Box C/D ribonucleoprotein particles (RNPs) are 2′-O-methylation enzymes required for maturation of ribosomal and small nuclear RNA. Previous biochemical and structural studies of the box C/D RNPs were limited by the unavailability of purified intact RNPs. We developed a bacterial co-expression strategy based on the combined use of a multi-gene expression system and a tRNA-scaffold construct that allowed the expression and purification of homogeneous archaeal and human box C/D RNPs. While the co-expressed and co-purified archaeal box C/D RNP was found to be fully active in a 2′-O-methylation assay, the intact human U14 box C/D RNP showed no detectable catalytic activity, consistent with the earlier findings that assembly of eukaryotic box C/D RNPs is nonspontaneous and requires additional protein factors. Our systems provide a means for further biochemical and structural characterization of box C/D RNPs and their assembly factors. S-adenosyl-methionine (SAM) molecule to the 2′-hydroxyl group of the target RNA nucleotide. The Nop5 or Nop56/Nop58 proteins form a homo (archaea) or a hypothesized hetero (eukarya) dimer that is regarded as the scaffold of the complex Box C/D small nucleolar ribonucleoprotein particles (snoRNPs) are one of the two major types of RNPs required for ribosome and spliceosome maturation. Large majorities of box C/D snoRNPs catalyze 2′-O-methylation of ribosome and spliceosome RNA by an RNA-guided mechanism Structural and biochemical studies of the box C/D RNPs depend on our ability to assemble and to purify the functional complexes. These processes have been more difficult with eukaryotic than with archaeal box C/D RNPs, owing to challenges in preparation of soluble eukaryotic proteins. Furthermore, heterogeneously assembled box C/D RNPs in cells hinder the purification of a single native box C/D RNP. As a result, current structural models of box C/D RNPs come from crystallographic and electron microscopic studies of archaeal box C/D RNPs reconstituted from individually purified components. These studies yielded two very different organizations of the particle. In the mono-RNP model, the L7Ae-bound box C/D RNA lies along the homodimer of the Nop5-fibrillarin complex in which fibrillarin is placed near each guide-substrate duplex E. coliTo facilitate studies of intact box C/D RNPs assembled under more native conditions, a bacterial expression system that permits co-expressing and purifying all proteins and the associated box C/D RNA is explored. This development is based on the co-expression strategies that greatly increase the stability and solubility of both proteins and RNA Archaeoglobus fulgidus (Af) Nop5, fibrillarin, L7Ae and an sR3 RNA. To construct the human box C/D RNP plasmid, pQhsCDtRNA+, pQLink was inserted with the coding sequences of Homo sapiens (Hs) NOP56 (amino acids 1 to 411), NOP58 (amino acids 1 to 401) and fibrillarin (amino acids 83 to 316), 15.5 K, and U14 snoRNA. Each protein-encoding gene was first cloned into pQLink-N plasmid using BamH1 and NotI sites. The plasmids containing two different protein-encoding genes were then digested by Swa I and Pac I, respectively, treated with LIC qualified T4 polymerase (dCTP for Pac I digest and dGTP for Swa I digest) and then annealed at 70°C. Each clone was identified by digestion of the recombined plasmid with Pac I. This process was repeated for additional coding sequence until all were inserted.We used pQlink cloning system E. coli initiator tRNAMet scaffold under the control of lpp promoter. The fused mini gene sequences contain the Xho I site, the LINK1 sequence (required for pQLink-N fusion), the lpp promoter, tRNA scaffold part I, sR3 sRNA/U14 snoRNA, tRNA scaffold part II, the rrnC terminator and the Hind III site  strain and those encoding the Hs box C/D components were transformed into the Nico21 (DE3) cells 2HPO4, pH 7.6, 500 mM NaCl, 10% glycerol, 0.5% Triton X-100, 2 mM phenylmethylsulfonyl fluoride (PMSF), 15 mM β-mercaptoethanol (β-ME). Cell debris was cleared by centrifuge at 21000 rpm for 45 minutes and supernatant was loaded on to a Ni-NTA column equilibrated with the lysis buffer. The column was washed extensively using the lysis buffer containing 50 mM imidazole and the bound RNPs were eluted using the lysis buffer containing 250 mM imidazole. The elutant was further purified by gel filtration chromatography using a Superdex 200 column equilibrated with a buffer containing 25 mM Na2HPO4, pH 7.6, 500 mM NaCl, 10% glycerol and 15 mM β-ME. To purify Af box C/D sR3 sRNP, cells were lysed by sonication in the same lysis buffer above without Triton X-100 followed by heating at 70°C for 30 minutes. The cell debris was cleared by centrifugation and the sRNP was purified similarly as U14 snoRNP.The plasmids encoding the Af box C/D components were transformed into 2, 10 mM Tris-HCl, and pH 7.4 was added to the cells suspended in 180 µl lysis buffer . After 20-minute gentle agitation at room temperature, the aqueous phase was separated by centrifugation. Residual phenol was then removed by addition of equal volume of chloroform. RNA in the aqueous phase was ethanol precipitated by addition of 0.1 volume of 4.0 M NaCl and 2 volumes of cold dehydration ethanol. To verify the presence of box C/D RNA in the purified RNP, the Ni-NTA-affinity purified protein complex was phenol/chloroform extracted and the RNA was ethanol precipitated as described above. Each extracted RNA was reverse transcribed using SuperScript VILO cDNA Synthesis kit (Invitrogen). Quantitative PCR was performed using an Applied Biosystems 7500 Fast Real-Time PCR System, with Invitrogen SYBR Green PCR Master Mix and primers at 0.5 µM each in a 20 µl reaction. The reverse-transcription amplified PCR products were cloned into the pCR2.1 vector using Invitrogen TA-cloning kit and verified by DNA sequencing.To test the expression of recombinant box C/D RNA, total RNA from RNP-expressing cells was extracted by phenol/chloroform extraction. A 200-µl aliquot of phenol saturated with 10 mM MgCl32P]-labeled cytidine at methylation sites were prepared by in vitro T7 transcription and were used for methylation assays. Transcribed RNAs were purified by phenol/chloroform extraction and ethanol precipitation. Methylation reactions were initiated by adding 10 nmol sRNP/snoRNP and ∼5000 cpm [α-32P]-labeled target RNAs to a 20 µl reaction mixture containing 20 mM HEPES (pH 7.0), 500 mM NaCl, 1.5 mM MgCl2, 5 mM DTT, 1 mM SAM, and 10% glycerol. After incubation at 70°C for Af sRNP or 4°C, 22°C, and 37°C for Hs U14 snoRNP for 2 hours, RNA in the reaction system were extracted by phenol/chloroform again. Reacted and extracted RNA sample was then treated with 2 units of nuclease P1 at 37°C overnight. The final product was resolved on a PEI-cellulose thin layer chromatography (TLC) plate in isobutyric acid: NH4OH: H2O . TLC plates were exposed at −80°C on to a phosphor screen  and results were analyzed on a Storm 860 system (Molecular Devices).Target RNAs containing [α-Archaeoglobus fulgidus (Af) sR3 and human U14 RNA were used for co-expression with their respective box C/D proteins , Saccharomyces cerevisiae Nop58p (Sacc_N58), Pyrococcus furiosus Nop56/58 (Pyrococcus), and Sulfolobus solfataricus Nop56/58 (Sulfolobus). Red boxes highlight residues that have strict sequence identity and blue boxes indicate residues that have sequence similarity. Secondary structure elements of Pyrococcus furiosus Nop56/58 are derived from its crystal structure (PDBid: 3NMU).Sequence alignment of human NOP56 (human_N56), human NOP58 (human_N58) against their homologs: (DOCX)Click here for additional data file.Figure S2Validation of human U14 RNA by cloning the reverse PCR product obtained from co-purified human box C/D RNP followed by DNA sequencing. Human U14 snoRNA sequence cloned in vector pCR2.1 is highlighted in yellow.(DOCX)Click here for additional data file.


PET-based texture features have been used to quantify tumor heterogeneity due to their predictive power in treatment outcome. We investigated the sensitivity of texture features to tumor motion by comparing static (3D) and respiratory-gated (4D) PET imaging.18F]FDG-PET scans before the chemo-radiotherapy. The acquired 4D data were retrospectively binned into five breathing phases to create the 4D image sequence. Texture features, including Maximal correlation coefficient (MCC), Long run low gray (LRLG), Coarseness, Contrast, and Busyness, were computed within the physician-defined tumor volume. The relative difference (δ3D-4D) in each texture between the 3D- and 4D-PET imaging was calculated. Coefficient of variation (CV) was used to determine the variability in the textures between all 4D-PET phases. Correlations between tumor volume, motion amplitude, and δ3D-4D were also assessed.Twenty-six patients (34 lesions) received 3D and 4D [−3), Coarseness  and Contrast  compared to 3D-PET. Nearly negligible variability was found between the 4D phase bins with CV<5% for MCC, LRLG, and Coarseness. For Contrast and Busyness, moderate variability was found with CV = 9% and 10%, respectively. No strong correlation was found between the tumor volume and δ3D-4D for the texture features. Motion amplitude had moderate impact on δ for MCC and Busyness and no impact for LRLG, Coarseness, and Contrast.4D-PET increased LRLG , Busyness , and decreased MCC ( = 1%–2%, p<7.5×10Significant differences were found in MCC, LRLG, Coarseness, and Busyness between 3D and 4D PET imaging. The variability between phase bins for MCC, LRLG, and Coarseness was negligible, suggesting that similar quantification can be obtained from all phases. Texture features, blurred out by respiratory motion during 3D-PET acquisition, can be better resolved by 4D-PET imaging. 4D-PET textures may have better prognostic value as they are less susceptible to tumor motion. Motion induced image blurring in static PET images (3D PET) can lead to reduction in tumor uptake and over estimation of metabolic tumor volume With the growing interest of texture features and tumor heterogeneity, the impact of tumor motion on PET-based quantification needs to be studied as it is still yet unknown. In this study, we compared the quantification of texture features between 3D and 4D PET imaging. Although numerous texture features can be found in the literature et al in 1973 The NGTDM texture features were originally designed to resemble human perception and were first proposed by Amadasun and King (1989) 18F]FDG-PET/CT, a free breathing chest CT, and a 4D [18F]FDG-PET scans were acquired 1–2 weeks prior to the therapy. There were sixteen patients with adenocarcinoma and ten patients with squamous cell carcinoma. The internal tumor volumes (ITV), which encompassed tumor motion, of thirty-four lesions  were delineated by an experienced radiation oncologist on a 4D planning CT. 3D PET and 4D PET scans were performed on a Siemens Biograph PET/CT scanner . Attenuation correction of 3D PET images was performed using the whole body 3D CT images, while 4D PET images were corrected by the free breathing chest CT images. 3D PET scans were acquired approximately 100 min after injection of 16.7–22mCi of [18F]FDG in the patients. For the 3D PET scan, the images were acquired for 3–5 min/bed position in six to seven bed positions. The 3D PET images were reconstructed with ordered-subset expectation-maximization (OSEM) with 4 iterations, 8 subsets, 7 mm full-width-half-maximum (FWHM) post-filtration, and sampled onto a 168×168 grid comprised of 4.06×4.06 mm2 pixel. The image acquisition of 4D PET followed immediately after the completion of the 3D PET scan.This study was conducted under the Dana-Farber Cancer Institute institutional review board (IRB) approved protocol (protocol #: 06-294) and written consents were obtained from all patients. Twenty-six patients  with NSCLC received a treatment planning CT (both 3D and 4D) two weeks before the start of radiotherapy with or without concurrent chemotherapy. 3D FDG even 2 hours after injection 18F]FDG-PET injection, 4D PET imaging was acquired between 118–135 min after injection. Therefore, the increase in [18F]FDG-PET seen in our study may not be due solely to respiratory motion. Dong et al (2013) found a significant correlation between SUVmax and textures (entropy and energy) derived from PET intensity histograms in patients with esophageal cancer max was also found to be highly correlated to entropy and energy in a study conducted by Orlhac et al (2014) maxHowever, there is one limitation of our textures and SUV comparison as it has been shown that malignant tumor tissue can continuously increase the uptake of [All the PET images in our study underwent attenuation correction using the free breathing CT images. The blurred anatomical mismatched of the PET/CT scans due to respiratory motion may affect the quality of the attenuation corrected 4D PET images, and subsequently the quantification of texture features Texture features, representing tumor heterogeneity, are blurred out by respiratory motion during 3D PET acquisition. 4D PET imaging reduces motion blurring, enabling PET-based features to be better resolved. Significant differences were found in MCC, LRLG, Coarseness, and Busyness between 3D and 4D PET imaging. When measuring tumor heterogeneity characteristics with PET imaging, reduced motion blurring by 4D PET acquisition enables significantly better spatial resolution of texture features. 3D PET textures may lead to inaccurate prediction of treatment outcome, hindering optimal lung cancer patient management. 4D PET textures may have better prognostic value as they are less susceptible to tumor motion.


Squamous cell carcinoma (SCC) is the most common neoplasm in organ transplant recipients (OTR) on long-term immunosuppression and occurs 60- to 100-fold more frequently than in the general population. Here, we present the receptor for advanced glycation end products (RAGE) and S100A8/A9 as important factors driving normal and tumor keratinocyte proliferation. RAGE and S100A8/A9 were transcriptionally upregulated in SCC compared to normal epidermis, as well as in OTR compared to immunocompetent patients (IC) with SCC. The proliferation of normal and SCC keratinocytes was induced by exposure to exogenous S100A8/A9 which in turn was abolished by blocking of RAGE. The migratory activities of normal and SCC keratinocytes were also increased upon exposure to S100A8/A9. We demonstrated that exogenous S100A8/A9 induces phosphorylation of p38 and SAPK/JNK followed by activation of ERK1/2. We hypothesize that RAGE and S100A8/A9 contribute to the development of human SCC by modulating keratinocyte growth and migration. These processes do not seem to be impaired by profound drug-mediated immunosuppression in OTR. Squamous cell carcinoma is a common skin neoplasm characterized by infiltrative, destructive growth and metastasis. It is the most common malignant neoplasm in organ transplant recipients on long-term immunosuppression and occurs 60- to 100-fold more frequently than in the general population . StudiesRAGE is a multi-ligand member of the immunoglobulin superfamily of cell surface molecules –13 and iUp to now the role of RAGE-S100A8/A9 signaling in keratinocytes in SCC formation has not been investigated. Here, we analyze the involvement of these proteins in the development of human SCC.The mRNA expression level of RAGE in whole skin was similar in normal skin, IC SCC and OTR SCC. S100A8 and S100A9 mRNA expression was increased only in SCC of IC versus normal skin . In the The expression of RAGE and the S100A8/A9 complex on protein level was analyzed by immunohistochemistry. Although the analysis is semi-quantitative, both proteins were clearly upregulated in invasive or in situ SCC of IC and OTR in comparison to healthy skin . We did Using ELISA specific for S100A8/A9, we detected that normal keratinocytes secrete S100A8/A9. In comparison to SCC-derived keratinocytes, normal keratinocytes showed lower levels of spontaneous S100A8/A9 secretion . The dirCells were seeded in 96 well plates and exposed for 24 hours to purified S100A8/A9 at concentrations between 0.01–1μg/ml. All normal, AK and SCC-derived primary keratinocytes responded with increased proliferation ranging from 30% to 70% after 24 hours exposure to S100A8/A9 as revealed by BrdU assay . InducedKeratinocyte proliferation following S100A8/A9 exposure was abolished by blockade of RAGE using a functional blocking antibody . A reducin vitro was investigated using scratch assay. An increase in the number of migrated cells was detected 15 h after scratching (between 60–100%) in both normal and SCC-derived keratinocytes in the presence of 0.01 and 0.1μg/ml S100A8/A9 . This is probably due to the heterogeneous cell population present in whole skin. As reported, S100A8 and S100A9 are predominantly secreted by myeloid cells, but also by epithelial cells and keratinocytes during inflammation , 44–46. In summary, RAGE and its ligand S100A8/A9 seem active in SCC both in the setting of immunocompetence and immunosuppression, where immunosuppressive agents do not subdue the RAGE-S100A8/A9 axis but might potentially accentuate it.To investigate the functional role of S100A8/A9 and RAGE, we generated primary keratinocyte cell cultures derived from normal skin and from lesions of patients with in situ (AK) or invasive SCC. Similar to other reports, we observed that low, rather than high concentrations of exogenous S100A8/A9 were sufficient to induce cellular proliferation , 50. A pNormal and SCC-derived keratinocytes showed distinct phosphorylation of JNK after S100A8/A9 stimulation, which is probably due to a different response to S100A8/A9, based on their origin . Studies concerning the role of RAGE-S100A8/A9 axis in the induction of MAPK, JNK or p38 pathways show that depending on the cell type, a differential phosphorylation pattern of these kinases is observed. In breast and colon cancer cells, S100A8/A9 is found to induce phosphorylation of ERK and JNK , 35, 52.S100A8/A9 may act as a growth factor similar to chemokines. A chemokine-like function of S100A8/A9 was described in terms of enhancement of leukocyte recruitment to inflammatory sites –56. KeraOrgan transplant recipients suffer from SCC with a 60- to 100-fold increased incidence compared to the general population. In spite of their chronic immunosuppressive medication, a considerable amount of inflammation can still be observed in the local tumor microenvironment . While lIn summary, we present evidence for a direct effect of S100A8/A9 on the proliferation and migration of normal human keratinocytes and keratinocytes originating directly from SCC lesions, suggesting a prominent role of S100A8/A9 on SCC formation, with RAGE as important target. This role of S100A8/A9 seems maintained in OTR. Targeting S100A8/A9 or RAGE may be of clinical benefit for SCC prevention and treatment.The use of clinically indicated biopsy material for the study was approved by the ethical committee of the Canton of Zürich, Switzerland and was previously published: –64nvitrogen, Basel, Switzerland), and stored at −80°CInvasive SCC from organ transplant recipients (n = 13) and immunocompetent patients (n = 19) as well as in-situ SCC from OTRs and immunocompetent patients (1 and 5 respectively) were obtained at the time of surgery. Normal skin was obtained from abdominoplastic reconstructive surgery (n = 5). All specimens’ diagnoses were confirmed by a board-certified dermatohistopathologist. 4mm punch biopsies from the SCC or normal skin area were placed in preheated PBS at 60°C for 45 seconds, and then chilled on ice in for 1 minute, followed by mechanical separation of epidermis and dermis. The epidermis was then homogenized in TRIzol Reagent  following the protocol provided. After 1:4 dilution with sterile water, cDNA was stored at −20°C and used as template for subsequent quantitative real-time polymerase chain reaction (RT-PCR).Total RNA was isolated using TRIzol Reagent following the instructions provided by the manufacturer. cDNA was synthesized using a Reverse Transcription System kit R&D Systems Europe LtD) and anti-human S100A8/A9 mouse monoclonal antibody (clone #27E10)  in a working dilution 1:50 (anti-RAGE) and 1:100 (anti-S100A8/9) in Antibody Diluent . Overnight incubation was performed in a humid chamber at 4°C. For detection of antibody binding to S100A8/A9 the Dako REAL Detection System AP/RED was used, and for detection of antibody biding to RAGE the Vectastain ABC-APKit  was used according the manufacturers` protocol. The slides were briefly soaked in hematoxylin and then left under running tap water for 10 min. The slides were mounted with Mounting Medium (Dako).Four μm thick sections were deparaffinized by 2x10 min incubation in Xylol and then rehydrated in a descending ethanol series (from 100% ethanol to PBS). For antigen retrieval, citrate buffer pH 6 (0.05% Tween 20) was pre-heated to 95°C, and the sections were kept at this temperature for 15 min. They were then allowed to slowly cool down to room temperature, and were then transferred to PBS. Blocking of nonspecific antibody binding was achieved by incubating the sections in PBS containing 2.5% of normal rabbit serum for RAGE staining or 2.5% normal goat serum for S100A8/A9. The staining was performed using anti-human RAGE polyclonal goat antibody . For sufficient separation of the epidermis and dermis the punch biopsies were incubate overnight at 4°C in keratinocyte medium (Progenitor Cell Targeted (PCT) epidermal keratinocyte medium CnT07 (CELLnTec) with Dispase II (Roche). Afterwards the epidermal part of the skin was incubated in trypsin  to insure efficient isolation of single keratinocytes. Freshly isolated cells were washed with medium and then transferred into CnT07 medium for a continuous incubation .Single keratinocytes were isolated from 3–4mm punch biopsies following the standard protocol for generation of primary keratinocyte cultures (Promega). Specific primers for RAGE , S100A8 , S100A9 , 36B4  and K14  were used. Real Time PCR was performed using Light Cycler FastStart DNA Master Sybr Green 1 kit .RNA was extracted using the TRIzol reagent  following the protocol provided by the manufacturer. cDNA was synthesized from 0.5μg total RNA using a Reverse Transcription system kit  method . StatistR&D Systems) and visualized by specific donkey-anti goat FITC labeled secondary antibody  in PBS at 4°C for 1h under gentle shaking conditions. The analysis was performed on 10 000 gated live cells together with a control (unstained and the secondary-FITC conjugated antibody) using FACSDIVA software .The assessment of the surface RAGE expression after induction with S100A8/A9 was performed using FACS analysis. Cells were detached using 4mM ETDA in PBS, washed with PBS, incubated with a specific goat anti-human RAGE antibody (Millipore). Before the staining cells were fixed with 4% PFA for 20min, followed by permeabilization with 0.5% Triton X, washed 3 times and proceeded for staining.Double staining for RAGE and BrdU was performed using the specific RAGE antibody and specific mouse anti BrdU antibody (3/well. Incubation with purified S100A8/A9 protein was performed using concentrations between 0.01–1ug/ml for 24h. Blocking goat anti-human RAGE antibody (R&D systems) was added 1h prior to S100A8/A9 exposure. Cell proliferation was measured using the BrdU proliferation assay (Millipore) according to the manufacturer`s instruction. The percentage of cell proliferation was calculated using the equation: (mean OD of treated cells/mean OD of control cells) X 100. Purified S100A8/A9 protein was provided by Thomas Vogl, , [Cells were seeded in 96 well plates in serum-free CnT07 medium in a cell density of 4.10ermany), , 66. Humconventional recipe). Cell lysates were collected after each time interval and subjected to PAGE, followed by immunoblotting. The phosphorylation of ERK1/2, p38 and SAPK/JNK was detected by specific rabbit anti phospho-ERK1/2, -p38 and -SAPK JNK antibodies . The non-phosphorylated form of the kinases were detected using specific rabbit anti- ERK1/2, p38  and anti SAPK/JNK . The loading control actin was detected by specific anti actin antibody .Normal and SCC-derived keratinocytes were grown in 6cm dishes in 60% confluence. Afterwards cells were stimulated with S100A8/A9 (10ng/ml) for different time intervals (0–60 min). Cells were lysed in RIPA buffer (Millipore).RAGE expression in normal and SCC keratinocytes was confirmed by using of specific human anti RAGE antibody (Santa Cruz Biotechnology). Positive clones were selected by puromycine selection. Stimulation of shRAGE and sh control expressing cells with S100A8/A9 protein was performed when the cells have reached an appropriate density. For determination of the effect of RAGE knockdown on cellular proliferation with a following S100A8/A9 stimulation, BrdU proliferation assay was performed.Primary keratinocytes were infected with RAGE sh lentiviral particals (sc-36374-v) and control sh viral particals (sc-108080), following the protocol conditions (CELLnTEC). Scratches were performed using a blue pipette tip. Cells were washed afterwards 2 times with basal keratinocyte medium. Exposure to exogenous S100A8/A9 was performed in a dose dependent manner using concentrations between 0.01–1μg/ml. Exposure was performed in triplicates for each concentration and control cells. After 15 and 24 hours, respectively, cells were fixed with 4% Paraformaldehyde , stained with Diff-Quik kit , and counted in the whole scratch. The percentage of migrated cells was calculated as % of control.Normal and SCC-derived keratinocytes were grown in 24 well plates until they reached full confluence and starved for further 15 hours in basal keratinocyte medium without supplements  and blocked (1% BSA) prior staining with the specific RAGE antibody. As a control isotype control antibody was used. b: RAGE expression in normal and SCC derived keratinocytes. Cell lysates from primary normal and SCC keratinocytes were set to SDS PAGE electrophoresis and western blotting was performed using specific primary anti RAGE and secondary HRP-conjugated antibodies. As a loading control additional staining against actin was performed using specific anti actin antibody.(TIF)Click here for additional data file.S2 Figa: Spot density-based quantification of pERK1/2 versus ERK1/2 and versus loading control (actin). b: Spot density-based quantification of pSAPK/JNK (pp54/p46) versus SAPK/JNK and versus loading control (actin). c: Spot density-based quantification of pp38 versus p38 and versus loading control (actin).(TIF)Click here for additional data file.S3 FigThe RAGE expression after knockdown was analyzed on transcriptional level by qPCR using specific primers for RAGE and compared to sh control.(TIF)Click here for additional data file.S4 FigS100A8/A9 has been extracted from granulocytes of buffy coats. The purity, quantity and quantity of the extracted S100A8/A9 were analyzed by Coomasie blue staining after SDS gel electrophoresis.(TIF)Click here for additional data file.


C57BL/6 is the most often used laboratory mouse strain. However, sometimes it is beneficial to cross the transgenic mice on the C57BL/6 background to the other strain, such as FVB. Although this is a common strategy, the influence of crossing these different strains on homeostatic expression of cytokines is not known. Here we have investigated the differences in the expression of selected cytokines between C57BL/6J and C57BL/6JxFVB mice in serum and skeletal muscle. We have found that only few cytokines were altered by crossing of the strains. Concentrations of IL5, IL7, LIF, MIP-2, and IP-10 were higher in serum of C57BL/6J mice than in C57BL/6JxFVB mice, whereas concentration of G-CSF was lower in C57BL/6J. In the skeletal muscle only the concentration of VEGF was higher in C57BL/6J mice than in C57BL/6JxFVB mice. Concluding, the differences in cytokine expression upon crossing C57BL/6 and FVB strain in basal conditions are not profound. Laboratory mouse is one of the central model organisms in modern biology, especially since the sequencing of mouse genome. Mouse fanciers and scientists created first inbred strains in the early XX century . Till noThe strain ontology can be assessed with the analysis of single nucleotide polymorphisms (SNP)  combinedHmox1−/−). Hmox1/−− mice were created on the background of C57BL/6J mice by Poss and Tonegawa [ Hmox1/−− animals were smaller than their wild type littermates, had microcytic anemia, reduced serum iron levels while iron accumulated in kidney and liver, and showed chronic inflammation and splenomegaly [ Hmox1+/− mice bred poorly and only less than 5% of newborns from Hmox1+/− parents were Hmox1/−− homozygotes [ Hmox1/−− homozygotes in 160–200 pups) [ Hmox1/+− mice breeding , Hmox1+/− mice were backcrossed 4-5 times to wild type FVB mice. Such Hmox1/−− C57BL/6JxFVB mice could be more easily bred, but maintained other characteristics of original Hmox1/−− strain [One of the examples where crossing different strains turned out to be beneficial was observed in case heme oxygenase knock-out animals  . Therefo− strain . On the  Hmox1 can be driven by nuclear factor (erythroid-derived 2)-like 2, also known as NFE2L2 or Nrf2, in response to oxidative stress [−/− mice were generated in 1997 on the ICR mouse strain background [ Hmox1/−− mice show impaired regeneration in response to the hind limb ischemia [−/− mice show better revascularization due to the inflammatory angiogenesis [Expression ofe stress . Nrf2−/−ckground  and thenckground . Hmox1−ischemia  while Nrogenesis . However Hmox1 deficient mice were initially kindly provided by Dr. Anupam Agarwal, University of Alabama, Birmingham, USA.Animals were handled in a strict accordance with good animal practice as defined by the relevant national and local animal welfare bodies. All animal work was approved by the Local Ethical Committee for Animal Research at the Jagiellonian University. Breeding heterozygote pairs ofFemale 5-month-old C57BL/6J and C57BL/6JxFVB mice were euthanized by overdosage of xylazine and ketamine. Blood was collected from the vena cava and after clot formation centrifuged for 10 minutes at 1000 g. Serum was collected and frozen at −80°C for further analysis.Luminex assay  was performed according to manufacturer's instructions. The samples were diluted 1 : 1 in Assay Buffer and incubated with Premixed Beads overnight at 4°C. Signal detection was done using FLEXMAP 3D system.μL saline intramuscularly (to both legs). Mice were euthanized by isoflurane overdose and gastrocnemius muscles were excised and homogenized in lysis buffer  in tissue lyser (Qiagen).Female and male C57BL/6J and female C57BL/6JxFVB 2- to 3-month-old mice were injected with 25 μL of 1 : 50 mixture of CuSO4 and bicinchonic acid was added to 5 μL of sample on 96-well plate, incubated 30 min at 37°C and absorbance was measured at 562 nm on Tecan microplate reader.Luminex assay  was performed according to manufacturer's instructions. The samples were diluted in lysis buffer and incubated with Premixed Beads overnight at 4°C. Signal detection was done using FLEXMAP 3D system. Total protein concentration was measured by BCA assay. Briefly, 100 t-test was applied. Results were considered as statistically significant, when P ≤ 0.05 .Statistical analysis was done with GraphPad Prism software. Data are presented as mean + SEM of at least 4 measurements. Two-tailed unpaired P = 0.0021, We observed increased concentration of granulocyte colony-stimulating factor (G-CSF) in the serum of C57BL/6JxFVB mice in comparison to C57BL/6J mice  and interferon γ (INFγ) in C57BL/6JxFVB and C57BL/6J  of proinflammatory cytokines, tumor necrosis factor C57BL/6J .α, IL9, IL10, IL12p40, IL13 and IL17 were similar in both strains, whereas concentrations of IL1β, IL2, IL3, IL4, IL6, IL12p70 were below the detection limit. Most of the interleukins were at levels of 1–10 pg/mL, whereas IL1α and IL13 were more abundant .Out of the whole panel of interleukins measured, only few were in different concentrations between the strains. IL5, leukemia inhibitory factor (LIF) and IL7 concentrations were lower in the serum of C57BL/6JxFVB than in C57BL/6J mice , howeverβ (MIP-1β), RANTES, or eotaxin was different between the compared strains. However two CXCL chemokines, macrophage inflammatory protein-2 (MIP-2) and interferon gamma-induced protein (IP-10), were decreased in serum of C57BL/6JxFVB when compared to C57BL/6J mice . Concentrations of other CXCL chemokines, KC, LPS-induced CXC chemokine (LIX) and monokine induced by gamma interferon (MIG), were the same in both strains (α (MIP-1α) was not detectable in any of the strains.Next we have analyzed the concentration of selected chemokines in serum. None of the measured chemokines from the CCL family, monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein-1  strains . MacrophFinally, we evaluated the expression of several cytokines in the lysates from the gastrocnemius muscle. Cytokines which were in different concentrations in plasma in C57BL/6JxFVB and C57BL/6J, IL5 and IP-10, were at the same concentration in the muscle in both strains. We observed no differences in the concentrations of GM-CSF, IL1b, IL10, MCP-1 and KC in the muscles of C57BL/6JxFVB and C57BL/6J. However, the expression of vascular endothelial growth factor (VEGF) was lower in C57BL/6JxFVB than in C57BL/6J. At the same time concentration of another proangiogenic cytokine, basic fibroblast growth factor (bFGF), was unchanged .The presented study revealed the degree of changes within concentration of inflammatory mediators in serum and muscles between C57BL/6J strain and mixed C57BL/6JxFVB strain.It is widely accepted that various mouse strains are phenotypically different. The differences between commonly used mouse strains are also visible on genomic level. Significant gene content variation characterizes the genomes of inbred mouse strains , 29 and Several phenotypical differences were observed between C57BL/6 and FVB strains. C57BL/6 mice are more susceptible to anesthetic agents, pentobarbital, ketamine, and nitrous oxide in comparison to FVB mice . MammaryDifferences in expression of cytokines between the strains were also reported. For example, profiles of serum cytokines were different both in control mice, as well as after induction of experimental immune thrombocytopenia between C57BL/6J and BALB/c . Hmox1+/− mice was improved after crossing with FVB [ Hmox1+/− mice which were predominantly on the C57BL/6 background resulted in Hmox1−/− offspring in the number of 2–6.25% of expected mice from Mendelian ratio [ Hmox1/−− pups are 5.1% of delivered newborns when crossing C57BL/6JxFVB Hmox1 heterozygotes, what is in consistence with the original report [ Hmox1−/− mouse generation to 20.1% of delivered pups when Hmox1−/− males are crossed with Hmox1+/− females. One should be aware, however, that manipulating the background may influence the phenotype of the transgenic animal. Different phenotype of the same mutation in different strains gives the opportunity to choose the strain which better resembles the human disease. In the model of hereditary inclusion body myopathy (HIBM) the GneM712T/+ transgenic mice of C57BL/6 strain developed severe kidney dysfunction and died within few days after birth [M712T/M712T glomerular disease was less pronounced and mice survived longer, retaining skeletal muscle pathology [C57BL/6 strain is commonly used for generation of novel knock-out animal models. However, this strain possesses features that may be unfavorable in given experimental settings comparing to other strains. FVB/N strain is frequently used as a background for transgenic mouse models due to its high fertility and large litters , 35. Thewith FVB . Crossinan ratio . In our l report . We couler birth . Kidney athology . Hmox1 deficiency and glomerular disease of transgenic GneM712T/M712T mouse we addressed this question in the presented work.Despite the fact that crossing genetic mouse models on C57BL/6 background with FVB mice helps to avoid limitations, is it has not been studied how such strategy influence the expression of inflammatory mediators. Given that crossing to FVB strain partially rescued the embryonic lethality ofWe found several differences in cytokine concentration in serum between the C57BL/6J and the mixed strain C57BL/6JxFVB. IL5 concentration was lower in C57BL/6JxFVB in comparison to the C57BL/6J. However we have not seen any differences in the levels of the same cytokine family members, GM-CSF and IL3. Out of the common cytokine receptor gamma-chain family, only IL7 was affected, concentrations of IL2, IL4, and IL9 remained unchanged. We observed smaller concentration of LIF in serum of C57BL/6JxFVB than in C57BL/6J mice. Level of IL6, member of the same family as LIF, was similar in both mouse strains. However concentrations of these cytokines are very small, on border of detection limit and therefore they may not have the biological significance. The important difference seems to occur in case of G-CSF. C57BL/6JxFVB mice had 1.7x higher concentration of G-CSF than C57BL/6J mice . G-CSF is important for neutrophil precursors proliferation and differentiation, and mature neutrophils release from the bone marrow to the blood . DiffereM712T/M712T transgenic mice was significantly improved by crossing the original C57BL/6 background with FVB strain [165b in the muscle biopsies from patients with idiopathic inflammatory myopathies, among them sporadic inclusion body myositis (IBM) [M712T/M712T transgenic mice on the mixed background.In the earlier mentioned work by Valles-Ayoub et al. survival of hereditary inclusion body myopathy GneB strain . Interesis (IBM) . Our res in vivo experiments. It should be also taken into consideration when the cells isolated from mice are cultured ex vivo in the presence of cytokines. Possibly, for cells isolated from some of the strains different concentrations of supplemented cytokines may be needed to induce differentiation or functional activity.Differential expression of cytokines can influence the results of variousConsidering significant genetic differences between various mouse strains it could be expected that concentration of proinflammatory cytokines in often used C57BL/6JxFVB mixed strain will be also affected. However our study showed that crossbreeding of C57BL/6J with FVB mice did not affect majority of cytokines in serum and skeletal muscle. The only cytokines that were significantly different between C57BL/6J and mixed C57BL/6JxFVB strain include G-CSF concentration in serum and VEGF concentration in gastrocnemius muscle. Therefore findings of the study should be considered when using this mixed background in hematological studies due to different G-CSF concentrations in serum and myopathy and muscle regeneration studies due to the different concentrations of VEGF in muscles.


Both protein families show structural similarity but limited amino acid identity. Moreover, the organization of the inverted repeat (IR) and the loop that shape the nic site differs in both proteins. Arguably, replicases cleave their target site more efficiently, while relaxases exert more biochemical control over the process. Here we show that engineering a relaxase target by mimicking the replicase target, results in enhanced formation of protein-DNA covalent complexes. Three widely different relaxases, which belong to MOBF, MOBQ and MOBP families, can properly cleave DNA sequences with permuted target sequences. Collaterally, the secondary structure that the permuted targets acquired within a supercoiled plasmid DNA resulted in poor conjugation frequencies underlying the importance of relaxase accessory proteins in conjugative DNA processing. Our results reveal that relaxase and replicase targets can be interchangeable in vitro. The new Rep substrates provide new bioconjugation tools for the design of sophisticated DNA-protein nanostructures.During bacterial conjugation and rolling circle replication, HUH endonucleases, respectively known as relaxases and replicases, form a covalent bond with ssDNA when they cleave their target sequence ( HUH proteins are enzymes widespread in all three domains of life, where they process DNA during initiation of rolling circle replication (RCR) of certain phages and eukaryotic viruses, conjugative transfer of plasmid between cells and transposition of insertion sequences and helitrons . These enic sites) with nanomolar specificity and form a protein-DNA covalent bond in presence of divalent cations. The linkage is a phosphotyrosyl intermediate between the catalytic tyrosine and the 5’ phosphate of the nic site. Moreover, they both carry out a second nucleophilic attack to the newly synthesized ssDNA strand, which results in recircularization of one unit of the ssDNA genome. Relaxases and replicases display structural similarity [nic-containing ssDNA loop [nic-cleavage reaction [nic site as ssDNA within the active center of the protein (nic efficiently unless relaxase-accessory proteins (RAPs) allow the generation of ssDNA. On the other hand, RAPs are not required for replicase activity. Thus, in order to facilitate the relaxase nic-cleavage reaction, we relocated the nic site within the loop of the IR, mimicking a replicase substrate. This substrate was further improved by exchanging the relative position of IR and nic sites. The results obtained provide a strategy for the design of more efficient substrates to be used in vitro for relaxase bionanotechnological reactions.Relaxases and rolling-circle replicases belong to the HUH-endonuclease superfamily . Replicamilarity  but alsomilarity . The catmilarity . Thus, tDNA loop ,8. On threaction . The rel protein . Neverthnic site in their cognate substrate is structurally different in replicases, relaxases and other HUH endonucleases [nic sites are located 3´ to the hairpin  and a proximal arm (P) with respect to the nic site (N). Moreover, there are eight nucleotides between P and N that are bent with a U-shape (U). The sequence (S) downstream N is tethered to the relaxase after cleavage  substrates  containing the nic site (N) and a dsDNA stem of 6 bp. The length of the loop (U+S) was tuned by including different number of nucleotides in the S region before the proximal arm (P)  called TrwCR hereafter.Location of the ucleases . Despite hairpin  while re hairpin . The wt cleavage . In ordebstrates . To crea (P) see . Oligonuin vitro nic-cleavage reactions. This reaction generates a protein-DNA covalent complex that can be quantified by its lower mobility using SDS-PAGE as described in Materials and Methods. TrwCR was able to cleave and remain covalently bound to oligonucleotides containing IR2-nic (W(25+18)) or P-nic (W(14+14)), . Interestingly, TrwCR could also cleave a Rep-like oligonucleotide containing a loop with only 2 nucleotides in the S region (H(14+8)) (R cleavage activity on H(14+8) was similar to that on W oligonucleotides. Incubation of TrwCR with Rep-like hairpins containing longer loops, such as H(14+10), H(14+12) or H(14+13), resulted in a band with reduced mobility, as expected by the formation of a TrwCR covalent complex with a decamer (Lane 5), dodecamer (Lane 6) and tridecamer (Lane 7), respectively. Similar results were obtained with substrates H(14+14), H(14+15) or H(14+17) . Labeled Rep-like oligonucleotide H14+14 was also found to be effectively cleaved by TrwCR using TBE-urea gel electrophoresis analysis (The effect of the DNA substrate length and secondary structure on relaxase cleavage was investigated through H(14+8)) , lane 4.analysis .R protein was previously shown to cleave oligonucleotides containing the R388 nic site and to perform an in vitro strand transfer reaction to a second oligonucleotide also containing the nic site [R cleaves Rep-like oligonucleotides and remains covalently attached to the oligomer downstream nic (R and oligonucleotide H(14+8), 29% of the labeled oligonucleotide shifted to a position corresponding to a F(25+8) oligonucleotide  to the proximal arm (P) would be critical for cleavage, since only certain loop lengths would allow the nic site to reach the active site of the protein. As similar nic-cleavage efficiencies were observed regardless of loop length (R interacts with Rep-like conformation substrates. Increasing concentrations of TrwCR were incubated with 50 nM of a green-fluorescent labeled Rep-like oligonucleotide H(14+14) or with a red-fluorescent labeled wt oligonucleotide W(25+8). Although the EC50  was the same with both oligonucleotides, only one shifted band was observed with W(25+8), while several shifted bands appeared with H(14+14). Both unbound oligonucleotides produced a single band  seems to form a unique complex, while different complexes are formed with H(14+14).We thought that the distance (S) from p length , gel shiR was further analyzed by gel filtration. Consistent with the EMSA result, several peaks with higher molecular weight than the predicted peak for 1:1 complexes appeared when either Rep-like H(14+14) and H(16+16) (S = 8), H(14+15) (S = 9) or H(14+17) (S = 11) oligonucleotides were incubated with TrwCR , H(23+26), H(24+24), H(24+27), H(24+31)). Now, a sole band of protein-DNA was shown in chromatography, while SDS-PAGE gels revealed that the covalent complexes are still being formed with longer stem substrates .R- and TraIR -DNA complex structures was also observed in the structure of Y1 relaxase NES [nic [R in this study) complexed with a 33-mer oligonucleotide and compared this model with TrwCR bound to the 23-mer [R. To study the influence of IR length, we designed a shorter wt substrate, containing a 6 bp IR WQ(23+7) (R.Relaxases contain either one Y1) or two (Y2) catalytic tyrosines [ or two (xase NES –15. Howexase NES . In ordeNES [nic . Mozingoe 23-mer . AccordiWQ(23+7) . AnalysiWQ(23+7)  Lane 2 oWQ(23+7)  Lane 3 gnic-site sequence were designed as for R388 nic (R cleaved the Rep-like substrate HQ(18+16) to 13%  to 13%  Lane 4. ) to 13% . These rnic. In our study, a substrate with a 6-bp hairpin conformation WP(24+8) was designed. As shown in R). Covalent complex formation by the wt substrate was slightly lower than when WP(15+6) was used    Lane 5 nR wt substrate W(25+8), the stem loop is non-covalently bound by the relaxase after cleavage, while the 8-mer downstream nic is covalently attached to the catalytic tyrosine. There is a cleavage-ligation equilibrium with wt oligonucleotides, because the 25-mer oligonucleotide remains in the protein DNA binding domain after cleavage and, therefore, the 8-mer can be easily religated (nic (U-N-S) to the 5´end of the hairpin, the 5´side of nic (the single strand U sequence upstream the nic site) would lose binding affinity (nic site.In TrwCeligated . We thouaffinity . As a coaffinity . Under tnic) or a fragment (up to seven nt), which would be liberated after cleavage. Besides, S length varied from 8 to 11 nucleotides, to allow the correct disposition of the hairpin into the relaxase. As shown in the SDS-PAGE gel of R388 reverse oligonucleotides were designed using either the whole U ssDNA (8 nt before R _RP4 (R could not cleave similarly designed oligonucleotides RQ(8+28), RQ(8+34) or RQ(8+40)  was recognized and cleaved by TraIR _RP4  Lane 4. R _RP4 . InteresR with the novel substrates supports the notion that both Rep-like and reverse oligonucleotides could modify the in vivo efficiency of relaxases. The importance of the DNA stem loop structure for efficient relaxase-mediated DNA processing in vivo was investigated by using plasmids containing synthetic oriTs. Synthetic R388 oriTs were designed by substitution of the wt nic-cleavage site (IR2+nic) by either H(14+14) Rep-like or R(8+24) Reverse-like sequences (The stable interaction of TrwCequences .OriT-containing plasmids (CmR) can be mobilized to a recipient cell  in the presence of the helper plasmid pSU2007 (KmR). Mobilization frequencies of the synthetic oriT-containing plasmids were measured as the number of mobilized plasmids (CmRSmR) per donor cell (CmRNxR) . Interestingly, the conjugation rate of ΔDistal Arm oriT plasmid dramatically dropped below detection levels (<10−6 transconjugants/donor).(CmRNxR) . The Repnic site without any binding sites for IHF and TrwA, named minimal Rep-like. Mating carried out with minimal Rep-like oriT did not generate transconjugants. Similarly, reverse-like plasmids, which have the nic site placed upstream IR2, also resulted in a substantial reduction of plasmid conjugation.We also checked the conjugation ability of a plasmid containing just the Rep-like nic site layout. Thus, we designed Rep-like substrates with secondary structures mimicking the stem-loop recognized and cleaved by rolling circle replicases  although, unexpectectly, the yield of covalent complex formation with the novel substrates did not improve  and WQ(23+7)  , becausein vitro reactions were enhanced by novel relaxase substrates, we analyzed their behavior in whole cell experiments. With the goal of improving the efficiency of relaxases within living cells, synthetic plasmids in which the relaxase target was converted to either Rep-like sequence H(14+14) or Reverse R(8+24) were tested. Conjugation rates critically fell for all synthetic plasmids tested  and IHF (ibs) binding sites. Moreover, ssDNA hairpin formation by the modified IR region is different in recipient cells and could affect the termination reaction. It has been described for relaxases as well as replicases that the sequence requirements for the termination are different than the requirements for initiation [oriTs to obtain transferable plasmids in the absence of RAPs.Once demonstrated that s tested . Rep proverse R8+ were tesng ssDNA –27. In titiation . Furtherin vitro results derives from the finding of new cleavable substrates for HUH proteins by rational design. Results show that relaxase TrwCR acting on reverse nic sites is more efficient in cleavage than when acting on its cognate sites. Relaxases are bifunctional proteins, having an N-terminal relaxase domain composed of roughly 300 residues fused to a C-terminal domain with various biochemical activities . The relaxase domain can be fused with heterologous proteins  without losing activity. Since relaxases (and relaxase proteins fused to fluorescent proteins) attach covalently to a specific sequence of DNA, even on DNA nanostructures [The significance of the ructures . Relaxasructures . As a seructures . The preructures . Here weR, amino acid residues 1 to 293) was purified as previously described [R expression from plasmid pSU1588 (pET3a:trwcR) in Escherichia coli strain C41 was induced with 0.5mM IPTG for 3 h. Cells were harvested by centrifugation and stored at -80°C. Frozen cells were thawed at 37°C, resuspended in a solution containing Tris 100 mM (pH = 7.6), NaCl 500 mM, EDTA 1mM, PMSF 1% and lysed by 10 min. sonication in pulses. The lysate was centrifuged at 40,000g for 15 min at 4°C. Supernatants were applied to a P11-phosphocellulose column equilibrated in buffer A  containing 200 mM NaCl and eluted with buffer A with 600 mM NaCl. Fractions containing TrwCR were pooled, diluted to 200 mM NaCl, loaded onto a Hi-Trap SP HP column , and eluted with a linear NaCl gradient (200 mM–1000 mM NaCl) in buffer A.The N-terminal relaxase domain of TrwC , was cloned in pET3a expression vector between NdeI and BamHI restricition sites (pSU10064). Expression of MobAR was achieved using E.coli C41 strain. After induction for 3 h with IPTG, cells were harvested and stored at -80°C. Purification was performed according to the procedure described for TrwCR.The N terminal fragment of RSF1010 go et al. and hereR) was PCR amplified from plasmid RP4 and subcloned in pET3a using NdeI and BamHI restriction sites, resulting in plasmid pSSP10. C41 strain carrying pSSP10 was induced for 3 h with IPTG. Cells were collected by centrifugation and stored at -80°C. RP4 TraIR was purified as described above for TrwCR, except the Hi-Trap SP HP column was substituted by a Hi-Trap Heparin column, from which TraIR was eluted with a linear 200–1000 mM NaCl gradient.The N terminal domain encoding the 270 residues of RP4 TraI  for TrwCR, 32430 M-1 cm-1 (Abs 0.1% of 1.551) for MobAR and 20190 M-1 cm-1 (Abs 0.1% of 0.636) for TraIR. After Superdex75 column chromatography, relaxase containing fractions were pooled and stored at -80C for further use.Gel filtration of all relaxases was carried out in a Superdex75 column 10/300 GL  equilibrated in 100 mM Tris–HCl (pH 7.6), 500 mM NaCl, 0.1 mM EDTA. All three relaxases eluted as a monomers of about 30 kDa for TrwCobAR see . ProteinUnlabeled DNA oligonucleotides were obtained from Sigma-Aldrich . Their sequences are listed in R with 15 μM of each oligonucleotide at 37°C for 1 h in 10mM Tris-HCl, pH7.6, 5mM MgCl2, 375 mM NaCl and 15 μM EDTA [R (7 μM) was incubated with 15 μM of each substrate in 25 mM Tris-HCl pH 8.0, 5 mM MgCl2, 225 mM NaCl and 15 μM EDTA [R, the optimized reaction mixture was 10 mM Tris pH 7.6, 5 mM MgCl2, 225mM NaCl and 15 μM EDTA [R was incubated with 15 μM oligonucleotide in each reaction. Reactions were stopped by adding SDS and boiling the samples. The cleavage activity was checked by the lower mobility of the protein-DNA covalent complexes in SDS-PAGE. There was always a 2:1 molar excess of oligonucleotides to guarantee that all the protein is in complex with the oligonucleotide. DNA nicking activity was expressed as percentage of covalent complexes generated (the intensity of the product band divided by the sum of product and free protein band intensities). Each data point represents the average of three reactions. Data were processed using Quantity One .Cleavage reactions were carried out by incubating 6.3 μM TrwC μM EDTA . Similar μM EDTA . For Tra μM EDTA . 1.5 μM R in 10 mM Tris-HCl, pH7.5, 5 mM MgCl2, 100 mM NaCl. After incubation for 1 h at 37°C, reaction mixtures were digested with 0.6 mg/ml proteinase K and 0.05% (w/v) SDS for 20 min at 37°C. Reactions were run through a denaturing TBE-urea polyacrylamide gel at 200V for 70 min to separate cleaved product DNA from the substrate. Oligonucleotides were visualized using an Odyssey Infrared Image System (LI-COR Biosciences).Cleavage reaction mixtures (20 μL) contained 50 nM IRDye-labelled oligonucleotide W(25+8) or H(14+14), 1 μM TrwCR in 10 mM Tris- HCl pH 7.6, 5 mM MgCl2, 110 mM NaCl and 15 μM EDTA. After 30 min, 50 nM IRDye800-labelled oligonucleotide R388 W(25+0) (5´- CGCACCGAAAGGTGCGTATTGTCT) was added to the reaction mixture. After incubation for 30 min at 37°C, reaction mixtures were digested with 0.6 mg/ml proteinase K and 0.05% (w/v) SDS for 20 min at 37°C. Oligonucleotide separation was performed by electrophoresis in a denaturing 18% Acrylamide, 8 M Urea gel at 200V for 70 min. Images were processed with Odyssey Infrared Image System (LI-COR Biosciences) and quantified by Quantity One software (Biorad).For oligonucleotide strand-transfer reactions, mixtures contained 0.25 μM of Rep-like oligonucleotides  and1 μM R binding to IRDye800-labelled oligonucleotide R388 W(25+8) (CGCACCGAAAGGTGCGTATTGTCT/ATATTGTCT), or IRDye700-labelled R388 H(14+14) (GGTGCGTATTGTCT/ATAGCCCGCGCACC) was analyzed by EMSA. Binding reactions contained 50 nM IRDye-labeled oligonucleotide and increasing concentrations of TrwCR in buffer . Reaction mixtures were incubated for 30 min at room temperature and loaded onto a 10% non-denaturing polyacrylamide gel. After electrophoresis at 200 V during 15 min, images were processed and quantified using Odyssey Infrared Image System (LI-COR Biosciences).TrwCR was determined by gel filtration in a Superdex75 PC 3.2/30 . 2.0 μl of each oligonucleotide (100 μM) were diluted in 18 μl of buffer solution  and injected in the column loop of 25 μl. The elution buffer was 100 mM Tris 7,6, 200 mM NaCl, 1 mM EDTA. When we analyzed the protein, 20 μl of stock solution (42 nM of TrwCR) was injected alone. The complexes of TrwCR with oligonucleotides were loaded after 30 min incubation at room temperature, with an excess of oligonucleotide (ratio 1:1.5) to ensure maximum complex formation.Analysis of intra- and intermolecular structures in the oligonucleotides library created for the determination of the best substrate for TrwCR in presence of 10 mM Mg2+ during 1 h was analyzed by gel filtration and native electrophoresis. After high resolution gel filtration column chromatography of the binding mixture, 10 μl of the fractions were loaded into a 5% non-denaturing acrylamide gel, and the fluorescent label of the oligonucleotide was detected with Odyssey infrared scanner.To study the new balance achieved with Rep-like substrates, IRDye700 labeled oligonucleotide R388 H(14+14) mixed with TrwCoriT plasmids were design and ordered to GeneArt (Life technologies-Invitrogen). The ΔDistal Arm plasmid resulted from pSU1186 PCR-amplified with primers containing the deletion of the distal arm of IR2. The linearized plasmid was self-circularized with T4 ligase. For minimal Rep-like plasmid, two oligonucleotides with the 28 base pairs Rep-like sequence between BamHI and HindIII sites were cloned in the pSU19 digested vector.The Rep-like and Reverse-like R) cells containing the helper plasmid pSU2007 (a KmR derivative of R388) [oriT-containing plasmids to be checked (CmR) were mated with strain UB1637 (SmR) as a recipient strain. Saturated cultures of donor and recipient strains were mixed in a 1:1 ratio and mated on a LB agar surface. After 1 h at 37°C, serial dilutions were plated. Mobilization frequencies were expressed as number of CmR transconjugants (CmRSmR) per donor cell.Matings were carried out by the plate-mating procedure described in . DH5α (Nof R388) ,33 and tS1 FigR from R388 plasmid bound to its target (PDB 1OMH)) and RCR replicases (AAV with RBE (PDB 1UUT)). Relaxases and RC-Rep suffered a circular permutation in the primary sequence that localizes the catalytic tyrosine at the N-terminal in the relaxases, but close to the C-terminal in Rep proteins. Relaxases recognize the nic site (red triangle) 5´ to an inverted repeat, while Reps cleave a nic site within a stem-loop. B) Ribbon structure of relaxases (left) and RC-Reps (right). TraIR from plasmid pCU1 (PDB 3L57) and TraIR from plasmid F (PDB 2AOI) belong to MOBF family of relaxases whereas NES from pLW1043 (PDB 4HT4) and minMobA frm R1162 (PDB 2NS6) belong to MOBQ family of relaxases. RepB, the RC initiators of plasmid pMV158 (PDB 3DKX) and Rep from geminivirus (PDB 1L5I) also have a similar folding. All these proteins possess a core of five antiparallel β-strand, where the HUH motif is located at the third β-strand, near the α-helix in which the catalytic tyrosine(s) is held.A) Organization of the catalytic centre in Relaxases (TrwC(PDF)Click here for additional data file.S2 FigR. Wt 5’IRDye700-labelled W(25+8) oligonucleotide is shown in lanes 1 and 2, whereas Rep-like 5’IRDye800-labelled H(14+14) oligonucleotide is shown in lanes 3 and 4. Oligonucleotides were incubated with TrwCR, treated with proteinase K and SDS, as described in Material and methods. A 25-mer (lane 2) and a 14-mer (lane 4) oligonucleotide appeared in samples treated with TrwCR.—represents the control DNA substrate (lanes 1 and 3).Denaturing TBE-Urea gels showing the products of cleavage of wt and Rep-like substrates by TrwC(PDF)Click here for additional data file.S3 FigR cleaves oligonucleotides containing the R388 nic-site and performs the strand transfer reaction to a labeled oligonucleotide W(25+0). In green are shown the oligonucleotides that remain covalently attached to TrwCR. The gels show the appearance of a labeled transfer product. (B) Denaturing gels showing the strand transfer reaction catalyzed by TrwCR with wt, Rep-like and reverse substrates. 5’IRDye-labelled W(25+0) oligonucleotide is shown in control lanes 1 and 7. A 25+x labeled oligonucleotide was obtained after strand transfer reaction with the wild type oligonucleotides W(25+18), W(18+18), W(14+14) and W(8+8) in lanes 2, 3, 4 and 5, respectively. Strand transfer reaction to W(25+0) was also analyzed for Rep-like H14+x oligonucleotides. Lane 6, H(14+8); lane 8, H(14+10); lane 9, H(14+12); lane 10, H(14+13); lane 11, H(14+14); lane 12, H(14+15) and lane 13, H(14+17). Reverse substrates were also analyzed for strand transfer in lane 14, R(7+27); lane 15, R(8+24); lane 16, R(8+27) and lane 17, R(8+14).(A) Schematic representation of the strand transfer reaction. TrwC(PDF)Click here for additional data file.S4 FigR was incubated during one hour in presence of EDTA with a 1.5:1 molar excess of each oligonucleotide. Then 20 μl of the samples were injected in a S75 column using the ETHAM system (GE Biosciences). Free oligonucleotides at the same concentration were also injected separately for comparison. Chromatograms of oligonucleotides are shown as dashed lines while TrwCR with oligonucleotides are shown as continuous lines. Several shifted peaks and broaden peaks are observed in the cases of short hairpins H(14+14), H(14+15) and H(14+17). TrwCR interacts with oligonucleotides with longer stems H(16+16), H(23+23), and H(23+26) in a similar fashion. Reverse oligonucleotides R(8+27), R(8+24), R(7+27) show a slight shift when TrwCR is bound to it, similar to the one obtained with wt oligonucleotides (data not shown). Oligonucleotides tested were Rep-like oligonucleotides with D = P = 6 and different loop lengths; H(14+14) S = 8, H(14+15) S = 9 and H(14+17), Rep-like oligonucleotides with S = 11 and different stem lengths: H(16+16) D = P = 8 S = 8, H(23+23) D = P = 15 S = 8, and H(23+26) D = P = 15 S = 11 and Reverse oligonucleotides; R(8+27) D = P = 6 U = 8 S = 11, R(8+24) D = P = 6 U = 8 S = 8 and R(7+27) with D = P = 6 U = 7 S = 11. Absorbance at 260 nm was used during chromatography to determine the presence of DNA. All the chromatograms are normalized and shifted peaks are shown by stars.6.3 μM TrwC(PDF)Click here for additional data file.S5 FigR was incubated during one hour at RT in presence of 10 mM MgCl2 with a 2:1 molar excess of IRDye H(14+14) oligonucleotide. Then 20 μl of the sample was injected in a S75 column using the ETHAM system (GE Biosciences). The chromatogram shows the obtained four A260 peaks with the Ve of each peak. After gel filtration column chromatography, 20 μl of 19 fractions collected within these four peaks were loaded into an acrylamide native gel. Lane M contains free oligo. Shifted oligonucleotides are shown by arrowheads. Gels were scanned in Odyssey (LI-COR).6.3 μM TrwC(PDF)Click here for additional data file.S6 FigR was incubated with 15 μM of different oligonucleotides. Lane 1, no oligonucleotide; lane 2, H(14+14) P = D = 6 S = 8; lane 3, H(14+17) P = D = 6 S = 11; lane 4, H(23+23) P = D = 15 S = 8, lane 5, H(23+26) P = D = 15 S = 11, lane 6 H(24+24) P = D = 16 S = 8; lane 7, H(24+27) P = D = 16 S = 11. Lane 8, SDS-low range molecular ladder.6.3 μM TrwC(PDF)Click here for additional data file.S7 FigR, TraIR and MobAR. 3D structures of TrwCR and MobAR were determined by x-ray crystallography (PDBs 1OMH and 2NS6) while TraIR was modelled with RaptorX (raptorx.uchicago.edu). HUH relaxases have the catalytic tyrosine within an α-helix (depicted in blue in the ribbon structures) and the motif H+HUH located in two juxtaposed β-sheets (depicted in light pink and wheat respectively). (C) SDS PAGE gels showing the protein purity after HPSP  or Heparin  column chromatography and the protein purity after S75 gel filtration column chromatography (lanes 2). M, standards of the Low Range Protein Ladder (BioRad). Overlay of S75 chomatograms of the gel filtration molecular weight markers Bovine-Serum-Albumin  and Ribonuclease A  (blue); TrwCR (green); TraIR (yellow) and MobAR (orange). All the relaxases elute as monomers with an apparent calculated molecular weight of 30 kDa for TrwCR and TraIR, and 21 kDa for MobAR.(A) Location of the catalytic tyrosine and HUH motif in relaxases and replicases. The alignment of Y and HUH motifs in the model relaxases used in this study is shown. (B) Three dimensional structure of HUH relaxases TrwC(PDF)Click here for additional data file.S8 Fig1 to IR4) are highlighted and their sequences underlined. New IR2s obtained in Rep-like and reverse plasmids are shown by orange arrows. The nic site is indicated with a yellow triangle. Stars show point mutations included in some of the R388 synthetic plasmids to create BglII, BamHI, KpnI and PstI restriction sites.The four inverted repeats (IR(PDF)Click here for additional data file.


Trigonopsisvariabilis DAAO (TvDAAO) variant F54Y for oxidative deamination of CPC. A single amino acid substitution at the 54th position of TvDAAO from phenylalanine (F) to tyrosine (Y) resulted in a 6-fold improvement in catalytic activity towards CPC and in thermostability after heat treatment at 55°C [β-Lactam antibiotics  are used in clinical practice to combat microbial infections. Commercial cephalosporins are all semi-synthetic and chemically derived from 7-aminocephalosporanic acid (7-ACA), a cephem nucleus that is traditionally produced by multi-step chemical reactions from cephalosporin C (CPC). However, the procedures are environmentally damaging, costly, and low yield . The inc at 55°C . The pot at 55°C . To furtEscherichiacoli BL21(DE3)pLysS. E. coli cells were harvested by centrifugation  at 4°C and lysed in ice-cold 20 mM sodium phosphate (pH 7.0) supplemented with 1 mM phenylmethylsulphonyl fluoride by sonication . The cell-free extract was clarified by centrifugation  at 4°C, filtered through a 0.20 μm nylon membrane, and purified by a diethylaminoethyl-cellulose column and a HiLoad 16/60 Superdex 200 prep grade gel filtration column. Purity of the enzyme was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, and its concentration was determined by Bradford assay. The catalytic activity of the purified enzyme was determined by measuring the formation of GL-7-ACA at 22°C with agitation [The W113 (tryptophan) residue of the TvDAAO F54Y variant was converted to tyrosine by the overlapping polymerase chain reaction using mutagenic primers. The mutations in the resultant TvDAAO variant, TvDAAO F54Y/W113Y, were confirmed by automated DNA sequencing. The variant was expressed in The TvDAAO F54Y/W113Y variant was prepared and purified to homogeneity. Catalytic activity was increased by ~10% with respect to the original TvDAAO F54Y variant, but was deactivated by heat treatment.The W113 of TvDAAO plays a pivotal role in thermal deactivation. W113Y substitution may alter the local spatial arrangement of the substrate binding cleft, and hence increase the vulnerability of the nearby oxidative-sensitive C108 , promoti


DNA methylation at imprinting control regions (ICRs) is established in gametes in a sex-specific manner and has to be stably maintained during development and in somatic cells to ensure the correct monoallelic expression of imprinted genes. In addition to DNA methylation, the ICRs are marked by allele-specific histone modifications. Whether these marks are essential for maintenance of genomic imprinting is largely unclear. Here, we show that the histone H3 lysine 9 methylases G9a and GLP are required for stable maintenance of imprinted DNA methylation in embryonic stem cells; however, their catalytic activity and the G9a/GLP-dependent H3K9me2 mark are completely dispensable for imprinting maintenance despite the genome-wide loss of non-imprinted DNA methylation in H3K9me2-depleted cells. We provide additional evidence that the G9a/GLP complex protects imprinted DNA methylation by recruitment of de novo DNA methyltransferases, which antagonize TET dioxygenass-dependent erosion of DNA methylation at ICRs.  •ESCs lacking G9a and GLP display loss of DNA methylation from ICRs•The enzymatic activity of G9a/GLP is dispensable for imprinted DNA methylation•G9a/GLP stabilize imprinting by recruitment of de novo DNA methyltransferases to ICRs•Recruitment of DNMTs to ICRs antagonizes TET-dependent loss of DNA methylation Loss of allele-specific DNA methylation from imprinting control regions leads to unbalanced gene expression and disease. Here, Zhang et al. show that the KMT enzymes G9a and GLP stabilize imprinted DNA methylation in embryonic stem cells by recruitment of de novo DNA methyltransferase enzymes, which counteract TET dioxygenase-dependent demethylation pathways. The matG9a or Glp genes in mice results in widespread loss of H3K9me2, growth retardation, and lethality of homozygous null embryos at E9.5–E10 (Snrpn ICR was reported for G9a−/− ESCs (G9a−/− ESCs (Snrpn has not been determined.G9a and the G9a-like protein GLP (EHMT1) form a G9a/GLP heterodimer in ESCs and function cooperatively to establish and maintain the abundant repressive H3K9me2 modification, in addition to modifying several non-histone proteins . The G9aE9.5–E10 . ImportaE9.5–E10 . Both G9E9.5–E10 , and los−/− ESCs . Interes−/− ESCs  indicatiHere, we report that pluripotent stem cells null for either G9a or GLP display a widespread loss of imprinted DNA methylation, which can be reproduced by a small hairpin RNA (shRNA) knockdown of G9a in wild-type ESCs. Although the G9a-dependent H3K9me2 preferentially marks chromatin at methylated ICRs, we demonstrate that H3K9me2 is not essential for stable maintenance of imprinted DNA methylation in ESCs. Furthermore, we show that the G9a/GLP complex maintains imprinting by recruitment of de novo DNA methyltransferases, which antagonize the TET-enzyme-dependent DNA demethylation pathways.G9a−/− ESCs were reported to display global and locus-specific DNA hypomethylation , while DNA methylation at control promoters, such as Ankrd50, was only mildly affected  in two wild-type ESC lines with different genetic background (TT2 and early passage E14) and generated clonal cell lines derived from single cells  with anti-H3K9me2 antibodies from wild-type ESCs followed by bisulfite DNA sequencing revealed that, unlike in the input DNA, predominantly the methylated ICRs were present in the H3K9me2 antibody-precipitated chromatin A and 3B.The experiments described above demonstrate that the G9a/GLP complex, but not its enzymatic activity, is required for maintenance of DNA methylation at ICRs. G9a was reported to interact directly, via its ankyrin repeat domain (ANK), with DNMT3A and DNMT3B and also indirectly, via GLP, with DNMT3A . The N tTo test this hypothesis, we generated ESC lines stably expressing shRNA-resistant either wild-type G9a (shR-WT) or mutant forms of G9a, which were either unable to bind H3K9me1/me2 (shR-ANKm) or lacked the entire ANK domain (shR-ANKΔ), respectively A. We theTwo TET family enzymes, TET1 and TET2, are highly expressed in ESCs  and may Tet1/Tet2 double-knockout (DKO) ESCs  ESCs   ESCs A and exaype ESCs . Howeverype ESCs , DNA mettr cells C and 5D.terparts A. TogethG9a−/− ESCs, and these cells are also characterized by significantly higher levels of 5hmC  suggesting that G9a/GLP and ZFP57/KAP1 complexes may act cooperatively and reinforce each other’s function as neither complex on its own is sufficient to fully maintain the imprinted DNA methylation.Notably, DNA methylation at specific loci, including promoters , satelliOur dissection of the H3K9me2-independent function of G9a in maintenance of imprinted DNA methylation led us to conclude that the ANK region, which was previously shown to interact with DNMT3A and DNMT3B , as wellWhat attracts G9a/GLP complex to methylated ICR and sequences elsewhere in the genome is currently unknown and requires further investigation. Recruitment of G9a by non-coding RNA has been reported and might be important for the establishment and maintenance of DNA methylation and heterochromatin . Given tGiven that H3K9me2 is not essential for maintenance of imprinted DNA methylation in ESCs, it is likely that multiple mechanisms operate simultaneously to ensure that the G9a-dependent DNA and histone methylation at ICRs are preserved. It will be important to investigate further the role of G9a/GLP-interacting C2H2 zinc finger proteins and the largely overlooked role of H3K9me1 in the nucleation, spreading, and propagation of G9a-dependent DNA methylation and heterochromatin maintenance. Moreover, it will be essential to determine whether the maternally contributed and the zygote-expressed G9a and GLP stabilize and protect the imprinted DNA methylation in developing embryos.Embryonic stem cells were grown in minimal essential medium (Life Technologies) supplemented with 10% fetal bovine serum (Thermo Scientific), non-essential amino acids, sodium pyruvate, β-mercaptoethanol, and leukemia inhibitory factor on gelatine-coated flasks (Greiner). Where indicated, the cells were grown in the presence of 500 nM of G9a/GLP inhibitor UNC 0638 (Sigma-Aldrich).Knockdown of G9a in ESCs and expression of mutant forms were performed by stable integration of electroporated plasmids. Detailed procedures are described in the E-MEXP-2872.MAP of methylated DNA, hybridization to NimbleGen promoter microarrays, and data analyses were described previously . The datMethylated DNA immunoprecipitation (MeDIP) was performed essentially as described  with antGenomic DNA was treated with EpiTect Bisulfite conversion kit (QIAGEN) and then used as a template for PCRs with specific primers listed in the Chromatin immunoprecipitations (ChIPs) were performed as described previously  with antNuclear proteins were extracted as in Antibodies against G9a, GLP (R&D Systems Europe) and non-specific mouse IgG (Sigma-Aldrich) were used for immunoprecipitations from 400–500 μg of nuclear extract according to standard protocols. The amount of extract was doubled when dimerization-deficient forms of G9a were immunoprecipitated. The immunoprecipitated complexes and 1/10 of the input were resolved on 7% SDS-PAGE gels, transferred to nitrocellulose membranes, and detected as described above.Statistical methods used to analyze the promoter microarrays data A were deI.S. and T.Z. designed the project. T.Z. performed most experiments. A.T. carried out MBD affinity purifications, microarray analyses, and validation. X.X.B. helped with bisulfite DNA sequencing. J.C. and B.R. performed 5mC quantification. B.O. purified G9a-associated proteins. F.d.L.A. carried out mass spectrometry analyses. J.R. provided assistance with mass spectrometry. I.S., B.R., and T.Z. wrote the manuscript.


The increased number of outbreaks of H5 and H7 LPAI and HPAI viruses in poultry has major public and animal health implications. The continuous rapid evolution of these subtypes and the emergence of new variants influence the ability to undertake effective surveillance. Retroviral pseudotypes bearing influenza haemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins represent a flexible platform for sensitive, readily standardized influenza serological assays. We describe a multiplex assay for the study of neutralizing antibodies that are directed against both influenza H5 and H7 HA. This assay permits the measurement of neutralizing antibody responses against two antigenically distinct HAs in the same serum/plasma sample thus increasing the amount and quality of serological data that can be acquired from valuable sera. Sera obtained from chickens vaccinated with a monovalent H5N2 vaccine, chickens vaccinated with a bivalent H7N1/H5N9 vaccine, or turkeys naturally infected with an H7N3 virus were evaluated in this assay and the results correlated strongly with data obtained by HI assay. We show that pseudotypes are highly stable under basic cold-chain storage conditions and following multiple rounds of freeze-thaw. We propose that this robust assay may have practical utility for in-field serosurveillance and vaccine studies in resource-limited regions worldwide. The increased number of outbreaks of H5 and H7 low pathogenicity avian influenza (LPAI) and high pathogenicity avian influenza (HPAI) viruses in poultry has major public and animal health implications and significant economic impact. The evaluation of evidence of influenza infection or vaccination efficiency in poultry species is assessed via the measurement of immunological responses against avian influenza viruses and serological assays representing an important tool for serosurveillance studies particularly in new outbreak locations. Although vaccination, combined with improved biosecurity has successfully prevented significant mortalities and production loss, on-going evolution of the virus requires the development of surveillance systems to ensure that vaccination continues to be effective .Influenza A viruses infecting poultry are divided into two groups (HPAI and LPAI) on the basis of their ability to cause disease and with different human and animal health implications. Evidence , 3 suppoAll avian sera were provided by the FAO, OIE, and National Reference Laboratory for Newcastle Disease and Avian influenza  and consisted of ten sera H5 positive collected from chickens vaccinated with the inactivated H5N2  vaccine (no. 1–10), ten sera H7 positive no. 11–20) collected from turkeys during an Italian outbreak caused by an LPAI H7N3 virus , ten sera positive for both H7 and H5 collected from chickens vaccinated with an inactivated bivalent vaccine produced with the LPAI H7N1 (A/chicken/1067/1999), and H5N9  strains. Forty negative sera were included in the study and were obtained from chickens tested AI antibody-free by enzyme-linked immunosorbent assay (ELISA) and agarose gel immunodiffusion (AGID) assay using standard protocols described previously  collecte. AdditioAll avian sera employed in the study were tested by HI at FAO, OIE, and National Reference Laboratory for Newcastle Disease and Avian influenza, Istituto Zooprofilattico Sperimentale delle Venezie with different reference antigens routinely used for avian influenza surveillance in Italy, namely, H5N2 , H7N3 , H7N1 (A/Africa starling/England/983/1979), H5N9 , and H7N1 . For the HI tests, standard protocols were used as described previously [Lentiviral vector  pseudotyped with HA envelope glycoproteins derived from the HPAI H5N1 viruses (clade 1 A/Vietnam/1194/2004 and clade 2.1.3.2 A/Indonesia/5/2005) and the HPAI H7N1 virus  were produced as described previously , 24, excμL) were twofold serially diluted in culture medium (DMEM GlutaMAX supplemented with 15% FBS and 1% Penicillin/Streptomycin) and mixed with pseudotype virus  at a 1 : 1 v/v ratio. After incubation at 37°C for 1 hour, 1 × 104 HEK 293T/17 cells were added to each well of a white 96-well flat-bottomed tissue culture plate. 48 hours later, pseudotype transduction titres obtained at each of a range of dilution points were expressed as RLU/mL, and an arithmetic mean was calculated. For each serum sample, RLUs were normalized and compared with the signal detected in the absence of pseudotype virus  and the signal of the negative control . The 50% inhibitory doses (IC50) were determined as the reciprocal of serum dilution resulting in a 50% reduction of a single round of infection .Serum samples (5 μL) were dispensed together with cell culture medium (DMEM GlutaMAX supplemented with 15% FBS and 1% Penicillin/Streptomycin). After 48 hours, the neutralizing antibody responses against each subtype were detected by using the Dual-Glo reagent (Promega) which differentiates between the two reporter genes as detailed in the manufacturer's instructions, so that neutralizing antibody titre for each influenza pseudotype could be recorded for each serum sample concurrently.To allow detection of neutralizing antibody responses against two different influenza viruses (H5 and H7) in the same well of a 96-well flat-bottomed tissue culture plate, fixed amounts  of both influenza pseudotypes (one containing the firefly reporter gene and the other the Renilla reporter gene) were added to each well in which twofold serially diluted serum samples  was transformed to logarithmic scale. Subsequently, the geometric mean of duplicate observations was calculated. Statistical analyses for all the data and correlation coefficients  were performed using GraphPad Prism. The radial tree and HA amino acid identity grid were constructed with MATLAB (MathWorks).Data analyses were undertaken using Excel and GraphPad Prism (Version 6). Antibody titres observed for H5 (A/Vietnam/1194/2004 and A/Indonesia/5/2005) and H7  influenza pseudotypes when used in the monoplex and multiplex assays were expressed as geometric mean titer (GMT). Firstly, the ICWe have constructed H5N1 and H7N1 pseudotypes  encoding the firefly luciferase reporter gene, and additionally, for use in a multiplex assay, an H7N1 pseudotype  encoding a Renilla luciferase reporter. The phylogenetic relationship between these pseudotype serological antigens (and the other antigens utilized in this study) can be visualized on a radial tree in 5 IFU/mL and >80% infectivity remained after five cycles of freeze-thaw  or simply may have no access to a −80°C freezer. We therefore undertook a series of A/Viet Nam/1194/2004 HA pseudotype virus stability investigations by storage of this virus at the higher temperatures of −20°C (standard freezer), +4°C (standard fridge), and room temperature and by subjecting the pseudotype virus to multiple freeze-thaw cycles. The initial titre of the H5 lacZ pseudotype was 4.3 × 10eze-thaw . In paraeze-thaw . In relaThree panels of sera  were initially tested using a monoplex pseudotype-based format. An initial pilot study was carried out where H5N1 hyperimmune (SH454) and H7N1 (02/294) sheep sera were tested for the ability to neutralize influenza pseudotypes bearing the HAs from H5N1 A/Vietnam/1194/2004 and H7N1 A/chicken/Italy/13474/1999. The H5 influenza pseudotypes were neutralized by the SH454 sera (100% inhibition of pseudotype entry at 1 : 1280 serum dilution) but not by the 02/294 sera whilst the H7 pseudotypes were neutralized by 02/294 (with a 100% inhibition at dilution 1 : 1280) but not by SH454. This lack of cross-neutralizing antibody response between H5 and H7 subtypes was consistent with the different clustering, within Group 1 and Group 2, of HA-subtypes based on phylogenetic relationship analysis of influenza subtypes. According to this analysis, H5 HA belongs to Group 1 “cluster 1”  and H7 HA belongs to Group 2 “cluster 7” (together with H10 and H15) . Subsequr = 0.87, P < 0.0001) and clade 2.1.3.2 A/Indonesia/5/2005  despite the fact the HAs used in the two serological assays were not optimally matched. The percentage amino acid identities between the pseudotype antigens, the HI antigen, and the vaccine antigen are shown in As shown in P value <0.0001 and r = 0.93 when analyzed using Student's t-Test (paired data set)  .Next, a panel of ten sera obtained from turkeys naturally infected with an H7N3 strain with titres ranging from low (1 : 32) to high (1 : 128) as tested by HI  were subsequently tested using H7 pseudotypes A/chicken/Italy/13474/1999 and H5 A/Vietnam/1194/2004. All ten sera (no. 11 to no. 20) were found positive when tested against H7 pseudotypes with GMT range from 1 : 28 to 1 > 2560 while only one serum sample was positive (GMT of 1 : 28) against H5 A/Vietnam/1194/2004 .P < 0.001) between antibody titers obtained with both assays. The correlation coefficient between pp-NT and HI for the panel of H7 positive was 0.72  and the Pearson's correlation test. The results of this analysis revealed a highly statistically significant correlation  produced with the avian influenza vaccine strains H7N1  and H5N9 , monoplex (as described before) and multiplex (by using firefly/Renilla) pp-NT assays were undertaken against H5 A/Vietnam/1194/2004 and H7 A/chicken/Italy/13474/1999 pseudotypes. As shown in P < 0.0001; Pearson's correlation; Subsequently, neutralizing antibody titres, obtained when standard and dual H5/H7 assays were undertaken . Similar results were observed for antibody responses against H7 A/chicken/13474/1999 tested in monoplex and multiplex assays   mirrored those obtained when the same panel of sera were tested against H5 A/Vietnam/1194/2004 (firefly gene) mixed with H7 A/chicken/13474/1999 carrying the Renilla gene ( 0.0002) . The mag 0.0002) .Since 1997, H5 and H7 outbreaks in domestic poultry have been increasing in frequency and it is likely to be due to a complex set of factors such as improved diagnostic tools, climate fluctuations, and changes in trade flows of poultry products . One log gag-pol core structural proteins, HA and NA expression plasmids, and retroviral vectors incorporating the reporter gene. Firefly and Renilla luciferases were employed in this study, but potentially a wide range of reporter genes can be used in these assays (green fluorescence protein (GFP), red (RFP)/yellow (YFP), secreted embryonic alkaline phosphatase (SEAP), and lac-Z) [ lac-Z (pCSLZW) [Recent studies have provided the impetus that the future of avian serology, rather than moving towards a single assay approach, is the implementation of a strategy that involves conventional and novel technologies to be used in conjunction with validated and standard tests. Comparative serology aims to achieve a more holistic view of the serological response and newer assays like the pseudotype-based neutralization assay presented in this study are key , 23. We d lac-Z) , 34, 35.(pCSLZW) , 24. Of (pCSLZW) . Our resP = 0.001) and H7 (P = 0.0002) influenza strains with results from the monoplex mirroring those obtained with the multiplex assay  pseudotypes per 96-well plate is required. It is possible with the multiplex pp-NT assay to measure neutralizing antibody responses against large panels of H5/H7 influenza viruses and drift variants faster and more accurately than laborious wild-type virus microneutralization, thus providing comprehensive data on antigenic evolution of avian influenza viruses.Results collected from pp-NT assays were statistically significant when performed in monoplex and multiplex from both H5 (ex assay . This syMoreover, the major limitations to the use of HI assay are that it is not practical for general influenza A screening with significant level of intralaboratory variability as demonstrated in human serology . It requ