Trichoderma harzianum. The enzyme, comprised of 572 amino acids, shares high homology with PAF-AH proteins from T. koningii and other microbial species. The optimum enzymatic activity of PAF-AH occurred at pH 6 in the absence of Ca2+ and it localized in the cytoplasm, and we observed the upregulation of PAF-AH expression in response to carbon starvation and strong heat shock. Furthermore, PAF-AH knockout transformant growth occurred more slowly than wild type cells and over-expression strains grown in SM medium at 37°C and 42°C. In addition, PAF-AH expression significantly increased under a series of maize root induction assay. Eicosanoic acid and ergosterol levels decreased in the PAF-AH knockouts compared to wild type cells, as revealed by GC/MS analysis. We also determined stress responses mediated by PAF-AH were related to proteins HEX1, Cu/Zn superoxide dismutase, and cytochrome c. Finally, PAF-AH exhibited antagonistic activity against Rhizoctonia solani in plate confrontation assays. Our results indicate PAF-AH may play an important role in T. harzianum stress response and antagonism under diverse environmental conditions.We investigated the properties of platelet-activating factor acetylhydrolase (PAF-AH) derived from  These enzymes hydrolyze ester bonds at the sn-2 position of glycerophospholipids to release free fatty acids and lysophospholipids, which often trigger signal transduction pathways in animal or human cells. For example, arachidonic acid is the precursor of eicosanoids, prostaglandins, and leukotrienes, all of which play important roles in the inflammatory response Saccharomyces cerevisiae significantly enhances the yeast's resistance to oxidative stress The platelet-activating factor acetylhydrolase (PAF-AH) belongs to the phospholipase ATrichoderma, which is present in diverse habitats, has been used as a biocontrol agent for many decades e.g., hsp70 and hex1) to adapt to varied environments Trichoderma with its host, such as chitinase, glucanase, and PAF-AH Trichoderma koningii (T30) transformants generated by restriction enzyme-mediated integration under different stress conditions and found that PAF-AH transformants were susceptible to H2O2 and other stresses. We currently observed Trichoderma harzianum (T28) PAF-AH expression was significantly upregulated under heat stimulus and that T28 PAF-AH transformants grew more slowly than the wild type strain. These results indicate PAF-AH plays an important role in Trichoderma stress response, but little information is available regarding its physiological function(s). Therefore, in this work, we analyzed the PAF-AH gene derived from Trichoderma harzianum and investigated its function in stress response and antagonism.T. harzianum T28 (CCTCC AF 2013026) was stored in the Laboratory of Resource and Environmental Science. Wild type and transformants were maintained in PDA  at 28°C until sporulation occurred. Escherichia coli DH5α and BL21 (DE3) were purchased from Invitrogen  for plasmid propagation and protein expression, respectively. Bacterial strains were grown in Luria-Bertani (LB) broth or LB agar plates supplemented with kanamycin sulfate (0.1 mg/mL) or ampicillin (0.1 mg/mL).The wild type strain HindIII, XbaI, KpnI, or SacI , respectively, and gel purified and ligated to modified plasmid pCAMBIA1300 (provided by Chu Long Zhang of Zhejiang University). Agrobacterium tumefaciens-mediated transformation was performed as previously described trpC promoter and terminator sequences from the vector pSilent-1 A genome walking kit  was used to clone the PAF-AH gene, resulting in a transformant referred to as P6 hyg gene (for hygromycin B resistance) was amplified with primers Hyg-F/Hyg-R; (2) the PAF-AH deletion was verified by attempting to amplify PAF-AH; (3) T-DNA insertion numbers were determined by using primers RB-F/Hyg-R; and (4) primers PAF-AH-UP-F and Hyg-R were used to confirm the hyg cassette position. In addition, 40 µg genomic DNA from T28 and transformants was digested with HindIII and BamHI . Finally, Southern blotting was carried out to confirm the transformants as described previously TrpC-F/Terminator-R to amplify the PAF-AH ORF over-expression cassette, and Southern blotting was again used to determine the copies of PAF-AH over expression cassette present. Over-expression PAF-AH transformant (OE1) strains were identified with one T-DNA insertion site (The genomic DNA of transformants were isolated by a modified CTAB method ion site .into E. coli BL21 (DE3). Gene expression was induced with addition of 1 mM IPTG. Transformants were collected, suspended in lysis buffer , and subjected to sonication. The solution was centrifuged at 5000 rpm for 10 min, and resulting supernatants were transferred to Ni-NTA agarose columns , and PAF-AH enzymatic activity was analyzed by the PAF-AH assay kit . For immunoblotting assays, protein samples were separated by SDS-PAGE and transferred to a nitrocellulose membrane. After electroblotting, filters were saturated with 5% non-fat dry milk in TBS with 0.1% Tween for 1 h at room temperature. Monoclonal 6×His-tag PAF-AH-specific antibodies  were used as primary antibodies, and an anti-mouse peroxidase conjugate  used as secondary antibody for detection of PAF-AH. For chemiluminescence detection, ECL Plus Western Blot detection reagent  was used.The PAF-AH gene was amplified by PCR and inserted into vector pET-28a (+) , and the construct was transformed in vivo fluorescence analysis, knockout (KO40) transformants expressing PAF-AH C-terminally fused with GFP under pdg promoter For 6 mL−1) from wild type T28 and transformant strain KO40 were added to a 500 mL sterile flask with 200 mL SM medium 2O. The mycelia were ground by mortar and pestle with liquid nitrogen, and TCA-acetone was used to extract the mycelial proteome. Total protein concentrations were determined by the Bradford method t-test at P<0.05) indicated significant differential expression of PAF-AH. Gel analysis and protein identification were performed at the research laboratory of Fudan University.One hundred microliters conidial suspension (1×10actin gene. Expression levels of genes hex1 (Accession no. KF356403), cu/zn superoxide dismutase (sod) (Accession no. JX481779), and cytochrome c (Accession no. JX481780) from strains T28 and KO40 were tested using the following primer sets: hex1-F/hex1-R, cu/zn sod-F/cu/zn sod-R, and cytochrome c-F/cytochrome c-R . Quantitative real-time PCR (qRT-PCR) was performed using a SuperReal PreMix (SYBR Green) kit  with the synthesized cDNA as template. The PCR conditions were as follows: 95°C for 30 s for initial denaturation and 40 cycles each consisting of 95°C for 20 s, 58°C for 30 s, and 72°C for 20 s. For this experiment, an FTC-3000 Real-Time PCR System  was used according to the manufacturer's instructions. The qRT-PCR was carried out with primers Actin-F and Actin-R for amplifying the reference control rome c-R , respect6 conidia mL−1) from wild type T28 and transformant KO40 were incubated in 200 mL SM medium at 28°C with 180 rpm shaking for 6 days. The mycelia were filtered and washed with sterile ddH2O three times and then lyophilized for 3 h. The mycelia (0.5 g) were ground by mortar and pestle with liquid nitrogen, and fatty acids were extracted by the standard chloroform/methanol method. After saponification in methanol with 1 M NaOH in a 75°C water bath for 30 min, the fatty acid methyl ester was dissolved in 1 mL normal hexane. The fatty acids and sterols were analyzed by GC/MS according to the previously described method m/z 33 to 500 at full scan, and the acceleration voltage was initiated after a solvent delay of 3 min. The data were then compared with reference spectra from the NIST 2011 library.Conidial suspensions  tree was constructed with the MEGA 5.1 software. SOD and catalase activity assay were carried out with the same amount of tissue proteins that dissolved in 0.1 M PBS, and the strains were cultured the same as in 2-DE protein extraction. Data were measured with Microplate Reader.P≤0.05). Graphs were prepared with GraphPad Prism 5.All experiments were performed in independent biological triplicates. Data were analyzed statistically using SAS 8.0 software (significance indicated by Trichoderma spp. (teleomorphic Hypocea spp.), yeast, and humans  was amplified from synthesized T28 cDNA. The 1783 bp gene contains 65 bp of intronic sequence and a 1719 bp ORF that encodes 572 amino acids. A neighbor-joining tree was constructed after aligning PAF-AH protein sequences with those from different organisms, including other d humans  to inferBamHI and EcoRI and then ligated with plasmid pET-28a. The presence of pET-28a-PAF-AH was verified by PCR and DNA sequencing. Then, pET-28a-PAF-AH with N-terminal fusions and metal-binding 6× His tag was transformed into E. coli strain BL21 (DE3), to which 1 mM IPTG was added for induction of gene expression. A soluble fusion protein was used for SDS-PAGE and Western blot analysis  transformants were generated to investigate the PAF-AH gene's function in sistance . We usedsistance . The hyghyg gene as a probe, transformant KO15 showed another T-DNA insertion compared with transformant KO40 . Moreover, levels of PAF-AH expression were also upregulated after exposure to maize inbred line (Huangzhou 4) roots . FurtherP<0.05), indicating they are typically associated with PAF-AH expression.Differences between fatty acid and sterol profiles in the transformants and wild type strains were analyzed with GC/MS. Ten distinct types of fatty acids and ergosterol were found and are shown in cis-trans isomerase, hypothetical protein CHGG_10818, HEX1, and Cu/Zn superoxide dismutase, as well as seven unknown proteins were identified by MS/MS  on agar plates previously colonized by T. harzianum but was higher (P<0.01) on agar plates previously colonized by KO40  and Cordyceps militaris (Accession no. EGX90692.1), respectively , possibly due to reduced secreted enzymes that are important to T. harzianum's mycoparasitic interactions with R. solani. Another reason for reduced antagonism of KO40 may be related to deficient PAF-AH-induced sensitivity to R. solani-secreted enzymes or secondary metabolic products that affect cell membrane integrity of Trichoderma.We also determined PAF-AH is necessary for the antagonistic ability of Trichoderma spp. exhibit remarkable features, such as fast growth, colonization, and efficient utilization of nutrients in the environment. These characteristics are common in fungi; for instance, the mRNA of TbSP1 (an important gene from phospholipases A2) of T. borchii is significantly upregulated when carbon or nitrogen sources are scarce, and the expression of two other PLA2 mRNAs is elevated in Aspergillus oryzae under carbon starvation and oxidative stress conditions T. harzianum was notably upregulated in response to carbon source starvation and temperature stress. Furthermore, we determined interactions between T. harzianum and maize roots led to increased PAF-AH expression. Because PAF-AH might interact with maize seedling roots in rhizosphere, during which nitrogen and carbon supplies may be limited, we propose increased PAF-AH activity would favor Trichoderma growth under carbon starvation conditions in barren soil.T. harzianum stress response and antagonism. This protein mediates tolerance to abiotic stress and the microbe's antagonistic ability toward R. solani. Additionally, PAF-AH might be involved and in scarce nutrient utilization of Trichoderma and interactions with maize seedling roots in the rhizosphere. We conclude PAF-AH has versatile function in Trichoderma, and its gene expression is likely triggered at different environments to aid in Trichoderma's adaptation and survival.In summary, this present work was the first report regarding the biological role of PAF-AH in Figure S1Screening of PAF-AH overexpression transformants. (A) Physical map of overexpression cassette. (B) Amplified fragment of Hygromycin B gene. (C) Amplified fragment of promoter trp C. (D) Amplified fragment of PAF-AH. (E) Amplified fragment of terminator trp C. (F) Amplified fragment of PAF-AH overexpression cassette. (G) Southern blot to confirm PAF-AH overexpression transformants. The genome DNA of WT and transformants were digested with Xba I. The 450 bp fragment amplified from Hygromycin B gene was labeled with 32P-dCTP as probe.WT: wild-type strain. P: plasmid. 1, 2, 3: PAF-AH overexpression transformants. M: DNA maker.(TIF)Click here for additional data file.Figure S2PAF-AH transcription level analysis in KO15 and KO40 transformants. (A) Electrophoresis of PCR products of PAF-AH ORF. (B) Electrophoresis of PCR products of 500 bp fragment of the PAF-AH. The PCR template was 1 µL cDNA of WT T28 and the KO transformants respectively. M: DNA marker, WT: Wild Type (T28), KO15, KO40: PAF-AH KO transformants.(TIF)Click here for additional data file.Figure S3PAF-AH activity assay of T28, KO40, and OE1. The experiment was carried out with the same amount of tissue protein dissolved in 0.1 M PBS, and these strains were cultured the same as in 2-DE protein extraction. Data were measured with Microplate Reader. * indicated significant difference of enzyme activity compared with the wild type, * showed P≤0.05. Results were means±SD (n = 3).(TIF)Click here for additional data file.Figure S4Different strains grew on different pH SM plate agar. The photograph was taken after 24 h inoculation at 28°C, T28 (wild type), KO40 (PAF-AH KO transformant), OE1 (PAF-AH overexpression transformant).(TIF)Click here for additional data file.Figure S52O2.Different strains grew on SM plate agar containing different concentration of H The photograph was taken after 24 h inoculation at 28°C, T28 (wild type), KO40 (PAF-AH KO transformant), OE1 (PAF-AH overexpression transformant).(TIF)Click here for additional data file.Table S1Primer used in PCR reaction of this paper.(DOC)Click here for additional data file.Table S2qRT-PCR efficiency of PAF-AH at different condition.(DOC)Click here for additional data file.Table S3qRT-PCR efficiency of hex1, cu/zn sod, cytochrome c.(DOC)Click here for additional data file.


FAU) gene is down-regulated in human prostate, breast and ovarian cancers. Moreover, its dysregulation is associated with poor prognosis in breast cancer. Sponges (Porifera) are animals without tissues which branched off first from the common ancestor of all metazoans. A large majority of genes implicated in human cancers have their homologues in the sponge genome. Our study suggests that FAU gene from the sponge Suberites domuncula reflects characteristics of the FAU gene from the metazoan ancestor, which have changed only slightly during the course of animal evolution. We found pro-apoptotic activity of sponge FAU protein. The same as its human homologue, sponge FAU increases apoptosis in human HEK293T cells. This indicates that the biological functions of FAU, usually associated with “higher” metazoans, particularly in cancer etiology, possess a biochemical background established early in metazoan evolution. The ancestor of all animals possibly possessed FAU protein with the structure and function similar to evolutionarily more recent versions of the protein, even before the appearance of true tissues and the origin of tumors and metastasis. It provides an opportunity to use pre-bilaterian animals as a simpler model for studying complex interactions in human cancerogenesis.Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV) ubiquitously expressed ( The evolution of cancer is still not fully understood. However, it is known that the genes of cellular cooperation that evolved along with multicellularity, when malfunctioned, contribute to cancer development . The majO-methylation of target rRNAs), H/ACA box snoRNAs  and small Cajal-body-specific RNAs (scaRNAs) . snoRNAs are believed to be the most ancient non-coding RNAs (ncRNAs) [Ribosome assembly is a complex process requiring coordinated activation of more than 200 non-ribosomal factors, including numerous small nucleolar RNAs (snoRNAs). Their role in modification of ribosomal RNAs (rRNAs) was shown to be essential for the correct assembly of the ribosome ,8. Three(ncRNAs) . Many ex(ncRNAs) . snoRNAs(ncRNAs) .FAU gene (Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV) ubiquitously expressed) encodes the ribosomal protein S30 (RPS30) fused with an ubiquitin-like protein FUBI [v-fos and fox, can induce osteosarcomas in susceptible mice [Fox is an antisense sequence to the cellular gene FAU, which indicates a putative tumor suppressor role for FAU. Moreover, the FAU gene is down-regulated in human breast, prostate and ovarian tumors and its down-regulation is strongly associated with poor prognosis in breast cancer [Ribosomal proteins (RPs) are an evolutionarily conserved component of ribosome in every cell of every organism. Analysis of 66 complete genomes revealed that 34 RPs are common to all living organisms . Many RPein FUBI . Retrovible mice . Fox is t cancer ,17,18. Pt cancer . Since tFAU gene from the sponge Suberites domuncula probably reflects the characteristics of the FAU gene from metazoan ancestor that changed only slightly during animal evolution. FAU protein from sponge possesses pro-apoptotic activity and increases apoptosis in human HEK293T cells, same as its human homologue. This suggests that biological functions of FAU, usually associated with the origin of cancer in “higher” metazoans, possess biochemical background established early in metazoan evolution. Our results implicate that the ancestor of all animals possessed FAU protein with the structure and the function similar to its evolutionarily recent versions, even before the appearance of true tissues and the origin of tumors and metastasis.Our study suggests that FAU gene encodes an ubiquitin-like protein (FUBI) fused to the ribosomal protein S30 (RPS30). This type of gene organization seems to be conserved throughout metazoan evolution  and Drosophila melanogaster . Both invertebrate model organisms underwent recent accelerated evolution [volution A. Spongevolution . Since sFAU promoter, we compared over-represented motifs in RPG promoters of the sponge Amphimedon queenslandica with FAU sequence. In the majority of analyzed human RPG promoters, YY1 motifs were found downstream of the transcription start site (TSS) [FAU (p = 3.00 × 10−7) , while E-box palindromic core CACGTG was also identified in FAU promoter (p = 3.12 × 10−6). Among many transcription factors that recognize the E-box, oncoprotein c-Myc (MYC) is known to bind to it [cis-regulatory element in transcriptional regulation and has a role at the translational level [FAU promoter indicates that the foundations of the human FAU cis-regulation were established early in metazoan evolution.Synchronized expression of ribosomal protein genes (RPGs), driven by the promoters of similar strength, ascertains equimolecular amount of ribosomal proteins in the cell, which is prerequisite for the ribosome assembly ,22. Humate (TSS) . This mo × 10−7) B. Two SPnd to it . c-Myc end to it . This imal level ,24. In cal level . The preFAU gene orthologues was used to characterize intron dynamics by comparing the intron positions  , SNORA62 can change its genomic location. In S. pagurorum, S. domuncula and S. ficus this snoRNA is present in the same intron, but apparently is missing in the same conserved intron in S. massa (FAU) evolution even on shorter evolutionary time scales.The accumulating genomic data strongly confirm the tendency of snoRNAs to colonize RPGs and ribosome related genes in eukaryotes . The totA62 (E2) A. SNORA6A62 (E2) B. The spA62 (E2) A. Its exA62 (E2) C. Phylogain/loss . It is kain/loss . Since adynamics . HoweverS. massa . Our resRhizaxinella pyrifera, a single snoRNA with a potential target rRNA was found  when compared to cells with pcDNA3 control (p = 0.014) increased caspase activity as compared to pcDNA3 transfected cells . Both host RPGs possess an extraribosomal function involved in the maintenance of cellular viability through the caspase-dependent regulation of apoptosis [SNORA62 is the most upregulated gene in diallyl sulfide (DAS)-induced apoptosis in HeLa cells, as a part of p53, mitochondria- and caspase- pathways [From all presented data we can conclude that the structure and the functions of the recent elopment . Numerouelopment ,40. Our poptosis ,41. Intepathways . It woulpathways , or tumopathways  and if tpathways . If SNORSuberites domuncula  and Sycon raphanus  were collected in the Northern Adriatic Sea near Rovinj, Croatia, and stored at −80 °C. Specimens of Oopsacas minuta  were collected in the cold water pit in the Middle Adriatic and specimens of Rhizaxinella pyrifera  were collected in the Middle Adriatic Pit, around 200 m depth and stored in 96% EtOH. Suberites ficus and Suberites pagurorum were collected in Ireland, while Suberites massa in the English Channel.Live specimens of the sponge http://www.ncbi.nlm.nih.gov). Sequences were analyzed using Lasergene . Multiple sequence alignments (MSA) were performed with CLUSTALX [http://www.psc.edu/biomed/genedoc).Homology searches and sequence retrievals were done using BLAST  and p-value  were extracted from MEME. The location of transcription factor binding sites relative to the transcription start site was also extracted from MEME results for FAU sequence within a set of sequences with the same identified motif. Obtained motifs were then compared using TOMTOM [Over-represented motifs were searched using MEME , as descg TOMTOM  against g TOMTOM .FAU gene was amplified by PCR, using LA Taq DNA polymerase  and primers specific for ends of the coding sequence of: Demospongiae (5′-GCAAGTTTTCGTACAAGGAGGGGC-3′ and 5′-GGTGCATTTGAGTTTGGTCCAC-3′), Hexactinellida (5′-ATGCAAGTGTTTGCACAAAC-3′ and 5′-CTAACTAGCTGAGTTTGAG-3′) and Calcarea (5′-CAGATTTTCATTCAGGCCCAGGC-3′ and 5′-GCGTTAGAGTTGGGTC-3′). The amplified fragments were separated on a 0.8% agarose gel, purified and cloned into the pGEM-T vector . Positive clones were sequenced using T7/pUC primers.For genomic DNA preparation, sponge specimens were cut into pieces, frozen in liquid nitrogen and grounded to fine powder from which total DNA was isolated using the Genomic DNA Purification kit . Sponge FAU genes [http://snoopy.med.miyazaki-u.ac.jp/) and the snoRNA-LBME databases [S. domuncula were cut into pieces, frozen in liquid nitrogen and grounded to a fine powder. mirPremier microRNA Isolation Kit  was used, according to the manufacturer’s protocol for plant tissue. Polyadenylation was performed by incubation with E. coli Poly(A) Polymerase (BioLabs). Poly(A) tailing reaction mixture was then reverse transcribed using the SuperScript II Reverse Transcriptase  and a modified poly-d(T) primer (5′-GCGTAAGTGACTAGCGTGTTTTTTTTTTTTVN-3′). The resulting cDNA was used for PCR with a reverse primer (5′-GCGTAAGTGACTAGCGTG-3′) and forward primer specific for predicted SNORA62 (5′-CCCCATAGTGTTTGTTAGTAGC-3′). The product was cloned into the pGEM-T vector (Promega). Positive clones were sequenced using the ABI PRISM BigDye Terminator v3.1 Ready Reaction Cycle Sequencing Kit and T7/pUC primers.snoSeeker was used to identify snoRNAs in introns of AU genes . Rfam [5AU genes , snOPY  supplemented with 10% fetal bovine serum  and GA-1000 (30 mg/mL Gentamicin and 15 µg/mL Amphotericin)  at a 1:1000 ratio in humidified chamber with 5% CO2 at 37 °C.Human HeLa cell line (ATCC) and HEK293T cell line were cultured in Eukaryotic expression vector pcDNA3 (Invitrogen) was digested with BamHI and EcoRI. The insert containing the FLAG sequence and sponge FAU was cloned using these sets of primers: 5′-GTCTAGGGATCCACGAGATGGACTACAAGGACGACGACGATAAGATGCAAGTTTTCGTA-3′ and 5′-CTAGACGAATTCTCATCACTGAGGTGCATTTG-3′, and human FAU using: 5′-GTCTAGGGATCCACGAGATGGACTACAAGGACGACGACGATAAGATGCAGCTCTTTGTC-3′ and 5′-CTAGACGAATTCTTATTAAGAGTTGGCATTGG-3′ For localization assay, sponge FAU was cloned in fusion with GFP using pEGFP-C1 (XhoI/BamHI restriction sites) with the following set of primers: 5′-CCACTCGAGCTATGCAAGTTTTCGTACAAGG-3′ and 5′-GACGGATCCTCATCACTGAGGTGCATTTG-3′, and human using pDsRed-C1 with 5′-CCACTCGAGCTATGCAGCTCTTTGTCCGCGC-3′ and 5′-ACAGGATCCTTAAGAGTTGGCATTGGGGCCC-3′ set of primers.4 cells were seeded onto 24-well culture slides containing DMEM supplemented with 10% FBS to obtain 80% confluence. Cells were transfected with 500 ng of plasmid DNA. Forty eight hours post transfection, the cells were washed with PBS pH 7.5, fixed in 4% formaldehyde, and mounted in SlowFade Diamond Antifade Mountant with or without DAPI .Lipofectamine 3000 reagent (Invitrogen) was used for HeLa cells transfections, according to the manufacturer’s instructions. Twenty four hours before transfection 5 × 10Fluorescent images were obtained by Leica SP8 X FLIM laser scanning confocal microscopy equipped with HC PL APO CS2 63×/1.40 OIL objective. GFP was excited by 488 nm laser line, CFP using 433 nm, DAPI using 405 nm and DsRed at 560 nm laser lines.The cell lysates were loaded on SDS-PAGE and electrotransferred to a PVDF Hybond-P membrane . The membranes were incubated with anti-FLAG M2 antibody (1:5000) (Sigma) for detection of FLAG-FAU or anti-PARP antibody (1:5000) (ab137653). Protein bands were visualized using chemiluminescence detection .3 viable cells suspended in 100 µL 10% FBS DMEM were added to each well of 96-well plates. HEK293T cells were transfected using standard calcium phosphate method [HeLa cells were transfected as described above, trypsinized 24 h post-transfection and 10e method . Cell pr3 viable cells suspended in 100 µL 10% FBS DMEM were seeded in 96-well culture plates. Caspase-3/7 activity was measured using the Caspase-Glo 3/7 Assay Kit (Promega) according to the manufacturer’s instructions with luminometer Infinite 200 (TECAN).Cells were transfected as described above, trypsinized 24 h post-transfection and 5 × 10t-test. Probabilities of less than 0.05 were considered statistically significant.Statistical analysis was performed using Student’s


PD-L1 regulates CD8 T cell/Th1 immune responses. PD-L1 expressed in the tumor microenvironment can bind to PD-1 or B7.1 on activated T cells and mediate cancer immune evasion. MPDL3280A is a human mAb containing an engineered Fc-domain designed to optimize efficacy and safety that targets PD-L1 and blocks it from binding to its receptors.Immunologic pharmacodynamics effects were evaluated in tumors and bloods from patients treated with MPDL3280A. MPDL3280A was administered IV q3w in >300 pts with locally advanced or metastatic solid tumors. PD-L1 and CD8 were measured by IHC. PD-L1 expression was evaluated in tumor and intra-tumoral immune cells. CD8 was assessed in the tumor center, periphery and invasive margin. The expression of ≈90 immune-related markers was evaluated at baseline (BL) and on-treatment using a custom-designed immunochip. BL tumor samples were available for 125 pts, and matched on-treatment samples were available for 31 pts. Further, blood-based biomarkers and circulating immune subsets were serially measured in 114 patients by modified ELISA and FACS, respectively.On treatment, responding tumors showed increase in expression of tumor cell PD-L1 and infiltration of CD8+ T-cells and a Th1-dominant immune infiltrate, providing evidence for adaptive PD-L1 up-regulation. Non-responders showed minimal tumor CD8+ T-cell infiltration and an absence of T-cell activation . We also profiled circulating biomarkers for their association with clinical outcomes. A sub-population of patients, including those with RCC and melanoma, exhibited elevated PD-L1 expression on circulating T-cells at BL that was associated with response to MPDL3280A. On treatment, we observed a delayed increase in frequency of CD4+ICOS+ and CD4+PD1+ T-cells in patients responding to MPDL3280A monotherapy. In contrast, frequency of CD8+HLA-DR+Ki67+ T-cells increased shortly following the first dose of MPDL3280A and returned to baseline levels by the end of cycle 2 when assessed in all patients, representing a transient pharmacodynamic measurement of PD-L1 inhibition. On-treatment, increase in plasma IL-6 was associated with disease progression. Associations with other circulating markers of inflammation including CRP with clinical outcomes will be presented.Our data show that changes in pharmacodynamic immune markers are associated with clinical outcomes to MPDL3280A. These data provide mechanistic insights into immune checkpoint inhibition in cancer and identify potential biomarkers that may be monitored as on-treatment markers of clinical activity for patients treated with inhibitors of PD-L1/PD1 pathway.


The glycoprotein tapasin represents a target for immune evasions and functions within the multimeric peptide-loading complex to stabilize empty class I molecules and promote acquisition of high-affinity peptides. All three B*35 subtypes showed a tapasin-independent mode of peptide acquisition. HLA-B*35-restricted peptides of low- and high-binding affinities were recovered in the presence and absence of tapasin and subsequently sequenced utilizing mass spectrometry. The peptides derived from B*35 variants differ substantially in their features dependent on their mode of recruitment; all peptides were preferentially anchored by Pro at p2 and Tyr, Phe, Leu, or Lys at pΩ. However, the Trp at residue 156 altered the p2 motif to an Ala and restricted the pΩ to a Trp. Our results highlight the importance of understanding the impact of key micropolymorphism and how a single AA mismatch orchestrates the neighboring AAs.Defining permissive and non-permissive mismatches for transplantation is a demanding challenge. Single mismatches at amino acid (AA) position 156 of human leucocyte antigen (HLA) class I have been described to alter the peptide motif, repertoire, or mode of peptide loading through differential interaction with the peptide-loading complex. Hence, a single mismatch can tip the balance and trigger an immunological reaction. HLA-B*35 subtypes have been described to evade the loading complex, 156 mismatch distinguishing B*35:01 and B*35:08 changes the binding groove sufficiently to alter the sequence features of the selected peptide repertoire. To understand the functional influences of residue 156 in B*35 variants, we analyzed the peptide binding profiles of HLA-B*35:01The online version of this article (doi:10.1007/s00251-015-0896-4) contains supplementary material, which is available to authorized users. It becomes obvious that a stringent HLA matching between donor and recipient is the most important criteria to be considered in transplantation. It could be demonstrated that best outcomes post-transplantation have been achieved using HLA identical sibling donor:recipient pairs, whereas HLA mismatching might lead to serious transplantation complications including graft-versus-host disease (GvHD), graft rejection, and/or mortality  is widely used as a curative therapy for various hematological malignancies and immune-related disorders  resident transmembrane glycoprotein that exists as a disulfide-linked heterodimer with ERp57  . The allotypes HLA-B*35:01 and B*35:08 have been described to bind peptides of viral  or Epstein-Barr virus (EBV)) origin; the existence of T cells recognizing these pHLA-B*35 could be verified indicating a role of this HLA subtypes in viral immunity. Natural presentation of the described viral peptides by HLA-B*35 alleles has not been observed, yet. However, probable epitopes were mapped using various cell-based assays to demonstrate the function of T cells against the viral pHLA-B*35 complexes , 721.221 (HLA−/TPN+/TAP+), and T2 (HLA+/TPN+/TAP−) were used for transduction of recombinant HLA-B*35/156 constructs. The cell lines were maintained in RPMI 1640 medium  supplemented with 10 % heat-inactivated fetal calf serum  and 200 mM glutamine . Human embryonic kidney 293 T cells (HEK293T) were cultured in DMEM  supplemented with 10 % heat-inactivated FCS, 20 mM L-glutamine, 1 mg/ml penicillin-streptomycin , and 1 mg/ml G418 . All cell lines were maintained at 37 °C in an atmosphere of 5 % CO2.The lymphoblastoid cell lines (LCLs) 721.220 (HLAFor expression of full length (m) HLA-B*35:01 (exons 1–7) molecules, HLA-B*35:01+ cDNA was amplified by PCR using the primers HLA-B1-TAS (5′-GAGATGCGGGTCACGGCG-3′) and HLA-B-TAAS-E7 (5′-TCAAGCTGTGAGAGACACATCAG-3′). The PCR product was cloned into the lentiviral vector pRRL.PPT.SF.pre.V5-His as previously described HLA-B*35/156 variants were generated from pRRL.mHLA-B*35/156 vectors by introducing a stop codon using the primers Sdm_B35_sE4_F (5′-CCTCACCCTGAGATGAGAGCCATCTTCCCAGTC-3′) and Sdm_B35_sE4_R (5′-GACTGGGAAGATGGCTCTCATCTCAGGGTGAGG-3′).® Plasmid Maxi Kit .Verification of HLA-B*35/156 inserts was performed by DNA sequencing using an ABI Prism 3730 DNA Analyzer . Subsequently, endotoxin-free plasmid DNA was purified using the EndoFree® 2000 , HEK293T cells were transfected with the plasmids encoding for mHLA-B*35/156 or sHLA-B*35/156 for production of lentiviral particles. The target cells were transduced with the respective lentivirus encoding for HLA-B*35/156 constructs.LCLs 721.220 and 721.221 or T2 cells were transduced with lentiviral particles encoding for recombinant mHLA-B*35/156 (exons 1–7) or sHLA-B*35/156 (exons 1–4) molecules as described previously .For quantitative verification of sHLA molecules in the supernatant, a double-antibody sandwich (DAS)-ELISA was applied. The mAb W6/32 was used as a capture antibody and a rabbit anti-human β2m pab was utilized as the detection antibody. The clones with the highest expression of sHLA-B*35/156 molecules were used for large-scale production.2m, and peptide) were eluted using 0.1 M glycine/HCl buffer (pH 2.7).sHLA-B35*/156 producing B-LCL cells were expanded in the two-compartment bioreactor CELLine classic 1000. Supernatants were harvested weekly and monitored for sHLA production. sHLA molecules were affinity purified using N-hydroxysuccinimide (NHS)-activated HiTrap columns coupled to mAb W6/32. Affinity purification was performed on the BioLogicDuoFlow system . Trimeric complexes . Database queries for peptide sequences and peptide source were carried out by Mascot software .Following affinity purification, trimeric complexes were filtered through an Amicon Ultra-15 Filter Unit  with a molecular weight cutoff (MWCO) of 10 kDa and the peptides detected in the flowthrough were considered to be of low-binding (LB) affinity. The retentate was further acidified by treatment with 0.1 % trifluoroacetic acid (TFA) and filtered through a MWCO 10-kDa filter to elute high-binding (HB) affinity peptides. Subsequently, the peptides were sequenced using an Eksigent Nano-LC Ultra 2D HPLC coupled to LTQ Orbitrap XL mass spectrometer . The mass spectrometric data were then utilized on Mascot server via a Mascot Daemon interface   were shared among the HLA-B*35/156 variants Fig. . More peOnly 3.72 % of the peptides were shared between HLA-B*35:08 and HLA-B*35:62, while 7.77 % were shared between HLA-B*35:01 and B*35:62. HLA-B*35:01 and B*35:08 share an overall peptide repertoire of 8.78 % Fig. . BetweenAnalysis of HLA-B*35/156-restricted peptides demonstrated Pro at p2 Fig. .Fig. 5p2At pΩ, a preference for Tyr, Phe, Leu, or Lys could be observed Fig. . Unlike The majority of sHLA-B*35/156-restricted peptides were found to be of canonical length (8–10 AAs). However, peptides of non-canonical length (>10 AAs) could also be recovered by sHLA-B*35/156 variants ; this is in contrast to HLA-B*35:62-restricted peptides (28 %).Taken together, in the absence of TPN, both canonical and non-canonical sHLA-B*35/156-restricted HB peptides were observed. The majority of HLA-B*35:01- and B*35:08-restricted LB peptides were of canonical lengths. Contrary, most of the HLA-B*35:62-restricted LB peptides acquired in the absence of TPN were of non-canonical length (71.79 %).sHLA-B*35:62-restricted peptides that were acquired in the absence of TPN are predominantly anchored by a C-terminal Trp. Residue 156 that distinguishes the three allelic variants is not part of pocket F and thus has no direct influence on the peptide C-terminus. To understand the unexpected alteration of the C-terminal peptide anchor, YASARA/FoldX software was utilized for generating a model of HLA-B*35:62. Crystal structures of HLA-B*35:01 (2AXG) , since the association between TAP and the HLA hc is mediated through TPN. TPN plays an important role in steady state expression of TAP  displayed the majority of them being HB peptides. It was expected that the absence of TPN would lead to the presentation of a higher quantity of LB peptides. However, no significant differences between the percentages of HB and LB peptides were observed from peptides presented by these alleles in the absence of TPN. Especially, it was expected that HLA-B*35:08, which was found to be relatively more dependent on TPN for surface expression, would present peptides of low affinity in the absence of TPN. Our findings suggests that the probable dependence of HLA-B*35:08 on TPN is on egress of pHLA complex from ER to cell surface and their stabilization on cell surface rather than inside the cell. The results could be comparable to the findings observed in peptides associated with HLA-B*08:01 and HLA-A*02:01, where the peptides acquired in absence of TPN were found to be of unexpected higher affinity than those acquired in its presence  and further studies are needed to better understand the reason for its failure to be selected in the course of host-pathogen co-evolution.Moreover, the presence of Trp at position 156 in HLA-B*35:62 was shown to confer a TAP-independent mode of peptide loading that could be suggestive of conferring the ability of peptide presentation via non-classical pathways and its potential role in immune response against viral infections. However, such differences on the mode of peptide loading can have interference on the alliance with ER quality control factors, stabilities of antigenic peptide associations with HLA-B molecules, and hence the abilities of HLA-B molecules to mediate immune responses during infections. HLA-B*35:62 is a rare allele occurring sparsely in Hispanic population ESM 2(DOCX 23 kb)ESM 3(DOCX 226 kb)


P = .502) compared to subtype B . In multivariate analysis, female sex was significantly associated with a 2.7 times higher chance of achieving CD4+ T-cell recovery  and both baseline CD4+ T-cell count (P = .001) and viral load (P = .001) were important predictors for CD4+ T-cell recovery. Immunological recovery correlated significantly with female sex, baseline CD4+ T-cell counts and viral load but not subtype.Human immunodeficiency virus type 1 (HIV-1) subtypes have been shown to differ in the rate of clinical progression. We studied the association between HIV-1 subtypes and the rate of CD4+ T-cell recovery in a longitudinal cohort of patients on combination antiretroviral therapy (cART). We studied 103 patients infected with CRF01_AE (69%) and subtype B (31%) who initiated cART between 2006 and 2013. Demographic data, CD4+ T-cell counts and HIV-1 viral load were abstracted from patient medical charts. Kaplan-Meier was used to estimate the time to CD4+ T-cell count increase to ≥350 between subtypes and effects of covariates were analysed using Cox proportional hazards. An 87% of the study population were male adults (mean age of 38.7 years old). Baseline CD4+ T-cell counts and viral loads, age at cART initiation, sex, ethnicity and co-infection did not differ significantly between subtypes. A shorter median time for CD4+ T-cell count increase to ≥350 cells/μL was observed for CRF01_AE (546 days; 95% confidence interval [CI], 186–906 days;  The HIV-1 epidemic involves the co-circulation of genetically-diverse subtypes of group M, comprising of 11 subtypes or sub-subtypes and 72 circulating recombinant forms (CRF). In Southeast Asia, it is estimated that approximately 1.8 million people live with HIV/AIDS (PLHIV) and CRF01_AE is the major circulating subtype  besides subtype B  due to inadequate number of cases. Important clinical data collected included baseline and serial assessments of CD4+ T-cell counts (every six months) and plasma HIV-1 RNA viral load (once or twice yearly), follow-up duration, history of cART prescription and co-infection with either tuberculosis or hepatitis B/C (HBV/HCV). Basic patient demographic information included age at the initiation of cART, sex, ethnicity and route of disease transmission. Baseline HIV-1 genotyping was performed using our in-house nested PCR amplification and population sequencing of the analyses . All labP < .05 is considered significant). Kaplan-Meier and log-rank test were used to estimate the time to CD4+ T-cell count increase to ≥350 cells/μL upon cART initiation between the infecting subtypes. Effects of covariates were analysed using Cox proportional hazards and important covariates  were included in the subsequent multivariate analysis [Patients’ baseline clinical and demographic characteristics were compared between HIV-1 subtype groups using an independent t-test and Mann-Whitney U test as appropriate , of Chinese ethnicity (75.7%), and with a mean age of 38.7 years old (ranging from 30.7–46.7 years old). The basic demographic and relevant clinical data were summarized in Approximately 70.9% of patients had a baseline CD4+ T-cell count of ≤200 cells/μL prior to cART initiation. At the end of the study follow-up period , 59.2% achieved a CD4+ T-cell recovery to ≥350 cells/μL and within this group, 57.4% (n = 35/61) achieved a CD4+ T-cell count of ≥500 cells/μL and the remaining 42.6% did not achieve CD4+ T-cell count of ≥500 cells/μL. On the other hand, 40.8% (n = 42/103) of the population did not achieve CD4+ T-cell count of ≥350 cells/μL at the end of follow up.P = .502) compared to subtype B  (P = .458) compared to CRF01_AE (3090.0 days)  . The cur.0 days) . HoweverP = .001 and log-transformed HIV-1 RNA viral load (P = .257) and absence of co-infections (P = .050) were significantly associated with the rate of CD4+ T-cell recovery to ≥350 cells/μL. In the multivariate analyses, female sex is significantly associated with a faster rate of CD4+ T-cell recovery to ≥350 cells/μL  compared to the males. Both baseline CD4+ T-cell count  and log-transformed HIV-1 RNA viral load  and the follow-up period (P < .001) were strongly associated with the rate of CD4+ T-cell recovery in this population. In the adjusted analyses, non-Chinese ethnicity (P = .173), age (P = .448) and timing of cART initiation (P = .060) and co-infections (P = .067) were not significantly associated with the rate of CD4+ T-cell recovery.Next, we evaluated the effects of other covariates on HIV-1 disease progression and response to treatment . In the Since the advent of cART in the 1990s, a longer life expectancy and better prognosis are now attainable among 12.9 million people living with HIV worldwide. However, the number of individuals who had access to therapy is a staggering proportion of the total 35 million PLHIV within that year (UNAIDS Fact Sheet 2014). In Malaysia, nearly half, that is 17,369 individuals of the total 38,420 PLHIV received cART in 2013 . In the present study, we studied the immunological response in HIV-1 patients infected with different subtypes in Kuala Lumpur, Malaysia by measuring the time from initiating cART to CD4+ T-cell recovery to ≥350 or ≥500 cells/μL within a median follow-up period of 2.8 years. We have shown that the rate of CD4+ T-cell recovery to either ≥350 or ≥500 cells/μL did not differ significantly between patients infected with either subtype B or CRF01_AE, hence suggesting that immunological response in our study population was relatively independent of the infecting subtypes. A recent systematic review on published findings on the impact of non-B subtypes towards disease progression in patients on cART corroborated our findings, in which it was concluded that the difference in disease progression was minimalized in cART-experienced patients [In a study examining the association between the main co-circulating subtypes in Singapore and disease progression, HIV-1 seroconverters were followed from the estimated date of seroconversion to the time when cART was initiated . CRF01_A4–105 copies/mL, HIV-1-infected women had a better clinical outcome compared to men [Similar to previous studies, both baseline CD4+ T-cell counts  and plasd to men . In anotd to men . In thisThe differential rates of disease progression in HIV-1-infected persons is complex as it involves an interplay of the host and viral factors in the population. Our present study found that CRF01_AE and subtype B were not important predictors of CD4+ T-cell recovery in a cART-experienced population. On the other hand, female sex and both baseline CD4+ T-cell counts and HIV-1 viral load were strong predictors of CD4+ T-cell recovery during cART. Therefore, an earlier cART initiation as recommended by the 2013 WHO treatment guidelines for CD4+ T-cell count <500 cells/μL may prove to be useful in promoting CD4+ T-cell recovery in patients receiving cART. Furthermore, current treatment strategies and prevention efforts should take into account the sex-based differences in immunological response to therapy in order to greatly reduce HIV-1/AIDS-associated morbidity and mortality.


Regulatory T cells (Treg) in allografts are important for the prevention of graft-versus-host disease (GVHD) post-transplantation. The aim of this study was to compare the contents of Tregs and effector T cells in granulocyte colony-stimulating factor (G-CSF)-primed bone marrow grafts (G-BM) and peripheral blood grafts (G-PB).+CD25highFoxp3+ Treg cells, were analyzed using flow cytometry. The levels of interferon-γ (IFN-γ) and interleukin-17 (IL-17) secreted by T cells stimulated with PMA and ionomycin were also determined by flow cytometry.G-BM and G-PB were obtained from 20 allogeneic donors. T-cell subgroups, including conventional T cells and different types of Treg cells, as well as the percentage of Ki67 expression on CD4+CD25highCD127-/dimCD62L+ Treg cells was significantly higher in the G-BM group, with higher proportions of CD45RA+ naïve Treg cells and higher expression of CD69 on Treg cells in G-BM (P < 0.05). The percentage of Ki67 expression in CD4+CD25highFoxp3+ Treg cells in G-BM was significantly higher than that on G-PB. The suppressive functions of Treg cells in inhibiting T-cell activation were comparable between G-BM and G-PB. The proportions of CD4+CD25−CD69+ Treg subsets as well as Th1 cells in G-BM were also significantly higher than those in G-PB (P < 0.001). The proportions of conventional T cells and Th17 effector cells were comparable in G-BM compared with those in G-PB. Thus, the ratio of conventional T cells and CD4+CD25highCD127-/dim regulatory T cells were lower in G-BM than that in G-PB (P = 0.014).The percentage of CD4In addition to the much higher T-cell counts in G-PB grafts that may contribute to more severe GVHD, the higher frequency of Treg cells and lower ratio of conventional T cells to Treg cells in G-BM compared with G-PB grafts might reduce GVHD post-transplantation in G-BM compared with G-PB transplantation.The online version of this article (doi:10.1186/s12967-015-0507-z) contains supplementary material, which is available to authorized users. Allogeneic hematopoietic stem cell transplantation  is the only curative method available for malignant hematologic diseases. However, its broad application is limited by the high incidence of graft-versus-host disease (GVHD). The current allo-HSCT procedures consist mostly of bone marrow (BM) cells or granulocyte-colony stimulating factor (G-CSF)-primed peripheral blood stem cells (G-PB) or G-CSF-primed bone marrow (G-BM). Although both G-BM and G-PB contain large numbers of mature donor T cells that could cause GVHD , clinica+CD25highCD127-/low regulatory T cells (Treg cells) have been demonstrated to have immunosuppressive ability and to be key players in the regulation of immune responses .We isolated human Treg . Therefore, the percentage of CD4+CD25highCD127-/dimCD62L+ regulatory T cells was significantly higher in G-BM compared to G-PB  is an important T-cell homing receptor as well as a marker for T-cell development and that CD69 is an important activating receptor that exerts regulatory functions in the immune response of T cells, we also compared differences in the expression of CD62L and CD69 on CD4+HLADR−, CD45RA−HLADR+, CD45RA−HLADR−) in G-BM and G-PB [+HLADR− naive Treg cells among the Treg cells was significantly higher in G-BM compared to G-PB  and the CD45RA−HLADR−Treg subset  was significantly lower in G-BM compared to G-PB . The ratios of Th17 cells to CD4+CD25highCD127-/low regulatory T cells were comparable between G-BM and G-PB (P = 0.970).In addition to comparisons of individual subsets of T cells between G-BM and G-PB, it is important to examine the relative balance of the effector and regulatory arms of the immune system. The ratio of CD4+CD25highCD127-/low regulatory T cells and CD4+CD25highCD62L+ regulatory T cells in G-BM  and in G-PB .Positive correlations were found between the proportions of CD4+CD25highCD127-/low regulatory T cells , and FOXP3, produced no inflammatory cytokines and maintained robust suppressive activity after expansion [+HLADR−“naive” Treg cells compared with tolerant patients [+CD25−CD69+ T cells is correlated with a low risk of acute GVHD in allotransplants [+CD25−CD69+ T cells in G-BM compared to G-PB would support the lower occurrence of acute GVHD post-G-BM transplantation compared to G-PB transplantation [The CD4lunteers . Boyer Ol subset . Consisteg cells  and posseg cells ,26. CD69xpansion . The Trepatients . Therefopatients . The hignsplants . Thus, t+CD25highCD127-/low Treg cells and CD4+CD25highCD62L+ Treg cells but not in CD4+CD25−CD69+ T cells between G-BM and G-PB, suggesting that the CD4+CD25highCD127-/low Treg cells and CD4+CD25highCD62L+ Treg cells in G-PB might derive from BM and might overlap each other; however, the development of CD4+CD25−CD69+ T cells in G-PB had no direct relevance to those in G-BM, which will need to be confirmed in the future.Correlations were found in CD4Although acute GVHD has been classically assumed to be Th1-mediated on the basis of findings in animal models, we now face a more complex scenario involving possible roles of newly identified Th17 cells as well as Treg cells in GVHD . The difIn summary, this study demonstrated that higher frequencies of regulatory T cells and a decreased ratio of effector T cells to regulatory T cells in G-BM would lay part of the immunological groundwork for a lower occurrence of acute or chronic GVHD post-G-BM transplantation compared to G-PB transplantation. Conducting a large cohort study in a prospective manner would be warranted to compare the different clinical outcomes between G-BM and G-PB transplants and their correlation with different Th and Treg subsets within G-BM or G-PB allografts.


TAL1 is amongst the most frequently deregulated oncogenes. Yet, over half of the TAL1+ cases lack TAL1 lesions, suggesting unrecognized (epi)genetic deregulation mechanisms. Here we show that TAL1 is normally silenced in the T-cell lineage, and that the polycomb H3K27me3-repressive mark is focally diminished in TAL1+ T-ALLs. Sequencing reveals that >20% of monoallelic TAL1+ patients without previously known alterations display microinsertions or RAG1/2-mediated episomal reintegration in a single site 5′ to TAL1. Using ‘allelic-ChIP’ and CrispR assays, we demonstrate that such insertions induce a selective switch from H3K27me3 to H3K27ac at the inserted but not the germline allele. We also show that, despite a considerable mechanistic diversity, the mode of oncogenic TAL1 activation, rather than expression levels, impact on clinical outcome. Altogether, these studies establish site-specific epigenetic desilencing as a mechanism of oncogenic activation.T-cell acute lymphoblastic leukaemias (T-ALL) are aggressive malignant proliferations characterized by high relapse rates and great genetic heterogeneity.  TAL1 is frequently deregulated in T-cell acute lymphoblastic leukaemias, but the mechanism remains largely unclear. Here the authors show that microinsertions upstream of TAL1 cause its epigenetic reactivation, and that the mode of TAL1 activation correlates with prognosis. T-cell acute lymphoblastic leukaemia’s (T-ALL) are malignant proliferations of immature T-cell progenitors. Although the outcome of T-ALLs has greatly improved in the last 10 years, ~30% of cases relapse within the first 2 years following diagnosis; moreover, acute- and long-term toxicities remain important issues for long-term survivors, underlining the critical need of better risk stratification of T-ALL patients, and the implementation of more adapted and/or targeted therapies. A major obstacle in the molecular dissection of these processes is that T-ALLs constitute a particularly heterogeneous group of disease, characterized by complex combinations of multigenic aberrations and oncogenic cooperation. The deregulation of over 40 distinct oncogenes and tumour suppressors has been reported, occurring through a large diversity of genomic aberrations and epigenetic mechanisms1TAL1 is one of the most frequently deregulated T-ALL oncogenesTAL1 is a regulatory gene that promotes access to alternative fates in haematopoiesis. Expressed in early haematopoiesis, its expression is maintained in the erythroid lineage, but normally irreversibly epigenetically silenced in the T-cell lineage45TAL1 in the T-cell lineage are thought to constitute strong drivers of T-ALL leukemogenesis. Known TAL1 dysregulation mechanisms consist of t translocations (1–2%) and SIL-TAL deletions (15–20%). Yet, over half of the TAL1+ cases lack TAL1 lesions, suggesting unrecognized (epi)genetic deregulation pathwaysTAL1 expression can be monoallelic, compatible with a direct alteration in cis within or around the TAL1 gene, or biallelic, likely reflecting indirect deregulation in transTAL1 ‘unresolved’ cases. Using ChIP-seq and an ‘allelic-ChIP’ assay allowing to discriminate histone marks separately on each allele, we describe a new process of oncogene activation, whereby the targeted eviction of polycomb H3K27me3 marks and concurrent recruitment of H3K27ac marks by micro- and macroinsertional events, trigger the monoallelic desilencing of TAL1. Incidentally, we also report the first example of oncogenic activation by recombination-activating gene (RAG)-mediated episomal reinsertion, a very elusive event predicted in vitro as a source of oncogenic activation over 10 years ago8TAL1 oncogene activation. Finally, we demonstrate that the mode of activation  rather than the level of TAL1 expression impacts on prognosis, with cis-mediated alterations significantly associated with adverse clinical outcome.Here we sought to gain insights into the (epi)genetic deregulation pathways of TAL1 promoter and encompassing part of the gene body was enriched in H3K27me3, suggesting the involvement of polycomb complex (PcG) repressive activity in TAL1 silencing  T-ALL cell-line and a control TAL1− sample (DA). Sample DA displayed the expected H3K27me3 deposition, coherent with TAL1 silencing in T cells . Accordingly, knockdown (by shEZH2) or inhibition (using the EZH2 inhibitor GSK126) (ref. TAL1 expression through T-cell differentiation.To investigate the (epi)genetic deregulation pathways of TAL1 ‘unresolved’ cases, we first took advantage of chromatin immunoprecipitation coupled to a high-throughput sequencing (ChIP-seq) data set describing active/inactive epigenetic marks in mouse developing thymocytes6ilencing . This pr T cells . Surpris of TAL1 , we reasL1  ref.  of the P26) ref. . SimilarTAL1 activation. While no macromolecular TAL1 alteration was identified by SNP array (0/134), sequencing revealed seven new cases of similar microinsertions (1–9 bp), all precisely located at the Jurkat insertion breakpoint , insertions were exclusively found in monoallelic cases , in agreement with a cis-mode of TAL1 activation  trial was split into TAL1 expression quartiles, and compared for disease-free (DFS) and overall survival (OS). The seven patients with identified insertions (three of whom were GRAALL treated), all belonged to the high-expression quartiles (Q3–4). However, no significant difference in survival was observed between the quartiles  affected the clinical outcome compared with trans-mediated events, associated with biallelic TAL1 expression. Clinical outcome was indeed found to be significantly improved in the biallelic group . Despite genetic heterogeneity, monoallelic cases also displayed higher blasts counts at diagnosis than biallelic cases and a significantly lower frequency of deregulation of recurrent oncogenes such as TLX1, CALM-AF10 and TLX3 , further disruption of the region 5′ of the insertion site  might be necessary to impose desilencing once TAL1 extinction is established in the T-cell lineageTAL1 silencing (DN1-2). Altogether, our data are in line with current models of permanent gene extinction of transcription factors during T-lineage commitmentTAL1 silencing. Their genetic disruption constitute a recurrent epigenetic mechanism of TAL1 deregulation in T-ALLs, contributing to a substantial fraction (>20%) of the TAL1+ monoallelic ‘unresolved cases’, and associated with adverse prognostic. That a cis-deregulation regrouping as diverse mechanisms as SIL-TAL deletions, translocations or insertional desilencing impact more on prognosis than TAL1 expression levels underlines the fundamental oncogenic difference between a deregulation targeting a single locus, and the wider effect of trans-acting factors. Transcription factors indeed often bind to a large number of target genes (hundreds to thousands) and their deregulation (whether gain or loss) will likely affect a complex set of cellular functions, some of which might antagonize tumour progression, or resistance to treatment. Recently, reports identifying loss-of-function mutations in polycomb-related components1921NOTCH and TAL1 deregulation can coexist, we find that while NOTCH patients are associated with a favourable prognosis, monoallelic, but not biallelic TAL1, expression tends to convert the clinical outcome towards more adverse prognosis in the Notch subgroup, and to further aggravate the bad prognosis of Notch1WT patients. This suggests that distinct modes of deregulation of the same epigenetic complex might coexist in a tumour cell, leading to complex and potentially conflicting clinical outcomes which ought to be clarified when considering epigenetic inhibitors for new lines of treatment1533This and the other insertional mutagenesis T-ALL cases described here also revealed a novel oncogenic activation pathway, whereby a genetic alteration drives a site-specific and monoallelic epigenetic deregulation. We demonstrate that such insertions drove a switch from H3K27me3 to H3K27ac deposition, leading to the maintenance and/or re-expression of ng assay  suggest raction >% of the ChIP was performed as previously describedq value cutoff=5.00e−02, larger scaled towards smaller, λ range=1,000–10,000 bps, Broad on.Before sequencing, ChIP DNA was quantified using the picogreen method  and quality controlled on a 2100 Bioanalyzer (Agilent). At least 1 ng of ChIP or input DNA was used for library preparation according to the Illumina ChIP-seq protocol. After end repair and adapter ligation, fragments were size selected on a gel before preamplification and clustering. The resulting fragments were again verified on a 2100 Bioanalyzer before clustering and 25 or 36 cycle sequencing on a Genome Analyzer II  according to the manufacturer’s instructions. Raw data were bowtie aligned and the tags elongated and further processed to wiggle files as describedTo identify a difference in H3K27ac enrichment in ChIP-seq between alleles with and without insertion (12 bp) in −(IP Ct or input Ct- IgG IP Ct). Allelic-ChIP was carried out using allele-specific primers (by substituting one of the germline primers with a primer located in or overlapping the insertion). EZH2 knockdown was achieved using a doxycycline-induced short hairpin RNA (sh-RNA)-targeting EZH2 . A non-silencing sh-RNA (pTRIPZ-NS) was used as control. Jurkat cells were electroporated and cells containing the pTRIPZ were selected on puromycine. Knockdown of EZH2 was obtained by the addition of doxycycline (2 μg ml−1) to the cells during 10 days. Western blot was performed using the anti-EZH2 BD Biosciences # 612666.Input and IP genomic DNAs were analysed by RT–PCR using power SybrGreen on a 7,500 Fast Real-time PCR system (Applied Biosystems). IgG control ‘cycle over the threshold’ Ct values were subtracted to Input or IP Ct values and converted into bound value by 2TAL1 exon 1 was mapped on both alleles by LRPCR/cloning and standard Sanger sequencing as previously describedTAL1 sequence and variants  included in GRAALL-03/05 trial (registration #NCT00327678 and #NCT00222027) were analysed for Cell lines used in this study were purchased from the ATCC collection and were mycoplasma free.TAL1 transcript with the following primers: TAL1 F: 5′-ACA-ATC-GAG-TGA-AGA-GGA-GAC-CTT-C-3′, TAL1 Probe: fam-5′-CTA-TGA-GAT-GGA-GAT-GGA-GAT-TAC-TGA-TG-3′-tamra, TAL1 R: 5′-ACG-CCG-CAC-AAC-TTT-GGT-G-3′, 40 cycles were run on ABI 7500HT (Applied Biosystem) as describedTAL1 transcript quantification was performed after normalization with the housekeeping gene ABL using the ΔCt method and results calculated according to the following formula 2Δ(CtABL–CtTAL1).RNA was reverse transcripted using MMLV (Invitrogen). We used a TaqMan assay to quantify TAL1 gene were identified by PCR amplification and direct sequencing of 100 ng of genomic DNA. Allelic expression analysis was performed by PCR amplification and by direct sequencing of RT–PCR products from heterozygous patient samples. Three different PCRs were made to cover nine most frequent SNP among the 11 SNP previously describedAllelic expression was performed as previously describedChIP. The power of t-test was estimated a priori using pwrR-package, and the expected variations between conditions evaluated. Differences in ChIP data between inserted and GL TAL1 alleles were analysed by unpaired t-test. Samples collections constituted of five or six technical replicates were first checked for normal distribution using Kolmogorov–Smirnov test and the equality of variances was tested using F test. Results of t-test are shown as two-tailed P values. The statistical power of executed t-tests was at least 80%. Errors bars on histograms represent s.e.m.OS/DFS. Patients’ characteristics were compared using the Fisher’s exact test. Median comparisons were performed using the Mann–Whitney U-test. OS and DFS were calculated from the date of prephase initiation. Events accounting for DFS were induction failure and first relapse from any cause in first CR. OS and DFS were estimated by the Kaplan–Meier method and then compared by the log-rank test. All calculations were performed using the SPSS software, version 15.0 and the GraphPad Prism, La Jolla, CA, USA.+ and GWAB−). A primary PCR amplification was performed using an adapter-specific primer (AP1) and primer specific for the different TCRβ gene segments (ext). A secondary PCR was performed using nested AP2 and TCRβ primers (int), and analysed on 1% agarose gel. Non-germline PCR products were purified and sequenced. The functional and non-functional V(D)J rearrangements from patient OC were obtained using Jβ2.7-extB/intB and AP1/2 primers. The breakpoints corresponding to the episomal insertion were then obtained using Vβ7.4-extB/intB, Dβ1-extA/intA and AP1/2 primers, and validated by direct PCR using TAL1.OC.1B and Vβ7.4-intB primers.Genomic DNA was extracted using QIAamp deoxyribonucleic acid Blood mini kit according to the manufacturer’s instructions (Qiagen). DNA (500 ng) was used for LM-PCR; DNA was digested by DraI, EcoRV, PvuII, SmaI, SspI or StuI restriction endonucleases. Purified digested DNAs were ligated with an adapter  according to the manufacturer’s instructions. Reverse transcription was performed with SuperScript III Reverse transcriptase (Invitrogen) and random primers (applied Biosytem). cDNAs were analysed by real-time quantitative PCR (RT–PCR) using power SybrGreen on an ABIPRISM 7500 (Applied Biosystems). All PCRs were performed in duplicate. 5′-RACE was performed using 2 μg of total RNA and the 5′/3′ RACE kit, 2nd generation (Roche). Modifications from the instruction manufacturer were the generation a poly(G) tailing of first strand cDNA and the use of an oligo d(C) anchor primer. PCR was performed using the Pfu Ultra II fusion HS DNA polymerase (Agilent technologies).−1 penicillin/streptomycin (Life Technologies) at 37 °C in the presence of 5% CO2. The day of transfection, 1 million cells were nucleofected according to the manufacturer’s instruction (Lonza), with 500 ng DNA donor sequence containing Neomycin-resistant gene and 2 μg of the Cas9/gRNA expression vector (Addgene #42230). The chimeric guide RNA targeted TAL1 insertion site, and was cloned according to Cong et al.4 cells per well and incubated in presence of 1,200 μg ml−1 geneticin G418 (Life Technologies) for 2 weeks. After selection and growing, a PCR was conducted to amplify the targeted region with genomic DNA derived from the surviving clones, and amplicons were separated on a 1% agarose gel then extracted with GEL/PCR clean up wizard (Promega) and sequenced (MWG-Biotech). CRISPR guide RNA: 5′-GAAAGACGTAACCCTACTTCC-3′.PEER T-ALL cells line were cultured in RPMI medium (Life Technologies) containing 20% fetal calf serum, 1% L-glutamine, 1% sodium pyruvate and 100 U mlPrimers list is available on request.ChIP-seq experiments were performed and analysed by L.C.P., R.F., M.A.M., M.G., I.G.G. and J.-C.A.; allelic-ChIP experiments were performed and analysed by J.-M.N., L.C.P., M.L., M.K., C.P., D.P.-B. and E.D.; T-ALL sample characterizations were performed and analysed by A.T., J.-M.N., L.C.P., S.L.N., S.J., L.S., E.A.M. and V.A.; TALEN/CRISPR assays were performed and analysed by J.-M.N., M.L. and D.P.-B. with the advice of C.G. and B.M.; episomal reintegration characterization was performed by J.-M.N., E.A.M., S.H.-B.-A., S.J.H., H.B.G. and A.J.T.; survival analysis was carried out by A.T., H.D., E.A.M., N.I. and V.A.; V.A. and B.N. conceived and directed the project, and B.N. wrote the paper.How to cite this article: Navarro, J.-M. et al. Site- and allele-specific polycomb dysregulation in T-cell leukaemia. Nat. Commun. 6:6094 doi: 10.1038/ncomms7094 (2015).Supplementary Figures 1-8, Supplementary Tables 1-2 and Supplementary References


VEGF gene has been reported to be related with many diseases and recurrent pregnancy loss in various studies. Concerning the role of VEGF polymorphisms in pregnancy losses, generally mothers genotypes have been analyzed. To evaluate the association between VEGF A +405G/C (rs2010963), −460T/C (rs833061), +936C/T (rs3025039) and - 2578A/C (rs699947) polymorphisms and spontaneous abortion, we studied the genotypes of spontaneously aborted fetuses, their mothers and healthy controls. 23 spontaneously aborted fetal materials, 22 mothers who had these abortions and 86 healthy controls were included in this study. rs2010963, rs833061, rs3025039 and rs699947 polymorphisms were analyzed by Real Time PCR technique after genomic DNA isolation from all subjects. The frequencies of VEGF A rs2010963 GG genotype and rs2010963 G allele were higher in fetuses compared both with mothers and healthy controls. VEGF A rs3025039 TT genotype and rs3025039 T allele frequencies were higher in fetuses comparing with mothers. VEGF A rs833061 CT and TT genotypes frequencies were higher in fetuses comparing with mothers. We ascertained that VEGF A rs2010963, rs833061 and rs3025039 are the risk factors for spontaneous abortion in fetal genotypes comparing with their mothers and healthy controls. Spontaneous abortion is defined as the loss of a fetus prior to 20 weeks of gestation. Approximately 15~20% of clinically recognized pregnancies are spontaneously aborted, most of them during the first trimester  induces endothelial cell proliferation and migration, enhances vascular permeability, reduces endothelial cell apoptosis and promotes stromal proteolysis . The VEGThe VEGF-A gene is located on chromosome 6p21.3 and contains eight exons and seven introns. Several single nucleotide polymorphisms (SNPs) have been identified in the VEGF-A gene and are believed to have functional activity. Among these, −460T/ C (rs833061) and -2578A/C (rs699947) in the promoter region, +405G/C (rs2010963) in the 5′-untranslated region (position+ 405 after transcription initiation site), and+ 936C/T (rs3025039) in the 3′- untranslated region are known to modulate the protein expression of VEGF-A , and control group including 86 fertile healthy controls (43 couples with no abortion history) were included in this study. Abortion materials were obtained from only spontaneous abortions. Control group subjects had at least two children and no spontaneous abortion. Maternally contaminated abortion materials were excluded from the study . Maternal contamination was detected with the correlation of fetus and mother STR markers. 131 cases including 23 abortion materials, 22 mothers and 86 controls were genotyped for VEGF A SNPs  and both abortion materials group and the corresponding mothers group as well as abortion materials group and healthy controls were compared.Mutation analysisTotal genomic DNA was isolated from 10-15 mg of abortion material tissue by the QIAGEN QIAamp DNA Mini Kit and peripheral blood samples containing EDTA from the mothers and the control group by the QIAGEN QIAamp DNA Blood Mini Kit. Four polymorphic regions of the VEGF A gene  DNA fragments were ampliﬁed by real-time polymerase chain reaction technique using LightCycler 2.0 (Roche ).+2, stock solution, Primer mix, and HybProbe mix), and DNA template   were used for real- time ampliﬁcation. The multiple PCR consisted of a denaturation step of 10 min at 950C, followed by 45 cycles of 10 s at 950C,10 s at 600C, and 15 s at 720C, and a melting step of 20 s at 950C, 20 s at 400C, a continuous mode at 850C, a cooling step of 30 s at 400C for all four regions. A software program  was used for the detection of the genotype proﬁles of the target gene. VEGF A +405 C/G (rs2010963) wild type +405 CC was analyzed in channel 530 and with Tm 65 0C. The mutant genotype +405 GG was analyzed with Tm 52 0C. VEGF A -460 C/T (rs833061) wild type -460 CC was analyzed in channel 530 with Tm 61 0C. The mutant genotype -460 TT was analyzed with Tm 54 0C  (R5'-GTC-TGTCTGTCTGTCCGTCA-3') (F5'-GGATGGGG-CTGACTAGGTAAGC-3') (R5'-AGCCCCCTTTT-CCTCCAAC-3'), (F5'-TGTGCGTGTGGGGTTG-AGCG-3').(R5'- TACGTGCGGACAGGGCCTGA-3'), and (F5'-AAGGAAGAGGAGA-CTCTGCGC-3') (R5'-TATGTGGGTGGGTGTGT-CTACAG-3'), respectively. Brieﬂy, LightCycler FastStart DNA Master HybProbes , master mix   wild type -2578 CC was analyzed in channel 530 with Tm 55 0C. The mutant genotype -2578 AA was analyzed with Tm 62 0C.VEGF A +936 C/T (rs3025039) wild type +936 CC was analyzed in channel 530 with Tm 66 Statistical analysisStatistical analysis was done using medcalc statistical program. Chi-square test was used to analyze the differences between the abortion materials, mothers and control subjects. Genotype and allele frequencies were compared between fetal  group and mother group, and fetal group and fertile control group. Odds ratio (OR) and p-values were used to estimate the risk for the VEGF A SNPs in the groups.VEGF A gene in study groups , and the results were compared between both fetus-mother group and fetus-healthy control group. The estimate risk was examined by odds ratio.By real time PCR technique, we evaluated four SNPs of the Peripheral blood-EDTA samples from mothers and healthy controls and abortion material tissue samples were examined for genotyping in the current study. The mean age of the mothers was 29.6 (21-40) years, and the mean age of controls was 46.5 (27-68) years. The mean abortion week was 9.4. Twenty abortions were in the first trimester and three abortions in the second trimester. There were no other diseases in the study groups.VEGF A +405 C/G polymorphism, in the statistical analysis GG genotype and G allele were found as risk factors for abortion in both groups  patients and 30 controls. They reported a relationship between -1154 G/A polymorphism and RPL and proposed that miscarriages would be due to VEGFs' importance in placental angiogenesis (VEGF -1154 G/A polymorphism and RPL (VEGF -634 G/C and +936 C/T polymorphisms and RPL (VEGF gene and RPL studies are found in the literature, but these studies were planned with mothers genotypes. In our study, we correlated fetus, mother and control subjects genotypes for spontaneous abortions. A haplotype study which compared the -2578 CA+ AA/ -634 CC and -1154 GA+ AA/ -634 CC combined genotypes of spontaneous abortion materials with control subjects reported these genotypes in the fetus as possible risk factors for spontaneous abortion (VEGF A +405 C/G +936 C/T, -460 C/T. Further studies and validations are necessary to determine the potential clinical impact of these findings.ogenesis . Su et a and RPL . Traina  and RPL . Lee et  and RPL . AlthougAuthors declare no conflict of interest.


S. cerevisiae. Among the X positive hits, the Leucin Rich Repeat (LRR) domain of Ipaf was found to interact with cN-II. Recombinant Ipaf isoform B (lacking the Nucleotide Binding Domain) was used in an in vitro affinity chromatography assay confirming the interaction obtained in the screening. Moreover, co-immunoprecipitation with proteins from wild type Human Embryonic Kidney 293 T cells demonstrated that endogenous cN-II co-immunoprecipitated both with wild type Ipaf and its LRR domain after transfection with corresponding expression vectors, but not with Ipaf lacking the LRR domain. These results suggest that the interaction takes place through the LRR domain of Ipaf. In addition, a proximity ligation assay was performed in A549 lung carcinoma cells and in MDA-MB-231 breast cancer cells and showed a positive cytosolic signal, confirming that this interaction occurs in human cells. This is the first report of a protein-protein interaction involving cN-II, suggesting either novel functions or an additional level of regulation of this complex enzyme.IMP/GMP preferring cytosolic 5'-nucleotidase II (cN-II) is a bifunctional enzyme whose activities and expression play crucial roles in nucleotide pool maintenance, nucleotide-dependent pathways and programmed cell death. Alignment of primary amino acid sequences of cN-II from human and other organisms show a strong conservation throughout the entire vertebrata taxon suggesting a fundamental role in eukaryotic cells. With the aim to investigate the potential role of this homology in protein-protein interactions, a two hybrid system screening of cN-II interactors was performed in  Cytosolic 5’-nucleotidase II (cN-II) is an IMP/GMP preferring 5’-nucleotidase whose activity, structure and expression has drawn the attention of several research groups from various fields such as biochemistry, crystallography, molecular biology, oncology, pharmacology and genetics. cN-II is a bifunctional enzyme , 2, operNT5C2 (the gene encoding cN-II) have been reported to drive pharmacological resistance in hematological tumors [Fluctuations of cN-II expression and activity have been associated with highly proliferating cells and neurological disorders , 13, andl tumors , 15. Morl tumors . In 2008l tumors . This fiin vitro and in human cells.The widespread distribution and the extreme conservation among vertebrates of the protein sequence  may suggWe believe that further studies of the interaction between cN-II and Ipaf could contribute to a better understanding of the innate immune response, as well as to new regulatory roles of cN-II in cell biology.TM Library-Universal Human , Clontech) was cloned into the pGADT7-RecAB vector . Plasmids pReceiver.B01, carrying the complete cDNA for the NOD-like receptor Ipaf (pRec.B01-Ipaf), pEZ-M15-YFP/Ipaf and pEZ-M02-Ipaf were purchased from GeneCopoeia  and verified by restriction fragments analysis and sequencing . Plasmid for Ipaf isoform B was obtained by deletion of the cDNA encoding the NBD domain in pRec.B01-Ipaf. This was performed by amplification of the LRR and CARD domains using two semi-complementary primers: T7 Fwd together with 5'-AGTCAGACCACTTTGTCCATTCAAGTCC-3', and B01 Rev together with 5'-GGACAAAGTGGTCTGACTGAC AGCTT-3', respectively. Fragments were hybridized and amplified with T7 fwd and B01 rev. PCR products and pRec.B01-Ipaf were digested with NotI and XbaI , separated in 0.8% agarose gel, purified with “Wizard SV Gel” and “PCR Clean-Up System”  and subsequently ligated  to form pReceiver.B01-IpafB. All procedures were conducted following the manufacturer instructions. pRec.B01-IpafB was controlled by PCR analysis with T7 prom forward primer and B01 reverse primer, restriction fragments analysis and sequencing (Bio-Fab). Plasmids pEGFP-C1-Ac-Ipaf and pcDNA3.1-myc-LRR were kindly provided by Pr. G. Swarup [AACCTCTTGGTCTGTGCACATTTCAAGAGAATGTGCACA GACCAAGAGGTTTTTTTGGAAA-3’, bottom 5’-TCGATTTCCAAAAAAACCTCTTGGTCTGTGCACATTCTCTTGA AATGTGCACAGACCAAGAGGTTGGG-3’, hybridized and cloned into pSUPERIOR.neo  with BglII and XhoI to obtain the pScN-II vector. A non-targeting control plasmid (pScont) was prepared in the same way with the following oligomers: Top 5’-GATCCCCCACTACCGTTGTTAAGGTGTTC AAGAGACACCTTAACAACGGTAGTGTTTTTA-3’, bottom 5’-AGCTTAAAAACACTACCGTTGTTAAGGTGTCTC TTGAACACCTTAACAACGGTAGTGGGG-3’.All cloning was performed according to standard procedures . Plasmid. Swarup . In our Saccharomyces cerevisiae strain Y187  by standard procedure using lithium acetate and ssDNA as carrier [S. cerevisiae strain AH109  was transformed with pBD-GAL-cN-II. Screening was carried out by mating the two yeast strains in complete liquid medium for 17 hours at 30°C under shaking, plating the culture in the medium lacking tryptophan and leucine to select for diploids . In parallel, the culture containing about 109 cells was plated onto medium lacking tryptophan, leucine, adenine and histidine to score for positive clones. Then, positive clones were streaked out onto medium lacking tryptophan, leucine, adenine and histidine to confirm the positiveness. Clones were cultivated in medium lacking leucine for 48 hours at 30°C and plasmids were extracted using the yeast plasmid miniprep kit  and transformed in competent E. coli for amplification. Plasmid DNA was extracted from E. coli by standard method and further analyzed. The cDNA from plasmids extracted from all positive clones was sequenced.The pGADT7-RecAB vector carrying the human cDNA library was transformed in the  carrier . Yeast mE. coli strains BL21 Rosetta (DE3) and BL21 (DE3) were used for Ipaf B and cN-II protein expression respectively, with transformation as described before [d before . The expE. coli BL21 (DE3) containing cN-II was loaded onto the “Perfect Pro Ni-NTA Agarose” beads  equilibrated in the lysis buffer. The beads were then washed with 10 column volumes of lysis buffer, 100 mM NaCl, 30 mM imidazole in 20 mM Tris-HCl, pH 8.0. Successively, supernatant from E. coli BL21 Rosetta (DE3) expressing Ipaf B was loaded onto the same Ni-NTA beads. Absorbance at 280 nm was monitored and when flow-through proteins stopped eluting the beads were washed with 10 volumes of 10 mM Tris-HCl pH 7.4 and finally recombinant proteins were eluted from the bead with 250 mM imidazole in 50 mM Tris-HCl, pH 7.4. As control, the same experiment was performed without loading of supernatant from E. coli BL21 (DE3) with cN-II.After overnight growth, bacteria were harvested and the pellet was suspended in lysis buffer . Lysis was achieved by freeze-thawing the cells in the presence of 2 mg/ml lysozyme. Lysates were then centrifuged at 35,000 g for 90 min. Supernatant from 2 / 95% air atmosphere. Transfections were performed with plasmids previously expanded and purified from E. coli DH5α using the The PureLink HiPure Plasmid Filter Maxiprep Kit (Invitrogen). A549 and MDA-MB-231 cells were stably transfected with pScN-II and pScont using lipofectin. Briefly, cells  were incubated for 5 hours with 5 μg of plasmid in presence of 12.5 μg of lipofectin (Invitrogen). After 72 h of growth in complete medium stably transfected cells were selected for three to four weeks with appropriate concentrations of neomycin (0.8 mg/ml). For transient transfection of HEK 293 T and MCF7 cells 70% confluent T75 flask were incubated for 5 hours with 15 μg of plasmids in presence of 37.5 μg of lipofectin  before complete medium was added.All cell lines were from ATCC and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% Fetal Bovine Serum, 100 U/ml penicillin and 100 μg/ml streptomycin  at 37°C in a humidified 5% CO2, 2 mM EGTA, 0.5% NP40, proteases inhibitors cocktail). Cells were disrupted by repeating aspiration trough a 21 gauge needle and incubated for 1 h at 4°C. Cellular debris were pelleted by centrifugation at 12000 g for 15 min at 4°C and 500 μg of proteins were incubated three times for 30 min with 20 μl of A/G plus-Agarose  previously equilibrated with lysis buffer without proteases inhibitors. After each incubation, samples were centrifuged at 2500 g for 5 min and beads were discarded. The remaining protein sample was incubated for 2 h with 2 μg of primary antibody  and subsequently incubated at 4°C on a rocker platform rotating device overnight after adding 20 μl of A/G plus-Agarose. Immunoprecipitates were collected by centrifugation at 2500 g for 5 min and washed three times with 1 ml of lysis buffer without proteases inhibitors. Finally, pellet was suspended in 50 μl of electrophoresis sample buffer and incubated for 5 min at 95°C, centrifuged at 2500 g for 5 min. A second elution with 15 μl was performed and the total amount of 65 μl of eluted proteins was separated by SDS-PAGE and analyzed with western and immunoblotting.Transiently transfected HEK 293 T cells were harvested and pellets were suspended in lysis buffer  using a Confocal Laser Scanning Microscope  equipped with an x63 objective lens (NA 1.32). The acquisition process contained three steps. First, images of GFP were acquired with an excitation wavelength of 488 nm and collected from 580 nm to 658 nm. Secondly, images of Cy3 staining were acquired with an excitation wavelength of 641 nm and collected from 678 nm to 703 nm. Finally, images of DAPI staining were acquired with an excitation wavelength of 405 nm and collected from 429 nm to 498 nm. As a technical control the same protocol was applied to cells exposed either only to one primary antibody or none of them.Cells  were seeded on Nunc Lab-Tek Chamber Slide System . Transient transfection of MCF7 cells was conducted for 48 h directly on the same support. After 48 h of transfection (for MCF7 cells) or the day after seeding (for A549 and MDA-MB-231 cells), medium was removed from the chambers and cells were washed twice with PBS 1X, fixed with 4% PFA and permeabilized with 0.1% Triton in PBS 1X. Unspecific proteins were blocked with PBS 1X, FBS 1% and BSA 0.1%. Cells were then incubated with primary antibodies against cN-II and Ipaf at 4°C overnight. The day after, cells were incubated with the appropriate DNA-linked secondary antibody furnished by the Duolink kit  and PCR S. cerevisiae, using the complete cN-II (1–561 aa) as bait. The screening was performed twice and gave 39 and 29 positive clones respectively. When eliminating clones that could not be sequenced, that contained mutations in the activation domain or that were not positive in the growth on medium lacking leucine, only 18 clones remained from the first screen and 29 from the second  and in human breast cancer cells (MDA-MB-231) since both interactors are well expressed in these cell models see . With thvertebrata taxon together with the growing body of data of multiple roles of cN-II in cell physiology, prompted us to investigate the possibility that cN-II could play a regulatory role dependent on protein-protein interactions in addition to its well-known catalytic activity. During the last decades, the catalytic function of cN-II has been extensively studied [The strong conservation of the primary cN-II sequence throughout the  studied –29 and i studied , 30, 31. studied –37. In s studied , 30, 31,We used a two hybrid system screening with the aim to detect possible cN-II interactors. The two independent screens performed identified several positive clones, but the majority turned out to be without interest. A clone containing the sequence corresponding to 97 aminoacids of the LRR domain of Ipaf was identified in the second run.Legionella pneumophila, Salmonella typhimurium, Shigella flexneri or Pseudomonas aeruginosa leading to macrophage cell death and the release of proinflammatory cytokines [et al. demonstrated an alternative pathway through which Ipaf leads to cell death by interaction with the component of the 26S proteasome Sug1 through caspase-8 recruitment and activation without any bacterial infection [stimuli, other roles of this protein remain possible. In view of the roles of this protein in cell biology and in particular in cell survival and induction of cell death, we found it to be an interesting potential candidate to explain some of the more recently described roles of cN-II.Ipaf belongs to the family of NBS-LRR (nucleotide-binding site and leucine-rich repeat) proteins and has been shown to take part in several intracellular signaling pathways involved in inflammatory responses. Four isoforms arising from alternate splicing of Ipaf mRNA have been identified in humans. The longest transcript termed Ipaf-A is 3.370 kb with an ORF encoding 1024 amino acids. Ipaf-B, C and D have exons corresponding to the nucleotide binding domain (NBD) spliced selectively to other exons forming shorter transcripts  which, iytokines . In addinfection . In the in vitro through co-purification of cN-II and a shortened form of IPAF. Furthermore co-immunoprecipitation experiments in HEK-293T demonstrated that Ipaf interacts with cN-II in human cell extracts through its LRR domain adding relevant information as to the interaction between proteins expressed and synthesized by human cells. The LRR domain is universally defined as the sensing domain through which Ipaf recognizes specific pathogen associated molecular patterns [The interaction between cN-II and Ipaf was confirmed patterns –45. Finastimuli, Ipaf is maintained in an inactive form Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 TableClones 1–18 were obtained in the first experiments, clones 19–47 in the second.(PDF)Click here for additional data file.


Picornaviridae. CV-A9 Griggs and HPeV-1 Harris (prototype) strains have been reported not to bind to heparin, but it was recently shown that some CV-A9 isolates interact with heparin in vitro via VP1 protein with a specific T132R/K mutation. We found that the infectivity of both CV-A9 Griggs and HPeV-1 Harris was reduced by sodium chlorate and heparinase suggestive of HSPG interactions. We analyzed the T132 site in fifty-four (54) CV-A9 clinical isolates and found that only one of them possessed T132/R mutation while the other nine (9) had T132K. We then treated CV-A9 Griggs and HPeV-1 Harris and eight CV-A9 and six HPeV-1 clinical isolates with heparin and protamine. Although infectivity of Griggs strain was slightly reduced (by 25%), heparin treatment did not affect the infectivity of the CV-A9 isolates that do not possess the T132R/K mutation, which is in line with the previous findings. Some of the HPeV-1 isolates were also affected by heparin treatment, which suggested that there may be a specific heparin binding site in HPeV-1. In contrast, protamine (a specific inhibitor of heparin) completely inhibited the infection of both prototypes and clinical CV-A9 and HPeV-1 isolates. We conclude that T132R/K mutation has a role in heparin binding of CV-A9, but we also show data, which suggest that there are other HSPG binding sites in CV-A9. In all, we suggest that HSPGs play a general role in both CV-A9 and HPeV-1 infections.Heparan sulfate/heparin class of proteoglycans (HSPG) have been shown to function in cellular attachment and infection of numerous viruses including picornaviruses. Coxsackievirus A9 (CV-A9) and human parechovirus 1 (HPeV-1) are integrin-binding members in the family  Heparan sulfate (HS) is a glycosaminoglycan chain found in heparan sulfate proteoglycans (HSPG). HSPGs are abundant on cell surfaces and widely distributed in animal tissues as part of extracellular matrix and integral membrane components. HS and a related heparin have highly sulfated disaccharide repeats, and hence they are negatively charged. By binding to numerous ligands and signaling molecules the role of HS is to act in cell adhesion, migration, proliferation and differentiation . HS alsoEnterovirus and Parechovirus genera, respectively, within family Picornaviridae [in vitro to integrins [Coxsackievirus A9 (CV-A9) and human parechovirus 1 (HPeV-1) belong to aviridae . In geneaviridae . CV-A9 aaviridae . They araviridae ,17. Bothntegrins –21. Othentegrins .McLeish et al.  has propThe human lung carcinoma (A549) cell line was obtained from the American Type Culture Collection (ATCC). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) and gentamicin 10 μg/ml. Culture medium for virus infections was supplemented with 1% FCS. CV-A9 (Griggs strain)  and HPeVin vitro assay was used anti-rabbit horseradish peroxidase (HRP) conjugate (Jackson ImmunoResearch).Polyclonal rabbit antiserum against CV-A9 and HPeV-1 were from laboratory collections ,26,28. ACells were seeded at 10,000 per well and grown on 96-well plates  to 80% confluency and viruses were used at m.o.i. of two. After 1 hour (h) of incubation, unbound virus was removed by washing with medium. Fresh medium was added, and the cells were transferred to 37°C. The infection was allowed to proceed for 6 h, fixed with 4% formalin and permeabilized with 0.2% Triton X-100. For immunofluorescence, cells were stained with rabbit polyclonal CV-A9 or HPeV-1 antiserum followed by combined staining with AF 488-labeled secondary antibody, and the nuclei were stained with DAPI. In the siRNA assay and in cell binding and inhibitor assays, the efficiency of infection was determined as the ratio of infected cells to the total cell number.2O was mixed with 0.2 μl of siLentFect (Bio-Rad) diluted in 25 μl of serum free medium and incubated for 30 minutes (min) at room temperature. A total of 30,000 cells were then added in 150 μl of serum-supplemented medium and cultured at 37°C for 48 h prior to infectivity assay (described above).Small inhibitory RNAs (siRNAs) were from Qiagen. Hs_EXT1-1 (SI 00002562) was targeted to exostosin glycosyltransferase 1 (NM_000127) ,30. Cont3 (Sigma-Aldrich). A549 cells were grown on 96-well plates in DMEM supplemented with or without 50 mM NaClO3 for 72 h prior to infectivity assay. Cell surface proteoglycans were digested by heparinase I (Sigma-Aldrich). Cells were incubated with 0.5, 2.5 and 5.0 U/ml heparinase I in serum-free DMEM for 2 h at 37°C prior to infectivity assay. Cell surface HS core was masked by protamine sulfate. Cells were incubated with 0.5 and 2.0 mg/ml protamine sulphate (Sigma-Aldrich) for 2 h at 37°C prior to infectivity assay. Virus particle HS binding sites were saturated with soluble low molecular-weight heparin (Sigma-Aldrich). Virus was incubated with 0.5 and 3.0 mg/ml heparin for 2 h prior to inoculation onto cells. After these treatments, virus infectivity assays were performed as described above.Incorporation of sulfates into the proteoglycans was inhibited with sodium chlorate, NaClOImages were acquired using a Zeiss Axiovert 200M microscope equipped with A-Plan 10x/0.25 Ph1Var1 objective (Zeiss). Brightness and contrast levels of the images were adjusted with Adobe Photoshop CS3 (Adobe Systems Inc.). The infection percentages were calculated as the ratio of infected cells to the total cell number from the images with open source software BioImageXD . The datFrom 54 clinical CV-A9 isolates, the putative HS binding site was identified and sequenced. These representative regions were aligned by ClustalW , translain vitro. They identified positively charged residue at VP1-T132 in two CV-A9 clinical isolates, and suggested that this site forms a putative HS binding site around five-fold axis of the capsid. Due to the slight discrepancy between the results, which may have arisen from different cell line used in the studies or passaged/cell-adapted CV-A9, we pursued further studies on the role of heparan sulfate /heparin in cellular infection of CV-A9 isolates. We also analyzed the same with HPeV-1 isolates, which also possess the RGD-motif.In the earlier studies to identify novel receptors for CV-A9 Griggs strain, we used siRNAs targeting known picornaviral receptors. We used human epithelial lung carcinoma cells (A549), which has proved to be useful in the analysis of integrin-dependent entry of CV-A9 . The cel3), which acts as metabolic inhibitor altering the biosynthesis of HS, followed by virus infection, staining and imaging. The result was measured as the number of infected cells against the total cell number in the image. As a result, CV-A9 (Griggs) infection was inhibited by 66% and HPeV-1 (Harris) infection by 59%, which were statistically significant (3 was used.In order to study further the role of HS in cellular infection of CV-A9 (Griggs) and HPeV-1 (Harris), we performed experiments with two strategies. Our first strategy was to alter the biosynthesis of HS. A549 cells were grown 72 h in media with or without 50 mM sodium chlorate , 81% (2.5 U/ml) and 76% (5.0 U/ml), and HPeV-1 infection by 23% (0.5 U/ml), 34% (2.5 U/ml) and 38% (5.0 U/ml), respectively . These eThe finding that CV-A9 (Griggs) and HPeV-1 (Harris) were inhibited by sodium chlorate and heparinase treatments suggested that these strains interacted with HSPGs ,24. To fPreviously, CV-A9 isolates possessing suggested T132R HS-binding site were shown to bind to heparin-agarose beads and blocked by soluble heparin , while HIn the case of HPeV-1, heparin significantly reduced the infectivity of most HPeV-1 isolates including Harris strain . Only onTo further examine the role of HS in the life cycle of CV-A9 and HPeV-1, we used protamine sulfate (PS), which is a drug that reverses the anticoagulant effects of heparin by binding to it. PS is an agent that contains clusters of positively charged amino acid residues and has the capacity to bind to negatively charged sulfate and carboxyl groups to antagonize protein interactions with heparin and HS. A549 cells were incubated with protamine sulfate prior to virus infection. Nine different CV-A9 isolates (including the Griggs strain) and seven HPeV-1 isolates (including the Harris strain) were used in PS assay. As indicated in the representative fluorescence images in Many viruses have been shown to utilize co-receptors for their initial attachment to the host cell, and to employ other molecules as principal receptors to gain entry into the cell ,37. BindTo determine the prevalence of T132R mutation, we analysed the region containing the T132 site in total of fifty-four (54) clinical CV-A9 isolates. We did not find VP1-T132R mutation to be common among CV-A9 clinical isolates. Instead, we identified nine isolates with VP1-T132K mutation, and only one isolate possessed T132R mutation. To analyse the role of HS further, we used four T132R/K mutants, four T132 and six HPeV-1 isolates together with the prototype Griggs and Harris strains in heparin and protamine sulfate cell blocking assay. Heparin was found to respond in a manner suggested by McLeish et al. : InfectiPrevious studies demonstrating the interaction of picornaviruses with HS suggest that the susceptibility to HS may be partially due to adaptation to specific cell line via mutations with capsid protein-encoding gene(s), suggesting that HS interactions may not have clinical importance . We used