figure S2 in File S1. Gating strategy. (A) Gating strategy for one-platform quantification of percentage and total numbers of CD3CD8-double positive T cells. From left to right: A gate is set on the fluorescent beads for calculation of absolute cell numbers. Flow Count over time is recorded in order to detect changes in the flow rate. The 3rd plot from the left shows gating on lymphocytes for exclusion of debris and other mononuclear cells. CD4CD8-double-positive cells are excluded. The percentage of CD3+CD4+ and CD3+CD8+ T cells is determined in the lymphocytes (beads and CD4+CD8+ cells excluded) and the absolute number per µL blood can be calculated. For A and G samples, beads were added to confirm constant flow rates and signal intensity. (B) Gating strategy for quantification of multimer-positive T cells. From top to bottom, staining in all 3 sample types is shown. The percentage of multimer positive cells is given as percentage of CD3+CD8+ T cells. FSc – forward scatter, SSc – side scatter, FITC - fluorescein isothiocyanate, PE - Phycoerythrin, PCy7 – Phycoerythrin-Cyanin 7, WBM – whole blood mobilized, A – material from apharesis filter, G – material from graft quality control, Lymph – lymphocyte gate. For multimer abbreviations, please refer to Table S1 in File S1. Figure S3 in File S1. In vitro application of G-CSF reduces IFN-γ production in PBMCs. The optimal G-CSF concentration for stimulation was determined by treating PBMCs with different concentrations (5 to 50 ng/ml) of G-CSF for one week and subsequent stimulation with anti-CD3 monoclonal antibodies on day 7. IFN-γ production was measured by intracellular staining and flow cytometry. Results of four independent experiments are shown and expressed as mean ± SD. Asterisks indicate significant differences (**p<0.01). The IFN-γ production in untreated PBMCs (controls) was used as the reference value (100%). The percentage of IFN-γ-expressing cells decreased after G-CSF. A strong effect was achieved using 10 ng/ml G-CSF. At this dose, only 38.73% of IFN-γ-producing cells were retrieved on day 7. Because there was no significant difference after treatment with higher concentrations of G-CSF, 10 ng/ml G-CSF was used in further experiments. Figure S4 in File S1. Staining with specific multimers is not significantly influenced by sample type. (A) Unspecific background staining (mNeg): The background detected (mNeg) is shown for each donor sample and the sample type is indicated on the X-axis. Groups were compared by Kruskal-Wallis analysis followed by Dunn's multiple comparison. (B) Exemplary analysis of mCMV_pp65_A02 staining in different samples  in CMV-seropositive donors. (C–E) Blots C to E show the comparison of detection of multimer positive cells in the mobilized samples  to freshly isolated whole blood (WB). Percentages detected in the mobilized samples only differed slightly from those in WB samples. Sample sources: (A) apheresis tubing set, (G) graft, (WB) whole blood, (WBM) mobilized whole blood on day of apheresis. Figure S5 in File S1. Multimer staining examples. Examples for multimer staining in the different sample types  in 5 donors are depicted. The total sum of multimer positive CD3+CD8+ T cells after substraction of background (mNeg) for CMV (ΣmCMV) and EBV (ΣmEBV) is given. These values are also reported in +CD8+ T cells. Figure S6 in File S1. Multimer staining with mCMV_pp65_A02 (tetramer) and mCMV_IE1_A02 (pentamer). Staining examples in CMV-seropositive  and CMV-seronegative  donors are given, for each donor, all 3 samples types are shown . Staining with mCMV_pp65_A02 (tetramer) gives clear populations in CMV+ donors and low background in CMV-donors. In contrast, staining with mCMV_IE1_A02 (pentamer) results in less defined populations, furthermore positive staining results are found in CMV-donors . Figure S7 in File S1. Functional assays for in vitro G-CSF-treated pCMV_pp65_A02-stimulated cells on day 7. (A) IFN-γ ELISpot reveals a decrease in IFN-γ secretion after G-CSF treatment. Unstimulated cells served as control. Means of spots per well (spw) are indicated. (B) Granzyme B ELISpot shows a reduction in granzyme B secretion in a target cell dependent manner. Unloaded target cells served as a control. Means of spw are indicated. (C) Representative staining results from the CD107a degranulation assay show a reduced degranulation activity of CD8+ T cells after G-CSF treatment. +/D+ patients.Figure S8 in File S1. Follow up in R (A–C) Number of CMV-CTLs detected in the patient and donor before HSCT and post-HSCT follow-up in patients UPN2104 (A), UPN2131 (B) and UPN2145 (C). CMV reactivation led to expansion of CMV-CTL in UPN2145 (C). Figure S9 in File S1. Overview of CMV-serostatus of recipients and donors at Hannover Medical School (MHH). Number of CMV-seropositive patients with seropositive donors  and seronegative donors  per year. Table S1 in File S1. Overview of multimers and antigens used.Table S2 in File S1. Variation of total multimer-positive population from baseline (fresh samples) after 24, 48 and 72 hoursof storage at 4°C or room temperature (RT).Table S3 in File S1. Donors with no, low and high percentages of multimer-positive T cells.Table S4 in File S1. Sequence alignment for the A02-restricted IE-1 epitope VLAELVKQI.(DOC)Click here for additional data file.


Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species.A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm  Site-specific recombinase (SSR) technology is an important molecular biotechnology that was developed during the 1980s. Through the genetic manipulation of the eukaryotic genome and exogenous DNA by SSR-mediated recombination between two recombination target sites (RTs), SSR can induce the replacement, inversion and tissue-specific knockout of target genes loxP from Escherichia coli phage P1 FRT from the 2-µm plasmid of Saccharomyces cerevisiaeatt from the Streptomyces phage ФC31 loxP system for generating a tissue-specific knockout mice model attP [34 base pairs (bp) long] and attB (39-bp long) is directional and irreversible et al. developed an efficient site-specific integrase-mediated repeated targeting (SIRT) method to target a single locus repeatedly and to facilitate targeted mutagenesis in Drosophilaatt system has also had an important role in the integration of transgenes into the mammalian genome and for the development of gene therapy FRT system has also emerged as a powerful tool to manipulate genomes of transgenic plants, mammals, insects and other higher eukaryotic model organisms. In recent years, the FLP/FRT system has been widely used in Arabidopsis thalianaOryza sativa) Mus musculus) Drosophila melanogasterCaenorhabditis elegansCurrently, the most commonly used SSR systems are Cre/FRT sites (FLP recombination target site) and mediate site-specific recombination reactions between two identical FRT sites FRT sites determine the outcome  of the FLP recombinase-mediated recombination reaction FRT system has been studied and used extensively for genome modification. FLP recombinase has been shown to catalyze site-specific excision of selectable marker genes from various transgenic plant species, including tobacco (Nicotiana tabacum) Zea mays) FRT system has also been used for gene knockout and conditional gene activation in mammalian cells and transgenic mice Drosophila genome FRT and Cre/loxP systems have been jointly applied to investigate the function of two genes at the same location in the Drosophila genome Drosophila, a strategy called FLP recombinase-mediated cassette exchange (FLP-RMCE) has been developed to avoid genomic position effects, which often confounds direct comparison of allelic transgenes Aedes aegypti) embryos Bombyx mori) FLP recombinase can identify specifically Bombyx mori, is a useful model lepidopteran that has been domesticated and reared for silk production for more than 5000 years B. mori genomes FRT system in silkworm by Tomita et al., who used the system to achieve site-specific excision of an extrachromosomal (plasmid-based) gene-containing DNA fragment in silkworm cells and embryos et al., who used the ФC31/att system to achieve site-specific recombination between attB and attP sites in cultured silkworm BmN4 cell lines The silkworm, FRT site-specific recombination system to achieve site-specific excision of a target gene at a predefined target site in transgenic silkworm genome. We demonstrate that injection of an FLP recombinase-expressing vector can induce site-specific excision in transgenic silkworms. This methodology will facilitate the development and application of the FLP/FRT system for the genetic manipulation of the silkworm and the potential use of this system for the analysis of silkworm gene function.In this report, we first describe a method using the FLP/B. mori bivoltine inbred strain Dazao has been maintained in our laboratory. It was necessary to change the diapause character of Dazao eggs for DNA pre-blastoderm microinjection. The 15°C-IMES germline transformation strategy used for the Dazao strain was based on the report of Zhao et al.The Chinese lineage piggyBac-derived vector pBac{3×P3-DsRed; FRT-3×P3-EGFP-SV40-FRT}  (ctcgagGTTCCCACAATGGTTAATTCG-3′) and SV40-R-SphI (5′-gactgcatgcTACGCGTATCGATAAGCTTTAAG-3′). The amplified fragment was double-digested with XhoI and SphI, and inserted between the XhoI/SphI site of the plasmid pSLfa1180fa FRT fragments were obtained by annealing the following sequences of the oligonucleotides: FRT-SpeI/XhoI-F (5′-ctagtGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCc-3′) and FRT-SpeI/XhoI-R (5′-tcgagGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCa-3′); FRT-SphI/BglII-F (5′-cGAAGTTCCTATTCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCa-3′) and FRT-SphI/BglII-R (5′-gatctGAAGTTCCTATACTTTCTAGAGAATAGGAACTTCGGAATAGGAACTTCgcatg-3′). The annealing conditions were as follows: initial denaturation at 94°C for 5 min; reduced by 1°C per 90 sec until 25°C; 25°C for 5 min; and then stored at 4°C.The equence)  was consFRT fragments were inserted between the SpeI/XhoI and SphI/BglII sites of the plasmid pSL{3×P3-EGFP-SV40}. The plasmid pSL{FRT-3×P3-EGFP-SV40-FRT} was generated. pBac{3×P3-DsRed; FRT-3×P3-EGFP-SV40-FRT} was then constructed by cloning a 1.3-kb AscI fragment from pSL{FRT-3×P3-EGFP-SV40-FRT} into AscI cut pBac{3×P3-DsRedaf} The two 48-bp gagctcATGCGCGTTACCATATATGGTG-3′) and A3-R-KpnI (5′-tataggtaccCTTGAATTAGTCTGCAAGAAAAG-3′). The amplified fragment was digested with SacI and KpnI, and inserted into the plasmid pSLfa1180fa to generate pSL-A3. The 0.37-kb silkworm A3 polyadenylation signal sequence (A3 polyA) was PCR-amplified from Dazao genome with an A3 polyA-F-SphI (5′-gcatgcatgcAGGAAGTGCTTCTAAGCGT-3′) and A3 polyA-R-BamHI (5′-gcatggatccGTGCTCCTAGCGTAACTGTC-3′) primer pair. The PCR product was digested with SphI and BamHI, and cloned into the plasmid pSL-A3 to generate pSL-A3-A3 polyA. The FLP recombinase gene was amplified by PCR from the plasmid pKhsp82-FLP ggtaccATGCCACAATTTGGTATAT-3′) and FLP-R-SphI (5′-gatcgcatgcTTATATGCGTCTATTTATGTAGG-3′). The 1.28-kb PCR product was inserted into the KpnI/SphI site of pSL-A3-A3 polyA to generate pSLA3-FLP.The FLP recombinase-expressing helper vector pSLA3-FLP  was consThe above sequences that are underlined show the restriction enzyme cutting sites. The sequences of the PCR products and resulting recombinant plasmids were confirmed by sequencing.piggyBac transposase. According to the 15°C-IMES germline transformation strategy Plasmid DNA for pre-blastoderm microinjection was purified using a QIAGEN Plasmid Midi Kit , and the prepared DNA solution was stored at –20°C until being used. pHA3PIG was used as the helper plasmid for the production of FRT system transgenic target strains (TTSs) were then produced.The expression of the red fluorescent protein (DsRed) and enhanced green fluorescent protein (EGFP) in G1 embryos, larvae, pupae and adults was detected using an Olympus MacroViewMVX10-AUTO fluorescent stereomicroscope  with a RFP or GFP filter, respectively. Filters passing light between 510 and 550 nm for DsRed, and between 460 and 490 nm for EGFP were used for excitation. The individuals with DsRed- and GFP-positive G1 offspring were identified as germline-positive transgenic silkworms. G1-positive larvae from different broods were reared (with each brood being a unit), and the FLP/HaeIII and circularized by overnight ligation at 16°C using T4 DNA ligase . The ligated DNA was treated with phenol/chloroform and then precipitated with ethanol. Approximately 50–100 ng ligated DNA was used as a template for inverse PCR. Primers were used to recover the flanking sequence of the piggyBac transposon as described by Ding et al.piggyBac left arm), the forward primer PLF (5′-CTTGACCTTGCCACAGAGGACTATTAGAGG-3′) and reverse primer PLR (5'-CAGTGACACTTACCGCATTGACAAGCACGC-3′) were used. For the 3′ junction (piggyBac right arm), the forward primer PRF (5′-CCTCGATATACAGACCGATAAAACACATGC-3′) and reverse primer PRR (5′-AGTCAGTCAGAAACAACTTTGGCACATATC-3′) were used. PCR was performed as follows: initial denaturation at 95°C for 5 min, then 35 cycles of 95°C for 30 sec, 63°C for 45 sec and 72°C for 3 min, followed by 72°C for 10 min.Genomic DNAs were extracted from G1 TTS moths and wild-type moths (as controls). DNA was purified using an improved phenol/chloroform method after proteinase K treatment PCR fragments were separated by electrophoresis in a 0.8% (w/v) agarose gel. Each single band was picked up from the gel and purified using a gel extraction kit . The purified fragments were cloned into the plasmid pMD19-T simple and sequenced with the M13F/R primer to identify the boundary sequence of the insertion site.www.ncbi.nlm.nih.gov) and the silkworm genome database [SilkDB (http://www.silkdb.org/silkdb/)]. Localization of the silkworm genomic insertion sites of the piggyBac-derived vector was completed using the SilkMap application (www.silkdb.org/silksoft/silkmap.html).Sequencing results were analyzed using NCBI BLAST searches  and P–R (5′-AATTCGAATGGCCATGGGACGTCGA-3′) were used to confirm individuals from FLP recombinase-mediated SSRSs of transgenic silkworms. The extracted genomic DNA from G1 TTS adults, G3 SSRS adults and wild-type adults were used as the template for PCR. The purified PCR fragments were cloned into the plasmid pMD19-T simple and sequenced with the M13F/R primer to identify the sequence of the FLP recombinase-mediated excision site.The primer pairs P–F  agarose gel. DNAs were transferred directly onto nylon filters  and immobilized by incubation for 30 min at 120°C. The probes were prepared as follows: a 720-bp EGFP fragment was amplified by PCR from pBac{3×P3-EGFPaf} with the primer pair pEGFP-f (5′-ATGGTGAGCAAGGGCGAGG-3′) and pEGFP-r (5′-CTACTTGTACAGCTCGTCCATGCCG-3′). A 678-bp DsRed fragment was amplified by PCR from pBac{3×P3-DsRedaf} with the primer pair pDsRed-f (5′-ATGGTGCGCTCCTCCAAGAACGT-3′) and pDsRed-r (5′- CAGGAACAGGTGGTGGCG-3′).25 µg genomic DNAs  were fully digested with These two PCR products were subjected to electrophoresis and recovered from the gel. Both fragments were labeled with DIG-High Prime reagent from the DIG High Prime DNA Labeling and Detection Starter Kit II . The DNA samples on the membrane were prehybridized for 1 h at 68°C, and hybridized overnight with the DIG-labeled EGFP probe and DsRed probe. The membrane was washed twice in 2×SSC containing 0.1% SDS for 15 min, and then washed twice at 65°C in 0.1×SSC containing 0.1% SDS for 15 min each time.The detection of hybridized DNA was done using a chemiluminescent method with ready-to-use CSPD  according to the manufacturer’s instructions. The blotting results were observed using a chemiluminescence imaging system .FRT site-specific recombination system in silkworm involves the following steps, as illustrated in piggyBac-mediated germline transformation of diapause silkworm strains FRT sites of the TTS genome was mediated by the FLP recombinase expressed by the helper vector pSLA3-FLP, resulting in the deletion of the 3×P3-EGFP expression cassette in G3 SSRSs  On FRT-flanked 3×P3-EGFP expression cassettes, 330 G0 non-diapause eggs from the wild-type B. mori strain Dazao were microinjected with the pBac{3×P3-DsRed; FRT-3×P3-EGFP-SV40-FRT} vector and helper plasmid pHA3PIG mixture. G0 adults were mated with each other or backcrossed with the wild-type Dazao strain. In total, we obtained 28 G1 broods, including two broods that had at least one DsRed- and one GFP-positive larva . The silkworm genomic sequences flanking the piggyBac transposon-derived construct in the genome of two TTSs were both single copies; the average frequency of FLP recombinase-mediated site-specific excision in two TTSs was 3.49%. To explore the feasibility and efficiency of FLP recombinase-mediated site-specific recombination in silkworm, heterozygous G1 male adults from each of the two TTSs were backcrossed to wild-type female adults to produce G2 non-diapause embryos for helper plasmid pSLA3-FLP microinjection. In total, 987 G2 embryos were injected, and 174 G2 DsRed- and GFP-positive adults were obtained. To screen for the individuals with germline site-specific recombination, 174 G2 DsRed- and GFP-positive adults were backcrossed with those from the wild-type Dazao strain and the 143 G3 broods obtained were analyzed for fluorescence phenotypes . FinallyFRT sites in the genome of these TTSs individuals was mediated by FLP recombinase.To confirm FLP recombinase-mediated site-specific excision of positive recombinant individuals, PCR was performed on genomic DNAs from G1 TTS adults, G3 SSRS adults and wild-type adults with the primers P–F and P–R . As showXhoI and BglII  Xenopus laevisFRT system-mediated site-specific gene excision was obtained in the current study, the recombination efficiency was similar to the piggyBac-mediated transgenic germline transformation of the silkworm FRT system is a potentially useful tool for the site-specific integration or knockout of transgenes in the silkworm.The results reported herein provide the first demonstration of the use of the FLP/karyotes . One posin vitro or in vivo, the risk of FLP toxicity still cannot be completely ruled out from the persistent expression of the FLP gene in hybrid offspring. Moreover, the FLP recombinase-mediated site-specific excision reaction between two FRT sites is reversible In our study, a direct injection method rather than sexual hybridization method was used to introduce and express the FLP gene in the pre-blastoderm of eggs from TTSs. The main disadvantage of the sexual hybridization method is that the FLP gene sequence would be introduced to the genome of the hybrid offspring, and unless it is crossed out, persistent FLP expression could negatively affect the presence or function of the target or donor genes. Although there have been no reports of FLP toxicity FRT system-based site-specific recombination technology has been widely used in Drosophila. Similar to Drosophila, B. mori is also a model organism for studies of the genetics of higher eukaryotes. The recombination activity of FLP recombinase in cultured cells and embryos of B. mori was confirmed as early as 1999, and the extrachromosomal recombination efficiency was approximately 20% in BmN4 cells and embryos FRT system in silkworms is that the optimum temperature of the FLP recombinase (30°C B. mori (approximately 25°C). The sophisticated Cre/loxP system is the most widely applied tool for genome manipulation at present, but the optimum temperature of Cre recombinase . Based on the results of our study, after the specific acceptor sites (FRT sites) have been generated by piggyBac-mediated transgenesis and suitable loci have been identified in silkworm, the introduction of transgenes by FLP recombinase-mediated site-specific recombination will be easy to generate and examine. By removing one or both of the two terminal sequences of the piggyBac transposon after integration, the insertion can be stabilized piggyBac-mediated random insertions into the silkworm genome, but also minimize the position effect in silkworm functional genomics research by means of creating and selecting appropriate TTS for subsequent germline transformation. Thus, once an appropriate and stable TTS with no or one of two terminal sequences of the piggyBac transposon after integration is established, this system would be a good candidate for site-specific transformation of B. mori. Furthermore, controlling for position effects by FLP/FRT system-based genomic targeting will also enable optimization of heterologous protein expression in B. mori for use as a protein bioreactor Strategies for genetic engineering generally require stable and inheritable modification of the target genome. Most methods for the introduction of exogenous DNA into the target genome are characterized by random integration. Random insertion mediated by transposons is particularly efficient in many different organisms, but the lack of control at the introduced DNA position leads to unpredictable variations in gene expression and undesirable mutagenesis of important genes. A germline transformation method using the B. mori genome manipulation. Our experiments have a huge improvement for B. mori genome manipulation using the FLP/FRT system since our experiments first obtained stable germline transformation in the individual level rather than the cell and tissue levels reported by Tomita et al, which only obtained the extrachromosomal (plasmid-based) site-specific excision FRT system in the genomic manipulation of silkworm and promote the establishment of a FLP/FRT system-based research platform for the functional analysis of unknown genes in silkworm. Moreover, the significance of this work is not confined to studies of silkworm functional genomics, but would also be relevant for the practical utilization of silkworm transgenic lines in sericulture, silkworm bioreactors and silkworm modeling.In conclusion, this study is the first to demonstrate the feasibility of FLP recombinase-mediated site-specific recombination for