We identify the regulatory kinase and a novel ubiquitin (Ub) modification on CIITA, Lys + T-cells. In the present study, we identified extracellular signal-regulated kinase (ERK)1/2 as the kinase responsible for phosphorylating the regulatory site, Ser280, which leads to increased levels of mono-ubiquitination and an overall increase in MHC II activity. Further, we identify that CIITA is also modified by Lys63-linked ubiquitination. Lys63 ubiquitinated CIITA is concentrated in the cytoplasm and following activation of ERK1/2, CIITA phosphorylation occurs and Lys=ubiquitinated CIITA translocates to the nucleus. CIITA ubiquitination and phosphorylation perfectly demonstrates how CIITA location and activity is regulated through PTM cross-talk. Identifying CIITA PTMs and understanding how they mediate CIITA regulation is necessary due to the critical role CIITA has in the initiation of the adaptive immune response.The class II transactivator (CIITA) is known as the master regulator for the major histocompatibility class II (MHC II) molecules. CIITA is dynamically regulated through a series of intricate post-translational modifications (PTMs). CIITA's role is to initiate transcription of MHC II genes, which are responsible for presenting extracellular antigen to CD4 Following antibody incubation, cells were washed twice with PBS, fixed with 2% paraformaldehyde and were stored at 4°C. MHC II cell surface expression was measured by LSR Fortessa and analysed using FlowJo. All samples were analysed using 10000 events per sample. One-tenth of the cell volume harvested for flow analysis was lysed in 1% NP40 buffer containing complete EDTA-free protease inhibitors (Roche) on ice. Proteins were denatured and separated by SDS/PAGE gel electrophoresis. Gels were transferred and individually immunoblotted with anti-Myc antibody (Abcam) or anti-Flag antibody (Sigma). HRP conjugates were detected with HyGlo (Denville) to determine expression of CIITA, S280A CIITA or ERK1/2 as indicated. Proteins were normalized using the Nano Photometer P-class (Implen) to determine equal loading in lysates.COS cells were plated at a cells density of 8×105 in 10 cm tissue culture plates. Following adhesion, cells were transfected as indicated. Twenty-four hours following transfections, cells were treated with 100 μM cycloheximide for 0–8 h. Following cycloheximide treatment, cells were harvested and lysed as described above. Lysates were normalized for protein concentration. As controls, COS cells were transfected as indicated and treated with 100 μM of cycloheximide and MG132 (EMD Biosciences) for 8 h. Proteins were denatured and separated by SDS/PAGE gel electrophoresis. Blots were individually immunoblotted. Proteins were normalized using the Nano Photometer P-class (Implen) and equal loading was determined.COS cells were plated at a cell density of 8×105 in 10 cm tissue culture plates. Following adhesion, cells were transfected with Myc–CIITA, Flag–ERK1, Flag–ERK2, Myc control (empty plasmid), HA–Mono Ub, HA–Poly Ub, HA–Lys63 Ub and HA–Lys48 Ub as indicated . Following treatments, cell fractionation was completed with ProteoExtract Subcellular Proteome Extraction Kit , following manufacturer's protocol. Following fractionation, proteins were normalized and immunoprecipitated with EZ view Red anti-Myc agarose beads (Sigma–Aldrich). Immune complexes were denatured and separated as described above. Blots were individually immunoblotted with anti-HA antibody (Santa Cruz Biotechnology). Lysate and fractionation controls were immunoblotted with anti-Myc (Abcam), anti-HSP 90, anti-H3 , anti-p44/42 MAPK  and anti-ERK1/2 rabbit polycolonal antibody (Proteintech).COS cells were plated at a cell density of 8×104 in six-well plates with glass coverslips. Eighteen hours following adhesion, cells were transfected with Myc–CIITA and HA–Lys63 Ub. Twenty-four hours following transfections, 10% FBS media was removed and washed with PBS and 1% FBS media was replaced were indicated for 6 h. Thirty minutes prior to fixation, 200 nM of PMA was added to 1% serum media to activate ERK1/2. All media was then removed and washed with PBS. To fix the cells on coverslips, ice-cold methanol was added and incubated at −20°C for 10 min. Methanol was aspirated and 500 μl of 2% BSA–PBS was added. To stain the coverslips, anti-CIITA (Santa Cruz Biotechnology), anti-Lys63 Ub (Abcam), anti-p44/42 MAPK (Thermo Scientific) or anti-ERK1/2 (Proteintech) was added to the indicated samples for 1 h. Following staining, antibody was removed and coverslips were washed 5× with PBS. Goat anti-rabbit Alexa 594 and goat anti-mouse Alexa 488 (Invitrogen) were added for 1 h in the dark. Following secondary antibody incubation, antibody was removed and samples were washed 5× with PBS. DAPI  was added to coverslips and incubated for 10 min in the dark. DAPI was removed and coverslips were washed 5× with PBS. Coverslips were mounted with ProLong Gold Antifade (Life Technologies). Samples were allowed to dry overnight in 4°C. Images were obtained using the LSM 700 Confocal Microscope (Zeiss) using 40× magnification.COS cells were plated at a density of 5×10280 that lies within the degron sequence [280 leads to subsequent mono-ubiquitination on three degron proximal lysine residues, leading to increased CIITA transactivity [357) that lies within a degron sequence phosphorylated by ERK1/2 [280, located within the degron sequence, was similarly targeted for phosphorylation by ERK1/2. First, to determine association of CIITA and the kinase complex; a co-IP assay was performed. Expression of wild-type (WT)-CIITA with ERK1/2 in COS-7 cells, followed by co-IP, demonstrates CIITA association with the individual components of the kinase complex, ERK1, ERK2 or with the ERK1/2 complex  [280 phosphorylation is required in order for additional sites to be phosphorylated. When all ERK1/2 phosphorylation is blocked, it is likely S280A CIITA is a non-viable and unstable protein.The phosphorylated form of CIITA migrates as a doublet at 145 and 149 kDa ,19. The 280 controls subsequent mono-ubiquitination that is required for increased CIITA transactivity and for increased MHC II transcription [280 is mutated from serine to alanine, CIITA's activity decreases, leading to an overall decrease in the levels of MHC II expression [280 by the kinase complex, ERK1/2. To determine if the association between CIITA and ERK1/2 drives CIITA transactivation and to determine any dependence of this interaction on CIITA Ser280, we performed luciferase reporter assays. WT-CIITA co-transfected with ERK1/2 leads to a 2-fold increase in CIITA transactivity and ability to drive MHC II transcription; conversely, the CIITA S280A mutant has a decreased ability to drive MHC II transcription. When the CIITA S280A mutant was co-transfected with ERK1/2, CIITA transactivity levels further decrease  ubiquitination, we conducted a co-IP experiment. In the absence of expressed ERK1/2, CIITA exhibits very low levels of global ubiquitination , CIITA was highly modified by Lys63 Ub in the cytoplasmic fraction , CIITA and Lys63 Ub co-localize to the nucleus  in which an additional Ub molecule is attached to the already existing Ub on the target protein [48-linked ubiquitination based on the lack of protein degradation of CIITA (Ubiquitination is a diverse modification that yields multiple types of poly-Ub conjugates and mono-ubiquitination. Poly-ubiquitination is determined by the Ub lysine residue added to a target protein (Lys protein . Our dat48-linked ubiquitination tags proteins for degradation via the 26S proteasome [63 ubiquitination of TNFR (tumour necrosis factor receptor)-associated factor 6 (TRAF6) links interleukin-1 receptor (IL-1R) signalling to the activation of NF-κB [63 ubiquitination allows additional proteins, including TAB2 (TGF-beta activated kinase 1/MAP3K7 binding protein 2), to bind directly to the Lys63 Ub chain resulting in additional downstream effects including phosphorylation and nuclear translocation [63 ubiquitination, resulting in multiple protein–protein interactions and downstream phosphorylation events in the NF-κB pathway [63 ubiquitination brings two-protein complexes together to act as a scaffold. Lys63 ubiquitination has also been implicated in endocytosis, protein trafficking and DNA damage repair [63 ubiquitination is the first of any isoform of CIITA and, in the present study, we show CIITA Lys63 ubiquitination followed by phosphorylation by ERK1/2, which leads to cellular localization changes  [To gain insight into the role of Lysy ERK1/2 P and 7T. B (Akt) .280, leading to a significant increase in CIITA mono-ubiquitination resulting in increased CIITA activity and MHC II expression and activity. Additionally, we are the first one to show a novel ubiquitination linkage on CIITA, Lys63. Further, we demonstrate a novel cross-talk between phosphorylation by ERK1/2 and Lys63 ubiquitination of CIITA IF3 and the combined role of these PTMs have in facilitating movement of CIITA from the cytoplasm to the nucleus and overall increase in activity (Because CIITA is dynamically regulated by multiple PTM's and the intertwined nature of these modifications has yet to be fully defined, it is important to decipher these modifications to gain a better understanding of the regulation of this critical adaptive immune protein. Phosphorylation of CIITA occurs on various residues ,16,28,29activity . Our fin


Using a pull-down approach, we observe that ULK2 interacts physically with Kapβ2 both in vitro and in vivo. Confocal microscopy confirmed the co-localization of ULK2 and Kapβ2. Localization of ULK2 to the nuclear region was disrupted by mutations in the putative Kapβ2-binding motif (P794A). Furthermore, in transient transfection assays, the presence of the Kapβ2 binding site mutant  was associated with a substantial increase in autophagy activity (but a decrease in the in vitro serine-phosphorylation) compared with the wild type ULK2. Mutational analysis showed that the phosphorylation on the Ser1027 residue of ULK2 by Protein Kinase A (PKA) is the regulatory point for its functional dissociation from Atg13 and FIP 200, nuclear localization, and autophagy. Taken together, our observations indicate that Kapβ2 interacts with ULK2 through ULK2’s putative PY-NLS motif, and facilitates transport from the cytoplasm to the nucleus, depending on its Ser1027 residue phosphorylation by PKA, thereby reducing autophagic activity.Uncoordinated 51-like kinase 2 (ULK2), a member of the serine/threonine kinase family, plays an essential role in the regulation of autophagy in mammalian cells. Given the role of autophagy in normal cellular homeostasis and in multiple diseases, improved mechanistic insight into this process may result in the development of novel therapeutic approaches. Here, we present evidence that ULK2 associates with karyopherin beta 2 (Kapβ2) for its transportation into the nucleus. We identify a potential PY-NLS motif ( Uncoordinated 51-like kinase 2 (ULK2) is a member of the serine/threonine kinase protein family, which plays an essential role in the regulation of autophagy in mammalian cells , since UThe central role of autophagy in normal cellular homeostasis and multiple diseases suggests that mechanistic insights into autophagy could drive the development of novel therapeutic approaches –8. Few eAlthough the mechanisms that regulate ULK2 in the autophagic process are not yet fully understood, precise control appears to be achieved through a combination of phosphorylation, localization, and interactions with ULK2 binding proteins . Unlike X2–5PY motif that Kapβ2 recognizes . The pre-cleaned lysate was incubated for 1 hour at 4°C with the appropriate antibody, and the resulting immune complexes were collected using Protein A-Sepharose beads . Immune complexes were then captured by centrifugation, washed extensively in lysis buffer, and solubilized with 2x sample buffer prior to loading onto a 10% SDS-PAGE gel.Whole cell lysates of HEK293 cells transiently expressing ULK2 were pre-cleaned with glutathione agarose beads, and 1μg of each glutathione agarose-tagged recombinant ULK2 or Kapβ2 preparations was subsequently added to separate samples followed by 2 hour incubation at 4°C with end-over-end rotation to allow for association between ULK2 and Kapβ2. Associated protein complexes were collected using the slurry of glutathione agarose beads, and washed extensively. After resuspension in 2x Laemmli sample buffer, samples were analyzed on a 10% SDS-PAGE gel, and western blotting was performed using Kapβ2 or ULK2 antibodies.Pulled-down or immunoprecipitated ULK2 (or Kapβ2) was resolved on 10% SDS-PAGE gels and transferred to a nitrocellulose membrane. Membranes were incubated in blocking buffer (5% dried skimmed milk in PBS and 0.05% Tween-20), and subsequently probed with specific antibodies, followed by a horseradish peroxidase-conjugated secondary antibody. Immune complexes were detected using a commercial western blotting detection system .HEK293 cells were seeded overnight at 60% confluence onto culture slides coated with human fibronectin . The following day, cells were transfected with the ULK2/EGFP construct, and allowed to grow for an additional 48 hours. Cells were washed several times with ice-cold PBS and fixed in 2% paraformaldehyde for 10 minutes. Fixed cells were permeabilized with 0.1% Triton X-100 for 10 minutes and blocked for 2 hours in PBS containing 5% BSA  and 0.1% Tween. Following incubation with a polyclonal (rabbit) or monoclonal (mouse) antibody against Kapβ2(1:100), ULK2 (1:100), LC3 II (1:100) or WIPI (1:100)  overnight at 4°C, the slides were washed three times with 0.01% PBS-Tween. Alexa Fluor 568 or 488-conjugated donkey anti-rabbit (1:200) or anti-mouse (1:200)  was used as a secondary antibody. Confocal microscopy analysis was performed using an LSM710  at the Center for Research Instruments and Experimental Facilities of Chungbuk National University. Using Profile in the ZEN program which was provided by the manufacturer, the co-localizations of proteins were observed and confocal microscopic images scanned. The nuclear or cytoplasmic fluorescence intensity profile of ULK1/2 was evaluated from the fluorescence images, and the nuclear-to-total cell fluorescence (Fn/t) ratio was obtained using the ZEN program ,17,18. PCells were transfected with ULK2 (WT), PY mutant (P242A or P794A), or EGFP vector, and the rate of apoptosis was measured using the Annexin V-PE apoptosis detection kit , according to the manufacturer’s instructions. Cells were vortexed gently, incubated for 15 minutes at 25°C in the dark, and 400μl binding buffer added to each tube. Within 1 hour, fluorescence-activated cell sorting (FACS) was performed using a FACS Calibur (BD Sciences) at the Core Facility of Chungbuk National University.To measure autophagy, we used the LC3 western blotting method (Bio-Protocol.org) following the manufacturer’s guide, in the same manner as described in the “Immunoblotting” section above using an antibody against LC3. Each assay was performed 5 times.Numerical results were described as means ±SD from at least four independent experiments performed in triplicate. Differences in the measured variables between control and experimental groups were analyzed using a Student’s t-test. A statistically significant difference was accepted at * P < 0.05 or ** P < 0.01.(2–5)PY) , and it is possible for it to be phosphorylated by PKA  or inhibitor (H89)  27]. Co. Co27]. PCC between ULK2 and Kapβ2 was measured as shown in Therefore, we assumed that because the domain for protein-protein interaction with Atg13 or FIP200 overlaps with the ULK2 Ser1027 residue, which can be phosphorylated by PKA , the subEven though unknown proteins interacts with the C-terminus of ULK2, the phosphorylation of the Ser1027 residue  by protein kinases (such as PKA) promoters the dissociation of Atg13 and FIP200 from ULK2, and the PY-NLS of ULK2 is exposed to Kapβ2 in order to facilitate transport to the nucleus , otherwise the protein complex stays in the cytoplasm as an active form . The proWe measured cell viability using FACS analysis to determine whether the subcellular localization of ULK2 influences cell viability . As showCells were transfected with EGFP-ULK2 WT, its PY mutants  or the EGFP vector, and the rate of apoptosis was measured by FACS. The EGFP-ULK2 PY-NLS (P794A) or S1027A mutants, which are localized in the cytoplasm, promote cell apoptosis significantly more than does ULK2-WT, P242A, or S1027D, which show nuclear localization. The apoptotic effect of the ULK2 P794A and S1027A mutants was roughly half that of the ULK2 WT, P242A, or S1027D. For details, see the 774gpGfgssppgaeaapslRyvPY795) in the S/P space domain resulting in nuclear localization  with Kapβ2, which resulted in nuclear localization of ULK2, and the interaction between the two proteins was regulated by PKA phosphorylation on the Ser1024 residue of ULK2 (774gpGfgssppgaeaapslRyvPY795) in ULK2.The role of ULK2 (together with ULK1) in signal transduction has been most clearly characterized in the context of autophagic kinase signaling, in which it functions as a hub protein kinase . In the  of ULK2 . Using c of ULK2 . In addi774gpGfgssppgaeaapslRyvPY795 changed to 774gpGfgssppgaeaapslRyvAY795) affects kinase activity, regardless of protein-protein interactions with Kapβ2. It is interesting to note that one of the Kapβ2 binding motifs (223LrmfyeKnRslmpsipRetsPY243) present in ULK2 near its kinase domain (466padtaqtvgrRlstgssrPY475) that does not match the consensus PY-NLS motif (R/H/KX(2–5)PY) [Most Kapβ2s directly bind the PY-NLS of their cargo ,14–16. Ae domain  is foundIn comparison with the subcellular localization of ULK1 (which lacks a PY-NLS motif) in other studies –5, ULK2 Due to the fact that the phosphorylation of Ser1027 in ULK2 by protein kinases including PKA (S1027D mutant) inhibited the autophagic activity of ULK2, and decreased sensitivity to starvation and stress by promoting nuclear localization of ULK2 Figs  and 7, iEven though the ULK2 S1027A/D mutants showed a dramatic subcellular difference compared with WT, the subcellular localization of the ULK2 S468 A/D mutants did not change (unpublished data). The explanation for this event is the protein-protein interaction of Atg13 and FIP200 with the C-terminus of ULK2 . DespiteThe ULK2 S1027A/D mutant proteins play a dominant negative role in cell survival. While the ULK2 S1027A mutant forms more protein complexes with Atg13 and FIP200, less endogenous ULK1/2 binds with Atg13 and FIP200 , leadingRecently, ULK1 and ULK2 were reported to have distinct functions in the regulation of lipid metabolism in adipocytes . Nuclear995aAllleglsRilqdPa1010), where the binding site of Atg13-FIP200 overlaps near to the PKA phosphorylation site (S1027 residue). Therefore it seems to be the case that ULK2 phosphorylation by PKA affects both the nuclear localization and the autophagic activity of ULK2. However, even though the nuclear import of ULK2 by the interaction between its PY-NLS motif and Kapβ2 seems to be regulated by PKA phosphorylation on its S1027 residue, its mechanism of export to the nucleus is unknown. The possible nuclear export sequence (NES) motifs containing the leucine rich segments are also noticed in ULK2 with a NES consensus motif [It is also noteworthy that ULK2 contains another putative Kapβ2 interaction site (us motif . We are 774gpGfgssppgaeaapslRyvPY795) in its S/P space domain (220qdlrmfyeKnRslmpSipRetsPY243). Unlike ULK1, which is localized predominantly in the cytoplasm . S3. Data forFig 6A, 6B and 6C. Subcellular localization and autophagic ability of ULK2 Ser1027 mutants. The nuclear-to-total fluorescence ratio (Fn/t). S4. Data forFig 6N, 6O and 6P. The endogenous ULK2 in HEK293 cells was visualized with a PKA inhibitor (O) and activator (P). Control (N). The nuclear or cytoplasmic fluorescence intensity profile. S5. Data forFig 6N, 6O and 6P. The endogenous ULK2 in HEK293 cells was visualized with a PKA inhibitor (O) and activator (P). Control (N). The nuclear-to-total fluorescence ratio (Fn/t).(XLSX)Click here for additional data file.


The increasing catalogue of high-quality ice-penetrating radar data provides a unique insight in the internal layering architecture of the Greenland ice sheet. The stratigraphy, an indicator of past deformation, highlights irregularities in ice flow and reveals large perturbations without obvious links to bedrock shape. In this work, to establish a new conceptual model for the formation process, we analysed the radar data at the onset of the Petermann Glacier, North Greenland, and created a three-dimensional model of several distinct stratigraphic layers. We demonstrate that the dominant structures are cylindrical folds sub-parallel to the ice flow. By numerical modelling, we show that these folds can be formed by lateral compression of mechanically anisotropic ice, while a general viscosity contrast between layers would not lead to folding for the same boundary conditions. We conclude that the folds primarily form by converging flow as the mechanically anisotropic ice is channelled towards the glacier. A range of mechanisms has been proposed for large-scale folding in polar ice sheets. Here, using new three-dimensional reconstructions of such folds in the onset region of the Greenland Petermann Glacier, the authors show that these formed due to flow convergence and the high mechanical anisotropy of ice. The polar ice sheets of Greenland and Antarctica consist of layer on layer of buried snow, compacted to ice. The ice sheets flow towards their margins under their own weight. Hence, their shape resembles that of a flat dome, with young ice at the top and old ice at the base that can be accessed by ice-core drilling3126Under the framework of Operation IceBridge (NASA), dense radar grids (ca. 8-km spacing) have been flown for selected areas67116717Here we show that the processes leading to our observed structures are most likely initiated due to the anisotropic nature of ice. To account for the anisotropy, several fully anisotropic flow models have been developed and numerically implemented141516171819141618A distinct horizon (A) could be traced over the whole study area . This ho24Folding style changes and fold intensity increases with depth. In deeper layers, we identify overturned folds as well as sheath folds. These were traced in a selected area . Figure 6Surface velocity data derived from the MEaSUREs data set24268During very strong shearing, all linear structural elements passively rotate towards the shear direction, resulting in fold axes that are parallel to the flow direction and tubular sheath folds282930et al.Slippery patches can develop where the ice sheet is intermittently or not at all frozen to the bedrock, and localize where the pressure melting point is reached or a deformable sediment layer lies between ice and hard bedrockλ) of such folds is predicted to be about ten times the thickness of the higher viscous layer38λ≈5–10 km, the observed folds would need a layer thickness of ∼0.5–1 km, which is roughly one- to two-thirds of the ice sheet thickness in the studied area upstream of the Petermann Glacier. This applies to an isolated layer in an infinite matrix. However, our finite-element modelling shows that the bedrock, which in the model approximately mimics real conditions and acts as a fixed, zero vertical velocity boundary at the base, would hinder the development of folds with the observed amplitudes, even at a viscosity contrast of 100 . It has to be noted that the simulation of natural fold geometries is not possible in our 2D model. Therefore, some features, such as overturned folds, were not reproduced. The model, however, does show the expected initial fold geometry in a strongly anisotropic material. Fold geometries are expected to be modified in the complex 3D flow field  are interpreted to be the result of the strong bedrock-parallel simple shear component due to basal friction  and 3. F153865043Radargrams from CReSIS were imported as sections into the software 3D-Move (Version 2014), using Polar Stereographic as the coordinate system . We combet al.The 2D finite-element method was used to simulate non-linear viscous deformation in plane strain of a single layer embedded in a softer matrix, as described by Llorens Plane strain pure-shear deformation of ice was modelled using a full-field crystal plasticity code454647How to cite this article: Bons, P. D. et al. Converging flow and anisotropy cause large-scale folding in Greenland's ice sheet. Nat. Commun. 7:11427 doi: 10.1038/ncomms11427 (2016).Supplementary Figures 1-6, Supplementary Table 1 and Supplementary References.Fly-through movie for the reconstructed folds in the onset region of the Petermann Glacier. The movie shows the geometry of folds in horizons A-D in relation to the ice surface, bedrock and funnel-shaped inlet of the glacier.


The upstream stimulatory factor 2 (USF2) is a regulator of important cellular processes and is supposed to have also a role during tumor development. However, the knowledge about the mechanisms that control the function of USF2 is limited. The data of the current study show that USF2 function is regulated by phosphorylation and identified GSK3β as an USF2-phosphorylating kinase. The phosphorylation sites within USF2 could be mapped to serine 155 and threonine 230. In silico analyses of the 3-dimensional structure revealed that phosphorylation of USF2 by GSK3β converts it to a more open conformation which may influence transactivity, DNA binding and target gene expression. Indeed, experiments with GSK-3β-deficient cells revealed that USF2 transactivity, DNA binding and target gene expression were reduced upon lack of GSK3β. Further, experiments with USF2 variants mimicking GSK3β phosphorylated USF2 in GSK3β-deficient cells showed that phosphorylation of USF2 by GSK3β did not affect cell proliferation but increased cell migration. Together, this study reports a new mechanism by which USF2 may contribute to cancerogenesis. The usf1 and usf2 genes are ubiquitously expressed with varying ratios in different organs The upstream stimulatory factors (USFs) are involved in the transcriptional regulation of various genes whose products contribute to the stress and immune response, to cell cycle and proliferation as well as to lipid and carbohydrate metabolism −/− mice exhibited a rather normal phenotype, USF2−/− mice had a reduced lifespan, decreased fertility and were 20–40% smaller than their wild-type litter mates Generation and observation of USF knockout mice revealed that USF2 seems to be the more important USF variant. While USF1Although the above-mentioned studies indicate that USF2 has a crucial role in the organism, especially during cancerogenesis, there is not much knowledge about the mechanisms regulating the activating or suppressive functions of USF2. Eukaryotic cells feature numerous different mechanisms for the regulation of transcription factor function. One versatile option to reversibly regulate the activity of transcription factors is protein phosphorylation. While there is some evidence that USF1 can be phosphorylated by various kinases participating in different signaling pathways Our own previous work on the role of USF2 on the expression of the cancer marker plasminogen activator inhibitor-1 (PAI-1) We demonstrate that USF2 can be phosphorylated by GSK3β at two distinct sites. Our data show that these phosphorylation events have the ability to regulate the function of USF2 by altering the DNA binding capacity, the transactivity and the protein stability. Furthermore, we demonstrate that cell migration is influenced by GSK3β-mediated phosphorylation of USF2.All biochemicals and enzymes were of analytical grade and purchased from commercial suppliers. As GSK3 inhibitors, LiCl (Sigma-Aldrich), 6-bromoindirubin-3-oxime (BIO), and 1-Azakenpaullone (both Enzo Life Sciences) were used in the concentrations indicated. BIO and 1-Azakenpaullone were dissolved in DMSO, LiCl in water.5′-CTGAGAAGCTTATGCTGGACCCGGG-3′ and reverse primer 5′-CTGAGTCTAGACTGCCGGGTGCCC-3′ and a previously described USF2a plasmid 5′-CAGGGAATTCATGGACATGCTGGAC-3′ and the reverse primer 5′-GTATACTCGAGCTGCCGGGTGCCC-3′ were used to clone a wild-type USF2 product into the EcoRI and XhoI sites of the pGEX-5×1 plasmid . The plasmid pcDNA6A-Gal4 containing the Gal4 DNA binding domain was generated by cutting the DNA encoding the Gal4 DNA binding domain from the pSG424 vector 5′-GTATGGTACCTAGGATCCATATGGACATGCTGGACCCGGGTCTCGATCCCGCTGCCTC-3′ and the reverse primer 5′-CGCGTCTAGAGAATTCCTAGGGTGTTCTGG TTCCATC-3′ were used. For the generation of deletion and point mutants, the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) was used. The expression vectors for pcDNA3-GSK3β-WT, -S9A and -K85A were described before The expression vector for FLAG-myc-tagged wild-type USF2a (USF2-WT) was generated by PCR using the forward primer +/+ and GSK3β−/− mouse embryonic fibroblasts (MEFs), a kind gift from J. Woodgett, were cultured in Dulbeccós modified Eaglés medium . These immortalized MEFs were derived according to approved protocols from GSK3β nullizygous embryos as described 2, 5% CO2, 79% N2 and 97% humidity. Transient transfections were conducted by the calcium phosphate precipitation method Amaxa MEF 1 Nucleofector Kit .HeLa and HepG2 cells were maintained in Earle’s minimum essential medium ; GSK3β5 HeLa cells were seeded in 2.5 ml growth medium in 60 mm cell culture dishes. The cells were transfected overnight with 3 µg of a Firefly luciferase gene construct, 1 µg of expression vector and 50 ng of the Renilla luciferase reporter plasmid (pRL-SV40). The next morning, the medium was replaced by fresh growth medium and the cells were cultured for further 26 h. Then luciferase assays were performed as previously described For luciferase assays the transfection efficiency was controlled by cotransfection of a Renilla luciferase expression vector. In brief, one day before transfection 2×10Applied Biosystems 7500 Real-Time PCR System  by using an Applied Biosystem Power SYBR green PCR master mix . The following primers were used: USF2 forward 5′-GCGTTCGGCGACCACAATA-3′, USF2 reverse 5′-GACTACGCGGTATGTCACCTG-3′, FAS forward 5′-GGAGGTGGTGATAGCCGGTAT-3′, FAS reverse 5′-TGGGTAATCCATAGAGCCCAG-3′, HO-1 forward 5′-GATAGAGCGCAACAAGCAGAA-3′, HO-1 reverse 5′-CAGTGAGGCCCATACCAGAAG-3′, PAI-1 forward 5′-GTGAATGCCCTCTACTTCAGTG-3′, PAI-1 reverse 5′-GCTGCCATCAGACTTGTGGAA-3′, ActB forward 5′-ATGCTCCCCGGGCTGTAT-3′, ActB reverse 5′-CATAGGAGTCCTTCTGACCCATTC-3′, Hprt forward 5′-CGAAGTGTTGGATACAGGCC-3′, Hprt reverse 5′-GGCAACATCAACAGGACTCC-3′. All primer sets were validated for their product and amplification efficiency using standard dilution analysis and melting curve analysis. β-Actin and Hprt (Hypoxanthine guanine phosphoribosyltransferase) were used as internal controls to normalize the variability in expression levels. The experiments for each data point were carried out in triplicate. The relative quantification of gene expression was determined using the ΔΔCt method Isolation of total RNA was performed using the Qiagen RNeasy Mini Kit  following the manufacturer’s instructions. One µg of total RNA was used for first strand cDNA synthesis with the iScript cDNA synthesis Kit . Quantitative real-time PCR was performed in an 3VO4, 25 mM β-glycerophosphate). The cells were then treated for 20 min in an ice-cooled ultrasonic water bath and subsequently centrifuged at 4°C and 13700×g for 20 min. The protein concentration was estimated by the Bradford method.Cells were washed with cold PBS and scraped in lysis buffer , 25 mM NaF, 1 mM NaFor the analysis of proteins by Western blotting, cell extracts were denaturated with 4x SDS sample buffer  by incubating at 95°C for 10 min. Then the proteins were resolved by 10% SDS-PAGE and blotted to a nitrocellulose membrane. For the analysis of p-USF2, an acrylamide:bisacrylamide ratio of 200∶1 was used in the resolving gel.As primary antibodies monoclonal antibodies against USF2 , HA-tag , myc-tag  and α-tubulin , as well as polyclonal antibodies against USF2  were used. Horseradish peroxidase (HRP)-conjugated goat anti-mouse  and goat anti-rabbit IgGs  were used. The enhanced chemiluminescence (ECL) system  was used for detection.+/+, and GSK3β−/− cells were prepared in lysis buffer ). After scraping, lysates were incubated with continuous shaking at 4°C for 20 min and then they were centrifuged at 12.000 g at 4°C for 15 min For immunoprecipitation experiments, protein samples from HeLa, GSK3βFor dephosphorylation experiments, cell extract (90 µg protein) was incubated with 40 u calf intestinal phosphatase  at 37°C for 5 min. In the appropriate control sample the enzyme was replaced by water.+/+, and GSK3β−/− cells were treated with 10 µg/ml cycloheximide  for the indicated time periods. Whole cell extracts were prepared as above and 100 µg of protein was loaded onto 10% SDS- polyacrylamide gel. After electrophoresis and electroblotting onto a nitrocellulose membrane, proteins were detected with antibodies against USF2 and quantified with Fiji (NCBI) software.In half-life studies, GSK3βImmunoprecipitated proteins (250 µg endogenous proteins or 150 µg overexpressed proteins) were denaturated and subjected to 10% SDS-PAGE. Phosphoproteins were stained with Pro-Q Diamond Phosphoprotein Stain (Life Technologies) and detected with a wavelength of 532 nm using the Typhoon 9400 . Subsequently, total proteins were detected by silver staining E. coli BL21 (DE3) at 30°C and purified with Glutathione Sepharose beads as described previously 2, 3 mM EGTA, 1.2 mM EDTA, 150 µM DTT, 6 mM β-glycerophosphate) containing 20 ng of active, recombinant full-length human GSK3β , 50 µM unlabeled ATP and 1.5 µCi [γ−32P] ATP at 30°C for 20 min. The proteins were denaturated by incubating with 4x SDS sample buffer at 95°C for 10 min. Following separation of the proteins by 10% SDS-PAGE, the gel was exposed to a Kodak phosphor imaging screen overnight and thereafter autoradiography was detected with a Molecular Imager FX using the Quantity One software . Afterwards, the total proteins were detected by Coomassie staining or silver staining.Using the pGEX-5×1 system , GST fusion proteins were expressed in −/− MEFs were seeded in 15 cm cell culture dishes and ChIP was performed as described previously 5′-GTCTAGACGACCGACCAGCCAAA-3′, PAI-1 reverse, 5′-GAAATGTCTGGGCTGCCCGC-3′. HO-1 forward, 5′-GCCTCCGGGCTGGATGTTG-3′ and HO-1 reverse, 5′-GGAGACCGTGAGCGAGCAGC-3′, FAS forward, 5′-GACGCTCATTGGCCTGGGC-3′ and FAS reverse, 5′-ACGCCGCTGCCGTCTCTCT-3′. As a positive control, a RNA polymerase II antibody was used to immunoprecipitate RNA polymerase II from an actively expressed housekeeping gene promoter; the mouse β-actin promoter was amplified with the following forward 5′-GTGAGTGAGCGACGCGGAGCCAA-3′, and reverse 5′-CTTACCTGGTGGCGGGTGTGGA-3′ primers −/− MEFs were calculated by the percent input method using the formula triplicate average Ct100*2∧(Adjusted input - Ct (IP)), where input adjustment is calculated using the formula raw Ct(Ct Input-log2 of the dilution factor) (dilution factor (DF) is equal to 1/fraction input).Wild-type (WT) and GSK3β−/− MEF cells were seeded onto 10 cm cell culture dishes and one day later the cells were transfected by using the Amaxa MEF 1 Nucleofector Kit  with 5 µg of expression vectors for USF2-S155A/T230A, USF2-S155D/T230D or an empty vector. After 20 h, 4×104 electroporated cells were seeded in 500 µl growth medium into a 24-well culture dish for the MTT assay, 1×104 electroporated cells were seeded in 100 µl growth medium into a 96 well culture dish for the BrdU assay and 1×104 electoporated cells were seeded for migration assay (see below). From another aliquot of the electroporated MEFs whole cell extracts were prepared for the control of transfection efficiency by Western blotting. Cells plated for the MTT assay and the BrdU assay were cultured for further 30 h. For the MTT assay cells were stained with 50 µl MTT solution (4 mg/ml in PBS) for 1.5 h and the absorbance was measured at a wavelength of 570 nm. The BrdU assay  was performed according to the manufacturer’s instructions and the absorbance was measured at dual wavelengths of 450–540 nm.For BrdU, MTT and cell migration assays GSK3β4 cells per well in 100 µl serum-free medium. After incubation in a humidified incubator with 5% CO2 at 37°C for 4 h, cells were fixed with 4% paraformaldehyde and the non-migratory cells were scraped off from the top of the Transwell with a cotton swab. The migrated cells attached to the bottom chamber were stained with 0.0075% crystal violet. Crystal violet absorption at 595 nm was measured in a Tecan Infinite M1000Pro microplate reader. For each migratory condition, two identical replicates were performed.Cell migration was measured by using medium-treated 24-well Transwell chambers (BD Bioscience) with 8.0 µm polycarbonate membranes. The bottom chamber was filled with 600 µl medium containing 10% fetal calf serum; cells were seeded into the top chamber at a density of 1×10A homology model of USF2 was built using the I-TASSER online software −1 nm−1, a short 100 ps in the NVT  ensemble was carried to equilibrate the given system. This was followed by a 15 ns production run in the NPT  ensemble. All simulations were carried out at 1.0 atm constant pressure using the Parrinello-Rahman barostat The resulting structures  were each solvated in a rectangular box containing SPC waters Each experiment was performed at least three times and representative data are shown. Data in bar graphs are given as mean values ± standard deviation (SD). Statistical differences were calculated by using the Student t-test with error probabilities of p<0.05 to be significant.All experimental procedures involving animal-derived cell lines were approved and performed only under permissions from the Animal Experiment Board of Finland following the regulations of the EU Directive 86/609/EEC, the European Convention ETS123 and the national legislation of Finland.−/− MEFs) and their wild-type counterparts (GSK3β+/+ MEFs) and visualized threonine and serine phosphorylated USF2 via immunodetection with phospho-threonine and phospho-serine antibodies. We found that threonine and serine phosphorylated USF2 could be detected in the wild-type cells whereas none of these forms could be detected in GSK3β−/− MEFs . Together, these data, in particular the data obtained from the GSK3βinvitro. In addition to full length USF2, we also purified 6 different deletion variants  separately and converted them into non-phosphorylatable alanine residues. Since the amino acids 218–222 (THPYS) within the USR perfectly match the GSK3β minimal recognition motif S/T-X-X-X-pS/pT, where the N-terminal S/T is the proper GSK3β phosphorylation site and the C-terminal S/T is the site of priming phosphorylation, we also generated a T218A/S222A double mutant. The kinase assays with these USR mutants and GSK3β revealed that T230 is the proper phosphorylation site within this domain, since replacement of this residue by alanine completely abolished the phosphorylation. All other USR single point mutants and the T218A/S222A double mutant were still phosphorylatable by GSK3β .The N-terminal domain of USF2 comprising the amino acids 1–161 contains 18 phosphorylatable serine and threonine residues. We screened this sequence for minimal GSK3β recognition motifs and found one of these motifs within this N-terminal domain. Based on this, S151 would be the proper GSK3 phosphorylation site and S155 would be the site of priming phosphorylation. Hence, both sites were replaced by alanine. Additionally, S155 was substituted by aspartate; also a S151A/S155D double mutant was constructed. Kinase assays with these proteins revealed that any modification of S155 totally abolished GSK3β-mediated incorporation of radioactivity whereas the S151A mutant was still phosphorylatable. Since mutation of S155 completely prevented GSK3β-mediated phosphorylation, we concluded that this residue is the exclusive GSK3β site in this N-terminal domain of USF2 .To verify the importance of S155 and T230 within full length USF2, we performed a kinase assay with recombinant GSK3β and full-length wild type USF2 or the respective S155 and T230 mutants which we expressed and immunoprecipitated from HeLa cells. The USF2-WT protein and the single mutants USF2-S155A, USF2-T230A, and as a control USF2-T228A were phosphorylatable by GSK3β. By contrast, when both S155 and T230 were substituted with alanine (S155A/T230A) phosphorylation by GSK3β was completely prevented .Together, these data show that there are two residues within USF2, namely S155 and T230, which can be phosphorylated by GSK3β.To test whether the identified phosphorylation sites are crucial for the transactivity of USF2 we performed luciferase assays. We took advantage of the Gal4 system to assess the impact of the detected phosphorylation sites on the USF2 transactivity independent from their effect on the DNA binding capacity. Therefore, we generated constructs in which the USF2 DNA binding b-HLH-LZ domain (aa 232–346) was replaced with the DNA binding domain of the transcription factor Gal4. In addition to the resulting construct encoding the wild type Gal4-USF2 fusion protein -WT), another plasmid encoding a fusion protein in which the two GSK3β phosphorylation sites were substituted with alanine -S155A/T230A) was generated. As a control constructs encoding only the Gal4 DNA binding domain were used. These Gal4 fusion constructs were then cotransfected with a plasmid containing the luciferase gene under the control of 5 Gal4 response elements (RE). Thus, changes in the measured luciferase activity reflect the transactivity of the transcription factor USF2. While the presence of the constitutively active GSK3β-S9A significantly increased the observed luciferase activity with the Gal4-USF2(1-231)-WT construct, no change in luciferase activity could be detected with the Gal4-USF2(1-231)-S155A/T230A double mutant . Western+/+ and GSK3β−/− cells and measured FAS, HO-1, and PAI-1 mRNA and protein levels. Indeed, FAS, HO-1, and PAI-1 mRNA levels , plasminogen activator inhibitor 1 (PAI-1), and fatty acid synthase (FAS). To test this, we used GSK3βA levels  as well A levels  were red/− cells . Thus, t+/+ and GSK3β−/− cells and performed chromatin immunoprecipitations with an USF2 antibody followed by quantitative PCR with primers encompassing the USF binding sites within the FAS, HO-1, and PAI-1 promoter +/+ cells. By contrast, binding of USF2 to the FAS promoter was almost lost in −/−GSK3β cells and binding to the HO-1 and PAI-1 promoter was reduced by 60% and 70%, respectively , USF2-S155A, USF2-T230A or the S155A/T230A double mutant. As a control, the promoter luciferase constructs were cotransfected with a USF2 plasmid in which T228 was substituted with alanine . The lucNext we aimed to understand whether the phosphorylation of USF2 at S155 or T230 affects the 3-dimensional structure of the protein. Since there is no crystal structure for full length USF2 available we performed a series of molecular dynamics simulations of USF2 when S155 and T230 remained non-phosphorylated (native protein) and when S155 and T230 were phosphorylated. The modeled molecular structure of the native non-phosphorylated USF2 protein indicated that the protein is composed of two domains . The two+/+ and GSK3β−/− cells were treated with the protein synthesis inhibitor CHX and the level of endogenous USF2 was monitored by Western blotting. While the USF2 half-life in the control cells was about 38 h, the half-life in cells lacking GSK3β was estimated to be about 31 h  incorporation into newly synthesized DNA. We found that none of the USF2 variants exerted an effect on cellular viability and DNA synthesis . These data suggest that phosphorylation of USF2 by GSK3β has no impact on overall cellular viability and proliferation.−/− cells and GSK3β−/− cells transfected with either the non-phosphorylatable USF2 variant S155A/T230A or the phospho-mimicking variant S155D/T230D. A significant increase in migration could be detected with GSK3β−/− cells expressing the phospho-mimicking variant S155D/T230D; which reflects GSK3 phosphorylated USF2 whereas the USF2 S155A/T230A variant slightly decreased migration  appears to be crucial for the function of USF since the simple use of USF2 variants consisting just of the leucine zipper (LZ), basic helix-loop-helix (bHLH) and basic region (BR) which are involved in dimerization, DNA binding and nuclear localization Interestingly, when inspecting the USF2 sequence for the presence of GSK3 sites, we found the existence of some S/T-X-X-X-S/T motifs, which are typical recognition motifs for GSK3β. Although not strictly required, priming phosphorylation seems to increase substrate phosphorylation by GSK3β In addition to our own experiments, the only data for USF2 phosphorylation so far came from a study of the bovine system investigating the effect of forskolin and protein kinase A (PKA) on the prostaglandin G/H-2 synthase promoter. That study showed that USF2 contains three PKA phosphorylation sites and that overexpression of PKA enhanced USF DNA binding in bovine granulosa cells. Interestingly, mutations of the putative PKA phosphorylation sites S259, S269 and S275 in USF2 reduced but did not completely abolish transactivation capacity It already has been shown that the protein stability of several proteins which play a role in cancerogenesis is altered upon GSK3β-dependent phosphorylation, among them are for example the transcription factors HIF-1α The induction of USF2 transactivity due to phosphorylation by GSK3β is complemented by the induction in USF2 DNA binding capacity and half-life. At the same time an increased expression of the USF2 target genes FAS, HO-1, and PAI-1 is initiated. This type of regulation, together with the findings of the migration assay is in line with previous reports indicating that GSK3β and USF2 have a role in cancerogenesis However, the exact function of both, GSK3β and USF2 in cancerogenesis appears to be variable and may depend on the cellular context. While a number of studies support the idea of GSK3β being tumor suppressive, others studies showed that GSK3β may promote cancer development In summary, our study identified GSK3β as a kinase that phosphorylates USF2 and thereby regulates its function. The GSK3β-mediated phosphorylation of USF2 at S155 and at T230 leads to increased transactivity, DNA-binding, and half-life of the transcription factor and to an induction of cell migration. These novel findings on the regulation of the transcription factor USF2 set the basis for further studies investigating how these mechanisms contribute to the development of different types of cancer.


Sphingomyelin synthase (SMS) plays an important role in plasma atherogenic lipoprotein metabolism, inflammation, and the development of atherosclerosis. To understand whether the impaired apoB secretion and inflammation response is a direct result from lack of SMS activity, in this study, we prepared a series of compounds that inhibit SMS activity. Further, we characterized Dy105, the most potent inhibitor. We found that Dy105 treatment significantly reduces SM levels in SM-rich microdomain on cell membranes. Moreover, we found that SMS inhibition reduces apoB secretion in a human hepatoma cell line and reduces the activation of NFκB and p38, a MAP kinase, in bone marrow derived macrophages. These studies provided further evidence that SMS activity regulates atherogenic lipoprotein metabolism and inflammatory responses. Pharmacologic inhibition of SMS may be a new therapy for atherosclerosis by reducing apoB secretion, and reducing inflammation. In the developed countries, coronary heart disease (CHD) is the major cause of mortality. Now, statin therapy is the main option for CHD clinical management. Despite its potent efficacy, statin therapy is not always responsive and sometimes patient is intolerant to this therapy In human, serum or plasma sphingomyelin (SM) is considered as a risk factor for CHD SM is produced by the transfer of phosphorylcholine from phosphatidylcholine to a ceramide in a reaction catalyzed by sphingomyelin synthases (SMS) SMS gene family consists of three members, SMS1, SMS2, and SMS related protein (SMSr). SMS1 expression is located in the trans-Golgi complex, while SMS2 is predominantly expressed in the plasma membranes In this study, we developed compounds that inhibit SMS activity and found that small compound-mediated SMS inhibition reduces cell plasma membrane SM levels, thus reducing hepatocyte apoB-containing lipoprotein (an atherogenic lipoprotein) secretion and reducing macrophage endotoxin-mediated inflammation. These studies indicated that inhibition of SMS by specific small compounds might be a promising approach in preventing the development of CHD.35S]methionine (specific activity >1175 Ci/mmol) were purchased from Perkin Elmer, Boston, MA. Chromatographically purified LPS from S. minnesota was from Sigma-Aldrich, St. Louis, MO. Dy105 and analogues was synthesized by Department of Medicinal Chemistry, School of Pharmacy in Fudan University, Shanghai, China. Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP Rabbit anti-body were from Cell Signal Technologies, Shanghai, China. NFκBp65 subunit antibody was from Epitomics, Burlingame, CA, USA. Male (C57BL/6J) mice aged 8 weeks were from Central of Animal experiment in School of Pharmacy in Fudan University, Shanghai, China. All experimental procedures were approved by the Institutional Review Board of Fudan University.Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were from Thermo Scientific HyClone, Shiyi Biotechnology, Shanghai, China. L-methionine in methionine-free DMEM with or without Dy105. The cells were washed twice with PBS and then chased in DMEM for 4 hours. After chase, the culture medium was collected and apoB from medium was precipitated by an anti-human apoB antibody (Abcam) together with protein A/G beads. [35S]apoB was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The band corresponding to [35S]apoB was identified by an autoradiograph.The method has been described previously Mice were injected with Poloxamer 407 to block the clearance of VLDL from the circulation. Plasma (150 µl) was collected at 0, 3, 6, 24 hours after injection. Plasma triglyceride and sphingomyelin levels were measured.The method has been described before BMM were incubated with various concentration of Dy105 for 17 hours before LPS treatment. The cells were collected and total proteins were extracted by RIPA Lysis Buffer supplemented with 1 mM PMSF, 10 µg/ml aprotonin, 10 µg/ml leupeptin, 1 mM DTT, pH 7.4. Protein samples were detected by Western blot and probed with the following primary antibodies: rabbit anti-human phosphor-p38 and β-actin antibody , followed by HRP-conjugated secondary antibody  and developed with ECL reagent .t test, and among multiple groups by ANOVA followed by the Student-Newman-Keuls (SNK) test.Data are typically expressed as mean ± SD. Data between two groups were analyzed by the unpaired, two-tailed Student’s Based on homology modeling and molecular dynamics simulation, we have reported a structural model of human SMS1 We next sought to evaluate the effect of Dy105 on Huh7 cells (a human hepatoma cell line) and bone marrow derived macrophages (BMM). We found that the compound significantly decreases Huh7 cell  and BMM We next sought to investigate whether Dy105 has SMS inhibitory specificity, we utilized SMS1 gene knockout (KO) mouse livers in vivo, we injected (i.v.) mice with Dy105 (1.2 mg/kg). Four hours later, the mice were sacrificed and livers were isolated. SMS activity in mouse liver homogenate was measured. We found that Dy105 significantly decreased liver SMS activity  is the major protein component of VLDL and chylomicron (CM) in vivo. Wild type mice were injected with poloxamer 407 to block the clearance of VLDL from the circulation. We collected plasma 120 minutes after injection and measured triglyceride and sphingomyelin levels. We found that Dy105 treatment significantly decreases poloxamer 407-mediated triglyceride and sphingomyelin accumulation, suggesting that Dy105 has an effect on liver triglyceride/sphingomyelin-rich particle, i.e. VLDL, production , on Dy105-treated macrophages after LPS stimulation. We found that phospho-p38, the active form of the kinase, is decreased in the compound treated macrophages .In this study, we have demonstrated that SMS inhibition by a small compound, Dy105, resulted into a significant decrease of plasma membrane SM levels, a significant reduction of BLp production from a hepatoma cell line, and a significant reduction of endotoxin-mediated NFκB and p38 activation in macrophages. These are useful tools to explore the impact of SM biosynthesis inhibition on the lipoprotein metabolism and the development of atherosclerosis.SM is enriched on BLp The increase of hepatic apoB-containing lipoprotein production is considered as the principal defect in subjects with familial combined hyperlipidemia SMS inhibition influence inflammatory responses via changing lipid-raft structure. One of the key regulators of inflammation, downstream of TLRs, is NFκB In conclusion, SMS physiologically contributes to de novo SM biosynthesis and plasma membrane SM levels. A potent SMS inhibitor potentially affects the balance of SM, resulted into less apoB particle production and blunted NFκB and p38 responses to inflammatory/immunologic stimuli. Thus, inhibition of SMS activity may have an important impact on anti-atherogenic processes.


A4M7(XO4)6 . The proposed structural model is based both on a careful investigation of the crystal data, as well as validation tools by means of bond-valence-sum (BVS) and charge-distribution (CHARDI) calculations.Tetrasodium hepta hexa(arsenate/phosphate) is a new member of the isostructural family of compounds with the general formula  4Co5.40Al1.07(As0.883P0.116O4)6, was prepared by a solid-state reaction. It is a new member of the family of isostructural compounds with the general formula A4M7(XO4)6  that is most similar to Na4Co5.63Al0.91(AsO4)6. The Co2+ ions in the title compound are substituted by Al3+ in a fully occupied octahedral site (site symmetry 2/m) and a partially occupied tetrahedral site (site symmetry 2). A third octahedral site is fully occupied by Co2+ ions only. With regard to the P and As atoms, one site (site symmetry m) is simultaneously occupied by As and P, whereas in the second site there is only arsenic. The alkali cations are, as in the isostructural compounds, distributed over half-occupied crystallographic sites, with a positional disorder of one of them. The proposed structural model is based both on a careful investigation of the crystal data, as well as validation by means of bond-valence-sum (BVS) and charge-distribution (CHARDI) calculations. The correlation between the X-ray refinement and the validation results is discussed.The title compound, tetrasodium hepta hexa(arsenate/phosphate), Na Li et al. 6 , was obtained during the exploration of the Na–Co–P–As–O system by solid-state reaction; as for many aluminophosphates, aluminum was incorporated from the reaction container. The chemical composition and crystal structure were determined by energy-dispersive X-ray spectroscopy (EDX) analysis 6   with eight cations per unit cell, two others (denoted MA and MB) are simultaneously shared by Co2+ and Al3+ ions, and one is fully occupied by Co2+ ions: the same distribution is observed in the homologous arsenate Na4Cox7−Alx0.67(AsO4)6 (x = 1.37) (II)  Fig. 2 containsV according to the BVS model and charges Q from the CHARDI analysis should agree with the oxidation states of the atoms  in (I).The l Table 1. If compMB site with a tetrahedral coordination, the Co2+/Al3+ distribution is based on the same observations as in (II), mainly if it is refined as partially occupied by just Co2+, the charge neutrality is not achieved, and then a fraction of Al3+ was introduced in the MB site yielding an overall occupancy distribution of Co0.605Al0.135□0.260, with □ expressing the vacancy. The validation results for this particular distribution are: V(MB) = 1.31 and Q(MB) = 1.58, the theoretical value is 1.61 (Table 1MC): P/As = 0.35/0.65; V = 5.21 and Q = 5.00.For the 1 Table 1. Finally4Co5.40Al1.07(As0.883P0.116O4)6. It is the first case in its homologous family which contains such a number of elements. The similarity to (II)  in the molar ratio Na:Co:P:As = 2:1:0.5:1 was dissolved in deionized water and then heated at 373 K to dehydration. After grinding, it was placed in a porcelain boat and first heated at 673 K in air for 24 h and then heated gradually to 1123 K for 1 d. Some pink parallelepiped-shaped crystals were isolated from the sample. A qualitative EDX analysis confirmed the presence of Na, Co, Al, As and O  I. DOI: 10.1107/S205698901600400X/br2258Isup2.hklStructure factors: contains datablock(s) I. DOI: 1462882CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Total serum IgE result from the combination of specific and non-specific pools. High specific/total IgE ratio may reflect high level of allergen-specific IgE on mast cells. No data regarding its applications to drug allergies is available. One hundred seventy-one patients with a history of immediate reactions to β-lactams, confirmed by positive skin testing, and 122 control subjects tolerant to β-lactams, were studied. CAP System was used for the detection of serum total and specific IgE antibodies. The specific/total IgE ratio was tested for diagnostic accuracy compared with conventional criteria. We finally performed a simulation study to expand our investigation of the performance of the specific/total IgE ratio index in a scenario in which the new CAP detection threshold is lowered further. Specific/total IgE ratio values ≥0.002 were observed more frequently in reactive than in controls. Seventy-four of 80 subjects with values ≥0.002 were allergic to β-lactams, yielding a positive predictive value of 92.5%. The application of specific/total IgE ratio significantly improves the positive likelihood ratio and the overall diagnostic performance. In addition, we showed the capability of this new criterion to identify true reactive patients even among subjects with high levels of total IgE (>200 kU/L). Significant increase in both receiver operator characteristic (ROC) curve and sensitivity were observed in imputed case of the simulation study. The β-lactams-specific/total IgE ratio may be an additional index compared to the common criterion of positivity to a single hapten in the allergological work-up of patients with β-lactams immediate adverse reactions. When a Serum total IgE levels are not usually assessed since they are not considered useful in the clinical diagnosis of allergy and thus the ratio between specific and total IgE is almost never computed . It has This ratio has been already used in allergological literature to predict the response to allergen-specific immunotherapy and to predict and prevent pediatric allergy ,8. HowevWe enrolled a total of 171 β-lactams allergic patients (ADR+) who were referred to our attention for a recent immediate ADR to β-lactams. The positive diagnosis of β-lactams allergy was confirmed by skin testing positivity. Evaluation of atopic phenotype was based on history, clinical symptoms and positive skin prick test (SPT) for common allergens. Thirty-nine patients (39.2%) were atopic as defined by at least one sensitization to inhalant allergens and by the presence of related symptoms, and amoxicillin was the most frequent (88.5%) culprit drug. Most of them experienced urticaria (57.3%) or urticaria plus angioedema (7%). Anaphylaxis was recorded in 61 out of 171 patients (35.6%). A negative control group (ADR- patients) was also enrolled comprising 122 subjects (77 atopic and 45 non atopic) who reported tolerance to β-lactams administration in the last year and displayed negative skin testing. Written informed consent was obtained from all patients. This study exempted from institutional review board approval (Prot. 23.455-302/2009). -5 M, Diater Laboratories, Spain), minor determinant mixture , amoxicillin  and ampicillin . In both prick and intradermal testing a minimum wheal area of 3 mm diameter or an increase of area >3 mm was considered positive compared to a negative response to the saline control.Skin testing was carried out as previously described with 0.02 ml of solutions [CAP System FEIA  based on ImmunoCAP technology has been used for the detection of both β-lactam-specific and total serum IgE antibodies according to the manufacturer’s instructions. All results were expressed in kU/l. Sera obtained from all patients were analyzed for IgE towards the hapten c1 (penicilloyl G), c2 (penicilloyl V), c5 (ampicilloyl), c6 (amoxicilloyl). Serum samples were considered positive when one or more hapten positivities occurred (detection limit of 0.10 kUA/l). Sera with a concentration of specific IgE antibodies below the detection limit were assigned an arbitrary value of 0.0 kUA/l. The specific/total IgE ratio was calculated making the sum of the measurements of β-lactam-specific IgE antibodies divided by total IgE.Receiver Operator Characteristic (ROC) curve was used to plot sensitivity against 1—specificity and compute the Area Under the Curve (AUC) using the percentile value method suggested by Janes and Pepe . SensitiIn the simulation study, we wished to expand our investigation of the performance of the specific/total IgE ratio index in a scenario in which the new CAP detection threshold is lowered further. Specifically, we assumed that immeasurable specific IgEs could in fact be a mixture of cases in whom specific IgEs were truly absent, as compared to others in whom they were simply undetected since they were below CAP detection threshold. Therefore, we assessed the new diagnostic performance of CAP after imputing the specific IgE and total IgE joint distributions for cases in which specific IgEs were immeasurable.As a first step we used Spearman correlation to assess whether the sum of specific IgEs correlated with total IgEs, separately by reactive and non-reactive patients. We plotted the sum of β-lactam vs. total IgEs, with both axes on a log scale, mainly because this transformation tends to normalize such skewed distributions and, secondarily, enables to plot the ratio index values as a straight line, given that the ratio of two variables equals a linear difference in their logs.We performed multiple imputation using chained equations methods to impute missing, i.e. immeasurable, levels of the sum of β-lactam IgEs . We simuWe evaluated the distribution of specific/total IgE ratio values in patients with a confirmed history of immediate adverse reaction to β-lactams, compared to negative control subjects. As shown in P < .001). In fact, as reported in In clinical practice CAP for β-lactams is considered positive when specific IgE antibodies were present for at least one hapten. It has been previously showed that the diagnostic accuracy of CAP assay is greatly influenced by total serum IgE levels . The diaP < .001). The small difference in specificity (98% vs 96%), causes a large, but non statistically significant LR+ reduction, due to missing 2 vs. 6 reactive patients with the conventional vs. the ratio criterion.At the specific/total IgE ratio cut-off associated with 95% specificity in this study (0.002), a larger number of allergic subjects can be classified as positive (n = 74) compared to previous combined criteria of at least one hapten positivity in the presence of total IgE<200 kU/l (n = 58); this is a modest, but statistically significant increase in sensitivity from 35% to 45%  resulted true positive. Twenty-one out of these 80 patients displayed high levels of total IgE (>200 kU/l). However, most of them  were really ADR+, thus showing the capability of this new criterion to identify true reactive patients even among subjects with high levels of total IgE. When specific/total IgE ratio >0.002 was applied to reactive patients with at least one hapten positivity and low levels of total IgE (<200 kU/l) (n = 58), we observed that all but one was correctly identified by the ratio criterion .Overall, there was a gain in the number of correctly classified patients using the ratio criterion. The Net Reclassification Improvement achieved adding the ratio to the single apten positivity criterion was 20.6% (p = 0.003). That obtained adding the ratio to the combination of apten positivity and total IgE<200 was 6.7% (p = 0.022). The Net Reclassification Improvement allowed by the ratio index in patients with total IgE>200 kU/l was 22% over at least one hapten positivity (p = 0.005)Using observed data, a very strong correlation was observed between total and specific IgEs for non-reactive patients  as compared to a modest correlation in reactive patients . This high correlation among non-reactive observed patients can be appreciated in Figs Specifically Our study shows that the ratio of the sum of β-lactam specific to total IgE is able to improve the diagnostic performance of CAP assay in the identification of reactive patients. In particular, our data suggest the clinical usefulness of the ratio at least in patients with serum total IgE>200 kU/l.The elevated value of the specific/total IgE ratio observed in our β-lactams reactive patients is in accordance with the biological background of the mast cells/basophils IgE-induced activation. In fact, these effector cells are activated anytime a threshold number of hapten-specific IgE-charged FcεRI on them is cross-linked by hapten molecules . The speCompared to inhalant and food allergens, β-lactam-specific IgE antibodies are generally lower and the dilution effect of total IgE levels may have a major impact on effector cells response. In β-lactam unreactive patients the low concentration of the pathogenic IgE antibodies cannot reach the threshold level able to trigger mast cells. Considering these data, one may take into account the total IgE and should interpret specific IgE levels differently in patients with high or low total IgE levels.The clinical significance of the ratio value is also suggested by the data showing the relationship between the size of the disease-relevant IgE fraction and total IgE, and the efficacy of anti-IgE  treatment . This isOur simulation study shows that the misclassification of the reactive status in patients with immeasurable β-lactam IgEs, almost all of whom have total IgE values <200 kU/l, is a major limitation of the accuracy of CAP testing. The very high correlation of β-lactam and total IgEs in non-reactive patients suggests that the source for false negative findings is that CAP wrongly measures a fraction of total IgEs as β-lactam IgEs. This fraction is consistently seen as less than 2/1000, our ratio cut-off, in Our study aims primarily to compare different diagnostic expressions of CAP results to be used in future research in this field, and to estimate the diagnostic accuracy of the new index—the specific/total IgE ratio. However, some limitations to the interpretation of the data presented regarding the estimate of diagnostic accuracy, should be acknowledged. We used patients’ history of reaction combined to positive skin testing results as a reference, in order to create well-defined groups of reactive and non reactive subjects. Thus, one can maximize CAP diagnostic performance with the aim of exploring differences among diagnostic criteria. This case-control design overestimates diagnostic accuracy of a test as compared to its use in a series of consecutive cases ,16. A weIn conclusion, the IgE-based criterion proposed here should be used in studies investigating the diagnostic performance of CAP to detect β-lactams allergy. Future diagnostic accuracy studies should recruit subjects as a consecutive series, avoiding the exclusion of cases with uncertain β-lactams allergy status. In particular, the analysis of ratio in confounding cases, such as patients with suggestive history but negative skin testing, should give additional information on the applicability of our finding to real-life patients management. Large-scale multicenter studies are advisable to obtain the necessary statistical power and ensure the generalization of results. Finally, we suggest there would be great improvement in using the ratio criterion provided that technological development succeeds in lowering CAP detection threshold.S1 Data(XLS)Click here for additional data file.


Transient elastography (TE) is widely used for non-invasive assessment of liver fibrosis in HIV-HCV co-infected patients. TE, however, cannot determine liver morphology. Acoustic radiation force impulse (ARFI) imaging is a novel procedure enabling assessment of liver fibrosis during a conventional ultrasonographic examination. This study evaluated the correlation between liver fibrosis measurements by TE and ARFI.Each of 46 HIV-HCV patients underwent both ARFI and TE within 6 months. Patients were evaluated by the “equivalent METAVIR” scoring system, using previously established cut-off values. Agreements between the ARFI and TE scores were estimated by Kappa coefficients, with Kappa values ≥0.40, ≥0.60, and ≥0.80 defined as moderate, good and very good agreement, respectively.ARFI and TE yielded "Equivalent Metavir" fibrosis scores of F1 in 26 and 31 patients, respectively; F2 in nine and seven, respectively; F3 in three and two, respectively; and F4 in eight and six, respectively. The two methods showed very good agreement in predicting overall stages [Kappa = 0.82] and for F ≥3 [Kappa = 0.80] and moderate agreement in predicting significant fibrosis F ≥2 [Kappa = 0.50]. Morphologic ultrasound analysis concomitant to ARFI detected two hepatocarcinomas.ARFI showed promising results in the non-invasive assessment of liver fibrosis in HIV-HCV patients, with liver fibrosis staging similar to that of TE. Moreover, ARFI can assess morphology and fibrosis during the same session. Since the introduction of highly active antiretroviral therapy (HAART), chronic liver disease has emerged as one of the leading causes of morbidity and mortality in HIV-HCV co-infected patients,2. ThereEvaluation of liver fibrosis is considered essential to the management of patients with chronic hepatitis C, providing prognostic information and assisting in making therapeutic decisions-7. To daTE (FibroScan®) is currently the most extensively used elastography technique in clinical practice-19. ManyTE, however, has several drawbacks, including the requirement for specific equipment that performs only elastography, its ability to assess only the right liver, and its inability to visually determine the site of measurement. Moreover, ascites and obesity are limiting factors. ARFI imaging has many advantages over TE. The technology used for ARFI has been incorporated into a conventional ultrasound system, allowing ultrasound analysis of liver morphology at the same time. In addition, ARFI allows elastography to be performed with a flexible metering box at variable depths, enabling the exact localization of the measurement site, and the examination can also be performed in patients with ascites. ARFI measurements have been found to correlate well with the stage of hepatic fibrosis-36. HoweThe study was approved by the institutional review board of our University hospital. Between July 2009 and May 2012, HIV-HCV co-infected patients regularly followed up at the HIV out-patient Clinic of the Department of Internal Medicine and Infectious Disease of our hospital were prospectively enrolled. All patients provided written informed consent before inclusion.3), plasma HCV- RNA concentrations (IU/ml), and HCV genotype. Men and women were considered heavy drinkers if they consumed >3 and >2 glasses of alcohol per day, respectively. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transferase (GGT), alkaline phosphatase (ALP), and bilirubin concentrations were also measured.The demographic, clinical and biological information of all patients was obtained from their medical records. Factors included patient age, body mass index (BMI), alcohol consumption, medications, specific antiretroviral drugs, plasma HIV-RNA levels (IU/ml), complete blood counts, absolute number of CD4+ T-cells  and TE were performed by two different observers, with each subject undergoing the two examinations no more than 6 months apart. The physician performing the second procedure was blinded to the results of the first procedure.TE was performed in the right lobe of the liver through the intercostal space as described,40. LiveARFI imaging is a method of quantifying the mechanical properties of tissues, without manual compression, by measuring the shear wave velocity induced by acoustic radiation and propagating in the tissue. This quantitative technique provides a single unidimensional measurement of tissue elasticity like TE, although the measurement area can be positioned on a two-dimensional B mode image. The region is a 1 × 0.5-cm rectangle, which can be freely moved in the two-dimensional B mode image to a maximum depth of 8 cm from the skin plane. Measurements are expressed as m/s, indicating shear wave speed traveling perpendicular to the shear wave source. The principle of ARFI is described in Figure All patients underwent quantitative elastographic examinations in fasting conditions with a Siemens Acuson S2000TM ultrasound system  and an abdominal curved transducer (4C1), using the virtual touch tissue quantification system.The first step assessed liver morphology and echo-structure, looking for cirrhosis, signs of portal hypertension and tumor lesions. In the second step, 10 ARFI measurements of the right liver through the intercostal space were performed by a single examination. To standardize these examinations, an area of liver tissue free of large blood vessels was chosen at a minimal distance of 2 cm below the liver capsule. ARFI measurements were expressed in m/s as median (M), mean and standard deviation, and interquartile range (IQR), with the IQR/M ratio used to evaluate the variability of the measurements. An IQR/M < 0.3 was considered a homogeneous set of measurements. Median ARFI value was expressed according to the ‘equivalent Metavir’ score using previously determined cutt-off values for HCV mono-infected patients of 1.34, 1.55, 1.80 m/s for liver fibrosis scores of F ≥ 2, F ≥3, and F = 4, respectively.Quantitative characteristics were expressed as mean (standard deviation), median (first-third quartile) and range, and compared using non-parametric Wilcoxon statistics. Qualitative characteristics were expressed as frequencies and proportions, and compared using Fisher’s exact test.Correlations between TE and ARFI measurements were estimated using Spearman’s correlation coefficient and its two-sided 95% confidence interval (CI), as calculated with Fisher's z transformation.Agreements between “equivalent Metavir” fibrosis stages on ARFI and TE were assessed by simple Kappa coefficients for predictions of F ≥ 3 or F ≥2, and F = 4, and weighted Kappa coefficients for predictions of F1 to F4 Metavir scores; two-sided 95% CIs were also estimated. Kappa values ≥0.60 and ≥0.80 were considered indicative of good and very good agreement, respectively. Additional analysis was performed in patients infected with HCV genotypes 1 and 4, who usually have a poorer response than patients infected with genotypes 2 and 3.Quantitative and qualitative variables associated with discordances between ARFI and TE staging were assessed by the Wilcoxon test and Fisher’s exact test, respectively. Only univariate analyses were performed because of the small number of subjects.Of the 86 examinations, 40 were excluded because of missing data (n = 8), too long delay (>6 months) between ARFI and TE (n = 24) or repeated examinations for the same patient (n = 8). When liver stiffness was measured repeatedly in the same patient, only the measurement with minimal delay between ARFI and TE was included in the analysis. Valid results of both ARFI and TE were obtained for 46 patients; their demographic and clinical and biological characteristics are summarized in Table Of the 46 patients, 28 did not receive any treatment for HCV, whereas the other 18 were treated with peginterferon and ribavirin (SOC). At the time of ARFI, eight had achieved a sustained virologic response, whereas the other 10 had failed treatment. No patient received any direct-acting antiviral agents (DAAs). Moreover, all 46 patients had received highly active antiretroviral therapy (HAART), including 28 who received third-generation protease inhibitors (PIs), three who received integrase inhibitors (INIs), eight who received non-nucleoside reverse transcriptase inhibitors (NNRTIs), and seven who received other treatment combinations  and three with benign liver lesions. The two HCCs presented with arterial hypervascularity and wash-out in venous and delayed phase on dynamic enhanced MRI. Their dThe median delay between ARFI and TE in the 46 evaluated patients was 10 days range, 0–25 days). Median liver stiffness on TE was 6.1 kPa (range 3.4–35.3 kPa) and median shear wave velocity (SWV) on ARFI was 1.29 m/s (range 0.93–2.86 m/s). Median liver stiffnesses on TE in patients with F1, F2, F3, and F4 fibrosis stages were 5.4 kPa (range 4.1–6.1 kPa), 7.2 kPa , 11.6 kPa  and 24.6 kPa , respectively. Median SWVs on ARFI in patients with F1, F2, F3 and F4 fibrosis stages were 1.15 m/s , 1.44 m/s , 1.60 m/s , and 2.25 m/s , respectively. Figure 5 days. MSpearman correlation analysis showed that the correlation coefficient between ARFI and TE measurements was 0.76 . Table 3) cells; HCV genotype; increased serum AST, ALT, ALP, and GGT (>2N) and bilirubin concentrations; steatosis; cirrhosis; HIV and HCV treatment; and response to HCV treatment. Only age was associated with the discordance between the two methods (p = 0.047) with older patients in the discordant group. Discordance also tended to be associated directly with hepatomegaly and inversely with overweight, cytolysis, and cholestasis. No significant difference was found with HIV (p=0.22) and HCV(p=0.55) treatment.Of the 46 patients, 14 (30%) showed discordant Metavir scores between the two methods, 11 in predicting F ≥ 2 and three in predicting F ≥ 3. ARFI yielded a higher fibrosis score than TE for 11 of these patients. As the discordance was more important for predicting F ≥ 2, factors possibly associated with these discordances were analyzed, including age; sex; BMI; alcohol consumption; plasma HCV-RNA levels; absolute CD4+  and for the diagnosis of liver cirrhosis (AUROC = 0.93). MoreoveWe observed a moderate agreement between the two techniques for predicting F ≥ 2. Earlier studies also showed that TE or ARFI was more accurate for predicting extreme stages (F1 or F4) than moderate (F2) fibrosis. For example, the AUROCs for ARFI were higher for predicting F=4  than F≥ 2 ,33,49.In addition, agreement between the TE and ARFI scores was determined in patients infected by genotype 1 and 4 who usually have poorer response than patients infected with genotype 2 and 3. Similarly, there was no difference in concordance level between patients with genotypes 1 and 4 and those with other genotypes. The TE and ARFI methods showed very good agreement in predicting overall fibrosis stage and F ≥ 3 in patients with genotypes 1 and 4.This study included a small number of patients (n = 46). However, it was designed as a pilot study, with intraclass correlation coefficients precise enough to draw conclusions by comparing the lower limit of the 95% CIs with 0.6, deemed to represent the lower limit of good agreement.ARFI has several advantages compared with TE. TE has been reported to have an average failure rate of 3.1% and to be highly dependent on BMI. Moreover, TE measurements were estimated to be unreliable in 15.8% of patients in a very large study. We founTE and ARFI showed a discrepancy in predicting fibrosis score in 30% of patients, especially in predicting F ≥ 2. Apart from age, no factor was clearly associated with these discrepancies. However, the study size was not powered for this aim. Liver biopsy should have been performed in patients with discordance, but was not except in one patient, in whom ARFI, but not TE, agreed with biopsy results. Moreover, when a discrepancy was observed between the two techniques, it was not possible to conclude whether TE or ARFI was more correct. These discrepancies may be linked to the cut-off values used, as these are not the same in all published studies. The TE cut-off values we used yielded Finally, although liver biopsy has been considered the gold standard for the assessment of liver fibrosis, we elected to use non-invasive methods for comparison. FibrosiThis pilot study showed good agreement between TE and ARFI in evaluating liver fibrosis in HIV-HCV co-infected patients, suggesting that ARFI may replace TE in this population. In addition to being a new and promising sonography-based method for non-invasive assessment of liver fibrosis, ARFI is cost effective, allowing routine ultrasound examination and fibrosis assessment during the same session.Written informed consent was obtained from all patients for publication of this paper; copies are available for review by the Editor of this journal.This study and its publication were approved by our local ethics committee: CPP Sud-Ouest et Outre Mer III Bordeaux.AASLD: American Association for the Study of Liver Diseases; ALP: Alkaline phosphatase; ALT: Alanine aminotransferase; ARFI: Acoustic radiation force impulse; AST: Aspartate aminotransferase; AUROC: Area under the receiver operating characteristic curve; BMI: Body mass index; CT: Computed tomography; DAA: Direct-acting antiviral agents; GGT: γ-glutamyl transferase; HAART: Highly active antiretroviral therapy; HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus; HIV: Human immunodeficiency virus; INIs: Integrase inhibitors; IQR: Interquartile range; LB: Liver biopsy; LSE: Liver stiffness elastography; MRI: Magnetic resonance imaging; ND: Not done; NNRTIs: Non-nucleoside reverse transcriptase inhibitors; PIs: Protease inhibitors; RNA: Ribonucleic acid; ROI: Region of interest; RT: Reverse transcriptase; RTE: Real-time elastography; SOC: Standard of care; SWE: Shear wave elastography; SWV: Shear wave velocity; TE: Transient elastography; US: Ultrasound.The authors state that they have no competing interests.NF, HT, and PM designed and supervised the study. NF supervised all ARFI measurements. NF, JD, and FM, extracted the data. PP and JA analyzed the data. NF, PM, PP, and JA interpreted the data. NF wrote the manuscript. PM, JA, PP, and HT drafted and finalized the manuscript. All authors revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript. All authors are permanent employees of the Bordeaux University Hospital, which funded this study and the preparation of this manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/14/405/prepub


Within the last 25 years, bacteriophage integrases have rapidly risen to prominence as genetic tools for a wide range of applications from basic cloning to genome engineering. Serine integrases such as that from ϕC31 and its relatives have found an especially wide range of applications within diverse micro-organisms right through to multi-cellular eukaryotes. Here, we review the mechanisms of the two major families of integrases, the tyrosine and serine integrases, and the advantages and disadvantages of each type as they are applied in genome engineering and synthetic biology. In particular, we focus on the new areas of metabolic pathway construction and optimization, biocomputing, heterologous expression and multiplexed assembly techniques. Integrases are versatile and efficient tools that can be used in conjunction with the various extant molecular biology tools to streamline the synthetic biology production line.  •Phage integrases are site-specific recombinases that mediate controlled and precise DNA integration and excision.•The serine integrases, such as ϕC31 integrase, can be used for efficient recombination in heterologous hosts as they use short recombination substrates, they are directional and they do not require host factors.•in vivo and in vitro.Both serine and tyrosine integrases, such as λ integrase, are versatile tools for DNA cloning and assembly •Controlled expression of orthologous serine integrases and their cognate recombination directionality factors can be used to generate living biocomputers.•Serine integrases are increasingly being exploited for synthetic biology applications. The families are identified by the eponymous catalytic residue, that is, tyrosine or serine recombinases. Both families of recombinases comprise members that can mediate integration, excision (resolution) or inversion.Escherichia coli chromosome by a single crossover was first proposed by Campbell attB site, and this recombines with a phage attachment site, attP  and more complex; in addition to the core complementary sequence, attP contains a series of overlapping regulatory binding sites for integrase and accessory proteins  and host-encoded Fis attR inducing a compact loop structure that reconstitutes a tetrameric integrase complex bound to the core sequences that resembles the complex present during integration, but with a modified configuration of the core DNA sequences  Francisella tularensisattB site ccdB, both of which are replaced during the initial cloning step by the desired insert using standard techniques, for example, ligation or Gibson assembly. The reaction mix is then directly transformed into the target cells. As pOSIP also contains an antibiotic resistance gene, an attP site and a cognate temperature-inducible integrase gene (induced immediately after the DNA is introduced by transformation), integration into a compatible chromosomal attB site occurs. The integrase and antibiotic resistance genes are flanked by FRT sites and thus can be removed by expression of Flp recombinase if required. The efficiency of this procedure was tested for five tyrosine integrases endogenous to E. coli  plus one heterologous serine integrase (ϕC31). Multiple orthologous integrases can also be used to mediate simultaneous or sequential integration of different plasmids into their respective attB sites. It is important to note that most of these integration vectors rely on the presence of endogenous attB sites and host-encoded factors; therefore, it is a common practice to source the integrase from a phage infecting the bacterial strain intended for genetic manipulation. Whilst convenient, the disadvantage of relying on the endogenous attB sites is the lack of control on where the attB site is located. One of the recombination systems developed in Clonetegration is the heterologous ϕC31 phage integration system, a serine integrase described below. The ϕC31 attB site was inserted at a pre-defined site in the chromosome, thus expanding the integration strategies available to the researcher These methods were fine-tuned in intB13 λ-like integrase of an integrating conjugative element (ICEclc) found in Pseudomonas knackmussii has recently been shown to catalyse the targeted integration of large cosmid and BAC substrates (up to 75 kb) into the genome of the related Pseudomonas putida species E. coli resulted in severe growth inhibition; therefore, integration vectors were constructed with a hybrid promoter containing lacO sites to tightly repress integrase expression in lacI+ hosts Pseudomonas species, which does not possess lacI, repression is alleviated and efficient integration can occur tnpA mini-transposon that can be mobilised into pre-existing vectors to rapidly produce integrating plasmids without the need for de novo construction.A key goal for synthetic biology is to be able to mobilise large (megabase pairs down to 100 s of kilobase pairs) fragments of DNA for use in diverse target organisms, many of which may not be amenable to standard molecular genetic techniques. Integrases can provide a means of efficient integration of large DNA fragments into specific genomic locations. For example, the in vitro application of λ integrase to facilitate rapid cloning of linear DNA into an “entry vector” and subsequent exchange into specialised plasmids for downstream use att sites; the identity of these att sites (attP/B/L/R) relative to the insert allows the transfer of target DNA to be precisely manipulated. For example, an insert flanked by two attB sites will be swapped with another insert flanked by two attP sites when incubated with purified integrase and IHF. Supplementation with excisionase protein will catalyse the reverse reaction to restore the original substrates. Use of a recombinase in this manner removes the problems associated with traditional cloning, for example, reliance on the presence and compatibility of restriction sites, time-consuming reactions and multiple purification steps. This simple application has appeared in hundreds of publications, demonstrating the huge potential for optimization and commercialisation of recombinase technology.The most widely used tyrosine integrase recombination system is the Gateway™ cloning method . Gateway™ is based on an attP substrate to favour efficient integration or excision reactions. Consequently, the attP site is long and complex to accommodate multiple integrase and accessory protein binding sites  and the need for host proteins that limit the potential for cross-species or cross-genera use.Although useful for relatively straightforward cloning or DNA mobilization applications, there are several major drawbacks of phage-encoded tyrosine integrases. First is a requirement for host-encoded accessory proteins to bend the ng sites . There aattB and attP sites are typically ≤ 50 bp with different inverted repeat sequences flanking 2 to 12 bp of complementary sequence, which includes the recombination crossover point  for integration. Most serine integrases are inert on attL and attR, although traces of the excision reaction have been observed in some systems attL and attR sites and thus activating excision, and second, by inhibiting synapsis between attB and attP sites, reducing integration Streptomyces phages ϕBT1 and TG1, and there are numerous mycobacteriophages with RDFs related to Bxb1 gp47 In phage integration reactions that depend on serine integrases, the only protein required to mediate att sites and accessory proteins mean that in vitro and in vivo strategies for DNA assembly and other applications are far easier to design and optimize. This point is particularly salient when the system is applied to heterologous organisms that may share very little in common with the native host. Furthermore, serine integrases do not require any of the substrates to be supercoiled so that linear fragments can be recombined efficiently in vitro in both integration and excision reactions.In practice, the minimalist requirements of serine integrases for Streptomyces, which is the natural host for ϕC31 and therefore contains an innate attB site for integration The original application of ϕC31 integrase was in the development of integration vectors for use in loxP” and the yeast “Flp/FRT” systems, both of which use tyrosine recombinases loxP and Flp/FRT regenerate the original substrates after recombination. Consequently, both systems are efficient at creating deletion mutants by excising DNA located between directly repeated recombination sites but their reversibility is problematic for stable DNA integration. This problem can be mitigated by recombination site mutations in either of the inverted repeats or the central spacer region  site whilst retaining compatibility with an identical mutant site loxP sites , cassette exchange is promoted and unwanted deletion events precluded loxP site and the right is mutated on its partner site, recombination will produce a WT site and an inactive double mutant, thus inhibiting further recombination.The use of serine integrases in heterologous hosts took hold very quickly after their biochemical properties were first described r region a that fan et al.. For exaatt sites and, for this reason, can also be used for applications that require efficient DNA integration in addition to cassette exchange. Simple modifications have been used to increase serine integrase activity in heterologous systems, for example, the introduction of a nuclear localization signal open reading frame) to an endogenous 3′ untranslated region to favour in vivo expression in zebrafish attP sites attP sites has been used in several medicinally relevant studies, for example, production of therapeutic levels of human factors VIII and IX in mice attP integrations in three human cells lines detected 101 pseudo-attP sites that could be used by ϕC31 integrase attP sites for site-specific integrations. First, integrations into the pseudo-attP sites are often non-reciprocal; that is, small deletions were a frequent occurrence in the flanking DNA attP sites or whether these events are simply “ectopic” or random integrations. Either way, the frequency of use of pseudo-attP sites is very low as integrase can be expressed constitutively in the presence of one of its attachment sites over many cell duplications without any observable gene rearrangements occurring at the cognate attachment site The major advantage of the serine integrases is that they can catalyse highly efficient irreversible recombination using simple attP knock-in. Pronuclear injection of plasmid DNA into zygotes for this mouse line yielded 40% of pups with the desired DNA located in the docking site In order to make use of the high efficiency of site-specific recombinases, a docking or landing site containing an attachment site is normally introduced into the chromosome of a cell line or animal that will recombine with the partner attachment site in the presence of integrase. The docking site/landing pad is inserted either randomly into the target genome using established methods of transgenesis or by homologous recombination, as demonstrated in mouse embryonic stem cells or in the chicken DT40 cell line DrosophilaAnophelesDrosophila has had a substantial enabling effect on genetic research in this model organism as the introduction of different alleles at a single position permits greater consistency between constructs and largely avoids transgene position effects Arabidopsis, barley and wheat Docking sites have also been successfully introduced into insects, including loxP site by a serine integrase from either ϕC31 or ϕBT1 et al. use a combination of recombinases to increase the range of genome engineering approaches in mice attP docking site creating both a null mutant and the opportunity to reinsert different alleles. The technique uses Flp/FRT to remove unwanted vector and marker sequences and leaves the researcher the option of using the Cre/loxP system elsewhere in the genome. This approach has been adopted by the North American Conditional Mouse Mutagenesis ProjectUse of multiple site-specific recombinases has become possible since the advent of ϕC31 integrase-mediated genome engineering. An iterative assembly method has been developed in mammalian cells whereby successive rounds of plasmid integration using Cre recombinase are followed by removal of the selection marker and E. coliMicrobes have long been used to manufacture useful transgenic compounds, a successful example of which is artificial human insulin production by Streptomyces phage ϕBT1 has been used to assemble in vitro DNA fragments encoding the genes required for large and complex secondary metabolic pathways. The use of the integrase-mediated DNA assembly modularizes the pathway such that parts can be easily changed without having to reclone the entire pathway from the beginning attB and attP sites in order for recombination to occur, therefore allowing six orthologous target sites. Using this system an ~ 62 kb epothilone synthesis cluster was constructed, including the large epoD gene (~ 22 kb), in two construction steps. At least seven fragments could be stitched together in a single in vitro reaction.The integrase isolated from the et al. but based on ϕC31 integrase  or non-directional tyrosine recombinases (Cre and Flp)  stimuli  [106,107in vivo and in vitro. For many organisms or cell lines, the level of homologous recombination is extremely low and is not a viable route to generating desired genetic constructs. In these situations, the greater efficiency of site-specific recombination is an extremely useful substitute as demonstrated by the many researchers who have deployed integrases for use in higher eukaryotes. Although some integrases, notably ϕC31 and Bxb1 integrases, appear to be “plug and play” and display high levels of portability from one organism to another, others are only active in bacteria attL or attRPhage integrases are tools for generating precise DNA rearrangements The most exciting and promising applications of phage integrases, particularly serine integrases, lie in the field of synthetic biology. These have been exemplified by their uses in metabolic pathway assembly and in the construction of digital counters and logic gates. What is needed in this field are more integrases that act orthogonally and can be multiplexed for greater versatility and increased efficiency. The use of integrases for modularising and assembling complex pathways will greatly facilitate optimization of metabolite production and allow these pathways to be easily transferred into different hosts. Moreover, the use of RDFs to retrofit metabolic pathways should also streamline the modification of previously constructed pathways. Serine integrases complement other ingenious DNA assembly methods currently available, in particular, the seamless DNA assembly methods such as Gibson assembly, SLICE and overlap extension polymerase chain reaction. It used to be the case that cloning a single gene would take at best a couple of weeks. We now hope that we can use that time more effectively in designing and reconstructing whole metabolic pathways and even whole chromosomes.