The apoptotic potential of TRAIL-R2 on hHSC was confirmed by lentiviral-mediated knockdown. A functional inhibitory role for TRAIL-R3/4 was revealed by shRNA knockdown and mAb blockade, showing that these regulatory receptors limit apoptosis of hHSC in response to both oligomerised TRAIL and NK cells. A close inverse ex vivo correlation between hHSC TRAIL-R4 expression and susceptibility to apoptosis underscored its central regulatory role. Our data provide the first demonstration of non-redundant functional roles for the regulatory TRAIL receptors (TRAIL-R3/4) in a physiological setting. The potential for these inhibitory TRAIL receptors to protect hHSC from apoptosis opens new avenues for prognostic and therapeutic approaches to the management of liver fibrosis.The TRAIL pathway can mediate apoptosis of hepatic stellate cells to promote the resolution of liver fibrosis. However, TRAIL has the capacity to bind to regulatory receptors in addition to death-inducing receptors; their differential roles in liver fibrosis have not been investigated. Here we have dissected the contribution of regulatory TRAIL receptors to apoptosis resistance in primary human hepatic stellate cells (hHSC). hHSC isolated from healthy margins of liver resections from different donors expressed variable levels of TRAIL-R2/3/4 (but negligible TRAIL-R1)  The resultant liver fibrosis can ultimately lead to cirrhosis, portal hypertension and liver failure, responsible for more than a million deaths annually worldwide2. Targeted therapies able to specifically halt the progression and/or promote regression of liver fibrosis are therefore urgently needed.Chronic liver inflammation and injury from a variety of insults trigger a dynamic, reversible wound-healing response, in which matrix deposition is accompanied by matrix degradation. If there is chronic or repetitive injury, persistent accumulation of extracellular matrix and insufficient tissue remodelling lead to the formation of scar tissue4. In the quiescent state they contain multiple retinoid-rich droplets; upon liver injury they are activated to differentiate into proliferative and contractile myofibroblast-like cells, that produce the extracellular matrix components of scar tissue3. Termination of their pro-fibrogenic activity requires that HSC undergo apoptosis, senescence or reversion to a quiescent state5. Degradation of extracellular matrix will then outweigh new deposition, allowing fibrosis resolution and restoration of liver architecture5.Hepatic stellate cells (HSC), liver-specific pericytes residing in the Space of Disse, are the main cellular mediators of fibrogenesis in the liver7. Amongst these, NK cells are of particular interest because of their striking enrichment within the human liver, including a large CXCR6+TbethiEomeslo liver-resident subset8. In animal models, depletion of NK cells results in severely accelerated fibrosis progression whereas their activation ameliorates it11, suggesting that they play a major role in limiting fibrogenesis. NK cells can interact with HSC through a number of receptor/ligand pairs and have been shown to be able to kill them in an NKG2D and TRAIL-dependent manner14. We have previously shown activation of the TRAIL pathway in the HBV-infected liver; the ligand is induced on NK cells, allowing them to target hepatocytes and HBV-specific T cells, both of which upregulate the death-inducing receptor TRAIL-R217.It is increasingly recognised that many different components of the immune system have the capability to either promote or limit HSC activation and survival21. However, TRAIL can also bind to regulatory (decoy) cell-bound receptors TRAIL-R3 (DcR1) or TRAIL-4 (DcR2) that may protect against cell death, although to date there has been a scarcity of physiological demonstrations of this phenomena (demonstrable in over-expression systems25). We have confirmed TRAIL-dependent killing of primary human HSC (hHSC) by using lentiviral-mediated shRNA knockdown of the death-inducing receptor TRAIL-R2. We found that hHSC also express TRAIL-R3 and TRAIL-R4, both directly ex vivo and after activation. Importantly, we show that knockdown or blockade of these regulatory receptors enhances the susceptibility of hHSC to killing by oligomerised TRAIL and by TRAIL-expressing NK cells from patients with chronic hepatitis B (CHB). The baseline level of expression of the regulatory receptor TRAIL-R4 dictates the wide variability in susceptibility to TRAIL-induced apoptosis amongst hHSC from different donors, suggesting a role for regulatory TRAIL-Rs in limiting the resolution of liver fibrosis.In this work we have therefore focused on the potential for the TRAIL pathway to regulate stellate cell apoptosis. The ligand TRAIL has the capacity to initiate cell death by engagement with receptors TRAIL-R1 and TRAIL-R2, bearing intracellular death domains26, was not detectable above isotype staining using flow cytometry , as monitored by their expression of GFP , to simulate the clustering of receptors achieved by cell-bound TRAIL27. Induction of stellate cell apoptosis, quantitated by flow cytometry using caspase-3, was compared in live GFP-positive cells transduced with either shCTR or shTRAIL-R2  in transduced hHSC from different donors; this was consistently abrogated completely by TRAIL-R2 knockdown  to include TRAIL-R3 and TRAIL-R4. Activated hHSC not only expressed TRAIL-R2 but also had variable levels of TRAIL-R3 and TRAIL-R4 detectable on their surface  on activated hHSC isolated from liver tissue from 5 different donors  should therefore be investigated.Our data highlight the need for further studies to fully elucidate the contribution of TRAIL receptors in determining the lifespan of hHSC and resultant progression versus resolution of liver fibrosis. The capacity we observed of TRAIL-R4, in particular, to calibrate activated hHSC apoptosis 39. A concern has been the risk of accelerating liver fibrosis by preventing clearance of activated hHSC by endogenous TRAIL-expressing NK cells; our new data suggest that short-term TRAIL blockade may not have a detrimental effect in patients whose regulatory receptors are already limiting stellate cell apoptosis. Conversely, TRAIL is being considered as an anti-fibrotic therapy in other causes of progressive liver fibrosis due to ongoing liver injury. A recent study showed promising effects of a pegylated form of TRAIL in ameliorating liver cirrhosis by eliminating activated HSC in a rodent model of liver fibrosis26. Our findings imply that engagement of TRAIL-R2 without TRAIL-R3/4 would optimise stellate cell killing and the regression of fibrosis. This could be achieved, for example, with TRAIL receptor agonists which have recently been engineered to bind only to TRAIL death-inducing receptors and are unable to engage the inhibitory TRAIL-R3/440. Such an approach is supported by the superior efficacy of a TRAIL-R2 targeted ligand over native TRAIL in eliminating LX-2 or activated rat stellate cells in vitro40; further studies are needed to see if such reagents achieve the predicted enhanced clearance of human HSC.Our results also have therapeutic implications. In hepatitis B, we have previously proposed the use of TRAIL blockade as an adjunct to therapeutic vaccination because of its potential to prevent NK cell-mediated deletion of TRAIL-R2-bearing antiviral T cells and hepatocytes and thereby favourably shift the balance of immunity and immunopathologyAll experiments were performed in accordance with the approved guidelines and regulations.Ethical board approval for the study was submitted through the Royal London Hospital and Royal Free London NHS Foundation Trust. All participants of the study have given written, informed consent. All HBV infected patients were anti-HCV and anti-HIV antibody negative. The patients were stratified on the basis of their disease status Table : viral l41. The cells were either stained directly ex vivo (identity confirmed by autoflourescence and flow cytometric exclusion of other liver cells) or expanded and activated by culture on plastic in SteCM media  as described previously42 before being cryopreserved for future experimental use. All hHSC were used for experimental assays between passage 2 and passage 5. The immortalised human HSC cell line LX2, kindly provided by S. Friedman, was cultured in SteCM media and used for experimental assays at passage 2, at a maximum confluency of 80%.Liver tissue was digested using enzymes (1ng/ml of collagenase IV and 0.1ng/ml of DNAse I) and mechanical mashing. Optiprep  double density gradient (17% and 11.5%) was used to isolate the top layer of retinoid-rich hHSCNK cells were isolated from whole PBMC by negative selection using an NK cell isolation kit  according to the manufacturer’s instructions. The purity of NK cells was checked using flow cytometry by gating on live CD56 + CD3- cells and was always greater than 95%.2. The hHSC were then detached using 0.05% Trypsin/EDTA  and plated on 48 well plates . Where LX2 were used in assays, they were treated the same way as pHSC.Cryopreserved hHSC were thawed and cultured in T-25 tissue culture flasks  for 4 days at 37 °C with 5% COTM LSR II flow cytometer  and analysed using FlowJo . The positive control for apoptosis experiments was 500 ng/ml of SuperKiller TRAIL . For these NK-hHSC apoptosis assays, the data has been represented as percentage of caspase 3 normalised to positive control  according to manufacturer’s instructions. The data were collected on BDrol Fig. . This isTotal cellular RNA was extracted using QIAzol Lysis Reagent and RNeasy Mini Kit  according to the manufacturer’s protocol. After RNA quantification and quality control using NanoDrop1000 System , cDNA was synthesized with MultiScribe reverse transcriptase, random primers, deoxyribose nucleoside triphosphate (dNTP) mix and RNase inhibitor  according to the following protocol: 2 min 50 °C, 10 min 95 °C, followed by 40 cycles of 15 seconds 95 °C and 60 seconds 60 °C.T method was used as described previously43 using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the internal control. The amplification efficiency of target and reference genes was approximately the same (slope < 0.1).6.25 ng of the cDNA sample was used to set up real-time quantitative PCRs reactions using TaqMan® gene expression assays Hs00366278_m1 TNFRSF10B and Hs02758991_g1 GAPDH  and 7500 Fast Real-Time PCR System following the manufacturer’s protocol. Each sample was tested in duplicate. To quantify gene expression, the comparative CTRAIL-R3  and TRAIL-R4  neutralizing monoclonal antibodies (mAb) were used to block these receptors on pHSC. The antibodies were added at a concentration of 5 µg/ml for 30 minutes to HSC cultures. Matched isotypes of these were used as controls. The neutralizing antibodies were washed off using PBS prior to setting up apoptosis assays.TM LSR II flow cytometer  and analysed using FlowJo .Freshly isolated hHSC were stained with mAbs against CD19-APC-Cy7, CD146-FITC , CD14-v500, CD45-FITC , CD68-FITC, Cytokeratin-FITC, CD3-PE-Cy7 , CD56-ECD , TRAIL-R1-PE, TRAIL-R2-PE, TRAIL-R3-PE and TRAIL-R4-PE  after FcR blocking reagent  blocking. Appropriate isotype controls were used where necessary. NK cells were stained for CD56-ECD , CD3-PE-Cy7 , CD16-APC, TRAIL-PE. All cells were stained with Live/Dead® Cell viability stain . Data were acquired on BDThe lentivector pSIN-GFP was digested with EcoRI and BamHI, and the promoter of the gene encoding phosphoglycerate kinase (PGK) was amplified by polymerase chain reaction (PCR) for introduction of EcoRI-BamHI restriction sites. The SFFV promoter from the plasmid pSIN-GFP was replaced with the promoter of the gene encoding PGK, cloned by blunt-end ligation, to generate the plasmid pSIN-PGK-GFP. A second cassette containing the U6 promoter was cloned in this backbone downstream the cPPT sequence between ClaI-EcoRI restriction sites. A BamHI site was introduced downstream of the U6 promoter to clone the short hairpin RNA (shRNA) of interest between BamHI and EcoRI restriction sites. The U6-shRNA cassette was placed upstream the PGK promoter and GFP. All constructs were engineered by standard cloning techniques.The following shRNA sequences were used:shTR2: 5′-CTCACTGGAATGACCTCCTTTTTCAAGAGAAAAGGAGGTCATTCCAGTGAG-3′shTR3: 5′-CTTCCAACAATGAACCTTCTTTTCAAGAGAAAGAAGGTTCATTGTTGGAAG-3′shTR4: 5′-GATGGTCAAGGTCAGTAATTGTTCAAGAGACAATTACTGACCTTGACCATC-3′shCTR: 5′-CCTAAGGTTAAGTCGCCCTCGTTCAAGAGACGAGGGCGACTTAACCTTAGG-3′Transduction was monitored by GFP expression using fluorescence microscopy. Lentiviruses were produced using a packaging cell line (293T-cells) and transfected with p8.91 , pMDG (encoding VSVg env) and transfer vectors pSIN shCTR or pSIN shTR2. The shCTR (shCONTROL), shTR4 (shTRAIL-R4), shTR3 (shTRAIL-R3) and shTR2 (shTRAIL-R2) carrying lentivirus were transduced into HSC plated in a 48 well plate in 500 μl volume of SteCM at an MOI (multiplicity of infection) of 40 for up to three days. Transduction and knockdown was confirmed using FACS.Wilcoxon signed rank test was performed for all paired data. While comparing non-paired data, Mann-Whitney U test was performed. For correlation data, Spearman’s r test was used.Supplementary Information


Vaccination of turkeys with rLS/AMPV-C F&G induced both AMPV-C and NDV-specific antibody responses, and provided significant protection against pathogenic AMPV-C challenge and complete protection against velogenic NDV challenge. These results suggest that the rLS/AMPV-C F&G recombinant virus is a safe and effective bivalent vaccine candidate and that the expression of both F and G proteins of AMPV-C induces a protective response against the AMPV-C disease.Avian metapneumovirus (AMPV) infects the respiratory and reproductive tracts of domestic poultry, resulting in substantial economic losses for producers. Live attenuated vaccines appear to be the most effective in countries where the disease is prevalent. However, reversion to virulence has been demonstrated in several studies. Therefore, the development of a stable and safe next generation vaccine against the AMPV disease is needed. In the present study, we generated a recombinant Newcastle disease virus (NDV) vectoring the fusion (F) protein and glycoprotein (G) genes of AMPV subtype-C (AMPV-C) as a bivalent vaccine candidate using reverse genetics technology. The recombinant virus, rLS/AMPV-C F&G, was slightly attenuated  Turkey rhinotracheitis was first reported in the late 1970s in South Africa3 and since then, the virus has spread to all major poultry-producing areas in the world, except for Australia2. Isolates of AMPV have been classified into four subtypes, A, B, C, and D, based on the level of genetic variations and antigenic differences8. Subtypes A and B are present in most countries in the world, excluding the USA. However, AMPV-C is present in the USA, some Asian countries, and, to a limited degree, France. Finally, AMPV-D has only been isolated in France to-date.Avian metapneumovirus (AMPV) causes turkey rhinotracheitis (TRT), an acute upper respiratory tract infection in turkeys, and is associated with swollen head syndrome (SHS) in chickens, resulting in substantial economic losses to the poultry industryMetapneumovirus within the family Pneumoviridae9. AMPV genome consists of eight genes flanked by a 3′ Leader and 5′ Trailer in the order 3′-N (nucleocapsid)-P (phosphoprotein)-M (matrix)-F (fusion)-M2 (second matrix)-SH -G (glycoprotein)-L (large polymerase)-5′ 11. The fusion (F) protein and glycoprotein (G) are two major membrane-associated structure proteins of AMPV, and are believed to play important roles in virus pathogenicity and immunogenicity16.AMPV is a non-segmented, single-stranded negative sense RNA virus, and belongs to the genus 19. Although these live vaccines appear to be effective where the disease is prevalent, the stability and safety of some of these live vaccines are of concern22. Outbreaks of TRT at vaccinated turkey farms have been reported and were found to be due to the reversion of the vaccine virus after shedding to naïve birds23. In addition, it was found that viral subpopulations in AMPV vaccines may not be the only cause of reversion24. Therefore, the development of a safe, stable, and readily administered next generation vaccine against AMPV disease is needed.Live attenuated vaccines have been developed through serial passaging AMPV field isolates in cell culture25. Although these vaccine candidates were stable and safe in embryonated chicken eggs and vaccinated turkeys, they conferred only partial protection against homologous AMPV challenge in turkeys. It suggests that the G protein alone may be a weak antigen and other viral immunogenic components may be needed in combination to improve efficacy against AMPV diseases. Presence of the other major AMPV surface glycoprotein, fusion (F) protein, has demonstrated protective immunity in turkeys when expressed by either DNA vaccine or recombinant fowlpox virus26. Therefore, in the present study, we re-engineered the recombinant LaSota/AMPV-C G virus25 to express the F protein of AMPV-C, in combination, in an effort to improve the vaccine efficacy against AMPV-C disease.Previously, we generated Newcastle disease virus (NDV) LaSota vaccine strain-based recombinant viruses expressing the glycoprotein (G) of AMPV-A, B, and C viruses as bivalent vaccinesA full-length cDNA clone encoding the complete antisense genome of the NDV LaSota vaccine strain and the F and G genes of AMPV-C inserted between the F and HN genes of NDV was constructed using molecular biology techniques Fig. . The resin vitro and in vivo using the MDT and ICPI tests and several titration assays. As shown in Table 50, TCID50, and HA, were comparable to the titers of the parental LaSota strain  of SPF turkeys that were immunized with the rLS/AMPV-C F&G virus produced AMPV-C-specific antibodies at 14 DPV and 100% at 28 DPV as measured by ELISA Table  and 0.6 Viral RNA shedding, or the presence of the challenge virus (AMPV-C) in the tracheal lumen, was detected in 100% of birds in the parental LaSota vaccine control groups at 3, 5, and 7 DPC Table . ChallenAs shown in Table 25. In the present study, we further engineered the recombinant LaSota/AMPV-C G virus to express the other major surface glycoprotein of AMPV-C, the F protein, using an IRES to promote the foreign gene expression within a native NDV transcription unit27. This recombinant virus, rLS/AMPV-C F&G, was slightly attenuated in young chickens and delayed the onset of infection in cell culture when compared to the parental LaSota strain. It was stable and did not show any apparent biological changes when measured by MDT and virus titration after 10 passages in SPF chicken embryos. These results demonstrate the safety, stability, and possible application of this recombinant vaccine candidate in young turkeys, which is the most vulnerable population for NDV and AMPV diseases29.Previously, we generated and evaluated NDV LaSota strain-based recombinant viruses expressing the AMPV G protein as bivalent vaccines, which provided partial protection against homologous AMPV challenge in turkeys26. A combination of the F, G, and/or other antigenic conponents of the virus may be needed to improve the immune response and confer protection. In this study, we showed that the NDV recombinant vectoring both the AMPV-C F and G protein genes, rLS/AMPV-C F&G, induced a stronger immune response in turkeys against AMPV-C challenge than the rLS/AMPV-C G virus expressing the G protein alone. All turkeys vaccinated with the rLS/AMPV-C F&G virus developed AMPV-C specific antibody by 28 DPV, whereas only 50% of the rLS/AMPV-V G virus-vaccinated turkeys seroconverted by this time. Turkeys vaccinated with the F&G virus showed less clinical signs and shed less challenge virus than birds in the control groups at 7 DPC, demonstrating the improved efficacy induced by the presence of both AMPV-C F and G antigens. However, less than 50% the turkeys vaccinated with the rLS/AMPV-C F&G virus still showed mild transient clinical signs between 5–7 DPC. This incomplete protection conferred by the rLS/AMPV F&G virus warrants the need to futher develop a recombinant AMPV-C vaccine candidate and program, perhaps by either relocating the AMPVgenes closer to the 3′ end of the NDV genome for increased expression of the antigens or employing a prime and boost vaccination stragegy.The F and G proteins are two major surface glycoproteins of AMPV and believed to play important roles in virus infection, pathogenicity and immunogenicity. Expression of the F or G protein as a vaccine antigen from DNA vaccine, fowlpox or NDV vector conferred only partial protection in homologous challenged turkeysIn summary, in the present study we successfully generated a NDV LaSota strain-based recombinant virus vectoring the F and G protein genes of AMPV-C. Turkeys vaccinated with this recombinant virus were protected against a lethal velogenic NDV challenge and partially protected against pathogenic AMPV-C challenge. Compared with the rLS/AMPV-C G virus, the addition of the F gene in the new recombinant vaccine candidate, rLS/AMPV-C F&G, induced a stronger immune response in turkeys, indicated by reduced clinical signs and viral shedding after challenge. The results suggest that the rLS/AMPV-C F&G may be a promising bivalent vaccine candidate against NDV and AMPV-C disease, yet needs further development before use in large clinical trials on a commercial scale.All methods used in this study were carried out in accordance with the guidelines and protocols approved by the Institutional Biosafety Committee (IBC), Southeast Poultry Research Laboratory, United States Department of Agriculture, Agriculture Research Service . All animal experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC), SEPRL, USDA-ARS .2 in DMEM supplemented with 10% allantoic fluid (AF) from 10-day-old specific-pathogen-free (SPF) chicken embryos for all subsequent infections unless otherwise indicated. The NDV LaSota strain was obtained from ATCC and propagated in 9-day-old SPF chicken embryos. The velogenic strain of NDV, California 2002 /S0212676/02), and the AMPV-C Colorado strain (AMPV-CO) were obtained from the pathogen repository bank at SEPRL, USDA-ARS . The virulent AMPV-C virus stock (AMPV-CO Tr) was prepared from the tracheal tissues of AMPV-CO strain-infected SPF turkeys and titrated in SPF turkeys to determine 50% infective dose (ID50) as described previously16. Viral RNA from the NDV-infected chicken embryo AF or DF-1 cells was extracted using the TRIzol-LS reagent according to the manufacturer’s instructions (Life Technologies). Total cellular RNA from tracheal tissues was extracted using a MagMAX™ AI/ND Viral RNA Isolation kit (Life Technologies) following the manufacturer’s procedures.HEp-2  and DF-1  cell lines were grown in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal bovine serum  and antibiotics . The DF-1 cells were maintained at 37 °C and 5% CO13, was used to construct a recombinant cDNA clone containing the F gene of AMPV-C. To facilitate the expression of the AMPV-C F gene, an internal ribosomal entry site (IRES) sequence was inserted between the open reading frames (ORF)s of the NDV F and AMPV-C F genes . Subsequently, the IRES and the AMPV-C F ORF sequences were amplified and subcloned downstream of the NDV F ORF in the pLS/AMPV-C G vector using the In-Fusion PCR Cloning Kit (Clontech). The resulting recombinant clone contains the AMPV-C F gene as the 2nd ORF in the NDV F gene transcription unit and the AMPV-C G gene as an independent transcription unit between the NDV F and HN gene. The full-length cDNA clone was designated pLS/AMPV-C F&G and amplified in Stbl2 cells at 30 °C for 24 hours and purified using a QIAprep Spin Miniprep kit (Qiagen).The backbone of the previously generated infectious LaSota/AMPV-C G clone, pLS/AMPV-C G30. The rescued virus was amplified by inoculating 100 µl of the infected cell lysate into the allantoic cavity of 9-day-old SPF chicken embryos and incubating the embryos at 37 °C. After 4 days of incubation, the AF was harvested and the presence of the rescued virus was detected by hemagglutination (HA) assay31. HA-positive AF was diluted in PBS and amplified in chicken embryos three times. The AF was harvested from the infected embryos, aliquoted, and stored at −80 °C as a stock. The nucleotide sequence of the rescued virus was determined by sequencing the RT-PCR products amplified from the viral genome as described previously13.Rescue of the recombinant, infectious LaSota/AMPV-C F&G virus was performed by transfecting the full-length cDNA clone, pLS/AMPV-C F&G, and supporting plasmids into HEp-2 cells as described previously50) assay on DF-1 cells, and the 50% egg infective dose (EID50) assay in 9-day-old SPF chicken embryos31. Pathogenicity of the recombinant virus was also determined using the standard mean death time (MDT) and intracerebral pathogenicity index (ICPI) assays31.Titers of the LaSota, rLS/AMPV-C G, and rLS/AMPV-C F&G viruses were analyzed and compared by the standard HA test in a 96-well microplate, the 50% tissue culture infectious dose  of 0.01. At 24 h post-infection, the infected cells were washed with phosphate buffered saline (PBS) and fixed with 10% zinc formalin  for 15 min at room temperature. The cells were permeabilized by adding 0.5% Triton X-100  to the monolayer for 10 min at room temperature. The permeabilized cells were blocked with 5% goat serum  for 30 min at 37 °C. After blocking, the cells were stained with a mixture of chicken anti-AMPV-C F serum  and mouse anti-NDV HN monoclonal antibody  for 1 hour at 37 °C. Cells were then washed with PBS and stained with a mixture of FITC-labeled goat anti-chicken IgG (H + L)  and Alexa Fluor 568 conjugated goat anti-mouse IgG  for 1 hour at 37 °C. Immunofluorescence of the infected cells was visualized and photographed using an inverted fluorescence microscope at 100X magnification with filter combinations for FITC or Alexa Fluor 568 .ad libitum access to feed and water. At the termination of the experiments, all birds were humanely euthanized in accordance with SEPRL’s Institutional Animal Care and Use Committee approved animal use protocols.To evaluate the efficacy of rLS/AMPV-C F&G vaccine against AMPV-C and NDV challenge, two animal experiments were performed. In experiment 1, SPF turkeys were used in the BLS-2E animal facilities at SEPRL to evaluate the protection against virulent AMPV-C challenge. In experiment 2, SPF turkeys were used in the BSL-3E animal facilities at SEPRL to evaluate the protection against lethal NDV challenge. All turkeys were housed in Horsfal isolators  with 7 TCID50/ml) via intranasal (IN) and intraocular (IO) routes as vaccine vector controls for a total of 100 µl. Birds in groups 3 and 4 were vaccinated with 100 µl of rLS/AMPV-C G (1.0 × 107 TCID50/ml) and birds in groups 5 and 6 were vaccinated with 100 µl of rLS/AMPV-C F&G (1.0 × 107 TCID50/ml) per bird via IN/IO routes. At 14 days post-vaccination (DPV), the birds in groups 1, 3, and 5 were challenged with100 µl of pathogenic AMPV-C (1 × 103 ID50/ml) via IN/IO routes. At 28 DPV, the birds in groups 2, 4, and 6 were challenged with the same dose of pathogenic AMPV-C via IN/IO routes. Immediately before challenge, blood samples were collected from each bird for detection of serum antibody responses. The challenged birds were monitored daily for clinical signs of AMPV disease for 14 days. Typical clinical signs of the AMPV disease, nasal exudates when squeezed (score 1), nasal discharge (score 2), and/or frothy eyes (score 3), were assessed using the scoring system of Cook et al.17. At 3, 5, and 7 days post-challenge (DPC), intra tracheal swabs were collected from each bird for detection of challenge virus shedding.Sixty one-day-old SPF turkey poults were randomly divided into six groups of 10 birds. Each bird in groups 1 and 2 was inoculated with 50 µl of the LaSota vaccine (107 TCID50/ml) [Group 2], rLS/AMPV-C G (1.0 × 107 TCID50/ml) [Group 3], and rLS/AMPV-C F&G (1.0 × 107 TCID50/ml) [Group 4] via IN/IO routes. At 14 DPV, the birds were challenged with a lethal dose of the NDV/CA02 virus as described previously32. Serum samples were collected immediately before challenge for NDV antibody detection using the standard hemagglutination inhibition (HI) assay31. After challenge, the birds were monitored daily for clinical signs of Newcastle disease and mortality for two weeks.Thirty one-day-old SPF turkeys were randomly divided into three groups of 10 birds. Birds were inoculated with 100 µl of PBS [Group 1], the LaSota vaccine (1.0 × 1031. The antibody response to AMPV-C was examined by an enzyme-linked immunosorbent assay (ELISA) as described previously16. Briefly, Sucrose gradient purified AMPV-C virus was used as antigen. Turkey sera were diluted (1:100) and individually tested in triplicates. Negative and AMPV-C positive sera were included in each plate of the ELISA test as controls. Turkey sera were deemed positive when the mean chemiluminescence relative light unit (RLU) value was greater than the mean RLU plus 2× standard deviation of the three dilutions (1:100–1:400) of the negative turkey serum pool. Viral shedding from turkey tracheal tissue following challenge with AMPV-C was detected by RT-PCR with AMPV-C N gene-specific primers as described previously16.The antibody response against NDV was determined by performing a standard HI assay using LaSota virus as antigen


Streptococcus pneumoniae is the main virulence factors required for effective colonization and invasive disease. The capacity to regulate CPS production at the transcriptional level is critical for the survival of S. pneumoniae in different host niches, but little is known about the transcription regulators of cps locus. In the present study, we isolated and identified the response regulator ComE, the master competence switch in transformation of S. pneumoniae, as a transcriptional regulator of cps locus by DNA affinity chromatography-pulldown, MALDI-TOF mass spectrometry (MS) and electrophoretic mobility shift assay (EMSA). Our results showed that phosphorylated mimetic of ComE (ComED58E) bound specifically to the cps locus prompter in vitro, and phosphorylated ComE negatively impacted both cps locus transcription and CPS production attenuating the pneumococcal virulence in vivo. Compared with D39-WT strain, D39ΔcomE mutant exhibited much thicker capsule, attenuated nasopharyngeal colonization and enhanced virulence in both pneumonia and bacteremia models of Balb/c mice. Furthermore, it was demonstrated that CSP-ComD/E competence system involved in regulating negatively the CPS production during the progress of transformation in D39. Our CSP1 induction experiment results showed that the expression of ComE in D39-WT strain increased powerfully by 120% after 10 min of CSP1 induction, but the CPS production in D39-WT strain decreased sharply by 67.1% after 15 min of CSP1 induction. However, the CPS production in D39ΔcomE mutant was almost constant during the whole stage of induction. Additionally, we found that extracellular glucose concentration could affect both the expression of ComE and CPS production of D39 in vitro. Taken together, for the first time, we report that ComE, as a transcriptional regulator of cps locus, plays an important role in transcriptional regulation of cps locus and capsular production level.The capsular polysaccharide (CPS) of  Streptococcus pneumoniae (pneumococcus), a Gram-positive opportunistic pathogen residing on the human upper respiratory tract, remains a leading cause of morbidity and mortality worldwide in young children and in immunocompromised elderly  and downstream type-specific genes  are highly homologous among serotypes of Wzy-dependent pathway, which seem to play a role in modulation of CPS synthesis. The proteins CpsA-D encoded by common genes have been shown to affect the level of CPS expression   and electrophoretic mobility shift assay (EMSA). In this way, seven proteins were initially chosen to be further studied as candidate transcriptional regulators of cps locus  medium or on blood agar plates supplemented with 5% defibrinated sheep blood. E. coli was grown on LB agar plates or in LB broth with shaking at 37°C. All growth mediums were supplemented with appropriate antibiotics as described in Table S. pneumoniae were reported previously  were used to amplified 193-bp and 591-bp partial complement DNA fragments from chromosomal DNA in D39-WT, which contained the mutagenesis site of comE DNAT174C. The two DNA fragments were fused by PCR (with primer pairs of comE-F and comE-R) to generate complete comED58E gene fragment, then was digested with BamH I and Xho I, and ligated to BamH I-Xho I-digested pET-28a (+) plasmid DNA. Then pET-28a-comED58E plasmid was transformed into E. coli B21 to generate the recombination comED58E expression strain. The comED58E DNA fragment was amplified by PCR with the primer pairs of comED58E-hb-F and comED58E-hb-R, and the PCR product was digested with Nhe I and Not I, subsequently, was ligated to Spe I- Not I-digested pJWV25 plasmid DNA. Then pJWV25::comED58E plasmid was transformed into D39 strain to generate the ComE over-expression strain D39::comED58E . 780-bp Erm gene fragment was acquired by PCR from S. pneumoniae CPM8 strain (with primer pairs of Erm-F and Erm-R). To acquire the transformation donor DNA, a third PCR was carried out to fuse the upstream-Erm-downstream fragment with primer pairs of comE-P1 and comE-P4. The comE gene was completely replaced with the erythromycin gene by homologous recombination according to an established protocol  was used as wild type (WT) strain. To construct the D39ΔcomE and D39ΔcomE::comEcps promoter, β-galactosidase and GFP reporter constructs of cps promoter region were prepared in the pEVP3 and pAE03 plasmid vectors, respectively. A cps promoter fragment containing 780-bp upstream of cps2A was cloned into pEVP3 (at the BgI II and Sma I restriction sites) and pAE03 (at the EcoR I and Not I restriction sites), respectively. The pEVP3-cps-promoter and pAE03-cps-promoter plasmids were then transformed into D39-WT strains to generate the D39-pEVP3-cps-promoter and D39-pAE03-cps-promoter mutants. D39ΔcomE-pEVP3-cps-promoter and D39ΔcomE-pAE03-cps-promoter mutants were constructed by homologous recombination according to an established protocol  was PCR amplified from the chromosomal DNA of D39-WT strain using the primer pairs of Pcps –F and Pcps–R  was carried out by sonication with freeze thaw. The cells lysate was then incubated with C2-probe-streptavidin-coated magnetic beads  and purified with different NaCl concentration elution buffer, using the blank streptavidin-coated magnetic beads without C2 probe-coated as the control group. The elution protein under different NaCl elution buffer was collected respectively, analyzed via 15% SDS-PAGE, and stained by SYPRO-Ruby. Compared with control group, the enriched proteins were detected, excised and identified by MAIDI-TOF mass spectrometry .To isolate and identify the transcription factors of comE and comED58E gene were PCR amplified from chromosomal DNA of D39-WT and constructed comED58E mutant fragment with the primer pairs of comE-F and comE-R, respectively. The PCR product was purified and digested with BamH I and Xho I, and ligated to BamH I-Xho I-digested pET-28a(+) plasmid DNA. The E. coli B21 (DE3) strain was then successively transformed with a pET-28a-comE or pET-28a-comED58E. Expression and purification of ComE and ComED58E proteins were then carried out as reported previously  at an OD600 of 0.8 with 120 rmp shaking at 20°C for 18–20 h. Cells were harvested by centrifugation at 10,000 × g for 30 min at 4°C, resuspended in 50 ml of binding buffer, and lysised completely by sonication. Soluble His-tagged ComE or ComED58E proteins retained on Ni-NTA column were then eluted with increasing concentration imidazole buffer. The enriched proteins were analyzed for purity by SDS-PAGE and concentrated by ultrafiltration, and then small packing stored at −20°C.To construct expression plasmids, cps2A in D39 (218 bp) was PCR amplified to produce the 5′ biotin modification labeled probe C2 and unlabeled probe C0 with primer pairs of Pcps-F/Pcps-R and Pcps–F2/Pcps–R, respectively. The PCR product was purified with the high pure PCR product purification kit . EMSA was carried out according to the protocol of LightShift® Chemiluminescent EMSA Kit . Briefly, DNA-binding assays were incubated at 37°C for 30 min in 20 μl reaction buffer containing 1 × binding buffer, 1 μg Poly (dI-dC), 2.5% Glycerol, 0.05% NP-40, 5 mM Mn2+, 5 mM Zn2+, 2–4 μg interest proteins, 1 ng labeled probe C2 or 100 ng unlabeled probe C0. Following incubation, binding reaction mixtures were analyzed by electrophoresis in 6% native TBE polyacrylamide gels at constant 100 V for 60 min. Then the gels were transferred to nylon membrane at 380 mA for 40 min, and crosslinked for 10 min by a hand-held UV lamp equipped with 254 nm bulbs, and subsequently biotin-labeled DNA binding were detected by chemiluminescence according to supplier's instructions.The upstream promoter regions of S. pneumoniae was taken and the microscopic surveying instrument was used to determine the capsule thickness of 100 randomly chosen capsule swelling cells.To compare the difference of capsule between D39-WT and D39ΔcomE mutant, Neufeld test, immunofluorescence microscopy and transmission electron microscopy (TEM) were carried out according to the method described earlier  was used to isolated total RNA from D39, and the cDNAs of D39 were prepared using the total RNA preparation with an iScript™ cDNA synthesis kit  according to the supplier's instructions. The mRNA level of the cps locus was quantified by quantitative real time PCRs using cDNA as the template and the primer pairs of cps2A-F/cps2A-R and 16S rRNA-F/16S rRNA-R  as described  as described previously .Relative CPS levels of D39-WT, D39ΔcomE, D39ΔcomEFor Western blot analysis, CPS was detected with a rabbit anti-serotype-2 serum  as primary antibody and peroxidase-conjugated goat anti-rabbit IgG as secondary antibody , respectively. The member of the CPS-antibody was visualized using Western ECL substrate (Bio-Rad) according to the supplier's instructions. Quantification was done by spot densitometry using the Image J software . The results of representative experiments were presented as means of three replicates ± standard deviations.For ELISA analysis, as described previously .D39-pAE03-cps-promoter and D39ΔcomE-pAE03-cps-promoter mutants were grown in 30 ml C+Y medium supplemented with 1 mmol CaCl2 and 2% g/L BSA at 37°C under 5% CO2, respectively. When the culture reached an OD600 of about 0.1, cells were induced to the competence by CSP1 (500 ng/ml). 5 ml sample was taken periodically out from the culture after induction: 0, 5, 10, 15, 20 and 25 min. Samples were taken out and chilled rapidly on dry ice, without freezing, and then kept at 4°C until harvested by centrifugation at 10,000 × g for 5 min at 4°C. ComE and GFP were detected by Western blot analysis as described above. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected as an internal control for constitutively expressed gene. D39-pAE03 strains were used as control strains which were transformed with blank pAE03 plasmid.To further verify the effect of comE on the transcription of D39 and D39ΔcomE mutant were induced by CSP1 as described above. ComE and CPS were detected by ELISA. Samples for ComE were standardized to 0.8 mg/ml by total protein quantification, and then were used to coat 96-well microtiter plates at dilutions of 1:200, which were subsequently reacted with the mouse anti-ComE serum at dilution of 1:800. The bound conjugate were detected following incubation with goat anti-mouse IgG-HRP (Bio-Rad) at dilutions of 1:5,000, and the absorbance value at 450 nm was recorded. The results of representative experiments were presented as means of three replicates ± standard deviations.2, to an OD600 of 0.3 in 5 ml C+Y medium supplemented with following different concentration glucose: 2.0, 4.0, 8.0 mM (the glucose concentration in C+Y medium) and 16.0 mM, respectively. The samples were harvested and prepared for analysis as described above. The ComE and CPS were detected by Western blot and ELISA, respectively. The results of representative experiments were presented as means of three replicates ± standard deviations.D39-WT and D39ΔcomE strains were grown at 37°C under 5% COAll of the animals used in this study were purchased from the Department of Experimental Animal, Chongqing Medical University [certificate no. SYXK(yu) 2007-0001]. All animal procedures were approved by the Ethics Committee of Chongqing Medical University (reference no.2011-032). The research described here was carried out in accordance with the Declaration of Helsinki and with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.600 of 0.5 at 37°C under 5% CO2. The cells were adjusted to 3.0 × 106 CFU/30 μl concentration. Groups of two 6-week-old female Balb/c mice (n = 18) were intranasally challenged with 30 μl D39-WT or D39ΔcomE mutant. The animals were killed after inoculation: 6, 12, 24, 36, 48, and 72 h. Colonized bacteria were harvested from nasal lavage fluid and enumerated by plating serial dilutions. The nasopharyngeal lavage was collected as described previously  were intranasally challenged with 30 μl D39-WT, D39ΔcomE or D39::comED58E mutant as described earlier  by intraperitoneal injection, respectively. Mice were subsequently monitored for 4 days, and survival was recorded every 12 h.For a bacteremia model of infection: D39-WT, D39ΔcomE and D39::comEt-test or the non-parametric Mann-Whitney U-test or Gehan-Breslow-Wilcoxon test with GraphPadPrism 5 . P < 0.05 was considered to be statistically significant.Differences between groups were statistically analyzed with a two-tailed cps locus are co-transcribed as an operon from a common promoter upstream of cpsA  that located immediately upstream of cpsA. We obtained an enriched protein at about 29 kDa molecular mass in SDS-PAGE, which was subsequently identified as response regulator ComE with 78.4% coverage rate by MALDI-TOF MS analysis , but the difference disappeared when the deleted comE gene was ectopic complemented in D39ΔcomE::comED58E , which constitutes, together with competence-stimulating peptide (CSP), the master competence switch , and there was no significant differences between D39-pEVP3-cps-promoter and D39ΔcomE-pEVP3-cps-promoter::comED58E  and 58.8% (**p = 0.0064) as compared with that of D39-WT and D39ΔcomE::comED58E, respectively, and there was no significant differences between complemented D39ΔcomE::comED58E mutant and D39- WT strains  and 24.3% (*p = 0.0171) as compared with that of D39-WT strains and D39ΔcomE::comED58E mutants, respectively. There was no significant difference between D39-WT strain and D39ΔcomE::comED58E mutant .To further confirm our observations, we surveyed the capsule morphology difference between D39-WT strain and D39ΔcomE mutant by Neufeld test, immunofluorescence microscopy and transmission electron microscopy (TEM). The D39ΔcomE mutant exhibited stronger immunofluorescence and much thicker capsule than that of D39-WT strain Figures . The meacps locus and negatively regulates the CPS production.Taken together, all these data has demonstrated that the ComE is a transcriptional regulator of S. pneumoniae  induces the transcription of ComD/E and virulence genes in r Figure .cps locus, we constructed the GFP reporter bacterium for cps gene expression, D39-pAE03-cps-promoter and D39ΔcomE-pAE03-cps-promoter mutant. ComE and reporter protein GFP were detected by Western blot. The results showed that the expression of ComE in D39-pAE03-cps-promoter started to increase after CSP1 induction 5 min, and reached peak value which increased 3.38-fold higher after CSP1 induction about 10 min , and then decreased promptly after CSP1 induction 15–20 min , and then restored after about 25 min. However, the expression of GFP in D39ΔcomE-pAE03-cps-promoter was almost unaffected by CSP1 in the development of competence from 0 to 25 min , but the over expression of ComE strains (D39::comED58E) showed the equivalent virulence to that of D39-WT strains  in the mice pneumonia model. In the bacteremia models, however, the D39::comED58E strains showed the weakest virulence.  survived for the duration of the experiment, but 33.3% of the D39-WT –infected mice (4/12) survived until the end of the experiment. As shown in Figure S. pneumoniae to survive in different niches of their hosts. Maximal expression of capsule is necessary for systemic virulence, but decreased capsule is beneficial for the adherence and colonization in the nasopharyngeal tract of the host  and ComE are transcriptional regulators of cps locus. Despite it has been demonstrated that both GntR and ComE regulate negatively the CPS production, it is worthy of further study whether there is some correlations in their regulation function.The DNA affinity chromatography-pulldown is well established effective methods to isolate and identify novel transcriptional regulator, which do not demand any knowledge of the DNA-binding protein's identification (Jutras et al., R Figure . Of noteS. pneumoniae D39 ComE negatively regulates the transcription of cps locus and impacts the CPS production, which leads to the attenuation of the virulence in pneumococcal. CSP-ComD/E competence system involves in capsular regulation in the progress of natural genetic transformation of S. pneumoniae. Extracellular glucose may affect the expression of ComE and regulate positively CPS production. Given the importance of capsule in pneumococcal virulence and invasive infection, and the regulation of CSP-ComD/E system on CPS production, it deserves to be further studied that CSP may be applicable to reduce the invasive infections by S. pneumoniae.Our results show that Conceived and designed the experiments: YZ, XZ, and YY. Performed the experiments: YZ, XW, LW, and JZ. Analyzed the data: YZ and XZ. Contributed reagents/materials/analysis tools: YZ, XZ, and XW. Wrote the paper: YZ and YY. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Peanut stripe virus (PStV) belongs to the genus Potyvirus and is the most important viral pathogen of cultivated peanut (Arachis hypogaea L.). The eukaryotic translation initiation factor, eIF4E, and its isoform, eIF(iso)4E, play key roles during virus infection in plants, particularly Potyvirus. In the present study, we cloned the eIF4E and eIF(iso)4E homologs in peanut and named these as PeaeIF4E and PeaeIF(iso)4E, respectively. Quantitative real-time PCR (qRT-PCR) analysis showed that these two genes were expressed during all growth periods and in all peanut organs, but were especially abundant in young leaves and roots. These also had similar expression levels. Yeast two-hybrid analysis showed that PStV multifunctional helper component proteinase (HC-Pro) and viral protein genome-linked (VPg) both interacted with PeaeIF4E and PeaeIF(iso)4E. Bimolecular fluorescence complementation assay showed that there was an interaction between HC-Pro and PeaeIF4E/PeaeIF(iso)4E in the cytoplasm and between VPg and PeaeIF4E/PeaeIF(iso)4E in the nucleus. Silencing either PeaeIF4E or PeaeIF(iso)4E using a virus-induced gene silencing system did not significantly affect PStV accumulation. However, silencing both PeaeIF4E and PeaeIF(iso)4E genes significantly weakened PStV accumulation. The findings of the present study suggest that PeaeIF4E and PeaeIF(iso)4E play important roles in the PStV infection cycle and may potentially contribute to PStV resistance. Peanut stripe virus  is one of the most widely distributed peanut viruses constraining peanut production. PStV has been detected in various countries, including China, the US, the Philippines, Thailand, Indonesia, Malaysia, and Korea , sesame (Sesamum indicum), cowpea (Vigna unguiculata), hyacinth bean (Dolichos lablab), white lupin , and patchouli . The polyprotein is cleaved by three virus-encoded proteases into 10 mature proteins and an additional protein called PIPO, which is embedded in the P3 cistron , which consists of C-proximal, central, and N-proximal domains. The C-proximal domain separates HC-Pro from the polyprotein precursor via proteolysis . HC-Pro Another important region of the potyviral protein is VPg, which is translated into the polyprotein, NIa, which is also known as VPg-Pro. During the potyvirus infection, VPg participates in replication and proteolysis and is composed of N-terminal and C-terminal protease domains . VPg is eIF4E, which initiates the translation of mRNA and regulates protein synthesis 4E are functionally redundant and one or both of them interact with HCpro and VPg, which are indispensable for viruses to complete their infection cycle; therefore, abolishing this interaction may prevent the viral infection (pvr2 is a two-nucleotide substitution of the amino acid of pepper eIF4E and is resistant to PVY (pot-1 confer resistance against PVY and Tobacco etch virus (TEV) in tomato. Barley rym4 and rym5 are also amino acid substitutions that confer eIF4E resistance to Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV) in Barley (pvr1(2) contains an eIF4E mutation and pvr6 is an eIF(iso)4E mutation; simultaneous mutations in eIF4E and eIF(iso)4E confer resistance to Chilli veinal mottle virus (ChiVMV) in pepper, and silencing eIF4E and eIF(iso)4E reduces the ChiVMV accumulation 4E controls the effect of PStV in peanut. To test this hypothesis, we investigated the effects of silencing the translation initiation factor, eIF4E/eIF(iso)4E, to confer PStV resistance in peanut. Moreover, we examined the interaction between HC-Pro and VPg of PStV with eIF4E/eIF(iso)4E using Y2H and BiFC. We also detected the expression of eIF4E/eIF(iso)4E in different peanut tissues.Arachis hypogaea) leaves using TRIzol , and cDNA was synthesized using an M-MLV RTase cDNA synthesis kit , following the manufacturer’s recommendations. To design primers for cloning the eIF4E and eIF(iso)4E genes of peanut, we compared and downloaded the eIF4E sequences of Medicago truncatula (XM_003593785), Pisum sativum (AY423375), Pisum sativum (DQ641471), Phaseolus vulgaris (EF571276), Phaseolus vulgaris (EF571275), M. tornata (HQ735878), and M. truncatula (HQ735877). The conserved sequences were used in designing the primer pairs PeaeIF4E2-R and PeaeIF4E2-F (Supplementary Table eIF4E gene. The PCR products showing the expected lengths were sequenced and compared. The product with the correct sequence was then used in designing primers (Supplementary Table PeaeIF4E. A 5′-RACE kit (Invitrogen) was used according to the manufacturer’s instructions to obtain the 5′ terminus of the PeaeIF4E gene. The 5′-RACE eIF4E outer and inner primers and 3′-RACE eIF4E outer and inner primer (Supplementary Table PeaeIF4E cDNA. The PeaeIF(iso)4E gene was amplified using the primers listed in Supplementary Table Total RNA was extracted from peanut (PeaeIF4E and PeaeIF(iso)4E. Based on the reported cDNA sequence of PStV , we designed primers for the amplification of segments that corresponded to PStV VPg and PStV HC-Pro 4E genes. All peanut tissues were sampled from three different peanut plants as biological replicates. For the analysis of gene silencing in peanut, total RNA was extracted from the leaves of eIF4E-silenced, eIF(iso)4E-silenced, or eIF4E-eIF(iso)4E-double silenced peanuts and used in RT-PCR as previously described. Quantitative real-time PCR (qRT-PCR) reactions were conducted by using a SYBR Premix Ex Taq PCR kit (Takara) on an ABI7500 real-time PCR system . The primer pairs YG4E-R/YG4E-F and YG4IE-R/YG4IE-F were used to detect the expression of PeaeIF4E and PeaeIF(iso)4E. The primer pair YGPStV-R/YGPStV-F was used to detect the accumulation of PStV after inoculation. The primer pair actin-R/actin-F was used to amplify the actin gene of A. hypogaea, which was used as a reference. The PCR reaction system consisted of a total volume of 20 μL, which included 2 μL of the RT product, 10 μL of Ex Taq, 0.8 μL of the primers  using the corresponding primer pairs, ORF4E-R/ORF4E-F, 4E(isoORF)F/4E(isoORF)R, PV-F/PV-R, and PH-R/PH-F, and then cloned into a pMD-18T easy vector (Takara) for sequencing. After confirmation of the correct clone from pMD-18T, these were then introduced into an entry vector pGWCm by TA cloning, and finally, via LR gateway recombination reaction (Invitrogen), was transferred to the plant expression vector, pHZM03. Plasmid DNA with green fluorescent protein (GFP) was transiently introduced into toplasts . After iEscherichia coli DH5α cells for subsequent DNA sequencing. Both the confirmed correct prey and bait vectors were then co-transformed into AH109 yeast cells. SD/-Leu-Trp, SD/-Leu-Trp-His, SD/-Leu-Trp-His-Ade, and SD/-Leu-Trp-His-Ade+X-α-gal (Clontech) were used as selective media to detect any interactions. Yeast that contained both empty pGADT7 and pGBKT7 were used as negative controls, and yeast containing both pGBK-p53 and pGAD-RecT were used as positive controls.Yeast two-hybrid screening was conducted using a Matchmaker Gold Yeast two-hybrid system . The coding sequences of PStV VPg, PStV HC-Pro, and eIF4E/eIF(iso)4E were PCR amplified by using Phusion high-fidelity DNA polymerase (Takara) with the primer pair listed in Supplementary Table Arabidopsis protoplasts for transient expression 4E were introduced individually into the entry vector pGWCm as previously described. pGWCm-VPg and pGWCm-HC-pro were transferred to the pEarleyGate202-CYFP vector, whereas pGWCm-eIF4E and pGWCm-eIF(iso)4E were transferred to the pEarleyGate202-NYFP vector via the LR Gateway recombination reaction (Invitrogen). Plasmid DNA with YFP fusion was introduced into pression . After iPeaeIF4E and PeaeIF(iso)4E were amplified by using the primers listed in Supplementary Table XhoI and BamHI (New England Biolabs) and ligated into vector ALSV-RNA2, which was digested with the corresponding enzymes. The plasmid constructs were then sequenced (Takara) to confirm that we obtained the correct inserts. Apple latent spherical virus (ALSV)-based VIGS method was used. The constructed ALSV-RNA2 and ALSV-RNA1 were transformed into E. coli DH5α cells for sequencing; then the confirmed correct plasmid was cultured in E. coli DH5α cells using TransGEN plasmid Maxi Kit (TransGEN) for purification. Approximately 1 μg/μL of modified ALSV-RNA2 and ALSV-RNA1 were mechanically inoculated into Chenopodium quinoa plants. Two to three weeks later, symptomatic leaves were collected, homogenized in three volumes of extraction buffer , and re-inoculated in C. quinoa plants. Then the total RNA of symptomatic leaves were extracted and diluted to about 0.02 μg/μL to be mechanically inoculated into peanuts . The eIF4E gene was 764-bp long, with a 5′ 39-bp untranslated region, a 696-bp ORF, and a 29-bp 3′ untranslated region, and encoded a putative 231-amino acid polypeptide. Moreover, it had 92% nucleotide sequence identity to its homolog in Pisum sativum cultivar (GenBank Accession No. AY423375.2) and 70% nucleotide identity to its homolog in M. truncatula (GenBank Accession No. BT134162.1).The sequence of the peanut eIF(iso)4E gene was isolated as described above. The obtained peanut eIF(iso)4E ORF was 612 bp (GenBank Accession No. KF956378) and was predicted to encode a 203-amino acid polypeptide. The cloned peanut eIF4E and eIF(iso)4E genes were designated as peaeIF4E and peaeIF(iso)4E, respectively. The identity of the peaeIF4E and peaeIF(iso)4E genes at the nucleotide sequence level was 53.55%, whereas that at the amino acid sequence level was 42.67%.The peanut Figure 1). Furthermore, peaeIF4E was clustered with the eIF4E subgroup, whereas peaeIF(iso)4E was classified into the eIF(iso)4E subgroup. Analysis using the conserved domain search service of NCBI confirmed that the two proteins contained the eIF4E family conserved sequence.The eIF4E and eIF(iso)4E protein sequences of other plant species were aligned for phylogenetic reconstruction. In the phylogenetic tree, eIF4E and eIF(iso)4E formed two distinct branches (PeaeIF4E and PeaeIF(iso)4E in various tissues, quantitative real-time PCR was performed. RNA was isolated from various tissues such as the roots, stems, leaves, flower buds, and leaf buds of “Huayu 20.” The expression of the peanut actin gene is constant under different conditions and in different tissues (PeaeIF4E and PeaeIF(iso)4E were similar in all tissues . The highest transcript levels, for both genes, were observed in the leaf bud and the lowest in flowers .To detect the expression levels of peanut  tissues , and wasArabidopsis protoplasts cell. The Arabidopsis protoplasts cell were cultured in the dark at 23°C for about 12–16 h and observed by the confocal laser scanning microscopy (Leica SP5). The results suggested that the PeaeIF(iso)4E and PeaeIF4E fusion proteins were present in both the nucleus and the cytoplasm .To test the subcellular localization of PeaeIF4E and PeaeIF(iso)4E, PeaeIF4E and PeaeIF(iso)4E were fused to the GFP by cloning of the ORFs of PeaeIF4E and PeaeIF(iso)4E into the entry vector pGWCm. Recombinant plasmids that expressed the PeaeIF4E-GFP and PeaeIF(iso)4E-GFP fusion proteins were introduced into Arabidopsis protoplasts as above. The results suggested that the VPg fusion protein was present in the nucleus, and HC-Pro fusion protein was observed in the cytoplasm .We obtained the VPg and HC-Pro protein cDNAs from PStV by RT-PCR, and their ORFs were fused to GFP as above. The fusion proteins were expressed in Figure 4). The interactions were further confirmed by using BiFC. In this system, the YFP was split into N-terminal and C-terminal fragments, and the PeaeIF4E and PeaeIF(iso)4E were attached to the N-terminal fragment of YFP(eIF4E-NY and eIF(iso)4E-NY). VPg and HC-pro were fused to the C-terminal fragment of YFP(VPg-CY and HC-pro-CY). The eIF(iso)4E-NY+VPg-CY, eIF4E-NY+VPg-CY, eIF4E-NY+HC-pro-CY, and eIF(iso)4E-NY+HC-pro-CY plasmids were then transformed into Arabidopsis protoplasts cells. A nuclear fluorescence signal was, respectively, observed in eIF(iso)4E-NY+VPg-CY and eIF4E-NY+VPg-CY combination and the signal was observed throughout the nucleus. Cytoplasmic fluorescence signals were observed in the eIF4E-NY+HC-pro-CY and eIF(iso)4E-NY+HC-pro-CY combinations, and the signals were observed throughout the cytoplasm . As expected, the negative controls, i.e., the combinations of eIF4E-NY+CY and NY+HC-Pro-CY did not emit fluorescence signals . Taken together, these results show that PeaeIF4E/PeaeIF(iso)4E interacts with VPg in the nucleus, whereas PeaeIF4E/PeaeIF(iso)4E interacts with HC-Pro in the cytoplasm.Yeast two-hybrid analysis was used to determine whether there was an interaction between viral proteins and peanut proteins. Yeast two-hybridization showed interactions between VPg and PeaeIF4E/PeaeIF(iso)4E, and between HC-Pro and PeaeIF4E/PeaeIF(iso)4E (PeaeIF4E and PeaeIF(iso)4E genes in PStV infection, gene silencing was performed. The PeaeIF4E (355-nt) and PeaeIF(iso)4E (326-nt) fragments were inserted into the ALSV-RNA2 vector. The recombinant viruses (ALSV-eIF4E and ALSV-eIF(iso)4E) were then inoculated into a peanut. Two weeks after inoculation, real-time RT-PCR analysis was performed, which demonstrated that the expression levels of PeaeIF4E and PeaeIF(iso)4E were significantly lower in the inoculated plants as compared with control , although no significant phenotypic alterations were observed in the transgenic plants . The expression level of PeaeIF4E decreased by 60% (upon inoculation with ALSV-eIF4E) while that of PeaeIF(iso)4E decreased by 65% (ALSV-eIF(iso)4E inoculation) as compared with control. When inoculated with ALSV-eIF4E+ALSV-eIF(iso)4E, the expression levels of PeaeIF4E and PeaeIF(iso)4E decreased by 53 and 57%, respectively, as compared with control . Peanut plants where either the PeaeIF4E or PeaeIF(iso)4E were silenced, showed mosaic symptoms of infection at about 10–14 days after inoculation with PStV. On the other hand, silencing of both PeaeIF4E or PeaeIF(iso)4E caused the symptoms to appear later at about 18–20 days after inoculation with PStV, and the symptoms were milder as compared with plants with only one gene silenced and the control . Real-time RT-PCR analysis indicated that silencing both PeaeIF4E or PeaeIF(iso)4E reduced PStV accumulation by 70% compared to control plants. No significant differences were observed between plants in which either PeaeIF4E or PeaeIF(iso)4E was silenced as compared with controls  suggesting that the two isoforms play overlapping or redundant roles in the virus multiplication cycle.To confirm the role of the eIF4E/eIF(iso)4E genes and analyzed their protein sequences. Phylogenetic analyses of these sequences demonstrated that PeaeIF4E and PeaeIF(iso)4E showed high homologies with orthologs from related plant species. PeaeIF4E and PeaeIF(iso)4E were closely related to their homologs from soybean (G. max) and kidney bean (Phaseolus vulgaris). The expression levels of PeaeIF4E and PeaeIF(iso)4E were similar in different peanut tissues with both genes being upregulated in leaf buds and roots and downregulated in flowers . Previous studies have also shown that these two genes are upregulated in young tissues and downregulated in mature tissues of Arabidopsis and plum, which corroborated our results (The cap-binding protein eIF4E/eIF(iso)4E confers resistance to some RNA viruses in specific plant species . In the  results .Arabidopsis cells . In Chrysanthemum morifolium, eIF(iso)4E was also localized in the nucleus, cytoplasm, and cytomembrane 4E has been observed in the cytoplasm and nucleus 4E were localized to both the nucleus and the cytoplasm, but whether the two proteins have different cellular locations during different growth stages needs further investigation. In animals, eIF4E has been detected in both cytoplasm and nucleus, and about 68% of the eIF4E was detected in mammalian nuclei. eIF4E plays different roles depending on its subcellular location; when it is localized in cytoplasm, it functions in translation initiation. When it is localized in the nucleus, it participates in the export of mRNAs that contain 4E-sensitive elements 4E were both localized in the nucleus and the cytoplasm of membrane . In Arab nucleus . In matuents SE; , 2008. TMaize dwarf mosaic virus) and TuMV (Turnip mosaic virus), HC-Pro was detected in the cytoplasm  infected plants (Wheat mosaic virus) infected plants, VPg occurred in two forms in the nucleus; one gathered into one or several irregular shape inclusions, whereas the other was evenly distributed across the entire nucleus 4E conferred moderate resistance against PStV in peanut, as evidenced by symptom delay and reduced virus accumulation. This findings strongly suggest that PeaeIF4E and PeaeIF(iso)4E play important roles to facilitate virus infection and that they are functionally interchangeable. The silencing of PeaeIF4E and PeaeIF(iso)4E hindered the interaction between the host and the virus, which in turn prevented infection and viral replication in the host. Furthermore, viral accumulation was lower in gene-silenced peanut plants. Silenced plants with decreased expression of PeaeIF4E and/or PeaeIF(iso)4E did not phenotypically differ from control plants. In tobacco, antisense depletion of either eIF4E and two eIF(iso)4E isoforms displayed normal development, but antisense depletion of both eIF4E and eIF(iso)4E resulted in semi-dwarf phenotype  and ChiVMV in pepper (eIF(iso)4E in peach plants results in peach resistance to PPV (eIF(iso)4E gene in plum confers resistance to PPV 4E during its infection because silencing only one gene does not confer resistance to PStV in peanut. Our study suggests that the eIF4E/eIF(iso)4E gene may be utilized in increasing PStV resistance in peanut by gene silencing, gene mutation, or the TILLING strategy 4E genes result in a decrease in viral resistance, such as resistance to n pepper . Furthere to PPV . Silencie to PPV  and silesistance . These rstrategy . We haveTherefore, the findings of the present study suggest that eIF4E/eIF(iso)4E plays important roles in the PStV infection cycle and may serve as a novel method for increasing the PStV resistance in economically important peanut cultivars. The two genes may also be used as genetic resources for improving PStV resistance in peanut breeding programs.MX, HX, JW, and JY: Design of the work, analysis, and interpretation of data. LX and YC: Final approval of the version to be published and agreement to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


ETV6/RUNX1 (E/R) is the most common fusion gene in childhood acute lymphoblastic leukemia (ALL). Multiple lines of evidence imply a “two-hit” model for the molecular pathogenesis of E/R-positive ALL, whereby E/R rearrangement is followed by a series of secondary mutations that trigger overt leukemia. The cellular framework in which E/R arises and the maintenance of a pre-leukemic condition by E/R are fundamental to the mechanism that underlies leukemogenesis. Accordingly, a variety of studies have focused on the relationship between the clones giving rise to the primary and recurrent E/R-positive ALL. We review here the most recent insights into the pathogenic mechanisms underlying E/R-positive ALL, as well as the molecular abnormalities prevailing at relapse. ETV6/RUNX1  fusion gene .–48.E/R-presponse , 49. In  subtype . The fre subtype , 50. The6 allele . The 9p tor PAX5 , 38, 40. and MGA , 47, 52.reported . The geneceptors . Severaleceptors . These tE/R-positive ALL and may be expected to drive the evolution of overt leukemic cells, culminating in a clinical diagnosis of ALL. However, the mechanisms triggering these second hits have not been completely elucidated. A study reported that the expression of E/R was associated with increased ROS levels [E/R-positive ALL [These secondary genetic events are major rate-limiting events during the process of formation of S levels . ElevateS levels , thus letive ALL .E/R-positive ALL requires knowledge of not only the specific contributory fusion and the secondary genetic hits but also the cellular framework in which they arise and function. Most of the leukemic cells in E/R-positive ALL can be characterized by phenotypes  and genotypes  indicative of a B precursor cell identity. However, the cell of origin in which the initial functional impact of the fusion gene occurs has been contentious. It may lie anywhere antecedent to B precursor cells in the lineage hierarchy [IgH, TCR and ETV6/RUNX1 are leukemia-specific molecular markers in E/R-positive ALL. Analysis of these markers could illuminate when ETV6/RUNX1 gene fusion occurs during B cell development.Understanding the pathogenesis of ierarchy . IgH, TCE/R-positive ALL, this fusion transcript can occur during B-cell development before rearrangement of IgH and TCR genes [E/R fusion premalignant clone may occur before the pro-B cell stage. Sequence analysis of the E/R fusion region further supports this possibility, as there are no binding motifs indicative of RAG1/2 or TdT activity [E/R fusion gene [E/R in early progenitors or hematopoietic stem cells (HSCs) may account for the expression of the myeloid markers CD13 and CD33 in up to 48% of E/R-positive ALL patients [E/R's requirement to be expressed in leukemic stem and/or progenitor cells is that lymphoid progenitors lack self-renewal and cannot live long enough to accumulate the mutations required for full transformation [E/R-positive pre-leukemic cells to preserve the regulatory control mechanisms of normal stem cells to persist in their niche and avoid exhaustion.Confirmation of the leukemic cell of origin is fundamental to identify the cellular and molecular mechanisms that underlie leukemogenesis. A popular hypothesis is that completely transformed cancer clones are settled in a hierarchical pattern and sustained by a few “cancer stem cells” . HoweverCR genes . These rCR genes . Thus, tactivity , 63. In ion gene . Besidesion gene , 65. Thepatients . The expormation . It is aIgH and/or TCR gene rearrangements in E/R-positive ALL have demonstrated that the fetal cell type in which clonal advantage is elicited by E/R can be propagated after B-lineage commitment [E/R fusion gene in murine and human models suggested that E/R could promote self-renewal of early B-cell precursors [lowCD19+ cells were present in E/R-positive ALL patients [Ig and TCR gene rearrangements in five pairs of monozygotic twins concordant for ALL. They proved that the E/R fusion initially expanded in an early B-cell lineage-committed progenitor, most likely at the pro-B, or possibly at the pre-B, cell stage [E/R at the pre-B or pro-B cell stage is intriguing, as it may prove that the E/R fusion gene can directly influence the phenotype of leukemic cells.However, studies on clonal mmitment , 57, 66.ecursors , 66. Humpatients , 67. Thipatients . RecentlE/R fusion. In addition, this is also relevant to cancer therapy, where specific targeting of tumor-propagating cells may be desirable. Thus, it seems safe to conclude that the jury is still out on this issue, and further research is still required.Understanding the cellular targets of primary mutations and the ensuing malignant transformation is fundamental to understanding the function of the E/R fusion gene induces a pre-leukemic phenotype that can remain covert for up to 15 years [E/R is not only responsible for the initiation of leukemia but also essential for disease progression and maintenance. Although the expression of E/R interferes with critical regulatory functions [E/R fusion itself is not sufficient to explain the evolutionary and clinical emergence of ALL [Observations on normal cord blood and monozygotic twins demonstrated that the 15 years . Therefounctions , maintene of ALL . Insteade of ALL , 70, 71.ETV6 and RUNX1 are known regulators of hematopoiesis. The E/R fusion gene includes the N-terminal pointed (PNT) dimerization domain of ETV6 and the N-terminal Runt homology domain (RHD) and the C-terminal transactivation (TA) domain of RUNX1 [RUNX1 target genes and potentially convert RUNX1 to a negative transcriptional regulator [ETV6 [E/R chimeric protein is assumed to act as transcriptional repressor of RUNX1 target genes, several studies have reported similar levels of gene repression and activation for protein-coding and miRNA genes [E/R fusion gene product can disrupt the transcriptional repression of wild-type ETV6 by dimerization via the PNT domain [Both of RUNX1 . The resof RUNX1 . It can egulator , 74. Maxor [ETV6 . AlthougNA genes , 71, 75.T domain .E/R rearrangement involves two well-characterized transcription factors, the molecular pathways affected by the translocation are also responsible for leukemia clone maintenance and propagation  activation, aiding to the self-renewal and leukemogenic activity of leukemia cells [STAT3 activation has been shown to transcriptionally induce MYC [SMAD7 [E/R leukemic cells. In addition, the E/R gene product can activate the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway [E/R-positive leukemia [E/R can directly bind to the EPOR promoter and increase cell survival through activation of the JAK2-STAT5-BCL-XL pathway [MDM2 promoter activity. MDM2 functions as a crucial negative regulator of P53, and ultimately represses P53 signaling [P53 signaling is a crucial event in the development of leukemic cell survival [Although the n Figure . First oc clones . The E/Ria cells , 78. STAduce MYC  and SMAD pathway , resultileukemia . Moreove pathway  and the  pathway . Last buignaling , 81. TheE/R-positive ALL, and showed the effects of methylation on the expression of 17 potential drivers of leukemogenesis [EPOR has been shown to have a functional impact on promoting leukemic cell survival [IGF2BP1 (IGF2 mRNA binding protein 1) is another hypomethylated and upregulated gene in E/R-positive ALL. As a powerful regulator of E/R mRNA stability, IGF2BP1 may be a prominent player in cell transformation events in E/R-associated leukemogenesis [E/R fusion gene [miRNA-320a was found to be both hypermethylated and downregulated, together with miRNA-494, in E/R-positive leukemia [miRNA200c is another hypermethylated miRNA gene that was shown to exhibit strong tumor suppressor properties, partially by interacting with the epigenetic regulator and polycomb group protein BMI1 [NSD2 histone methyltransferase [E/R-positive leukemia cells. For instance, hsa-mir-125b-2 is highly expressed in E/R-positive leukemia, and can promote cell survival under growth inhibitory and apoptotic conditions in a P53-independent manner [mir-181a-1 is significantly reduced in E/R-positive cases, and a double-negative loop involving mir-181a-1 and E/R may contribute to E/R-driven arrest of differentiation [Epigenetic alterations, including DNA methylation and histone modifications, have also been reported to contribute to ALL progression , 83. Steogenesis . Among tsurvival . The oncogenesis –86. Seveion gene . miRNA-3leukemia . Both miein BMI1 . In addiein BMI1  and mutansferase  may alsot manner . In contntiation .E/R-positive ALL. Both lnc-RTN4R-1 and lnc-NKX2-3-1 can be regulated by the E/R fusion protein and might be functionally implicated in leukemogenesis [E/R can downregulate mitotic arrest-deficient 2L1 (MAD2L1) to attenuate the mitotic checkpoint (MC) [E/R with the antiapoptotic protein BCL2 [Recently, a comprehensive analysis of the long non-coding RNA (lnc RNA) transcriptome has been conducted in ogenesis . In addiint (MC) . This maein BCL2  and the ein BCL2 , althougETV6/RUNX1-positive leukemia generally has favorable long-term remission and survival rates [ETV6/RUNX1 is the most common structural genetic aberration in childhood B-cell precursor (BCP) ALL at first relapse [E/R-positive relapses occur early, and very early relapses are rare. In most patients relapses occur several years after cessation of treatment [ETV6/RUNX1-positive leukemia is a clinically and biologically heterogeneous disease. A variety of studies have focused on the relationship between the clones giving rise to the primary and recurrent leukemias, as well as the molecular abnormalities responsible for relapse.Pediatric al rates , but as  relapse . Only 10reatment , and occreatment . RelapseE/R-positive ALL relapse, many studies have compared Ig/TCR rearrangements, genomic boundaries of unrearranged ETV6 allele, and CNA patterns of matched diagnostic and relapse samples [Ig/TCR markers in some cases [ETV6 allele indicated that the relapse clone, at least in some patients, derive not from a dominant clone but from a sibling clone present at diagnosis [−4 to 5×10−3 cells [To characterize the clonal origins of  samples , 100–103me cases , 105. Lame cases . The celme cases . Compariiagnosis –103. Whiiagnosis , 103, thE/R-positive ALL in particular [Recently, research on the clonal origins of relapse in childhood ALL in general or rticular , 109 hasrticular –110. Therticular , 109. Inrticular , 108. varticular , 108, whrticular , 109. InE/R-positive leukemia can originate from either a major or minor clone at presentation  and CDKN1B (12p13) genes, which may have an adverse prognostic impact [CDKN2A/B, which are cell cycle regulators, are associated with poor minimal residual disease response and early emerging relapses [BCL2L14, CDKN1B, CDKN2A/B and FOXO3A may create a genetic background that renders an E/R-positive leukemic cell population drug-resistant and favors the development of early relapses. mir-650 may be associated with time to relapse, as it shows higher expression in early relapse cases [PAX5, EBF1, and IKZF1, are associated with B-lymphocyte differentiation and are affected by CNAs in E/R-positive relapse as well [SLX4IP show a gender-specific distribution at relapse; gain of Xq and deletion of SLX4IP are restricted to male patients [E/R-positive patients who have a three-way translocation pattern, comprising the classic E/R translocation plus a fusion of the remaining parts of ETV6 and RUNX1 with another chromosome. This pattern might be an important risk factor for relapse in patients with the E/R fusion gene [A substantial number of CNAs in relapsed ns Table  [30, 97, regions . The ext regions . The rec regions . The genrelapses , 97, 108patients . The comrelapses . Thus, tse cases . Moreove as well . CNAs ofpatients , while lion gene .NR3C1 (5q31.3) encodes the human GC receptor, and its deletion is linked with impaired response to GC [NR3C1 deletions prevail at relapse and possibly account for the adverse prognosis of some E/R-positive relapses [BCL2 modifying factor (BMF) and components of the mismatch repair (MMR) pathways [E/R-expressing leukemic cells [E/R-positive relapse pathogenesis, an observation that may contribute to guide future therapies.Glucocorticoids (GC) represent an essential component in frontline and relapse treatment protocols for ALL. GC resistance seems to be associated with ineffective blast cell clearance, disease recurrence and treatment failure in childhood ALL , 115. Thse to GC , 112. NRpathways . BMF plaic cells , 116. Deic cells , 117. ThE/R fusion gene is the most frequent genetic abnormality in pediatric ALL. Although multiple studies have identified a variety of second genetic hits, their correlation with E/R and their effects on the pathogenesis of leukemia need to be further explored. The cell of origin in which the initial functional impact of the E/R fusion gene occurs has been contentious. It may lie antecedent to B precursor cells or after B-lineage commitment. Further investigation into the precise cell type of origin has prominent therapeutic significance, as these cells may be the most relevant targets for treatment. In addition, functional studies are needed to unveil the key mechanisms channeling the evolution of E/R-positive pre-leukemic cells into B-ALL. E/R fusion drives not only the leukemic transformation process but also the maintenance and propagation of leukemia cells. The specific molecular alterations, including components of affected signaling pathways and epigenetic regulators, constitute ideal therapeutic targets. For instance, inhibitions of the aberrantly activated PI3K/AKT/mTOR pathway, STAT3 signaling and/or MDM2/P53 interaction represent promising therapeutic strategies.The Ig/TCR rearrangements on matched diagnostic and relapse samples showed that relapse in E/R-positive ALL originates from either major or minor clones present at diagnosis. The CNAs specific to relapse contribute to not only the development of relapse but also to drug resistance. GC-associated drug resistance is important in the pathogenesis of E/R-positive ALL relapse. Consideration of the course of childhood ALL implies the need for further biological risk markers that enable a more detailed understanding of poor treatment response. These risk markers may contribute to rationalize specific treatment intensification before stem cell transplantation in poor responding E/R-positive relapses.Analyses of CNAs and


The tumor to normal brain ratios (T/N ratios) were calculated, and the relationships between T/N ratio and World Health Organization tumor grade or MIB-1 labeling index were evaluated. The diagnostic values of T/N ratios were assessed using receiver operating characteristic (ROC) curve analyses to differentiate between high-grade gliomas (HGGs) and low-grade gliomas (LGGs).We investigated the relationship between metabolic activity and histological features of gliomas using fluorine-18α-methyltyrosine . Both T/N ratio (p = 0.016) and maximum standardized uptake value (p = 0.033) of 18F-FDG PET showed significant differences between HGGs and LGGs. ROC analysis yielded an optimal cut-off of 3.37 for the T/N ratio of 18F-FAMT PET to differentiate between HGGs and LGGs . Positive predictive value was 84%, and negative predictive value was 62%. T/N ratio of 18F-FAMT PET was not correlated with MIB-1 labeling index in all gliomas, whereas T/N ratio of 18F-FDG PET was positively correlated . Significant positive correlation was observed between T/N ratios of 18F-FDG and 18F-FAMT , but median T/N ratio of 18F-FAMT PET was significantly higher than that of 18F-FDG PET in all grades of glioma.Median T/N ratio of 18F-FAMT uptake has high positive predictive value for detection of HGGs. 18F-FAMT PET had higher T/N ratio, with better tumor-normal brain contrast, compared to 18F-FDG PET in both LGGs and HGGs. Therefore, 18F-FAMT is a useful radiotracer for the preoperative visualization of gliomas.The T/N ratio of  Further dynamic study using 18F-FAMT PET will be necessary in the future.More reliable grading may be possible with dynamic or grade . HGGs arr uptake . The difylalnine . Therefo18F-FAMT PET and 18F-FDG PET imaging is the ideal method of comparison. Since both tracers are labeled with fluorine, the tracer half-life requires a suitable interval between these PET studies. In these 38 cases, the interval between 18F-FAMT PET and 18F-FDG PET studies ranged from 1 to 38 days, and the median was 5 days. Recently, accumulation of 18F-FAMT was reported to be strongly correlated with the expression of LAT1 in cancers [18F-FAMT transport and LAT1 expression was not examined in this study. A further study will be needed to investigate the mechanism of 18F-FAMT accumulations in gliomas. 18F-FAMT is a new radiotracer for brain tumor imaging. More experience with cases of gliomas or other brain tumors is needed. A comparative study with radiotracers other than 18F-FDG is also needed to clarify the diagnostic utility of 18F-FAMT PET.There were limitations to the present study. This study was based on relatively strict pathological and grading differentiations in astrocytomas and oligodendroglial tumors. Therefore, some pathological categories included a relatively small number of samples. Furthermore, simultaneous  cancers . However18F-FAMT is a useful radiotracer for the preoperative evaluation of tumor malignancy. 18F-FAMT PET provides clearer imaging with higher T/N ratio and better contrast compared to 18F-FDG PET in all gliomas. Therefore, 18F-FAMT is a useful radiotracer for the preoperative visualization of gliomas.


A library of diverse heparan sulfate (HS) oligosaccharides was chemoenzymatically synthesized and systematically studied using NMR. 66 HS and heparin oligosaccharides covering different sulfation patterns and sizes was prepared through an efficient method of chemoenzymatic synthesis. A systematic nuclear magnetic resonance spectroscopy study was firstly undertaken for every oligosaccharide in the library. In addition to the availability of different oligosaccharides, this work also provides spectroscopic data helpful for characterizing more complicated polysaccharide structures providing a safeguard to ensure the quality of the drug heparin. This HS/heparin library will be useful for activity screening and facilitate future structure–activity relationship studies.Heparan sulfate (HS) is a member of the glycosaminoglycans (GAG) family that plays essential roles in biological processes from animal sources. Heparin, a highly sulfated form of HS, is widely used as anticoagulant drug worldwide. The high diversity and complexity of HS and heparin represent a roadblock for structural characterization and biological activity studies. Access to structurally defined oligosaccharides is critical for the successful development of HS and heparin structure–activity relationships. In this study, a library of  Often there are only relatively subtle structural features, such as sugar epimers, sulfo group position, or differences in linkage position and anomeric configuration, unique to these impurities or contaminants.19N-glycans. In our previous efforts,p-nitrophenyl glucuronide (GlcA-pNP), which is ultraviolet detectable, is capable of generating a series of heparin oligosaccharides in good overall yield and possessed excellent anticoagulant activity. Herein, we have utilized another ultraviolet detectable, addressable acceptor, N-(6-azidohexanamidyl) p-aminophenyl glucuronide (GlcA-pNA-N3), for chemoenzymatic synthesis and a systematic NMR study, and produced the largest library of 66 HS and heparin oligosaccharides to date.There are generally two approaches for the target driven synthesis of a library of structurally defined HS and heparin oligosaccharides. The first approach relies on a purely chemical synthesis method, based on repetitive steps of protection, activation, coupling and de-protection, and is incredibly challenging for the preparation of oligosaccharides larger than pentasaccharides.I–VII  monosaccharide acceptor and extended with UDP-sugar donors using a chemoenzymatic approach. Heparosan synthase-2 (pmHS2) from Pasteurella multocidaI–VII. GlcNTFA containing backbones were then subjected to chemical de-N-trifluoracetylation and modification by N-sulfotransferase (NST) resulting in key intermediates II–IV and VII. Key intermediates VI and VII were subjected to additional enzymatic modification relying on C5-epimerase, 2-O-sulfotransferase (2-OST), 6-O-sulfotransferases (6-OSTs) and 3-O-sulfotransferases (3-OSTs). For example, to synthesize intermediates IV through VI, GlcA-pNA-N3 was first repetitively elongated using pmHS2 with UDP-N-trifluoroacetyl glucosamine (UDP-GlcNTFA) donor and UDP-glucuronic acid (UDP-GlcA), to form pentasaccharide, GlcA(1→4)GlcNTFA(1→4)GlcA(1→4) GlcNTFA(1→4)GlcA-pNA-N3 (N-sulfo groups using NST and 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to obtain intermediate IV. Treatment of intermediate (IV) with C5-epimerase and 2-OST afforded IdoA2S-containing intermediate (VI) for further modification. C5-epimerase is known to be a bi-directional enzyme, catalyzing both the forward and reverse reaction and affording an equilibrium mixture of GlcA and IdoA residues.5-epimerase to GlcA2S.V by treating with C5-epimerase alone. This resulting intermediate V could be applied into the preparation of more structurally complex IdoA-containing heparin oligosaccharides GlcNS(1→4)IdoA2S(1→4) GlcNS(1→4)GlcA-pNA-N3  three different GlcNS3S6S-containing oligosaccharides 14, 28, 46, were prepared , 6-O-sulfotransferase (6-OST-1 and 6-OST-3) and 3-O-sulfotransferase-1 (3-OST-1), GlcNS3S6S-containing hexasaccharide 14 was obtained  residue represents <1–5% of total uronic acid residues in HS and heparin isolated from natural sources and only limited information on the biological role of these rare saccharide residues has been reported.IV, GlcA(1→4)GlcNS(1→4)GlcA(1→4)GlcNS (1→4)GlcA-pNA-N3, was treated with 2-OST65, or two GlcA2S residues, 66, respectively, were obtained with good regioselectivity and their structures were confirmed by ESI-MS and NMR analyses (ESI).The 2-Using a similar approach, all the other compounds presented in 14 GlcNS6S (1→4)GlcA(1→4)GlcNS3S6S(1→4)IdoA2S(1→4)GlcNS6S(1→4)GlcA-pNA-N3, is presented in 14 eluted as a single peak from high-resolution anion exchange HPLC column, indicating that this compound was pure , identical to the calculated molecular mass of 1900.6. During chemoenzymatic synthesis the strong UV absorbance of the pNA-N3 aglycone facilitates the detection and purification of the synthetic intermediates.Gel filtration chromatography relying in Bio-gel P2 columns represents an excellent way of removing salts but is inefficient for oligosaccharide purification, since reaction byproducts, often introduced through incomplete glycosylation, have molecular weights similar to the target compounds. An HPLC based approach for rapid access to pure HS and heparin oligosaccharides was developed. The elongated products of the HS backbone prior to sulfation reactions were purified using a C-18 column, while all the sulfated products of the oligosaccharides were purified using Q-Sepharose fast flow column. Purity and structural analysis of the entire oligosaccharide library  was cond14 by NMR spectroscopy is presented in 1H NMR spectrum, six anomeric proton signals are assigned confirming that this compound is a hexasaccharide  resonate from 5.4 to 5.6 ppm and the signals at 4.56 and 5.07 ppm correspond to GlcA residues B and F. The signal at 5.07 ppm of residue F-1 is due to the de-shielding effect of the nearby aromatic ring. The 3JHH coupling constants of A-1, C-1 and E-1 were 4 Hz while B-1 and F-1 were 8 Hz, suggesting α-linkages and β-linkages, respectively. Interestingly, when conducting NMR analysis on our oligosaccharide library, we discovered that the anomeric proton of IdoA2S residues usually appeared as broad peaks in the 1H NMR spectra, and correlation signals in 2D spectra were often of very low intensity. For example, the anomeric proton of the IdoA2S residue (D-1) of hexasaccharide 14 showed a broad peak at 5.18 ppm and the  cross-peak was so weak that it was not observed in the 1H–1H COSY spectrum. However, a relatively weak correlation in the IdoA2S residue between D-5 and D-4 was detected . The broad anomeric signal for the IdoA2S observed in the 1H NMR spectrum is attributed to the inter-conversion of the multiple conformational forms of the IdoA2S pyranose ring D, 1C4-chair, 2S0-skew boat and 4C1-chair. The addition of ethylenediaminetetraacetic acid (EDTA) significantly sharpened the IdoA2S signals and often resulted in significant chemical shift variations, possibly due to the chelation of trace amounts of divalent cations.17The representative characterization of oligosaccharide ccharide A and 3A.14 are highly overlapped, requiring that we conduct 2D NMR experiments, including, 1H–1H TOCSY, NOESY and 1H–13C HSQC, to fully assign chemical shifts  residue is on the reducing end of a hexosamine residue, both proton and carbon anomeric signals for GlcNS and GlcNS6S are dramatically shifted to 5.24/97.3 and 5.33/96.2 ppm, respectively. Similarly, the anomeric signal of GlcNAc and GlcNAc6S have a comparable chemical shift of 5.32/96.8 ppm if a GlcA residue is present on the reducing end side of this residue. The shift of the H-1 signal is very similar for GlcNAc(6S) and GlcNS6S, only a COSY spectrum could easily distinguish these due to their large differences in the shift values of their H-2 signals. The 3-O-sulfo group also exhibits an effect on GlcNS3S6S with an apparent downfield shift signal to ∼5.43 ppm, compared to the GlcNS and GlcNS6S residues. This observation is helpful in identifying the →4)GlcNS3S6S(1→4)IdoA2S on its reducing end. The proton chemical shift for GlcA notably shifts to 4.54 ppm when the reducing end GlcNX residue carries a 6-O-sulfo group. It is noteworthy that the 6-O-sulfo group has a greater impact than N-sulfo group of the neighboring glucosamine residue on the reducing end. The anomeric position of the IdoA2S residue exhibits consistent chemical shifts of 5.18/99.0 ppm independent of whether it has a GlcNX or GlcNX6S at its reducing end. The anomeric position of the IdoA residue has very different chemical shifts 4.95/101.5 ppm.Uronic acid residues show characteristic anomeric Heparin is a widely used clinical anticoagulant for the prevention and treatment of arterial and venous thrombosis.Introducing chemically active functional groups is expected to expand the use of the library. In the current library, each oligosaccharide possesses an azido group at the reducing end. The azide functionality is well known to be inert within biological systems but can be readily tagged covalently with imaging probes or modified using an azide-specific reaction, such as Staudinger ligation with phosphines and the [3 + 2] cycloaddition with alkynes.31O-sulfoglucosamine (GlcNS3S±6S) effects these important roles in contributing to HS biological activity. The ready availability of multi-milligram quantities of oligosaccharide structural variants containing a GlcNS3S6S residue should facilitate further biological investigation.Although sulfation at the 3-OH position of glucosamine occurs infrequently in HS, this sulfation pattern is closely related to its biological functions, facilitating the entry of herpes simplex virus into host cells to establish infection,O-sulfoglucuronic acid (GlcA2S) in heparin and HS very little research has been focused on the SAR of this residue. Moreover, while 2-OST has been shown to act, albeit poorly, on GlcA in a polysaccharide substrateDue to the low abundance of 2-66 structurally defined HS and heparin oligosaccharides, including “rare” and critical moieties, such as GlcA2S, IdoA, GlcNS3S6S and AT-binding pentasaccharide sequence. The ultraviolet detectable tag and an HPLC-based purification method facilitate the rapid access to a diverse library containing tens of milligrams of high purity HS and heparin oligosaccharides. These oligosaccharides are not only potentially important for bioactivity evaluation but also good substrates to construct glycoconjugates or microarrays through azide chemistry. This work also describes a systematic NMR study on these HS and heparin oligosaccharides, paving the way to more complicated HS and heparin polysaccharide characterization and helping to safeguard the quality of the critically important drug, heparin.We have successfully developed a chemoenzymatic approach for the efficient preparation of R. J. L. and J. L. designed experiments, analyzed data and wrote the manuscript. X. Z. and V. P., H. M. J., A. M. L. and A. M. P. G. performed all the synthesis and purification of oligosaccharides described within this manuscript. T. Q. P. prepared the enzymes and cofactors for the synthesis.J. L. is a founder of Glycan Therapeutics. V. P., H. M. J., A. M. L. and A. M. P. G. are employees of Glycan Therapeutics. X. Z., T. Q. P. and R. J. L. declare no competing financial interests.Supplementary informationClick here for additional data file.