Taking advantage of the human cytomegalovirus-encoded TAP inhibitor US6 as a luminal sensor for conformational changes of TAP, we demonstrated that the 50PLL52 motif is essential for freezing of the TAP conformation. Moreover, hierarchical functional interaction sites on TAP dependent on 50PLL52 could be defined using a comprehensive set of human-rat TAP chimeras. This data broadens our understanding of the molecular mechanism underpinning TAP inhibition by ICP47, to include the 50PLL52 sequence as a stabilizer that tethers the TAP-ICP47 complex in an inward-facing conformation.The transporter associated with antigen processing (TAP) translocates antigenic peptides into the endoplasmic reticulum (ER) lumen for loading onto MHC class I molecules. This is a key step in the control of viral infections through CD8+ T-cells. The herpes simplex virus type-1 encodes an 88 amino acid long species-specific TAP inhibitor, ICP47, that functions as a high affinity competitor for the peptide binding site on TAP. It has previously been suggested that the inhibitory function of ICP47 resides within the N-terminal region (residues 1–35). Here we show that mutation of the highly conserved  Whereas self-peptides are typically ignored, antigenic peptides are recognized by surveilling cytotoxic CD8+ T-lymphocytes, which subsequently become activated and eliminate the infected cell. The MHC class I peptide ligands are produced in the cytosol by proteasomal degradation. Therefore, for loading onto MHC class I molecules the peptides have to cross the endoplasmic reticulum (ER) membrane by active transport. This process is performed by the transporter associated with antigen processing (TAP)3, with the six C-terminal transmembrane helices (TM1-6) of each subunit together forming the core of the peptide translocation pore4. The N-terminal TMs, or TMD0, link TAP to the MHC class I peptide loading complex by binding to the chaperone tapasin7. The TMD0 is dispensable for peptide transport5, but the interaction with tapasin is crucial for efficient MHC class I loading8.TAP belongs to the large family of ATP binding cassette (ABC) transporters that are responsible for translocation of a broad spectrum of substrates across cellular membranes. The characteristic structure of ABC transporters comprises two transmembrane domains (TMD) and two nucleotide binding domains (NBD). The TAP complex is heterodimeric, being composed of TAP1 and TAP2 subunits, each of which comprises a NBD and a TMD. The TMD spans the ER membrane 10 times11, a process involving precisely coordinated major structural rearrangements of the TMDs and the NBDs10. A transmission interface between the NBDs and TMDs is formed by the so-called coupling helices (CH1 and CH2), which are located in the extended cytosolic loops between the TMs 2 and 3 and TMs 4 and 512. However, TAP1 and TAP2 are heterologous, possessing approximately 33% sequence homology and play different roles during the translocation cycle14. TAP1 contains a degenerate ATP binding site with non-canonical variations in the Walker A and B motifs, rendering ATP binding and hydrolysis by the Walker A and B motifs of TAP2 crucial for the peptide translocation cycle17.The NBDs bind and hydrolyze ATP, which is absolutely required to accomplish active and unidirectional peptide transport19, US6 22, BNLF2a 23, UL49.5 (varicelloviruses)24, and CPXV12 (poxvirus)25. Initially observed as an HSV-1 early gene product inhibiting antigen presentation to CD8+ T-cells26, ICP47 was the first TAP inhibitor described19. ICP47 is a small (88 amino acids (aa)) protein that is localized within the cytosol26, where it is considered to function as a high affinity competitor28 that blocks peptide binding to TAP. Notably, ICP47 is highly species specific, and can effectively inhibit human but not mouse TAP28. Functional analysis of ICP47 truncation mutants revealed that the amino acids 2–35 are sufficient for efficient inhibition of peptide translocation30. The structure of ICP47-bound TAP has been resolved by cryo-EM analysis to an overall resolution of 6.5 Å31. TAP adopted a structure very similar to other ABC transporters such as MsbA and P-glycoprotein and was locked in an inward-facing conformation due to its interaction with ICP47. More specifically, the region of ICP47, that was modeled in to the electron density map (residues 3–50), adopted a helix-turn-helix hairpin-like structure that wedged into the closed pore formed by the core TMs 1–6 of TAP1 and TAP2. Here, the high binding affinity of ICP47, which exceeds that of peptide substrates27 was attributed to the buried surface of the TAP-ICP47 interface. However, precisely how ICP47 so efficiently holds TAP in an inward-facing conformation remains unclear.Large DNA viruses have developed numerous strategies to interrupt the MHC class I antigen presentation pathway. To date five virally encoded inhibitors have been reported to directly block TAP function: ICP47 29. Indeed, the most conserved sequence of ICP47 and its homologs within the alpha-herpesvirus family is found in the central portion of the protein (aa 34–52)32. We now present data demonstrating that this region of ICP47 is responsible for freezing of the TAP conformation. We were able to narrow down the responsible residues of ICP47 to the positions 50–52 (ProLeuLeu), which are localized in close proximity to the TAP2 CH1 coupling helix. Here we propose a revision of the mechanism of ICP47-mediated TAP inhibition, in which the N-terminus (1–35) prevents peptide binding to TAP, but the 50PLL52 motif can induce freezing of TAP conformation and thereby block its function completely.Although the N-terminal segment of ICP47 is sufficient to block peptide binding to TAP, it has been previously suggested that sequences located at the central or in the C-terminal segment of ICP47 could also be important for TAP inhibition30. In these studies ICP47 was produced synthetically or was recombinantly expressed and analyzed using microsomes or permeabilized cell systems. These findings have never subsequently been confirmed in intact cells. We hypothesized that the highly conserved region of ICP47  somehow retains TAP in an inward-facing state.d values of 126 and 50 nM, respectively30. This suggested that the residues 49–53 were able to increase the affinity of ICP47 for TAP. Notably, this region contained three highly conserved residues  expressed as a fusion protein is able to bind to TAP, but this binding is not sufficient to efficiently block TAP function in a cellular context. Instead, complete arrest of the transport activity was only observed when the amino acids 36–57 were also present. To exclude the possibility that the loss of function of ICP47-N-TG was due to conformational changes induced by the C-terminal linker sequence, we performed site-directed mutagenesis on ICP47 fused to a C-terminal HA-tag (ICP47-HA). In a previous study, in which the affinity of various synthetic C-terminally truncated ICP47 fragments to TAP was determined, a noticeable difference in affinity was observed between the fragments comprising the aa 1–48 and 1–53 with K50PLL52 in the central part of ICP47 are involved in the freezing of the TAP conformation.To test whether residues 50–52 are required for a full arrest of conformational cycling of TAP, HeLa cells were stably transduced with the ICP47-HA constructs and then transiently transfected with US6-HA. Co-IP experiments were then performed using anti-US6 antiserum, and TAP binding to US6 was visualized by detection of TAP1 via Western blot. As expected, WT ICP47-HA blocked US6 binding to TAP Fig. , however27. Taking advantage of the well-documented species specificity of ICP4723, we set out to analyze the influence of WT ICP47-HA and ICP47m-HA on mixed combinations of human (h) and rat (r) TAP subunits . We used a rat TAP1 subunit with a human C-terminus (starting in the NBD at residue 539) to allow for detection by the anti-human TAP1 mAb 148.3. The TAP2 subunits were tagged with a FLAG-epitope at the C-terminus. TAP1 and TAP2 subunits were expressed in TAP-deficient mouse CMT64.5 cells by lentiviral transduction using the selection markers puromycin and neomycin, respectively. Subsequently ICP47-HA and ICP47m-HA were transduced using a bicistronic construct encoding EGFP to allow gating on ICP47 expressing cells during FACS analysis.Next we sought to investigate whether residues 50–52 of ICP47 influence the manner by which ICP47 interacts with TAPDifferent combinations of human and rat TAP1 and TAP2 subunits induced the expression of MHC class I to a similar extent on the CMT64.5 cells Fig. . Express3 were overexpressed in the CMT64.5 cells, the TAP sequences in HeLa cells are not known. This issue should be revealed in future experimental efforts.The inhibition of hT1/hT2 by ICP47m-HA was surprisingly strong in this system Fig. . A possi50PLL52 sequence, impairment of peptide transport via human TAP2 was not possible, emphasizing the importance of this sequence for the inhibitory function.While the ICP47m-HA mutant showed a species-specific regulation of TAP, no inhibition of mixed TAP subunits (including rT1/hT2) was observed. This indicated that without the Next, we analyzed binding of ICP47-HA to combinations of human and rat TAP subunits by co-IP and found a clear correlation between the extent of inhibition and binding. Transduction levels of the ICP47-HA constructs varied, as indicated by the relative intensity of the bands detected by Western blot analysis Fig. . Combina35. We used the same chimeric constructs as in our previous study3, but now renamed them  that renders this inhibitor human TAP-specific and thus identified several US6 interaction sites on TAP1 and TAP2The TAP1 chimeras were tagged with a Myc-tag at the C-terminus and the TAP2 chimeras with a FLAG-tag. The chimeras were stably expressed in CMT64.5 cells with either a human or rat counterpart. The expression levels of MHC class I on these cells varied between the different combinations of chimeric subunits Fig. , howeverAnalysis of the TAP2 chimeras revealed more than one interaction site between ICP47 and TAP2. The chimera 1rT2 was similarly inhibited by ICP47-HA as WT hT2 in combination with either human or rat TAP1 see Fig. , indicatThe use of TAP1 chimeras revealed a more narrow interaction with ICP47 than observed for TAP2. The chimeric TAP1 molecule 4rT1 mimicked hT1 in all aspects Fig. , also in30. Subsequent structural data revealed that this region adopts a helix-turn-helix conformation that inserts in to the pore formed by the TMDs of TAP1/TAP234 at a location that overlaps with the putative peptide-binding site suggested by mutagenesis and crosslinking experiments39. Thus it is now well established that ICP47 physically blocks binding of peptides to TAP. However, Galocha et al. have speculated that an additional stabilizing function could be conferred by the highly conserved sequence (34–52) of ICP47, which lies outside of the defined active domain. These authors commented that such an effect would not have been possible to measure using their particular experimental setting29. Here we confirm the existence of residues downstream of aa 35 that have a functional impact on TAP inhibition. Firstly, we used fusion proteins to analyze ICP47 truncation mutants, which strongly suggested that the residues 1–35 (ICP47-N-TG) do not possess the full inhibitory potential of ICP47. However, since fusion constructs of truncated ICP47 might cause inadvertent effects, we also mutated the conserved residues 50PLL52 of ICP47 full-length HA-tagged protein to alanine residues that are supposed to be functionally neutral (ICP47m-HA). The fact that the mutant ICP47m-HA and the fusion protein ICP47-N-TG only poorly blocked MHC class I cell surface expression, but to a similar extent, indeed supported the hypothesis that the function lacking in the ICP47-N-TG fusion protein is conferred by residues 50–52 of ICP47.ICP47 encoded by HSV-1 was the first TAP inhibitor identified and its inhibitory mechanism has been extensively investigated. Previous studies indicated that the active domain of ICP47 is located within the first 35 N-terminal residues3. In addition, US6 binds to TAP in a peptide-bound state21, implying recognition of a conformation distinct to that stabilized by ICP47. We therefore hypothesized that US6 would not be able to bind TAP complexes, if ICP47 was allowed to first freeze their conformation. Indeed this was the case, which clearly demonstrates that ICP47 and US6 bind to TAP during different steps of the peptide translocation cycle. We also performed the reciprocal experiment and likewise observed that prior expression of US6 strongly blocked ICP47 binding to TAP  blocks the binding of peptides to TAP, whereas the central region (residues 50–52) contributes to a conformational freezing of the TAP translocation cycle. This model is entirely consistent with the near-atomic resolution (4 Å) structure of TAP bound to ICP47lix Fig. 12. Thus,50Leu51Leu52 to AlaAlaAla mutation of ICP47 led to a loss of the observed partial TAP inhibition via hTAP2 when combined with the rTAP1 subunit. Inhibition by the ICP47 mutant was observed exclusively when both TAP1 and TAP2 were of human origin. From this we conclude that ICP47 interacts with the TMDs of both TAP1 and TAP2. These interactions sites do not contribute equally to the inhibitory efficiency of ICP47, but seem to have hierarchical influences . These findings are in good agreement with the structural ICP47-TAP model. Interaction with the region of the TMs 2–4 of TAP2 is sufficient to mediate inhibition when concurrent binding to TAP1 and interaction via the 50PLL52 site occurs. However, if one of the latter two sites is missing, inhibition is lost. Additional interaction with the region of TMs 5–6 of TAP2, however, leads to an inhibitory effect even in the absence of either TAP1 or the 50PLL52 sequence.In the structural model by Oldham 34, which suggests that Gln456 from the TAP1 TM6 could potentially form interactions with Asn14 and Arg16 within the ICP47 interconnecting loop, although the resolution was not sufficient to accurately determine the side chain conformations in this region. Nevertheless, it is interesting to note that residue 456 in rat TAP1 is an arginine, and the presence of a basic residue in this position could potentially lead to charge repulsion with Arg16 of ICP47, thereby explaining why ICP47 does not bind to rat TAP1. Interestingly, in our previous study focused on US6 the 4rT1 chimera was efficiently blocked by US63. This raises the intriguing question as to whether ICP47 and US6 could target TAP1 at the same position with US6 possibly reaching downwards into the open pore in the TAP outward-facing conformation, and ICP47 contacting the same position from the cytosolic side but in the inward-facing conformation.Analysis of ICP47 binding and inhibition of TAP1 chimeras suggested a more selective interaction with TAP1. In particular, using chimeric human/rat TAP1 subunits, only the region of TMs 5–6 (4rT1) was found to be important for ICP47 inhibition. This finding is consistent with the recently published structural model28 but also an additional ICP47-TAP interaction that results in a freezing of TAP conformational change, likely via a stabilization of the TAP2 coupling helix. Understanding of the mechanisms by which ICP47 blocks conformational changes of TAP will give important insights into how ABC transporters transmit structural changes in the TMDs to the NBDs. Precise identification of the site targeted by the residues 50–52 of ICP47 on the TAP peptide transporter will possibly open new avenues for the development of general inhibitors of ABC transporters.In summary, our study for the first time demonstrates a complex two-tiered mechanism of TAP inhibition by ICP47, involving not only the previously described blocking of peptide binding to TAP41 and HEK293T (ATCC CRL-1573) cells were grown in DMEM supplemented with 10% FCS, penicillin and streptomycin. When indicated, cells were transfected using Superfect (Qiagen) following the manufacturer’s protocol. In this study the following antibodies were applied: rabbit anti-TagGFP (Evrogen); the mAb 148.3 for TAP1 detection 42; rabbit anti-calnexin (Stressgen) and mouse anti-β-actin (Sigma-Aldrich); the mAb W6/32 for the detection of fully conformed, peptide-loaded HLA-A,B,C molecules43; APC-coupled anti-mouse antibodies (BD Pharmingen); anti-mouse anti-HA antibodies for flow cytometry and Western blot analysis (Sigma-Aldrich); rabbit anti-HA for immunoprecipitation (Sigma-Aldrich); anti-c-Myc and anti-FLAG clone M2 (Sigma-Aldrich); the mAb 20-8-4S specific for H-2Kb and H-2Db 44. The polyclonal anti-US6 serum was described before3.HeLa (ATCC CCL-2), CMT64.545. The transduced cells were cultivated in normal medium for 1–2 days before treatment with puromycin  or with geneticin .Production of lentiviruses was performed as described previously4GGSGS and were cloned into NheI and BamHI sites after amplification using the forward primer 5′-gctgctagcatgtcgtgggccctggaa-3′ for all constructs and the reverse primer 5′-cgaggatccgctgcctcctgcagcggccgctccggacgcataatccggcacatcatacgg-3′ for ICP47-WT-TG, 5′-cgaggatccgctgcctcctgcagcggccgctccggactcacgcccccttttattgatctcatcg-3′ for ICP47-N-TG, and 5′- cgaggatccgctgcctcctgcagcggccgctccggagggagagcgcagcagagg-3′ for ICP47-eN-TG. To allow for lentiviral transduction, the ICP47-TagGFP constructs were subcloned into the NheI site of puc2CL6Ipwo vector after excising the fusion constructs with NheI and XbaI from pcDNA3.1. Cloning of ICP47-HA into pcDNA3.1 was described before46. ICP47m-HA was produced by site directed mutagenesis using the Q5 Site-Directed Mutagenesis Kit (NEB). US6-HA was cloned into NheI and BamHI of pIRES-EGFP using the primers 5′-cgtgctagcatggatctcttgattcgtctcg-3′ and 5′-gcaggatccttacgcgtaatctggaacatcgtatgggtaagcggagccacaacgtcgaatc-3′.The ICP47-TagGFP fusion constructs were cloned into pcDNA3.1. First TagGFP was cloned into BamHI and XhoI. ICP47 C-terminal truncation mutants were amplified without a stop codon and the sequence at the 3′-end encoding the linker SGAFor construction of HA-tagged MHC class I, the tapasin signal peptide sequence was amplified with a HA-tag sequence using the primers 5′-cgtctcgagatgaagtccctgtctctgctcc-3′ and 5′-cctactgcagctgcgtaatctggaacatcgtatgggtacaccgcgggtcctgctga-3′and cloned into XhoI and PstI sites of pIRES-EGFP (Tpn-SP-pIRES-EGFP). HLA-B*44:02 was amplified with the primers 5′-cgtatgcattaggaggctcccactccatgagg-3′ and 5′-cgaggatccttcaagctgtgagagacacatc-3′ and HLA-B*15:03 with 5′-cgtatgcattaggaggctcccactccatgagg-3′ and 5′-cgaggatcctcaagctgtgagagacacatcag-3′ and cloned into PstI (PCR products were digested with NsiI) and BamHI sites of Tpn-SP-pIRES-EGFP. Sequenced inserts were subsequently subcloned into the puc2CL6Ipwo lentiviral vector using the restriction sites NheI and BamHI. Lentiviral vectors were also used for transient expression of MHC class I molecules.3. The names of the constructs used in the previous study and the epitope tag added in this one are given in Supplemental Table Cloning of human and rat TAP1 and TAP2 subunits and chimeras thereof was described beforeCells were trypsinized and stained with antibodies diluted in 3% FCS/PBS. Cells were washed in 3% FCS/PBS, supplemented with DAPI and measured by FACS Canto II (Becton Dickinson). Acquired data was analyzed by FlowJo .6 cells were lysed in 0.5 ml of digitonin lysis buffer ) for 30 min and cleared from membrane debris at 13,000 rpm for 30 min at 4 °C. Lysates were incubated with antibodies for 3–4 h at 4 °C in an overhead tumbler before immune complexes were retrieved by protein A-Sepharose . Sepharose pellets were washed four times with increasing NaCl concentrations (0.15 to 0.5 M in lysis buffer containing 0.2% detergent). Retrieved proteins were separated by SDS-PAGE and blotted onto a nitrocellulose membrane. Proteins were detected after treatment with specific antibodies followed by peroxidase-conjugated secondary antibodies and the SignalFire ECL Reagent . The Biorad Image Lab software was used for quantification of band intensities measured with a Biorad Chemidoc XRS system. Blots were rotated and cropped for convenient presentation of data using Adobe Photoshop CS5. Examples of the procedure used for presentation of blots are shown in Figs Cells were detached by scraping and washed with PBS. Ca. 3 × 1047. 2 × 105 HeLa cells were semi-permeabilized using streptolysin O . For permeabilization with SLO HeLa cells were incubated at 4 °C for 15 min and then washed in PBS to remove residual SLO. Transport was carried out in the presence of 10 mM ATP/ADP, 5 nM NST-Cy5 in PBS buffer supplemented with 10 mM of MgCl2 for 15 min at 37 °C in 50 μl. The reaction was stopped by addition of 150 µl PBS supplemented with EDTA (20 mM). Samples were acquired with the LSR II (BD) and analyzed using FlowJo 7.6.5 software reporting the mean fluorescence intensity (MFI). For HeLa cells expressing the ICP47 fusion proteins, MFI was analyzed from GFP positive cells only. The NST-Cy5 (RRYQNSTC(Cy5)L, cysteine labeled with Cy5) peptide was synthesized by the platform “Peptide Synthesis” of the Helmholtz Centre for Infection.Peptide translocation was performed as previously describedSupplementary Information


The signal transducer and activator of transcription 5 (STAT5) regulates differentiation, survival, proliferation and transformation of hematopoietic cells. Upon cytokine stimulation, STAT5 tyrosine phosphorylation (pYSTAT5) is transient, while in diverse neoplastic cells persistent overexpression and enhanced pYSTAT5 are frequently found. Post-translational modifications might contribute to enhanced STAT5 activation in the context of transformation, but the strength and duration of pYSTAT5 are incompletely understood. We found that O-GlcNAcylation and tyrosine phosphorylation act together to trigger pYSTAT5 levels and oncogenic transcription in neoplastic cells. The expression of a mutated hyperactive gain-of-function (GOF) STAT5 without O-GlcNAcylation resulted in decreased tyrosine phosphorylation, oligomerization and transactivation potential and complete loss of oncogenic transformation capacity. The lack of O-GlcNAcylation diminished phospho-ERK and phospho-AKT levels. Our data show that O-GlcNAcylation of STAT5 is an important process that contributes to oncogenic transcription through enhanced STAT5 tyrosine phosphorylation and oligomerization driving myeloid transformation. O-GlcNAcylation of STAT5 could be required for nutrient sensing and metabolism of cancer cells. Both are linked to transformation due to deregulated kinase/phosphatase signaling or STAT5 gain-of-function (GOF) mutations.13Cytokine binding to the cell surface leads to phosphorylation of the receptor complex by receptor associated Janus kinases (JAKs) at tyrosine residues.14 Interestingly, >4000 proteins are modified by O-linked β-D-N-acetylglucosamine (O-GlcNAc).15 The dynamic addition and removal of O-GlcNAc has been described for multiple cytoplasmic and nuclear proteins and it affects the function of various signaling molecules and transcription factors.16 The cycling of O-GlcNAc is dependent upon UDP-GlcNAc, synthesized in the hexosamine biosynthesis pathway,17 which serves as substrate for the O-GlcNAc transferase (OGT). OGT transfers O-GlcNAc moieties from UDP-GlcNAc to serine and threonine residues of proteins and O-GlcNAcase (OGA) catalyzes the opposite reaction to remove O-GlcNAc.18 Both enzymes, OGT and OGA as well as multiple O-GlcNAcylated proteins are enriched at sites of active transcription in human cells. O-GlcNAcylation regulates the transcriptional activity of p53, c-Myc, FoxO1 and CREB, as well as components of the basal transcription machinery, for example, the C-terminal domain of RNA polymerase II.16 Cancer cells exhibit elevated levels of O-GlcNAc, possibly to adjust to stringent metabolic demands and O-GlcNAcylation is a key metabolic regulator of glucose metabolism.18Additional post-translational modifications, such as ubiquitination, sumoylation, acetylation and glycosylation have been detected in STAT5.Here, we addressed the biological function of O-GlcNAcylation of STAT5. We characterized glycosylation deficient and/or hyperactive STAT5 variants and compared their oncogenic potential. The inability of STAT5 to be O-GlcNAcylated resulted in impaired tyrosine phosphorylation and a decrease of its transactivation potential. Moreover, hyperactive GOF STAT5A proteins increased phosphatidylinositol 3-kinase (PI3K)-AKT and ERK kinase activity in a glycosylation-dependent manner.19 The positions of the eight alpha-helices in STAT5 were modeled based on the STAT4 N-terminal structure using Swissmodel23 (http://swissmodel.expasy.org/). The crystal structure was obtained from the RSCB Protein Data Bank (www.rcsb.org)24 PDB ID 1BGF25 and colored using ADOBE PHOTOSHOP CS5 EXTENDED Version 12.0.4 x32 . For the prediction of phosphorylation sites, PhosphoSitePlus (www.phosphosite.org) was used.26The human STAT5 N-terminal (accession number P42229) sequence was obtained from The Universal Protein Resource (UniProt).Stat5a-pMSCV-IRES-GFP retroviral vector was used to obtain viral particles and to infect cells. All mutants were obtained through PCR-mediated mutagenesis and DNA sequences were verified. Ecotopic, replication-incompetent, retroviral gpE+86 producer cell lines were generated and selected for high viral titers (~106 particles/ml) by fluorescence-activated cell sorting (FACS). Producer cells were maintained in Dulbecco modified Eagle medium . Bone marrow (BM) was isolated from the hind limb of 12-week-old male mice (B6129F1) and cultivated . Single cell suspensions were prepared from BM and spleen. Cells were stained with antibodies conjugated to phycoerythrin or APC  and analyzed with Becton-Dickinson FACScalibur. Immunohistochemistry of liver, spleen and lymph node sections was performed and cells were analyzed by histo-pathology as described.27 Blood smears were stained with Benzidine.28 The mouse studies were performed in accordance to ethical approval by Austrian government authorities according to license BMWF-66.009/0282-II/3b/2012.The ated see . BM cellΔN/ΔN mice without splenomegaly were isolated and transferred into culture as described.29 Isolated splenic T-cells were co-cultured on viral producer cells as described27 and stimulated with anti-CD3 antibody (145.2C11) . Different concentrations of interleukin (IL)-2 or IL-4  were used for proliferation assays measured by 3H-thymidine  incorporation using a Wallac 1450 MicroBeta liquid scintillation counter  18 h after adding 1 μCi/well.Splenic T-cells from 12-week-old wild type (wt) and STAT5The glycoprotein isolation kit  was used and samples were prepared according to the manufacturer’s instruction. Pull down and flow through were used for immunoblotting.Statistical calculations were performed using the GraphPad Prism 5.01 software . Results are presented as means±standard deviation (s.d.). As statistical test either one-way analysis of variance or two-way analysis of variance was performed, as indicated in the corresponding legend. The experimental design and mouse numbers take into account variability our laboratory has observed through years, and will be sufficient to obtain valid results, no recipient animal was removed or excluded from analysis. Block randomization method for animal groups was performed in regard to BM donation for transplant settings of male donors and wt female recipients of B6129F1 genetic origin. Histo-pathology analysis was blinded by two board-certified pathologist evaluators.Cell culture techniques, DNA-binding assay, real-time PCR, primer sequences, immunoblotting and immunoprecipitation analysis are described in 30 This threonine is located within the N-terminal oligomerization domain of STAT5. The S5-T92A construct was mutated at position 92 and the threonine residue was replaced by alanine, preventing O-GlcNAcylation. cS5 is a hyperactive STAT5A variant, with a GOF mutation at serine 710 to phenylalanine (S710F)8 and was combined with the T92A variant, producing the cS5-T92A construct.STAT5A (S5) contains a threonine at position 92 , which i+86 cells could be shown in separate WGA assay  in immunoblotting experiments to visualize their presence in human cancer cell lines. The specificity of the antibody was evaluated in a competitive immunoblot. Pre-incubation with 100 me signal . In a poe signal . We detee signal . The megTo investigate the functional properties of the STAT5 variant with the T92A mutation, we performed DNA-binding assays. In all, 293 T cells were transfected with the S5, cS5, S5-T92A and the cS5-T92A constructs together with the erythropoietin receptor (EpoR) followed by EPO stimulation. Immunoblots of whole-cell extracts revealed that tyrosine phosphorylation can be induced by EPO stimulation in all STAT5 variants . An elecWe used murine Ba/F3 cells to evaluate the phospho-tyrosine levels of the STAT5 variants and measured response to IL-3 induction. As expected, Ba/F3 cells expressing the cS5 variant exhibit high pYSTAT5 levels, even in absence of IL-3 . This isPim1. STAT5 target gene expression levels were similar to those found in the parental Ba/F3 cells in cS5-T92A-expressing cells and significantly decreased in cS5-T92A compared with cS5. Thus, the threonine 92 mutation neutralized the enhanced STAT5 target gene expression in cS5 upon cytokine deprivation. To further study the interaction of O-GlcNAc levels and tyrosine phosphorylation, transfected Ba/F3 cells were treated with Alloxan an OGT inhibitor.31 By inhibition of OGT the pYSTAT5 levels were decreased at 30 mM  stem-cell antigen 1 (SCA1+) KIT+ (LSK) cells revealed that cS5 mice had an increased number of differentiated cells in the lineage FACS panel .26 We used the hyperactive cS5 variant to study the influence of O-GlcNAcylation on the phosphorylation state, the strength of STAT5 activation with target gene induction upon cytokine stimulation, and functional properties for transformation. Overall, STAT5 tyrosine phosphorylation and O-GlcNAcylation are functionally intertwined. Non O-GlcNAcylated STAT5 molecules can still be activated by tyrosine phosphorylation, maintain their DNA-binding activity and form tetramers, but they exhibit a much lower transactivation potential since they are not as efficient tyrosine phosphorylated and, therefore, deactivated faster by tyrosine phosphatase recycling.Signaling through STAT molecules is modulated by post-translational modifications and cofactor interactions.ΔN/ΔN T-cells, that display severely impaired proliferation in response to IL-2 or IL-4.29 Both, cS5 and cS5-T92A complemented IL-2- or IL-4-diminished T-cell proliferation to similar extends. Second, upon BM transduction followed by transplantation the cS5-T92A variant remained stably expressed in the erythroid lineage up to 9 months analyzed post transplant (It could be that diminished tyrosine phosphorylation of STAT5 and the decrease of the transforming properties in the cS5-T92A variant are associated with changes in protein stability. We exclude a severe change in protein stability of the cS5-T92A variant compared with wt STAT5A due to two different cytokine signaling read out assays: First, we performed a T-cell rescue assay with STAT5ansplant .34 Second, JAK2 kinase could bind more efficiently to O-GlcNAcylated STAT5. Third, tyrosine kinase activity might be enhanced in cS5-expressing cells. JAKs are highly phosphorylated proteins, with described negative and positive phosphorylation sites for kinase activity, where pAKT and pERK activation as downstream substrates of activated JAK2 are well known.35 Fourth, the reported docking and activation of cS5 to PI3K-AKT could be changed.32 The strong and prolonged tyrosine phosphorylation of the cS5 variant is connected to an increase of IL-3 signaling, but also absence of IL-3 stimulation displayed enhanced AKT and ERK activation. Interestingly, threonine 92 of STAT5A is not only the major site of O-GlcNAcylation30 but it was also described to be phosphorylated.26 T92 is located in a repeat of a four amino acids ATQL motif, which is also present at T58, conserved in other STATs and shown to be glycosylated.30 If glycosylation at T92 impacts modification at T58 and vice versa remains unsolved.26 Although, T92 is conserved among the STAT5A/B proteins, there was no significant change in tyrosine phosphorylation in the T92A mutant in human STAT5B and the hyperactive N642H derivative.36 Therefore, impact of O-GlcNAcylation on pYSTAT5 levels could be restricted to STAT5A. STAT5 was highly O-GlcNAcylated in human leukemic cell lines, which corroborates previous studies that discovered elevated O-GlcNAc levels in cancer cells.37 Colorectal and breast cancer cells contained elevated O-GlcNAc and OGT levels37 when compared with normal tissue. The functional significance was established in OGT knockdown experiments in breast cancer cells, which resulted in diminished proliferation.38 When chronic lymphocytic leukemia cells were compared with normal lymphocytes, high intracellular UDP-GlcNAc and high OGT levels were found, accompanied by higher O-GlcNAcylation of c-Myc, p53 and AKT.39 Excessive STAT5 activation is oncogenic in several hematopoietic malignancies such as CML, MPN, ALL or AML. STAT5 is phosphorylated by tyrosine kinases such as BCR-ABL, mutated JAK, FLT3-ITD or KIT mutations.41 STAT5 inhibition led to growth inhibition and apoptosis.41 Expression of cS5 caused enhanced levels of STAT5 tetramers in transplant settings and mice developed myeloid neoplasia.8 These transforming properties of cS5 are due to the enhanced and prolonged tyrosine and serine phosphorylation, which also required the N-terminal region of STAT5 together with T92 glycosylation.43 Importantly, absence of O-GlcNAcylation abolished transforming properties of cS5 and increased pYSTAT5 and tetramer levels reverted back to wt levels. Serine phosphorylation of STAT5 was not affected, indicating normal shuttling. A schematic model is shown in We link pYSTAT5 levels to the state of O-GlcNAcylation. Different scenarios could account for enhanced tyrosine phosphorylation of cS5 in comparison to non O-GlcNAcylated STAT5: First, docking of STAT5 to the IL-3 receptor as a protein dependent on UDP-GlcNAc levels itself could be impaired.31 pYSTAT5 levels were also decreased by reduction of the donor substrate UDP-GlcNAc under 6-diazo-5-oxo-L-norleucine treatment. Both inhibitor treatments underline the correlation of O-GlcNAcylation and pYSTAT5, although off-target effects under inhibitor treatment cannot be excluded. Thus, future studies should perform direct deletion of OGT, for example, through CRISPR-CAS9 editing.The interaction of O-GlcNAcylation and tyrosine phosphorylation of STAT5 was further shown in inhibitor experiments. Tyrosine phosphorylation as well as O-GlcNAc levels were decreased under the treatment with Alloxan by inhibition of OGT.45 For OGT itself it is known, that besides nutritional sensing via UDP-GlcNAc levels, also tyrosine phosphorylation and O-GlcNAcylation control its activity.46 c-Myc is O-GlcNAcylated at T58, a mutational hotspot in lymphomas and this threonine can also be phosphorylated.47 In prostate cancer, c-Myc is stabilized by O-GlcNAcylation.48 OGT, O-GlcNAcase, phosphatase and kinase were found in an enzymatic complex and several kinases are described to be O-GlcNAcylated.45 In the colorectal cancer cell line HT29, O-GlcNAc levels were decreased by siOGT knockdown resulting in lower serine phosphorylation of PKM2.49 O-GlcNAcylation was recognized as a nutrient sensor, which is added to many cellular signaling proteins reversibly.18 Inappropriate regulation of the O-GlcNAc cycling process contributes to disease development in, for example, diabetes or cancer.17 This could establish a connection between energy sensing and extent of pYSTAT5 signaling. Future studies should address if O-GlcNAcylation of STAT5 could serve as a nutrient sensor controlling metabolic pathways essential for cellular physiology. Hematopoietic phenotypes, known to be regulated by STAT5 have high energy requirements. Therefore, manipulation of OGT or OGA activity might be a strategy for cancer treatment by manipulating metabolism.Functional interactions between glycosylation and phosphorylation have previously been described and the same threonine residues could be modified by both post-translational processes.


Due to its ubiquitous role in the energy metabolism, both the oxidation and reduction of the enzyme’s substrate co-enzyme Q has been studied vigorously. Here, this vast amount of data is reassessed after probing the substrate reduction steps at the Qi-site of the cyt bc1 complex of Rhodobacter capsulatus using atomistic molecular dynamics simulations. The simulations suggest that the Lys251 side chain could rotate into the Qi-site to facilitate binding of half-protonated semiquinone – a reaction intermediate that is potentially formed during substrate reduction. At this bent pose, the Lys251 forms a salt bridge with the Asp252, thus making direct proton transfer possible. In the neutral state, the lysine side chain stays close to the conserved binding location of cardiolipin (CL). This back-and-forth motion between the CL and Asp252 indicates that Lys251 functions as a proton shuttle controlled by pH-dependent negative feedback. The CL/K/D switching, which represents a refinement to the previously described CL/K pathway, fine-tunes the proton transfer process. Lastly, the simulation data was used to formulate a mechanism for reducing the substrate at the Qi-site.The cytochrome (cyt)  One e− is transferred to the prosthetic 2-iron 2-sulfur cluster of the iron sulfur protein subunit, which then passes it on to the heme c1 group in the cyt c1 subunit  simulations+ transfers of the cyt c oxidase6In our prior study, the binding modes of two substrate forms, QHi-site  has a conserved binding position close to the Qi-site 89. From  Qi-site 10. The C Qi-site . Utilizi Qi-site , a Q mol-site  The C1-group of the substrate needs to form a H-bond or a water bridge with the Asp252 side chain . With boOH; COO− .COOH. Unlike the X-ray crystallographic data ((2) The His217 and/or Asn221 side chains H-bond with the C4-carbonyl of Q, in order to orient the substrate’s quinone ring to H-bond with the Asp252hic data , the simhic data  and S4.COO−  If the C1-group is reduced first , Lys251 side chain can rotate inward to form a lasting salt bridge with the Asp252COO−  and partCOO− . The Lysinding Q  and 3C,D−/H+ transfers ((4) The C4-carbonyl of neutral SQ  needs to form a continuous H-bond with His217 . This inransfers , could bransfers .NH3+ can rotate directly into the Qi-site, form a salt bridge with the Asp252COO− i-site i-site . The watProton transfers require short distance between the ionizable groups and comparable pKa values for the residuesi-sites are occupied by non-protonated Q (or anionic SQ) together with the Asp252COOH , water seems to be able to compensate for the lost function  pathways involving protonable residues/lipids exist elsewhere as well.From evolutionary perspective, the inherent randomness of water-mediated Homplexes  and perhBased on the simulations , prior mi-site.(1) Q binding is driven by the polar interactions between the quinone ring and the residues flanking the Q−/H+ transfers, the C1- and C4-carbonyl groups H-bond with Asp252 and Asn221 side chains, respectively. Note that the X-ray crystallographic data suggest that here His217 would eventually be H-bonding with the C4-carbonyl instead of the Asn221 side chain. At this point Lys251 would point out of the Qi-site ((2) Upon Q binding, before e Qi-site .− transfer from the heme bH to the substrate (producing anionic SQ) drives the H+ transfer to happen between the C1-carbonyl and the Asp252COOH. Alternatively, the H+ transfer could involve a water molecule residing between the aspartic acid and the C1-carbonyl  protonated His217, in other words, the quinone ring positioning would be fully coordinated.(3) The first ecarbonyl . The Lys+ transfer could be reversible; i.e. the proton could move back-and-forth between Asp252COO− and C1-carbonyl until the Lys251 side chain rotates inward and/or the second e−/H+ transfer ensues. In fact, only the deprotonated SQ has been detected at the Qi-site in previous experiments121314−/H+ transfer and to prevent otherwise potentially rampant SQ-fuelled superoxide generation at the Qi-site. When the final e− comes from the heme bH, the C4-carbonyl of half-protonated SQ takes a H+ from the His217 side chain. As a result, a fully protonated substrate QH2 is formed at the Qi-site.(4) The H2 unbinds. H-bonding between the Asp252COO− and the C1-hydroxyl might postpone the H+ transfer from Lys251 to Asp252 for a while, but eventually the Lys251NH3+ could act as a “bouncer” that throws QH2 out of the Qi-site by donating a H+ to the Asp252COO−. The potential deprotonated state of His217 would also likely end quickly via water-mediated H+ transfers that also could promote the QH2 unbinding.(5) Finally, the reaction product QHi-site to upkeep this arrangement; transferring one H+ at a time from the peripheral CL molecule to the Asp252 side chain, which passes them to the solvent and via water also to His217 ((6) At the outset, both His217 and Asp252 side chains are protonated and ready for Q binding. Lys251 would rotate in and out of the substrate-free Qo His217 .i-site, because the Lys251 side chain participates in the neutral SQ binding (− transfers to be tightly coupled to H+ transfers should be tested in the future using for example quantum mechanics/molecular mechanics (QM/MM) calculations utilizing the new binding geometry seen in the simulations. The firmly coordinated binding of neutral SQ at the Qi-site, involving the inward rotamer of the Lys251 side chain, could be in part needed to curb the superoxide generation linked to aging-related cellular damage.The simulations indicate that the binding of half-protonated semiquinone (SQ) would acquire more coordinated binding pose than the deprotonated quinone (Q) at the Q binding . Thus, ti-site  simulations were performed with NAMD2.9http://www.chemaxon.com). The pKa predictions were done at pH 7.4 with default settings for the PDB structures using PROPKA3.139i-site residues was verified by analyzing selected snapshot structures with known protonation states .


It has been revealed that mutations that lead to an increase of kinase activity of LRRK2 protein are significantly associated with PD pathogenesis. Recent studies have shown that some Rab GTPases, especially Rab8, serve as substrates of LRRK2 and undergo phosphorylation in its switch II domain upon interaction. Current study was performed in order to find out the effects of the phosphorylation of Rab8 and its mutants on lipid metabolism and lipid droplets growth.Several mutations in The phosphorylation status of Rab8a was checked by phos-tag gel. Point mutant construct were generated to investigate the function of Rab8a. 3T3L1 cells were transfected with indicated plasmids and the lipid droplets were stained with Bodipy. Fluorescent microscopy experiments were performed to examine the sizes of lipid droplets. The interactions between Rab8a and Optineurin were determined by immunoprecipitation and western blot.Our assays demonstrated that Rab8a was phosphorylated by mutated LRRK2 that exhibits high kinase activity. Phosphorylation of Rab8a on amino acid residue T72 promoted the formation of large lipid droplets. T72D mutant of Rab8a had higher activity to promote the formation of large lipid droplets compared with wild type Rab8a, with increase in average diameter of lipid droplets from 2.10 μm to 2.46 μm. Moreover, phosphorylation of Rab8a weakened the interaction with its effector Optineurin.Y1699C mutated LRRK2 was able to phosphorylate Rab8a and phosphorylation of Rab8a on site 72 plays important role in the fusion and enlargement of lipid droplets. Taken together, our study suggests an indirect relationship between enhanced lipid storage capacity and PD pathogenesis. Rabs are a group of about 70 eukaryotic proteins located on different membranes that play important roles in all stages of vesicle trafficking including budding, motility and fusion , 2. The Rab8 has shown to play important roles in membrane trafficking. It plays irreplaceable roles in multiple biological processes including cellular morphology, cell polarity, cell movement, neural differentiation and ciliogenesis , 10. FurLRRK2 gene are related to PD have brought LRRK2 into main focus of research [LRRK2 mutations  are primary causes for PD pathogenesis. Till now, it remains unclear how LRRK2 mutations occurring in different functional domains predispose to PD. The most powerful PD-associated LRRK2 mutation is G2019S, which increases LRRK2 kinase activity two to three fold [LRRK2 (leucine rich repeat kinase 2) has been a hot spot in the field of neurology as its mutations have been associated with Parkinson’s disease (PD) , 15. PD research . LRRK2 iRecently, a subset of Rab GTPases has been identified as key LRRK2 substrates. Through a combination of phosphoproteomics and molecular genetics approaches, it has been proved that several Rab GTPases especially Rab8 can be phosphorylated by LRRK2 on an evolutionary conserved residue in switch II domain . Since s2. Methods for 3T3L1 preadipocytes differentiation and electrotransfection were similar as previously described [Two hundred ninety-three T cells and 3 T3-L1 pre-adipocytes were cultured in DMEM  containing 10% FBS , 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/ml streptomycin at 37 °C in humidified incubators containing 5% COescribed .DNA plasmids were transfected into 293 T cells and 3 T3-L1 pre-adipocytes using Lipofectamine 2000 following manufacturer’s instructions .Rab8a and Optinurin were amplified from cDNA of 3 T3-L1 adipocytes. These cDNAs were subcloned into pCMV5-HA or pCMV5-FLAG using specific restriction sites. Point mutations of Rab8a were cloned by a PCR-based site-directed mutagenesis method . The accuracy of each plasmid DNAs was tested by sequence analysis.Human LRRK2 and its mutant (LRRK2 Y1699C) plasmids were purchased from Addgene (code #17609 and #25364). Full-length cDNAs encoding mouse Methods for immunoprecipitation and western blot sample preparation were same as previously described . Anti-FL2 were added to a standard gel solution to the final concentration of 50 mM and 100 mM, respectively. Then gels were polymerized by ammonium per-sulfate and TEMED. Cell lysates used for Phos-tag SDS-PAGE were supplemented with MnCl2 at 10 mM to neutralize the effect of EDTA in the lysates. After running SDS-PAGE, gels were washed 3 times with transfer buffer containing 10 mM EDTA followed by a wash with transfer buffer (10 min each). Blotting to nitrocellulose membranes was carried out by following a standard protocol. Phos-tag acrylamide was kept at 4 °C in black tubes blocking out light because Phos-tag acrylamide is light-sensitive.Phos-tag acrylamide and MnClImages for analyzing lipid droplets sizes were acquired under an LSM710 confocal microscope (Carl Zeiss) with 63× oil immersion objective. Images were exported out in 16-bit TIFF format. Further processing of single images  was performed in Photoshop .Quantitative analysis of LD size in 3 T3-L1 preadipocytes was performed as described by Sun et al. . For quat-test. Differences were considered significant at P < 0.05. *** indicated P < 0.001.All statistical analyses were performed in GraphPad Prism Version 5 (GraphPad Software). Significance was established using a two-tailed Student’s In order to confirm whether Rab8a could be phosphorylated by LRRK2, we overexpressed FLAG-tagged Rab8a  and MYC-tagged LRRK2 (Wild-type form and Y1699C form) in 293 T cells. The phosphorylation form of Rab8a was detected using phos-tag gel. As shown in Fig. Our previous data has shown that Rab8a plays important role in Fsp27 mediated lipid droplet fusion and enlargement . Here, wSince point mutation from threonine (T) to aspartic acid (D) or glutamic acid (E) can mimic phosphorylation, different mutations on the T72 site of Rab8a were generated . T72A represents the non-phosphorylated form of Rab8a while T72D/E mimics the phosphorylated form of Rab8a. We wanted to investigate the role of above mentioned Rab8a mutants in lipid droplets enlargement. Overexpression of WT Rab8a could enhance Fsp27’s ability to promote the formation of large lipid droplets. Our results showed that overexpression of Rab8a (T72D) and Rab8a (T72E) increased the size of LDs from 2.27 μm to 2.87 μm when compared with overexpression of Rab8a or Rab8a (T72A) Fig. . AccordiNext, we investigated whether the phosphorylation of Rab8a on 72 site affects its GTP/GDP binding status. Optineurin has been reported to be the effector of Rab8a which binds to the GTP form but not to the GDP form of Rab8a . We co-eIn this study, we have shown that Rab8a could be phosphorylated by Y1699C mutated LRRK2. Previous studies showed that there had been an increase in enzyme activity of LRRK2 Y1699C compared with WT LRRK2 , 24, 25.LRRK2 knockout rats and mice have shown deformed lungs and kidneys due to possible defects in the autophagosome pathway regulated by activated Rab proteins [Earlier studies have shown that LRRK2 mutations  which increase the activity of LRRK2 are the most common causes of the inherited form of PD pathogenesis , howeverproteins . Recent Overexpression of Rab8a, Rab1 and Rab3a proteins alleviate PD through reduced α-synuclein-induced cytotoxicity , 29. As Another study has established that change in Rab homeostasis may contribute towards development of PD. As LRRK2 is the main regulator of Rab proteins, overactive LRRK2 results in increased Rab phosphorylation, altering the balance between cytosolic and membrane bound Rab, which in turns disturbs intracellular trafficking. In PD-associated LRRK2 mutations, the membrane-cytosol balance of Rabs is shifted towards the membrane causing accumulation of inactive Rabs in the membranes . MoreoveOur findings have proved that Y1699C mutated LRRK2, but not wild-type LRRK2, could phosphorylate Rab8a and that phosphorylation of Rab8a on site 72 plays important role in the fusion and enlargement of lipid droplet. Furthermore, our results show that phosphorylation of Rab8a weakens its interaction with its effector Optineurin. It will be interesting to investigate the direct role of lipid droplets accumulation with pathogenesis of PD in order to explore therapeutic interventions against this disease.


Case Presentation. Implant prostheses are a successful treatment for replacing missing teeth. However, this treatment modality can have biological and mechanical complications causing serious problems for the dentist, as demonstrated in this clinical case. The patient presented with a fractured screw and a severely damaged implant hex connection that corresponded to the second premolar, upper left, stating that she unsuccessfully tried to remove the prosthetic screw, which was most likely to have been loose. After clinical and radiographic review, it was decided to remove small fragments of the fractured prosthetic screw inside the implant head. Removal by conventional methods was unsuccessful but was eventually achieved through use of a bur. Then it was possible to make a cast post  and develop a fixed prosthesis , which were attached with luting cement. A cast post  was made and a fixed prosthesis was developed , which were attached with luting cement, the same ones that can present mechanical complications such as fractures between the third and fourth thread of the implant, loosening of the abutment, and/or the prosthetic screw in individual crowns, most frequently in partially edentulous patients, mainly in the premolar and molar regions of the maxilla. Conclusion. Therefore the present technique used in this case is very simple, noninvasive, and useful to readers. Since Branemark introduced the concept of osseointegration, dental implants have been successfully used as a viable treatment for fully and partially edentulous patients . AccordiThe most common complication for a single crown was a prosthetic screw and abutment screw loosening , but theHurson  states tAccording to Nergiz et al. , screw iJemt  states tParafunctional habits can be a risk factor related to implant fracture and screw loosening and can create uncontrolled and excessive occlusal loading forces . Indeed A higher frequency of screw loosening has been reported for replacement of single crowns in the premolar and molar area than in the anterior region and three times more in the maxilla compared with the mandible . The posOnce fracture has been diagnosed, it is possible to proceed to extraction, beginning with the simplest and most conservative method, and trying to respect, as far as possible, the implant head, external hex, and internal thread. Any change in these areas can lead to limitations in future prosthetic use. The methods used to recover the broken fragments or screw are determined according to the location above or below the head of the implant. If a cap screw fracture is above the head of the implant, an explorer, a straight, or a hemostatic probe can succeed, and the tip of the instrument is carefully moved in the opposite direction clockwise on the surface of the segment screw according to Satwalekar et al. . If thatFinally, although the frequency of fractures of implants is low, treatment planning should include avoiding occlusal overload, in some cases using an occlusal splint to protect the restorations .This report describes the rehabilitation of an implant with screw fracture and severely damaged hex by a cast post.A 66-year-old female patient who takes bisphosphonates for osteopenia presented at the dental practice in the Postgraduate School of Oral Rehabilitation, Central University of Ecuador, with fracture of the implant prosthetic screw and damaged hex platform .The first step was to attempt extraction of the fragment, beginning with the most conservative and simple method, through dental explorer, without success. Therefore, the fragment was destroyed with a fissure bur, touching the internal threads of the implant, as seen radiographically .The gum around the implant platform was cut minimally and an acrylic resin impression made of the inside of the implant  .The inside of the implant was cleaned, washed, and driedHydrofluoric acid gel at 9.6% (EUFAR Laboratories S.A.) was applied to the post for 60 seconds, then washed, and driedLuting cement  was mixed and placed both on the post and inside the implantThe post was photopolymerized with ultraviolet light (350 to 500 Nm) for 3 minutesExcess cement was removedOnce the post cemented, cord (Ultradent Ultrapak of two ESPA 00) is placed, printing was performed with heavy and light polyvinyl siloxane (Elite HD + from Zhermack IT)  was made and its adaptation with wax (Wax Disclosing Kerr) checked radiographically , it was mack IT) , and metmack IT) , by wax Finally the prosthetic crown is cemented, after occlusal control, which was allowed to be below 12 micrometers' occlusion  and the To Hurson  screw loIn the article by Zarb and Schmitt  in whichJemt  stated sIn this case, the patient had fracture of the prosthetic screw with damaged external hexagon but had noticed loosening of the screw several times before its fracture; however, it had been decided to maintain the implant within the mouth for further prosthetic rehabilitation as the patient was taking bisphosphonates. The treatment reported here is in contrast to Gargallo Albiol et al.  who condIt has been proposed to wear down or attempt to make a notch in the head fragment of the broken screw with a round bur at high speed, depending on the location of the prosthetic screw. In the case reported it was not possible to extract the fragment because it was below the implant head so it was ground down with a bur. The risk of puncturing the implant and bone was controlled radiographically. After achieving the objective, a metal and porcelain post and crown were made.Among the advantages of using noble alloys is that they have a lower elastic modulus allowing the occlusal forces to transmit more efficiently to the remaining teeth. Binding of noble metal and porcelain is better than base metal because the oxide layer is thinner. Disadvantage is the high economic cost .Finally, in a monitoring appointment after one year of treatment the patient reported no discomfort. Normality was clinically and radiographically checked.Within the limitations of this case it was possible to conclude that the situations with screws fractured abutment and devastated hexagon can be solved in a time and manner of saving money, with a very useful alternative without the invasive treatment such as removal of the implant and submerging. Therefore the present technique used in this case is very simple, noninvasive, and useful to readers.


Moreover, following GM-CSF stimulation, the levels of phospho-JAK2 and -STAT5 and a STAT5 luciferase reporter assay were all reduced in Cbl knockout cells and this effect could be rescued by Cbl expression. Mechanistically, Cbl can interact with, and ubiquitinate JAK2 FERM and kinase domains via the Cbl TKB domain. Using lysine-to-arginine site-directed mutagenesis, K970 in the kinase domain of JAK2 was identified as the ubiquitination site important for promoting full JAK2 activation by Cbl via K63-conjugated poly-ubiquitination. Our study suggests that GM-CSF-induced JAK2 activation is enhanced by Cbl-mediated ubiquitination of JAK2. Targeting ubiquitination of JAK2 might offer a novel therapeutic strategy against JAK2-mediated disorders.JAK2 activation is crucial for cytokine receptor signal transduction and leukemogenesis. However, the underlying processes that lead to full activation of JAK2 are unclear. Here, we report a positive role for ubiquitination of JAK2 during GM-CSF-induced activation. Upon GM-CSF stimulation, JAK2 ubiquitination is significantly enhanced through K63-linked poly-ubiquitination. Studies employing both knockout and overexpression of Cbl, an E3 ubiquitin ligase, led to the conclusion that Cbl specifically promotes JAK2 ubiquitination, and this was further confirmed  Given the diversity of type II cytokine biology, JAK2 actively participates in many biological processes, including hematopoiesis and innate immune responses3. In 2005, a gain-of-function somatic JAK2 mutation, V617F, was identified to be highly prevalent in myeloproliferative disorders4. Patients with this gain-of-function mutation have frequently been identified in polycythemia vera , essential thrombocythemia , and primary myelofibrosis 7. These findings extend the importance of JAK2 dysregulation to include hematopoietic malignancies, in addition to the conventionally- recognized inflammatory and immunological disorders.Janus kinase 2 (JAK2) is a member of the Janus kinase family, which belongs to the non-receptor tyrosine kinase superfamily. JAK2 is a key intracellular signaling molecule that couples type II cytokine receptors, including the receptors for growth hormone, erythropoietin, and granulocyte-macrophage colony-stimulating factor (GM-CSF), to downstream signaling pathways10. The JH1 domain is a bona fide protein tyrosine kinase that contains two tyrosine residues  in the conserved activation loop, which, in turn, control kinase conformation and activation when phosphorylated12. The structure of the JH2 pseudo-kinase domain highly resembles a kinase domain but contains a shorter activation loop14 and plays a negative auto-regulatory role on the kinase domain18.The architecture of JAK family proteins has been highly conserved through evolution. These proteins contain four conserved domains: FERM, SH2, JH2 pseudo-kinase, and JH1 kinase. The N-terminal FERM and SH2 domains interact with the cytoplasmic tails of cytokine receptors; this is an essential step in JAK kinase activationvia promoting conformational changes. On the other hand, some of these sites are involved in down-regulation of JAK2 activation, such as Y317, Y570, Y913, and Y119, which may ensure tighter control of cytokine signaling20.Intensive research efforts have been focused on understanding the significance of phosphorylated tyrosine residues in JAK2, principally using site-directed mutagenesis of such amino acids. The current model for JAK activation is that, upon cytokine stimulation, JAK2 is phosphorylated at multiple sites, some of which are required for kinase activation, including Y1007/8, Y637, Y868, and Y972/966, possibly 23. The SOCS1 SH2 domain associates with JAK2 phospho-Y1007 in the activation loop, thereby blocking JAK2 catalytic activity. This association also leads to ubiquitin conjugation of JAK2, ultimately leading to its proteasomal degradation. Casitas B-lineage lymphoma  is an E3 RING ubiquitin ligase that regulates the function of both receptor- and non-receptor tyrosine kinases, either through ubiquitination or via adaptor functions24. Cbl contains a tyrosine kinase-binding (TKB) domain at its N-terminus, followed by a linker region, a central zinc-binding C3HC4 RING finger motif, and a number of proline-rich motifs at the polypeptide C-terminus26.In addition to phosphorylation, other post-translational modifications, including ubiquitination, have also been reported to control JAK2 stability and localization. Suppressor of cytokine signaling 1 (SOCS1) has been reported to inhibit cytokine-induced JAK2/STAT5 signaling through the ubiquitin-proteasome pathway28. A germline Cbl mutation (Y371H) has been identified in 10–15% of juvenile myelomonocytic leukemia (JMML) patients. JMML is a disease characterized by overproduction of monocytic cells that are highly responsive to GM-CSF stimulation30. Another Cbl mutation, C384R in the RING finger domain, has also been identified in myelodysplastic and myeloproliferative neoplasms30. Subsequent studies have revealed that homozygous Cbl mutations are present in most acquired uni-parental disomy myeloid malignancies, and that gain-of-function of mutations in Cbl are not associated with loss of the ubiquitin ligase activity, which is currently thought to play a tumor suppressing role31. It is also reported that Cbl could serve as an adaptor for the GM-CSF receptor (GMR) β subunit and down-regulate levels of Src protein and its kinase activity, which, in turn, would limit GM-CSF-induced GMR activation33. It was further suggested that, through their adaptor function, mutated Cbl proteins increase their association with Lyn kinase, as well as with the p85 regulatory subunit of PI3K, and thereby promote activation of Akt-dependent survival signals34.Cbl is mainly expressed in hematopoietic cellsIn this study, we investigated the molecular mechanisms underlying Cbl-mediated JAK2 ubiquitination following GM-CSF induction. We demonstrate that GM-CSF stimulates the interaction between JAK2 and Cbl, which promotes JAK2 ubiquitination. Our results reveal that the K970 ubiquitination site on JAK2 is important for JAK2 phosphorylation, and for downstream signal transduction. Our studies identify a novel molecular mechanism that drives GM-CSF-induced JAK2 activation.35. Intersectin interacts with Cbl to enhance EGFR ubiquitination and degradation following EGF stimulation36. Moreover, recent studies have indicated that Cbl is involved in GMR-mediated JAK2 activation33, but the precise mechanism is not clear. To investigate the potential involvement of Cbl in the GMR/JAK2/STAT5 signaling axis, we sought to explore whether ubiquitination occurs during GM-CSF induced signaling. We transiently transfected Flag-tagged ubiquitin into HeLa cells stably expressing GMRα- and -β (GMR cells), and precipitated Flag-tagged ubiquitinated proteins. We observed that ubiquitination of JAK2 was clearly increased when JAK2 was phosphorylated upon GM-CSF stimulation, but was not elevated for GMRβ and STAT5 -1 and TUBE-2 beads (see Methods), which preferentially recognize K63- and K48-linked poly-ubiquitin, respectively, we observed that in the presence of proteasome inhibitor, MG132, ubiquitination of both endogenous phospho-JAK2 and JAK2 were significantly increased after GM-CSF stimulation in TUBE-1 immunoprecipitates (IPs) compared to TUBE-2 IPs  by CRISPR/Cas9-based technology (see Methods) and used these to examine the effects on ubiquitination of endogenous JAK2 following GM-CSF stimulation. CKO2 and CKO17 cells were stimulated with GM-CSF and cell lysates were then IPed with TUBE-1 or TUBE-2 beads. GM-CSF-induced K63-linked JAK2 ubiquitination was reduced in CKO2 and CKO17 cells, compared to parental TF1 cells Fig. . On the cytokine-inducible SH2-containing protein (CISH) gene (pGL4-CISH)37, into GMRαβ-expressing CKO HeLa cells to monitor the function of the JAK2/STAT signaling pathway. As shown in Fig. In line with the observation that JAK2 ubiquitination predominantly involves K63, we did not detect significant changes in JAK2 protein levels, with or without cytokine stimulation or treatment with proteasome inhibitor. To explore whether K63-linked ubiquitination of JAK2 is involved in JAK2 signaling, rather than in protein stability, we first investigated whether GM-CSF-stimulated signaling is affected in CKO cells. As shown in Fig. in vivo. HA-tagged Cbl, GMRβ, or Flag-tagged JAK2 were co-expressed in 293 T cells and their respective interactions were analysed by IP with anti-Flag, anti-HA, or anti-GMRβ antibodies. As shown in Fig. in situ (see Methods). As presented in Fig. We further explored the question of whether Cbl can interact with GMR or JAK2 via the TKB domain of Cbl. Conversely, HA-tagged full-length Cbl was co-expressed with various GST-tagged truncated JAK2 mutants: the N-terminal region containing the FERM and SH2 domains (JN), the C-terminal region containing the pseudo-kinase and kinase domains (JC), and the kinase domain only (K)  that mediate interaction between Cbl and JAK2. Along with full-length HA-tagged Cbl (FL), we generated three truncation mutants: the TKB domain plus linker region (TL), the TKB domain (T) only, and the C-terminal region (C) only and co-expressed these along with full-length Flag-tagged JAK2 Fig. . Followi(K) Fig. . When ce(K) Fig. , these cWe next examined which domain(s) of JAK2 are conjugated by ubiquitin. To address this issue, we co-expressed HA-tagged ubiquitin along with different Flag-tagged JAK2 domains . Because the JAK2 FERM and kinase domains are two main targets of ubiquitination, we focused on analysing the importance of five ubiquitination sites in FERM and seven in the kinase domain  that were identified at least twice in three independent experiments . Similar results were also seen for phosphorylation levels at Y637 and Y1007/1008 in J2K7R and J2K970. To further demonstrate that lysine 970 is important for JAK2 kinase activity, an in vivo auto-phosphorylation assay using overexpression of the kinase domain was employed in 293 T cells. We expressed various Flag-tagged JAK2 kinase domain-only mutants   in the kinase domain is important for JAK2 activation, we generated a series of lysine-to-arginine (KR) mutants containing eight, seven, and six mutations  or a specific Kity Fig. . Interes6R) Fig. , lane 2.via K63-linkage.To investigate whether ubiquitination on JAK2 K970 is Cbl dependent, HA-tagged ubiquitin and Flag-tagged JAK2 kinase domain-only mutants  were co-expressed in CKO or parental GMR cells. Following IP of HA-ubiquitin, the ubiquitinated smearing was detectable in the K6R mutant which preserved lysine 970 as well as the wild type kinase domain (K) in GMR cells, but this smearing was decreased in CKO cells  domain of JAK2, 38 reveals that K970 is located near the C-lobe of the kinase domain and is exposed on the exterior surface of the molecule. Presumably, this surface localization indicates that K970 is highly accessible for post translational modification, e.g. ubiquitination. Since the loss of K970 ubiquitination not only affects phosphorylation of the entire kinase domain, but also the phosphorylation of Y317 in the FERM domain and Y637 in the pseudo-kinase domain, it appears that K970 ubiquitination is required for certain conformational changes to occur in the kinase domain that further affect domain-domain interactions during phosphorylation.How K970 ubiquitination modulates the maximum level of JAK2 activation remains to be discerned. According to sequence and structure comparisons of the JAK protein family, K970 is non-conserved; the corresponding residues in JAK1 and JAK3 are both arginine (R), and a glutamine (Q) is found at this position in TYK2 , a disease that is characterized by GM-CSF hypersensitivity and resistance to chemotherapy, were identified containing somatic mutations in Cbl42 and in the bone resorption activity of osteoclasts43. Recently Stanley and co-workers presented evidence that a Cbl-dependent pathway is required for CSF-1R ubiquitination, which permits full receptor tyrosine phosphorylation in activated macrophages44, despite the fact that several groups have reported the process of Cbl-mediated CSF-1R endocytosis and degradation in macrophages via direct45 or indirect mechanisms46. The Cbl-mediated CSF-1R ubiquitination likely causes conformational changes in the kinase domain that allows for full phosphorylation of the receptor tyrosine kinase. Studies on the NF-kB pathway have also revealed a similar mechanism, whereby K63-linked poly-ubiquitination of TAK1 facilitates protein kinase dimerisation and activation, which can then, in turn, phosphorylate and activate IkB kinase47. Therefore, the mechanism we now report in JAK2 activation, whereby K63-conjugated poly-ubiquitination functions as a conformational switch, which allows the JAK2 tyrosine kinase to be fully phosphorylated and maximally activated, may represent a more general mode of action that applies to many other kinases.Although ample evidence shows that Cbl functions as a negative regulator of various activated receptor tyrosine kinases by mediating their poly-ubiquitination and degradation, as a multi-domain adaptor protein, Cbl also acts as a positive regulator in integrin signaling in macrophagesin vitro ubiquitination reaction by Cbl where GM-CSF receptor was not required, although the presence of receptor and GM-CSF stimulation can significantly enhance the interaction and ubiquitination of JAK2 by Cbl. We anticipate that this Cbl action is important for all JAK2-using cytokine receptor signaling. However, due to the complicated interaction networks between Cbl and receptors, signal adaptor proteins and Src-family tyrosine kinases, the final influence of Cbl on each cytokine receptor signaling would be expected to be receptor-specific and cell-type-specific.Although endogenous Cbl maximally interacts and ubiquitinates JAK2 when cells were stimulated by GM-CSF, Cbl can interact with the kinase domain of JAK2, which was expressed as a cytosolic protein. Therefore, we anticipate that Cbl can ubiquitinate both receptor-bound and cytosolic forms of JAK2, and that this does not need to occur in a receptor-dependent manner. This notion is also consistent with our JAK2 35. pGMRβ-clip was constructed by inserting the PCR-amplified clip coding region from pCLIP vector  into the C terminus of the GMRβ gene in pCDNA3-GMRβ. The Cbl cDNA was kindly provided by Dr. Wallace Y. Langdon , and sub-cloned into the pcDNA6 vector. The Cbl C381A mutant was generated using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit . The construction of plasmids encoding the sub-domains of Cbl was performed according to a previous publication48. The sequences of all the primers used for Cbl construction are listed in Table http://crispr.mit.edu) for CRISPR/Cas9 knockout experiments and then cloned into the pZGB-RGN3 vector to yield pZG22C02-Cbl or pZG22C03-JAK2, respectively, and the plasmids were purchased from Zgenebio Biotech, Inc., .pcDNA3-GMRα and pGMRβ-clip are pcDNA3 vector-based expression systems encoding GMRα and GMRβ-clip proteins, respectively, as previously described293 T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)  containing 10% fetal bovine serum (FBS) . HeLa cells stably expressing both GMRα and GMRβ-clip (GMR) were established by co-transfecting pcDNA3-GMRα and pcDNA3-GMRβ-clip plasmids, and transfected cells were selected by culturing in the presence of G418 (200 μg/mL). Cbl knockout (CKO) or JAK2 knockout (JKO) cells were generated by transfecting GMR cells with either pZG22C02-Cbl or pZG22C03-JAK2, followed by sorting of GFP-positive or mCherry-positive cells, respectively, by flow cytometry. HeLa-derived cell lines were maintained in the same medium as 293 T cells. TF1 cells were cultured in RPMI 1640 medium (Corning Inc.) containing 10% FBS and GM-CSF (1 ng/mL). CKO TF1 stable clones were established by transfecting TF1 cells with pZG22C02-Cbl, followed by sorting of GFP-positive cells by flow cytometry. CKO TF1 cells were maintained in the same medium as that used for parental TF1 cells.The following antibodies were used in this study: anti-GMRβ (1C1) and anti-STAT5(C-17) from Santa Cruz Biotechnology ; anti-JAK2 (D2E12), anti-pJAK2 , anti-pSTAT5 , anti-Cbl (#8447), anti-ubiquitin , and anti-pY (#9411) from Cell Signaling Technology ; anti-HA (GTX628489) from Genetex ; anti-Flag (M2) from Sigma-Aldrich ; anti-JAK2 (04-001) and anti-GST (06-322) from Merck Millipore . Anti-pJAK2Y317 and pJAK2Y637 antibodies were kindly provided by Dr. Martin G. Myers, Jr. .N-ethylmaleimide and iodoacetate were purchased from Sigma-Aldrich; GM-CSF was purchased from Gentaur .Anti-Flag M2 affinity gels and EZview Red HA-Agarose beads were purchased from Sigma-Aldrich. Tandem Ubiquitin Binding Entity (TUBE) −1 and −2 agarose beads were purchased from LifeSensors . G418, N-ethylmaleimide (20 mM) and iodoacetate (5 mM) were used in the lysis buffer.Cells were lysed in either 1% Triton X-100 or 1% NP-40 lysis buffer, supplemented with protease and phosphatase inhibitors, and whole cell lysates (WCLs) were IPed and analysed by Western blotting using the indicated antibodies. In order to detect protein ubiquitination, cells were treated with MG132 (10 μM) for 4 h before harvest, and Endogenous protein-protein interactions were detected using a Duolink® PLA kit (Sigma-Aldrich) according to the manufacturer’s instructions. Briefly, after serum starvation, TF1 cells were treated with or without GM-CSF (1 ng/mL) for the indicated times and were then attached to glass slides by cytospinning. The slides were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS, and blocked with Duolink® blocking buffer for 30 min at 37 °C. The rabbit anti-human Cbl antibody and either a mouse anti-human GMRβ or a mouse anti-human JAK2 antibody in Duolink® dilution buffer were incubated with the slides for 16–18 h at 4 °C. After washing three times, Duolink® PLA immune-probes targeted against the two different primary antibodies (rabbit and mouse) were added for 1 h at 37 °C. If the antibodies are in close proximity (<40 nm), these probes can hybridize to form a circular DNA molecule. The circular DNA molecules were then covalently ligated using the Duolink® ligation solution for 30 min at 37 °C. The covalent circular DNA was then amplified by rolling circle amplification using the Duolink® polymerase for 100 min at 37 °C. Amplified DNA was then detected by hybridization of Duolink® detection probes and signals were detected by confocal microscopy using a Zeiss LSM780 and ImageXpress Micro Confocal High-Content Imaging System.2, 1 mM DTT, 2 mM ATP, and 2 mM ubiquitin aldehyde) at 37 °C for 4 h. Biotin-labelled ubiquitin and E1 were purchased from Enzo Life Sciences ; GST-Ubc13 was a kind gift from Dr. Sheau-Yann Shieh . After incubation, the reactions were analysed by Western blotting.Flag-tagged JAK2 and HA-tagged Cbl proteins were IPed from Cbl knockout 293 T cells using an anti-Flag M2 affinity gel or EZview Red HA-Agarose beads, and eluted with 0.1 M glycine-HCl pH 3.5. Eluted proteins were incubated with recombinant E1, E2 (Ubc13), and biotin-labelled ubiquitin in ubiquitination buffer  and/or other indicated plasmids. After transfection, cells were maintained in DMEM culture medium for 24 h, and then changed to fresh serum-free DMEM with or without 10 nM GM-CSF for an additional 24 h. Cells were then harvested and assayed for luciferase activity using Luc-Pair™ firefly luciferase HS assay kit . Briefly, the cells were washed in PBS and lysed with Luc-Lysis II Buffer at room temperature for 15 min, and then analysed for firefly luciferase activity (FLuc assay working solution) and Renilla luciferase activity (RLuc assay working solution). Each assay was performed at least four times.GMRαβ or CKO HeLa cells were seeded into 24-well plates for transfection. Reporter plasmids pGL4-CISH or pCISHmut (50 ng) were co-transfected with the Protein phosphorylation levels from Western blots were quantified using ImageJ software .Data are expressed as the mean ± SEM from three or more independent experiments and analysed with either Student’s t-tests (for comparison between two groups) or one-way analysis of variance (ANOVA) using Microsoft Excel.supplementary information


Marine diatoms in tillites along the Transantarctic Mountains (TAMs) have been used to suggest a diminished East Antarctic Ice Sheet (EAIS) during Pliocene warm periods. Updated ice-sheet modelling shows significant Pliocene EAIS retreat, creating marine embayments into the Wilkes and Aurora basins that were conducive to high diatom productivity and rapid accumulation of diatomaceous sediments. Here we show that subsequent isostatic uplift exposed accumulated unconsolidated marine deposits to wind erosion. We report new atmospheric modelling utilizing Pliocene climate and derived Antarctic landscapes indicating that prevailing mid-altitude winds transported diatoms towards the TAMs, dominantly from extensive emerged coastal deposits of the Aurora Basin. This result unifies leading ideas from competing sides of a contentious debate about the origin of the diatoms in the TAMs and their link to EAIS history, supporting the view that parts of the EAIS are vulnerable to relatively modest warming, with possible implications for future sea-level rise. A long-standing debate regarding the Pliocene history of the East Antarctic Ice Sheet was spurred by the discovery of marine diatoms in the Transantarctic Mountains. Here the authors show that the diatoms were emplaced by wind following a retreat of the ice sheet into coastal basins and subsequent isostatic emergence. Once the occurrence of the diatoms in association with the high-altitude deposits was independently replicated2020The majority consensus that the key diatoms were emplaced by aeolian processes was widely interpreted as refutation of the ‘dynamic' EAIS hypothesis12131927144The ‘stabilist' view gained considerable support from an early generation ice-sheet model2331In contrast with earlier modelling efforts331783334363739Here we further evaluate aeolian processes and test the hypothesis that aeolian diatoms in the TAMs were the result of Pliocene EAIS retreat, rather than evidence against ice-sheet dynamic behaviour. We evaluate coastal isostatic response over a Pliocene Antarctic ice-sheet reconstruction derived from an ice-sheet model that incorporates ice-sheet grounding zone processes, including cliff failure and hydrofracturingSilt and sand-sized grains, including microfossils, are well known to be raised by saltation processes and then carried significant distances by wind. Diatom frustules and small clumps of diatomaceous sediment (microclasts) have an extremely high surface-area:mass ratio , making 42Once aloft, these particles can be carried great distances, as readily seen by the observed distribution of diatoms across the Antarctic ice-sheet surface. Pleistocene and modern long-distance transport and atmospheric precipitation of diatoms on the Antarctic ice sheet and the TAMs have been demonstrated through analysis of ice cores44444445The fact that marine diatoms typically dominate the diatom assemblages extracted from most Sirius tillite surfaces, despite a constant and ongoing rain of non-marine diatoms across these surfaces today, indicates that the accumulation of marine diatoms is not a recent phenomenon, but largely reflects a specific interval or intervals of significant aeolian input with distinct source beds of subaerially exposed Pliocene marine diatomaceous sediments. Diatoms are generally not lifted from ocean surfaceset al.20et al.The widespread distribution of aeolian marine diatoms in Sirius deposits  implies 2 and a warm austral summer orbitet al.373839A new version of an established Antarctic ice-sheet model PSUICE-3D)  transitions described in the ANDRILL-1B drillcore (defined as Motif 2)Following uplift, a subaerially exposed diatom-strewn landscape would have been highly susceptible to wind erosion, especially during summer with reduced snow cover , thus prThe entire process takes several thousand years following climate warming. With grounding line retreat and ice shelf collapse, a marine basin opens, with copious diatom production in open waters during summer with little sea ice. The outer basins shallow with the isostatic response following ice retreat, eventually leading to emergence of uplifted islands and coastal areas. Although the simulation in 533−1 in many locations adjacent to the freshly exposed sediments, most notably along the Aurora Basin coastline  over Antarcticaoastline . Wind spoastline  indicateIn contrast with surface winds, mid-tropospheric winds at 500 hPa, ∼5,000 m altitude, are generally cyclonic over the continental interior and show strongly favourable transport directions towards the TAMs and Sirius outcrop localities, from both Wilkes and Aurora basin marine sediment exposures , especiaet al.et al.Although ice-free bedrock patches also emerge around the islands of West Antarctica , both su39et al.Despite a paucity of exposed source beds today, there is widespread evidence for rare diatoms from coastal and Dry Valley sites being raised to the Polar Plateau, but these are dominantly of non-marine origin44445572 has now reached Pliocene levels and is expected to continue to rise. Scientific debates, such as the interpretation of Sirius Group diatom data, often turn on key details that if misinterpreted or interpreted through a rhetorical bias may obscure broader truths. The debate between ‘stabilists' and ‘dynamicists' focused on the configuration of the EAIS during the Pliocene, but none of the published studies presented data that was directly related to or indicative of EAIS geometry or thickness during the Pliocene. Furthermore, neither a ‘stable' nor ‘dynamic' ice sheet was adequately defined in context, and intermediate ice-sheet configurations, such as those suggested by new models, were not considered. The IODP Sites 1165 . Surface winds are from the lowest model level (σ=0.9950).Pliocene mean annual and summer (January) temperatures are calculated from the RCM . These hPlots for both the ice sheet and atmospheric model outputs were prepared using MATLAB software .Ice-sheet and RCM model outputs are available on request. Netcdf-format output file is available for the ice-sheet distribution in How to cite this article: Scherer, R. P. et al. Windblown Pliocene diatoms and East Antarctic ice sheet retreat. Nat. Commun.7, 12957 doi: 10.1038/ncomms12957 (2016).


Psoralea corylifolia seeds (PCS) extract contains compounds that inhibit monoamine oxidase-B.Monoamine oxidase-B (MAO-B) inhibitors are widely used in the treatment of Parkinson’s disease. They increase vital monoamine neurotransmitters in the brain. However, there is a need for safer natural reversible MAO inhibitors with MAO-B selectivity. Our previous studies showed that hMAO-A and hMAO-B) isozymes. The ability of these compounds to inhibit MAO-A and MAO-B were compared to that of PCS ethanolic extract (PCSEE) using spectrophotometric assays and confirmed by luminescence assays. The highly potent and selective MAO-B inhibitor, BIO-A, was further investigated for both isozymes reversibility and enzyme kinetics. Molecular docking studies were used to predict the bioactive conformation and molecular interactions of BIO-A with both isozymes.In this study, six of PCS constituents sharing a benzopyrone structure were investigated. The compounds Biochanin-A (BIO-A), isopsoralen, 6-prenylnaringenin, neobavaisoflavone, psoralen, and psoralidin, were tested for their ability to inhibit recombinant human MAO-A and B (hMAO-A and hMAO-B with different degrees as confirmed with the luminescence assay. BIO-A inhibited hMAO-B with high potency and selectivity in the present study (IC50 = 0.003 μg/mL) and showing 38-fold more selectivity than PCSEE  without affecting hydrogen peroxide. Furthermore, BIO-A reversibly and competitively inhibited both hMAOs with significantly lower inhibitory constant (Ki) in hMAO-B (3.8 nM) than hMAO-A (99.3 nM). Our docking studies indicated that the H-bonds and hydrophobic interactions at the human MAO-A and MAO-B active sites contributed to the reversibility and selectivity of BIO-A.The data obtained indicate that benzopyrones inhibited The data obtained indicate that BIO-A is a potent, reversible and selective MAO-B inhibitor and may be recommended for further investigation in its possible use in the therapeutic management of Parkinson’s and Alzheimer’s diseases. Human monoamine oxidases A and B isozymes (MAO-A and MAO-B) regulate neurotransmitters such as dopamine (DA), norepinephrine (NE), and serotonin (5-HT) . These m2O2) and aldehydes cytotoxic byproducts /Km)). SI of BIO-A was calculated from mean Ki values (SI = hMAO-A Ki/hMAO-B Ki). The significance of differences between the different groups was determined using one-way ANOVA followed by Dunnett's multiple comparisons test or two-way ANOVA followed by Sidak's multiple comparisons test.The VhMAO-A and hMAO-B inhibitory effects of the six PCS benzopyrones constituents, we determined their enzymatic H2O2 IC50s using a spectrophotometric assay , with IPS, PS, and NBI having a relative selectivity to inhibit hMAO-A (RSA). Meanwhile, hMAO-B IC50s showed BIO-A > 6-PN > IPS > NBI > PS > PSD , with BIO-A and 6-PN having a relative selectivity in favor to inhibit hMAO-B (RSB). In this assay, BIO-A was, by far, the most potent and selective hMAO-B inhibitor among the six tested benzopyrones.To investigate the hMAO-A and hMAO-B were further confirmed at a fixed concentration using MAO-Glo™ kit luminescence assay, a highly sensitive and an H2O2-independent technique , confirming the inhibitory array pattern of the spectrophotometric assay with even higher inhibitory effects. Consistent with our spectrophotometric assay selectivity data, IPS, PS, and NBI, were more effective against hMAO-A, and 6-PN was more effective against hMAO-B, while BIO-A selectivity was masked by its highly potent effects using luminescence. Thus, the relative selectivity of benzopyrones matching the spectrophotometric assay is an indication that these benzopyrones possess hMAO-A and hMAO-B inhibitory effects with different selectivities.All the six benzopyrones activities against hMAO-A and hMAO-B with PCSEE inhibitory potencies, we elucidated PCSEE effects on both isozymes H2O2 production using the same spectrophotometric assay   with an IC50 of 3.03 μg/mL (hMAO-B) compared to 52.36 μg/mL (hMAO-A). Nevertheless, the left shifted BIO-A curves compared to PCSEE curves display BIO-A significantly higher potency and RSB than PCSEE  Fig.  a. PCSEEig. B 17.-fold wit2O2 scavenging activities were tested to reveal BIO-A and PCSEE direct effects on the enzymatic H2O2 or other side interactions with the hMAOs assays . However, the high tested concentrations of BIO-A and PCSEE did not show any scavenging activities. Moreover, PCSEE increased the H2O2 signal (p ≤ 0.05) which may indicate other autoxidation reactions that occur only at a very high concentration of 250 μg/mL. The results suggest that the inhibition of enzymatic H2O2 by BIO-A and PCSEE were solely by the mechanism of inhibiting hMAO-A and hMAO-B, and not by H2O2 scavenging activities.Hays Fig.  c. The ap > 0.05). At 254 nm with BIO-A increased concentration, BIO-A dark band matched a non-fluorescent dark band in the PCSEE fingerprint developed bands. Thus, in addition to its identification of the used seeds, the chromatographic method supported the reported presence of BIO-A as one of PCS phytochemicals.BIO-A identification was verified in the PCSEE extract using the fluorescent silica gel TLC and our optimized developing system Fig.  d. BIO-Ai values for hMAO-A and hMAO-B, are presented in Figs. To determine the isozymes mode(s) of inhibition by BIO-A, reversibility of inhibition, Michaelis-Menten parameters changes, KhMAO-A and hMAO-B inhibitor, we tested the recovery of both isozymes from BIO-A inhibition after dilution , and 51% (with hMAO-B) instead of 90%, respectively. That is an indication of possible slow, partial, or no recovery of the isozymes from DEP inhibition at the experimental conditions. With hMAO-B, repeatedly unexpected observed high signal with DEP could be due to interactions with hMAO-B sample contaminants at very elevated levels of drug and enzyme. Conversely, in similar preincubation conditions of BIO-A with hMAO-A and hMAO-B, dilution allowed a complete or an almost complete recovery of activities. The hMAO-A  geometrically doubled  with increasing BIO-A IC50s fold of concentrations. Similarly, hMAO-B Vmax showed no significant changes while benzylamine Km values were significantly (p < 0.01) nearly linearly increased  with the BIO-A doubled IC50s. The obtained data indicate the competitiveness of BIO-A to inhibit both isozymes.To determine the mode of BIO-A reversible inhibition on both MAOs, we investigated BIO-A effects on Michaelis-Menten kinetics of ies Fig. . In Fig.hMAO-A and hMAO-B inhibitions, and consequently determine BIO-A Ki and its hMAO-B selectivity index (SI), we analyzed Michaelis-Menten data by best-fit enzyme inhibitory models using GraphPad Prism.  and a non-competitive mode for hMAO-B inhibition . Meanwhile, the same model supported that BIO-A impedes substrate-binding affinity to both isozymes . Thus, the analysis confirms BIO-competitiveness for both isozymes and rejects the non-competitive, uncompetitive behavior. Consequently, BIO-A Ki values showed that BIO-A SI has a tight affinity to bind to hMAO-B more than hMAO-A .To confirm competitiveness of BIO-A for sm. Fig.  d; Tablehuman MAO-A and hMAO-B crystal structure active sites at which 2Z5X and 2XFN interacted, respectively  is the most potent EE Figs.  and 3. TO-A Fig.  and 3. Srat MAOs inhibitory effects [2O2 with a lower representation of the biological reactions. The varied inhibitory potencies may differ with the enzyme source and methods used as it was previously reported with other MAO-BIs [The obtained results indicate the non-selectivity of the studied coumarins as MAOIs. These results are supported by previous reports which indicate PCS total furocoumarins had antidepressant effects in mice and showed in vitro  effects , and ano effects . Additio MAO-BIs .hMAO-A and hMAO-B -induced dopaminergic cell damage in rats [human cortical neurons [In addition to our results in  in rats , and LPS in rats , protect in rats , 37, and neurons .More importantly, BIO-A ameliorated age- and drug-induced cognitive and behavioral dysfunctions in mice model , improve2O2 and toxic aldehydes, which are generated by hMAO-B. The reversibility and competitiveness of BIO-A for hMAOs inhibitions can also eliminate the classical side effects related to MAOIs irreversibilities such as the cheese effect, psychosis, withdrawal and possible drug interactions. Consequently, BIO-A may safely reduce the aging brain oxidative stress by introducing a new mechanism for reversibly inhibiting both human MAOs with MAO-B inhibitory selectivity. There are other natural products with MAO-B inhibition proposed for neuroprotection [The present kinetics studies indicate that BIO-A can be a safe and effective MAO-BI. The relatively superior selectivity of BIO-A can eliminate oxidative stress produced by both Hotection –49, and human MAO-B inhibitor among the studied benzopyrones. BIO-A, and the variable hMAO-A and hMAO-B inhibitions of benzopyrones may partially explain the PCSEE MAO-B inhibition potency and selectivity. The results also indicated that BIO-A is a reversible and competitive MAOI with a high selectivity index and high affinity to inhibit MAO-B. The predicted interactions of BIO-A with the active site amino acids involve reversible H-bonds and hydrophobic interactions instead of irreversible covalent adducts. This combined mechanism of MAO-B selectivity and reversibility may provide a greater safety margin and prevent the classical MAOIs side effects and following drug restrictions. Supported by previous reports, our data highlights BIO-A potentials to be used for the management of age-related neurodegenerative diseases such as PD, and AD.It was concluded from the current study that BIO-A as a PCS benzopyrone constituent was found to be the most potent and selective