Together with the native stain EM structures of AMPNPP, AMPNPP and R5P, ADP and the apo states with distinct conformations, we suggest the hexamer is the enzymatically active form. Based on crystal structures, sequence analysis, mutagenesis, enzyme kinetics assays, and MD simulations, we reveal the conserved substrates binding motifs and make further analysis of all pathogenic mutants.Human PRS1, which is indispensable for the biosynthesis of nucleotides, deoxynucleotides and their derivatives, is associated directly with multiple human diseases because of single base mutation. However, a molecular understanding of the effect of these mutations is hampered by the lack of understanding of its catalytic mechanism. Here, we reconstruct the 3D EM structure of the PRS1  The inhibition of the pathways for the biosynthesis of nucleotides blocks transcription and consequently prevents the proliferation of cells. Nucleotides can be synthesized de novo or through salvage pathways that use 5-phosphoribosyl 1-pyrophosphate (PRPP) as both a common metabolic intermediate and a regulator ..2+, are Bacillus subtilis  on beamline 17U of the Shanghai Synchrotron Radiation Facility at the Shanghai Institute of Applied Physics, Chinese Academy of Sciences (detector: marccd). Data were processed using HKL2000 and programs in the CCP4 package. Phase determination was performed by molecular replacement using MOLREP in the CCP4 package with the human PRS1 D52H mutant (PDB entry 4F8E) as the initial search model. The final refinement was carried out with refmac5. All crystal structures of PRS1 and its mutants have been deposited into the RCSB Protein Data Bank under accession codes 3S5J, 4LYG, 4LZN, 4LZO, 4M0P and 4M0U. Figures were prepared using PyMOL (w/w) uranyl formate. We imaged tilted (−55°) and untilted (0°) pairs of PRS1 particles under low-dose condition using a FEI Tecnai F20 microscope  with an FEI Eagle CCD camera at 62,000× magnification. The original pixel size was 1.77 Å per pixel and two-fold pixel-averaged to 3.54 Å per pixel. Particles were selected using the TiltPicker software[Negative stained specimens of PRS1 and PRS1 incubated with different substrates or inhibitors were preserved as previously described. The AMPWe obtained the 3D reconstruction using the random conical tilt (RCT) method. In totaSpinacia oleracea KPRS1 and KPRS3, Escherichia coli KPRS, Bacillus subtilis KPRS, Zea mays PRS, Thermoplasma volcanium KPRS and Methanocaldococcus jannaschii KPRS using Weblogo [To define the consensus residues involved in substrate binding in all 3 PRS classes ,24, we a Weblogo ,26.All enzyme activity assays were assayed by determining AMP formation using a method similar to that used in the published paper. The fin+ ions were added to the system to neutralize the net negative charge of the system by replacing water molecules with the most favorable electrostatic potential. The final system was energy minimized using the steepest descent followed by the conjugate gradient method until the maximum force on any atom was less than 400kJ/(mol.nm). Verlet integration scheme[−1 nm−2. The initial atomic velocities were generated according to a Maxwell distribution at 310 K. The following production run was 100 ns long. The simulation was performed in a constant NPT ensemble, and the system was coupled to a temperature bath of 310 K through use of an velocity rescaling thermostat[–5 bar−1[MODELLER was usedon scheme was usedhermostat. The pre[–5 bar−1. Covalen[–5 bar−1. The cut[–5 bar−1, with a 42− complex was determined using the MR method to 2.0 Å resolution. More information about the 2.0 Å resolution structure could be found in the supplementary materials  exist among all 3 classes of PRS (Thermoplasma volcanium PRS and its substrate complexes (PDB code: 3LPN and 3MBI)  . To testactivity , which ind R199A . Moreove strands . Further strands . Consequ89 to Ile107), exists among all known PRS structures (Thermoplasma volcanium PRS-ATP complex structure (PDB code: 3LPN) help to confirm the ATP binding pocket, of which the key residues involved are highly conserved (35 (located in the β2–3 strands) and His130 were both single mutated to Ala (F35A and H130A) for activity assays. However, although the H130A or F35A mutation does not display normal AMP activity , we inferred that the flexible loop is able to sweep across the ATP α-β phosphodiester bond using Lys99 and cut it into AMP and PPi moiety , AMPNPP and R5P, ADP and M method . The 2D M method , demonst hexamer . However hexamer . The PRS hexamer . The PRS hexamer , which mapo states confirm the three-leaf clover or sunflower-like structures  Monomer structure of human PRS1. The N-terminus is colored green, the C-terminus is colored ruby and the C terminal 310 helix 2 is colored purple. The linkers between domains are colored yellow. (B) The dimer in the one asymmetry structure with subunit A colored as in A and subunit B colored silvery white. The 2Fo-Fc density map of 3 SO42−s with sigma 1.0 is colored dark blue.(TIF)Click here for additional data file.S2 FigSecondary structure compositions of PRS1.(TIF)Click here for additional data file.S3 Fig(A) A typical micrograph of PRS1 preserved under negatively stained . (B) Representative of single particles of PRS1. The scale bar corresponds to 100 Å. (C) Two-dimensional (2D) EM analysis of PRS1. The scale bar corresponds to 100 Å. (D) Two-dimensional (2D) EM analysis of PRS1 in three variable conformations. The scale bar corresponds to 50 Å. (E) Three-dimensional (3D) reconstructions of PRS1 in three variable conformations. The scale bar corresponds to 50 Å.(TIF)Click here for additional data file.S4 Fig(A) A typical micrograph of PRS1 with AMPNPP preserved under negatively stained. (B) Representative of single particles of PRS1 with AMPNPP. (C) A typical micrograph of PRS1 with ADP preserved under negatively stained. (D) Representative of single particles of PRS1 with ADP. (E) A typical micrograph of PRS1 with AMPNPP and R5P preserved under negatively stained. (F) Two-dimensional EM analysis of PRS1 with AMPNPP and R5P to show complicate conformations.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S1 VideoThe movie was generated using PDB 3LPN and 3MBI. The ligand in the movie is R5P. The movie was made using Chimera.(MPG)Click here for additional data file.S2 VideoThe movie was generated using PDB 3S5J, 1DKU, and 1DKR. The ligand in the movie is ATP. The movie was made using Chimera.(MPG)Click here for additional data file.


Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5.Flavivirus RNA replication occurs within a replication complex (RC) that assembles on ER membranes and comprises both non-structural (NS) viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent-RNA polymerase (RdRp) domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3) at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV), the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 3 in vitro enzymatic activities of the recombinant proteins.DENV causes widespread mosquito-borne viral infections worldwide and nearly 40% of the world’s population is at risk of being infected. Currently, no licensed vaccines or specific drugs are available to treat severe infections by DENV. NS5 is a large protein of 900 amino acids composed of two domains with several key enzymatic activities for viral RNA replication in the host cell and constitutes a prime target for the design of antiviral inhibitors. We succeeded in trapping a stable conformation of the full-length NS5 protein and report its crystal structure at a resolution of 2.3 Å. This conformation reveals the entire inter-domain region and clarifies the determinants of NS5 flexibility. The inter-domain interface is stabilized by several polar contacts between residues projecting from the MTase and RdRp domains of NS5. Several evolutionarily conserved residues at the interface play a crucial role for virus replication as shown by reverse genetics, although the analogous mutations mostly do not abolish the  Several flaviviruses such as Dengue virus (DENV), Japanese Encephalitis virus (JEV), West Nile virus (WNV), Yellow Fever virus (YFV) and Tick-Borne Encephalitis virus (TBEV) are major human pathogens. The mosquito-borne DENV serotypes 1–4 cause widespread epidemics and nearly 40% of the world’s population is at risk of being infected  8]  [8] O at  [8] O aresponse . A putatresponse ,11 but tresponse ,13. The response –17. NS5 response . In addiresponse . In DENVresponse ,23. The response –28. A puin silico docking guided by reverse genetics, a first model for NS5 from WNV was proposed -SAM in 50 mM Tris-HCl, pH 7.5, 20 mM NaCl, and 0.05% (v/v) CHAPS. The 2’-O MTase reaction comprised 25 nM protein, 40 nM GpppA-7mer RNA (Trilink), 320 nM [3H-methyl]-SAM in 50 mM Tris-HCl, pH 7.5, 10 mM KCl, 2 mM MgCl2, and 0.05% CHAPS. Buffer, RNA substrate, and enzyme were first mixed in a single well in a 96-well half- area, white opaque plates , and the reaction was initiated by addition of [3H-methyl]-SAM. The N-7 and 2’-O reactions were incubated at RT for 15 min and 1 hr, respectively. The reaction was stopped with 25 μl of 2× stop solution  and shaken for 20 min at 750 rpm at room temperature followed by centrifugation for 2 mins at 1200 rpm. The plate was read in a Trilux microbeta counter  with a counting time of 1 min per well. All data points were collected in duplicate wells.N-7 and 2’-escribed ,48. In gde novo initiation/elongation assay was described . In. In31]. Based on the present report and comparable studies on NS5 from JEV and other flaviviruses, it is possible that the NS5 protein from different flaviviruses may have evolved to adopt different sets of conformations, stabilized by distinct interfaces and leading to different allosteric mechanisms to regulate their enzymatic activities Figs. . This hyS1 TextS3 Fig. Structure alignments of DENV3 NS5 with NS5-MTase and NS5-RdRp domains. (A) DENV3 NS5 is in purple, DENV3 NS5-MTase (PDB code: 3P97) is in yellow, DENV2 NS5-MTase (PDB code: 1L9K) in orange, DENV3 NS5-RdRp (PDB code: 4C11) in Cyan, DENV3 NS5-RdRp (2J7U) in gray. N’ and C’ stands for N-terminal and C-terminal of NS5 protein; G: motif G, residues 406–417; F: motif F, residues 455–468; Zn1 and Zn2 are two zinc cations in their binding sites. (B) Zoom-in view of the linker region. Key residues are displayed as sticks and the Cα atoms are shown as spheres. S4 Fig. Structure alignments of JEV full length NS5 with JEV RdRp domains. (A) JEV NS5 (PDB Code: 4K6M) is in red and JEV RdRp (PDB Code: 4HDG) is in blue. The structures are aligned and displayed as ribbon. Motifs F and G are shown as connected spheres and each sphere represents one residue. (B) Enlarged view at the domain linker region. S5 Fig. Homology modeling of flavivirus NS5 structures. (A) Homology modeling of DENV3, WNV, YFV, and TBEV NS5 to JEV NS5 was performed using HHpred server (http://toolkit.tuebingen.mpg.de/hhpred). The figure below is the structure alignment at the JEV interface. The hydrophobic residues at the interface are labeled based on DENV3 NS5. These residues are within the close proximity, suggesting a likely interface, possible for NS5 from all flaviviruses. (B) Structure alignment of the homology models of NS5 from JEV, WNV, YFV, and TBEV based on DENV3 NS5 crystal structure (this work), at the interface area. Similarly, the key polar residues that are labeled based on DENV3 NS5. These residues are also within the close proximity, suggesting a likely interface, possible for NS5 from all flaviviruses. S1 Table. List of DNA primers. (A) Primers used for site-directed mutagenesis (SDM) of DENV4-F plasmid. (B) Primers used for PCR of T7pro-LUC-NS3 cDNA cassette from pACYCI DENV4-WT replicon. (C) Primers used for PCR of NS5 full length mutant cDNA cassettes from pACYCI DENV4-F mutant Replicon plasmids  into pET28a vector. (D) Primers used for site-directed mutagenesis (SDM) of DENV2 infectious clone. S2 Table. Summary of the MTase-RdRP interdomain interface analysis of NS5 from DENV3 and JEV. (A) Comparative analysis of MTase-RdRP in DENV3 NS5 and JEV NS5. The MTase domain and RdRP domain were defined as regions listed in the table. iNres: number of residues involved in MTase-RdRP interactions. Total buried area: calculated as the difference between total accessible surface areas of isolated and interfacing structures, divided by two, using PISA. ΔiG: Indicates the solvation free energy gain upon formation of the interface. The value is calculated as difference in total solvation energies of isolated and interfacing structures, using PISA. NHB/NSB/NvdW: number of putative hydrogen bonds, salt bridges and additional van der Waals interactions that contribute to the interface. (B) Pairs of residues from MTase and RdRp that are involved in inter-domain interactions.Deuterium uptake data shown in the heat map format where peptides are represented using rectangular strips below the portion of the protein sequenc they map back to and colors are used to categorize average deuterium incorporation for each peptide. The key shows colors assigned to % deuterium ranges. (A) DENV3 NS5 (full length), (B) DENV3 NS5-MTase (1–296), (C) DENV3 NS5-RdRp (273–900). (D) Heat map data overlaid onto a crystal structure for the DENV3 NS5-MTase (1–296), and RdRp (273–900). The color key indicates the extent of the deuterium uptake (%), in which blue means the lowest deuterium uptake. Peptides that colored gray are regions that information of deuterium uptake is missing. F: motif F; G: motif G. (E) Comparison of HDX extent of the peptides at the inter-domain interface. Peptide F65–77, which positions as one interface region in MTase, exhibited similar deuterium uptake activities in both MTase protein (12%) and full-length NS5 protein (12%). Another interface region, residues K95–118, displayed 18% and 20% average deuterium uptakes for all HDX time points. (F) These two peptides displayed comparable HDX behavior in solutions as indicated by the same patterns of MS isotopic distributions. The C terminal region of MTase (peptide T243–273) also showed high hydrogen bonding activities in both MTase domain and NS5 samples. (DOCX)Click here for additional data file.S1 MovieInitial state was based on JEV NS5 and the end state was based on DENV3 NS5 structure.(MPG)Click here for additional data file.S2 MovieMTase domain is displayed as cartoon and the RdRp domain is in surface view. Same color theme as in (MPG)Click here for additional data file.S3 MovieMTase domain is displayed as cartoon and the RdRp domain is in surface view. Same color theme as in (MPG)Click here for additional data file.


Only miR34A-5p is expressed in PBL-B, and significantly induced in activated B-cells and reactive lymph nodes. In PBL-B, the TP53 mutation and MIR34A methylation (“double hit”) and these patients have an exceedingly poor prognosis with a median survival of 9.4 months (P<0.0001), while neither TP53 mutation, MIR34A or MIR34B/C promoter methylation alone (“single hit”) influence on survival. The TP53/MIR34A “double-hit” is an independent negative prognostic factor for survival (P=0.0002). In 2 DLBCL-cell lines with both TP53 mutation and promoter methylation of MIR34A, miR34A-5p is upregulated by 5-aza-2'deoxycytidine. Thus, the TP53/MIR34A “double hit” characterizes a very aggressive subgroup of DLBCL, which may be treatable with epigenetic therapy prior to or in combination with conventional immunochemotherapy.Nine de novo DLBCL cases (n=150) carry both  TP53 mutation alone as a prognostic factor for survival in DLBCL have been inconsistent, however, a number of studies have shown that dual disruption of the p16/Rb and the ARF/p53 pathway, e.g. deletion of the INK4A/ARF locus, are strong negative prognostic factors for survival [MYC and anti-apoptotic BCL-2 or BCL-6  are associated with exceedingly poor prognosis [Diffuse large B-cell lymphoma (DLBCL) comprise a variety of morphological and clinical phenotypes, resulting from miscellaneous, underlying molecular defects. Gene expression profiles and their immunohistochemical surrogate markers have been used to classify DLBCL into prognostic subgroups , 2, stilrognosis , 8.Most previous studies of tumor suppressor pathways in DLBCL have focused on the disruption of the coding sequences of genes. However, epigenetic alterations and aberrant expression of microRNAs (miRs) may be equally important for growth control. As for protein encoding genes, the transcription of miR genes may be inactivated by promoter hypermethylation , 10.MIR34A and MIR34B/C , and the miR34s in turn act as mediators of p53 signaling [The members of the miR34 family  have been recognized as tumor suppressors, and implicated in a variety of cellular processes that control carcinogenesis including cell cycling, apoptosis, somatic cell reprogramming and metastasis –14. It hignaling , 16. In ignaling –19. ThesTP53 mutation or 17p deletion, and has negative prognostic impact on both treatment free survival [MIR34B/C cluster is downregulated by promoter methylation in a large proportion of cases at relapse [de novo DLBCLs, the miR34s have not previously been investigated, and in spite of their implication in several other lymphoproliferative malignancies, little is known about the role of the miR34s in normal B-cells.Several studies have implicated both miR34A –22 and msurvival  and survsurvival . In mult relapse . In low- relapse . HoweverMIR34A and MIR34B/C locate to regions of allelic loss in DLBCL  [MIR34A and MIR34B/C promoter methylation, TP53 mutational status, clinical presentation patterns, and outcome.Here, we investigated the expression of miR34A, miR34B and miR34C in normal and reactive B-cells. Given that ctively) , 26, andde novo DLBCLs has not been analysed in detail, and little is known about the role of the miR34s in normal B-cells.Several studies have shown the implication of the miR34s in CLL and multiple myeloma, and a single study implicated miR34A in the transformation of gastric MALT-type lymphomas to DLBCL –24. HoweTP53 mutational status and hypermethylation of the MIR34A and MIR34B/C promoters in a panel of 150 newly diagnosed DLBCL and in 6 DLBCL cell lines, and compared these data to those obtained from PBL-B and reactive lymph nodes.p53 has been shown to directly bind to and regulate the transcription of both miR34A and miR34B/C , and we MIR34A and MIR34B/C promoters. While miR34A-5p was expressed at low levels in normal PBL-B, no expression of miR34A-3p, miR34B-5p, miR34B-3p, miR34C-5p, and miR34C-3p could be detected in PBL-B ). In reactive lymph nodes the expression of miR34A-5p was induced in average 70 fold (P<0.001), while no significant induction was observed for the rest of the investigated miRs, including the homologous miR34B-5p and miR34C-5p, indicating that the assay is highly specific to miR34A-5p , but not of miR34B/C. In addition, normal CD3+ T cells showed no expression of miR34A, indicating that the response we observe in reactive lymph nodes most likely originate from activated B-cells  for the bivalent H3K27me3/H3K4me3 silencing mark at the MIR34B/C promoter  at the r Figure .Taken together, these results indicate that in PBL-B both miR34A and miR34B/C are expressed at low levels. The miR34B/C cluster is downregulated by polycomb, and in contrast to miR34A, does not seem to be reactivated upon stimulation.MIR34A and MIR34B/C promoters in DLBCL-cell lines, and compared these data with TP53 mutational status. In all DLBCL cell lines the MIR34B/C promoter was methylated  are upregulated irrespective of the TP53 mutation   cases , suggesting that the methylation of these miRs may be interdependent and occur in coordinated manner. The MIR34A methylated cases did not differ significantly from the MIR34A unmethylated cases with respect to age, sex, clinical stage, LDH, performance score or IPI (Table MIR34B/C methylated vs. unmethylated cases (data not shown).To investigate whether these combined molecular alterations also occur ) Figure , and MIRs Figure . All excTP53 gene revealed mutations in 24 out of 150 (16%) of the primary DLBCL cases . Since studies in CLL suggest that the MDM2 SNP309 GG genotype associate with low miR34A expression and poor prognosis, we investigated the role of this SNP in a subset (120) of these primary DLBCLs, but observed no prognostic impact .DGGE-based screening of exons 5-9 of the s Figure . These ms Figure , and werTP53 mutation and MIR34A promoter methylation at diagnosis. The TP53 mutations were both loss of function mutations (4) and missense mutations in the DNA binding domain (5)  MIR34A methylated cases also had TP53 mutation (P=0.19), indicating that these alterations do occur independently of each other. These data support our findings from the cell lines, that a subset of DLBCL has combined loss of p53 and miR34A function. We observed that the MIR34A methylated and TP53 mutated patients were associated with aggressive disease with Ann Arbor stage III-IV (P=0.030), elevated LDH level (P=0.015), and IPI group III-IV (P=0.023). In the cohorte of patients treated with Rituximab the MIR34A methylated and TP53 mutated cases were also significantly associated with Ann Arbor stage III-IV (P=0.025), elevated LDH level (P=0.002), and IPI group III-IV (P=0.027) (Table By combining the above data we identified a subset of nine primary DLBCL cases that carried combined  the 42 2% MIR34A MIR34B/C did not differ from cases with unmethylated MIR34B/C (P=0.803). Cases with TP53 mutations had significantly poorer survival (P=0.004), whereas cases with MIR34A methylation showed a tendency towards poorer survival (P=0.077). However, the 9 cases with disruption of both TP53 and MIR34A showed an exceedingly poor survival of only median 9.4 months (P<0.0001)  and in the Rituximab treated patients (P=0.021). Furthermore, elevated LDH, age, performance status and lack of Rituximab are independent prognostic factors in the entire cohorte, while only LDH and age retain negative prognostic impact in Rituximab treated patients . Thus, our data suggest that mechanisms different than promoter methylation may keep the unmethylated alleles silenced , which is a subject for further investigation.Surprisingly, our DLBCL cell line data shows that methylation of approximately half of the investigated alleles at the reported R34A TSS , is asso alleles . As oppoMIR34A methylation in lymphomagenesis. Analyses of a small sample  showed promoter hypermethylation primarily in NK/T-cell NHL [MIR34A methylation. For the MDM2 SNP309 our observations are in line with previous studies [Two previous studies focus on the role of cell NHL , and a scell NHL . However studies , 45, whiTP53 mutations in DLBCL is still debated [TP53 disruption is still a negative prognostic factor for survival after the implementation of Rituximab[TP53 mutation only had died . This was the only patient in the “double-hit” group to achieve an etoposide containing regimen (R-CHOEP). Interestingly, PMBL was recently shown to have particularly good outcome when treated with the same drugs (dose-adjusted EPOCH-R) [The prognostic role of  debated , 46–48 hEPOCH-R) .MIR34A nor TP53 mutation alone influence survival , while the MIR34A/TP53 “doublehit” is an independent factor for survival. This is quite surprising given that studies in CLL have indicated that miR34A acts in concert with p53 and MDM2, and may serve as a surrogate marker for disruption of these molecules. Furthermore, it has been suggested that the apoptosis inducing function of miR34A is p53 dependent [These data are supported by our multivariate analysis which show that neither methylation of ependent . Howeverependent  , and CD3+T-cells from 6 healthy donors were used as controls. All patients were treated with anthracycline containing regimens (CHOP or CHOP-like). Sixty-two of the patients also received Rituximab (R). The clinical data were obtained from the patient files and from the Danish lymphoma registry, LYFO. Approval of this study was obtained from the ethical committee.5 cells/ml and cultured in 20% HIFCS RPMI1640 with 100 units/ml penicillin, 100μg/ml streptomycin, 50 ug/ml lipopolysaccarides from Escherichia coli O55:B5 , and 0,6 ng/mL IL-1α (Genescript) to stimulate IgM and IgG3. The activated B cells were harvested on day 4.PBL-B purified from healthy donors by RoboSep human CD19 positive selection kit (Stemcell) were plated 5 x 105 cells/ml 24 hrs prior to treatment with 0.1μM and 0.5 μM of the DNA methyltransfrease inhibitor 5-Aza-2-deoxycytidine . The medium was changed and the drug removed after 24 hrs treatment, and cells were collected 1 day and 5 days later. Untreated cells and cells treated with cytosine arabinoside  20nM or 100nM were grown and harvested under similar conditions as control.DLBCL cell lines: DB1, Toledo, Farage, DOHH2, HT, and U2932 were cultured as above. HT and U2932 cells were seeded 2x10MIR34A and MIR34B/C using the primers in Supplemental PBL-B were crosslinked, nuclear extracts sonicated, and mixed with 10 μg of antibodies H3 (Abcam ab791), H3K27me3 (Millipore 17-622) and 4 μg of antibody H3K4me3 (Active Motif 39159). Parallel preimmune control precipitation was performed by normal IgG . DNA precipitated in the ChIP experiments was amplified by RT-qPCR at the promotors, transcription start sites (TSS) and genomic regions for TP53 gene were scanned for mutations by PCR and denaturing gradient gel electrophoresis (DGGE) as desribed [The coding sequences and splice sites of exons 5-9 of the desribed . All mutMIR34A and the MIR34B/C promoters was examined using methylation-specific melting curve analysis (MS-MCA) [SssI treated DNA (Millipore) was bisulfite converted and used as positive control. Bisulfite converted DNA from PBL-B was used as normal control.One μg of each DNA sample was bisulfite converted using the EZ DNA Methylation kit (Zymo Research). The methylation status of the (MS-MCA) . The DNA(MS-MCA) , 61. For(MS-MCA) . For priMIR34A alleles PCR products were cloned into the pCR2.1 vector using the TOPO-TA cloning kit (Invitrogen). Colonies were screened for the respective inserts, amplified using TempliPhi (Amersham Biosciences) and 10 individual colonies were sequenced. For primers see Supplemental To analyze the methylation status of individual 8 copies to 10 copies per RT reaction.Fifty ng of RNA was reverse transcribed (RT) using miRCURY LNA™ Universal RT (Exiqon). RT-qPCR for miR34A-5p, miR34A-3p, miR34B-5p, miR34B-3p, miR34C-5p and miR34C-3p was performed using miRCURY LNA™ Universal RT primer sets (nomenclature according to miRbase18), and U44 RNA was used for normalization . The RT-qPCRs were performed on the LightCycler 480 instrument (Roche diagnostics) using the conditions recommended by Exiqon. Absolute quantification was performed using a miR-34A-5p mimic in final amounts of 1x10MIR34A, MIR34B/C and mutation of TP53 were assessed using the Pearson chi-square or Fisher's exact tests. Overall survival was estimated using the Kaplan-Meier method and log-rank test. For assessment of independent predictors of survival a multivariate Cox regression hazard model with backward stepwise (likelihood ratio) entry was applied. Effects not meeting a p-value < 0.05 were removed from the model. Statistical analyses were performed in SPSS 19.0 for Windows (SPSS Inc.) and SAS version 9.20 . Any differences were considered to be statistically significant when the p-value was <0.05.Differences in clinical characteristics of patients with or without methylation of


Sequencing of the third hypervariable loop allows to identify genotype-based HIV tropism. R5-tropic viruses associated with early stages of infection are preferentially transmitted, while non-R5 HIV-1 tropism has been associated with severe immunodeficiency and lower lymphocyte CD4 nadir and may reflect delayed HIV diagnosis. In this study, we investigate the changes in tropism frequency from 2007 to 2013.Study included 194 patients with confirmed HIV infection linked to care in 2007–2013. Baseline plasma samples from treatment naive patients were used for HIV-1 genotypic tropism assessment based on triplicate V3 loop sequencing. Non-R5 tropism prediction thresholds were assigned using a false positive rate (FPR) of 10% and 5.75% FPR and associated with clinical and laboratory data . For statistics, chi-square and Mann–Whitney U tests were used, time trends were examined using logistic regression  for binary variables and linear regression for continuous ones.Overall non-R5 tropism frequency for the 5.75% FPR was 15.5% and 27.8% for 10% FPR. Frequency of the non-R5 tropism predicted using 5.75% FPR increased significantly from 2007 (0%) to 2013 (25%)  a. With 1R5 tropism predominates among the treatment naive individuals but increase in the frequency of non-R5 tropic variants may limit clinical efficacy of the coreceptor inhibitors. Increased prevalence of non-R5 HIV-1 may be related to late care entry and higher number of AIDS diagnoses in the recent years.


GAS8 shares strong sequence similarity with the Chlamydomonas Nexin-Dynein Regulatory Complex (NDRC) protein 4 (DRC4) where it is needed for proper flagella motility. In mammalian cells, the GAS8 protein localizes not only to the microtubule axoneme of motile cilia, but also to the base of non-motile cilia. Gas8 was recently implicated in the Hh signaling pathway as a regulator of Smoothened trafficking into the cilium. Here, we generate the first mouse with a Gas8 mutation and show that it causes severe PCD phenotypes; however, there were no overt Hh pathway phenotypes. In addition, we identified two human patients with missense variants in Gas8. Rescue experiments in Chlamydomonas revealed a subtle defect in swim velocity compared to controls. Further experiments using CRISPR/Cas9 homology driven repair (HDR) to generate one of these human missense variants in mice demonstrated that this allele is likely pathogenic.Ciliopathies are genetic disorders arising from dysfunction of microtubule-based cellular appendages called cilia. Different cilia types possess distinct stereotypic microtubule doublet arrangements with non-motile or ‘primary’ cilia having a 9+0 and motile cilia have a 9+2 array of microtubule doublets. Primary cilia are critical sensory and signaling centers needed for normal mammalian development. Defects in their structure/function result in a spectrum of clinical and developmental pathologies including abnormal neural tube and limb patterning. Altered patterning phenotypes in the limb and neural tube are due to perturbations in the hedgehog (Hh) signaling pathway. Motile cilia are important in fluid movement and defects in motility result in chronic respiratory infections, altered left-right asymmetry, and infertility. These features are the hallmarks of Primary Ciliary Dyskinesia . While mutations in several genes are associated with PCD in patients and animal models, the genetic lesion in many cases is unknown. We assessed the  in vivo, we created a Gas8 genetrap mutant mouse. Though no overt primary cilia phenotypes were evident in the Gas8 genetrap mutant mice, there were severe motility defects and the mice presented with Primary Ciliary Dyskinesia (PCD) like symptoms including situs inversus and hydrocephalus. We also identified two potential disease causing GAS8 missense variants (A391V and E199K) in humans. Utilizing CRISPR/Cas9 we generated a mouse to mimic the A391V allele. When we crossed the Gas8AV mutants with the Gas8GT mutant, the compound Gas8GT/AV heterozygous animals developed mild hydrocephalus. Rescue experiments using Chlamydomonas with mutations in the Gas8 homolog revealed only a modest decrease in swim velocity raising the possibility that the E199K allele is not pathogenic.Growth-Arrest Specific 8 (Gas8) is implicated in dual roles at both the primary cilium to regulate hedgehog signaling and in motile cilia to coordinate cilia movement. To investigate these roles  Primary cilia are solitary and immotile cellular appendages that serve as signaling hubs for pathways such as Hedgehog (Hh) during development . Motile Chlamydomonas reinhardtii, data indicate that the N-DRC functions to link the A microtubule of one doublet with the B microtubule of the adjacent doublet. It coordinates the activities of the outer and inner dynein arms to regulate flagellar beat frequency and waveform . Th. ThGT mud screen . We utilAV mice onto the Gas8GT background to create compound heterozygous (Gas8GT/AV) mice. To determine the impact on motile cilia and test possible cause of the hydrocephalus, we took brains from 6 week old mice and analyzed cilia beat and the ability of motile cilia to move fluid. While there were no differences in beat frequency, bead flow analysis shows a modest decrease in the ability of Gas8GT/AV cilia to move fluid compared to Gas8GT/WT cilia  display hydrocephalus at this age, the severity ranged from mild (arrowhead) to moderate (arrow). The phenotype in the Gas8GT/AV mice is not as severe as in the Gas8GT mice and hydrocephalus was not present in any (n = 2) of the Gas8GT/WT or (n = 2) of the Gas8WT/AV mice analyzed.To further assess the potential pathogenicity of the human allele, we created a mouse harboring the A391V mutation via homology driven repair with CRISPR/Cas9 technology. Sequencing confirmed the presence of the c.1172 C>T mutation resulting in an A391V amino acid change . We crosWT cilia . CompounWT cilia  but therChlamydomonas before proceeding to a potential mammalian model. Alignment of GAS8 and the Chlamydomonas orthologue DRC4 revealed that E199 in GAS8 aligns with D198 in DRC4  of the human E199K alleles in a null mutant background (pf2). Interestingly, transformation with DRC4-DK-GFP rescued the severe motility defects seen in the pf2 null mutant, but measurements of forward swimming velocities revealed a subtle defect in the swimming phenotype of the rescued strains  as well as a positive effector of Smoothened transport into cilia during Hh pathway activation [Defects involving cilia motility cause severe phenotypes in humans including infertility, hydrocephalus, respiratory defects, and reversal of left-right asymmetry. Much of our understanding about cilia motility has come from studies in organisms such as tivation ,13,19.GT mutant allele is translated into a large fusion protein between the N-terminal portion of Gas8 and β-geo, it does not localize to motile cilia. Gas8GT mutant mice present with hydrocephalus starting at postnatal day 5 (P5) that becomes more pronounced as the mice mature and eventually leads to mortality between P14-P21. Development of hydrocephalus is associated with severe impairment of cilia motility on ependymal cells lining the ventricles of the brain.To further evaluate the connection between Gas8 and PCD in mammals, we generated a mouse with a β-geo cassette inserted in intron 7 of the Gas8 gene. Insertion of the β-geo genetrap cassette effectively eliminated the presence of wildtype transcript and protein in mutants as verified by RT-PCR and western blot analysis. Though the Gas8Chlamydomonas showed that strains with mutations in PF2/DRC4, the Gas8 homolog, were associated with the loss of the majority of the N-DRC complex along with a subset of the IDAs [Chlamydomonas results, the N-DRC and IDA do not appear to be overtly affected in Gas8 mutant mice based on standard thin-section TEM analysis. However, loss of Gas8 does effect microtubule organization, as indicated by a higher percentage of cilia with disorganized microtubule doublets in Gas8GT mutant mice when compared to Gas8WT mice. Altered cilia microtubules were recently also recently observed in human Gas8 patients [et al 2014.Previous studies using image average procedures to analyze flagella ultrastructure in the IDAs ,16,17,33patients ,13. TogeGT mutant cilia failed to move, however those that were observed moving displayed a modest decrease in beat frequency. The most distinguishable phenotype observed in the cilia that moved was a very rigid and short wave pattern. This pattern has also been observed in other cilia motility mutants thought to affect the NDRC [GT cilia show a more random distribution of cilia orientation than their Gas8WT counterparts supporting the necessity of proper fluid flow in establishing cilia orientation.Most Gas8the NDRC –12. Thesthe NDRC ,35. Gas8GT allele was inserted in close proximity (K337) to the A334* mutation. The E199K mutation also affects a highly conserved region within Gas8 that is proposed to be a Microtubule Association Domain (GMAD) [Variants in Gas8 were recently identified in human PCD patients. These mutations resulted in a similar, albeit not significant, decrease in beat frequency along with an abnormally rigid ciliary waveform ,13. Thisn (GMAD) .GT and Gas8AV mutations develop age dependent, mild hydrocephalus, but did not present with situs defects (n = 6 Gas8GT/AV mice). The phenotype was associated with a reduced ability of ependymal cilia to move fluid. Interestingly, beat frequency was not significantly altered from that of controls, suggesting that the defect lies within a subtle waveform difference or in cilia orientation. These data suggest that the A391V allele is pathogenic though more in-depth analysis of ciliary defects will be necessary to determine the precise mechanism. Data from the Chlamydomonas rescue experiments suggest that the E199K allele may have very subtle effects on motility. The D198K rescued strain in Chlamydomonas showed a small but statistically significant reduction in forward swim velocity of approximately 10 percent. While statistically significant, additional work is needed to determine whether such small changes might impact ciliary motility and have pathogenic consequences in different tissues and different organisms. As this variant is commonly found in Latino populations, it seems more likely that this variant is a benign polymorphism.To test pathogenicity of the A391V allele, we used CRISPR/CAS9 homology driven repair (HDR) to generate a mouse line mimicking the human mutation. Mice compound heterozygous for the Gas8In vitro data indicated that the C-terminal region of Gas8 binds to Smoothened (Smo) and acts at the base of primary cilia as a regulator of Smo entry into the cilium following Hh pathway activation [in vitro findings, we expected to see Hh defects in our mutant mice. However, the Gas8 mutants survive to birth and have normal digit number and patterning as well as normal neural tube dorsal ventral patterning. Furthermore, there were no significant differences in Smo accumulation in cilia between Gas8WT and Gas8GT MEFs after SAG stimulation, suggesting that in this mutant model, Gas8 does not act as a regulator for Smo entry. The role that Gas8 plays at the base of primary cilia remains uncertain; however, we do not see any other pathologies that would suggest there is a defect in primary cilia such as cystic kidney disease.Gas8 was previously implicated as a modulator of the Hh pathway. tivation . These dGAS8 as a disease causing gene in humans and elucidate the mechanisms by which loss of Gas8 causes disease. We identified new independent, homozygous missense mutations and used model systems to test the pathogenicity of the alleles. Importantly, these results suggest the A391V allele is pathogenic while the E199K variant is not. Our results demonstrate the importance of testing the potential pathogenicity of human alleles in easily amenable model systems such as Chlamydomonas and further reveal the ease with which CRISPR/Cas9 has now made it possible to conduct similar tests in mouse models.The data presented here solidify GT embryonic stem cells were from the 129P2/OlaHsd genetic background and were injected into C57BL/6 blastocysts using standard procedures. Chimeras were bred with albino C57BL/6 females and germline transmission was confirmed by coat color and subsequent PCR genotyping.The Gas8 mutant mouse line was generated using embryonic stem cell line CH0760 (BayGenomics) in which a β-galactosidase neomycin resistance fusion cassette was inserted into intron 7 of Gas8. The insertion site was confirmed by genomic PCR and sequence analysis. PCR primers for genotyping were designed based on the insertion site and are as follows 5’-GGGACAAGCAGATTCTGGTC-3’, 5’-CAGGGTTACACACAGAGAAACC-3’, and 5’-CCGCAAACTCCTATTTCTG-3’. The Gas8All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) regulations at the University of Alabama at Birmingham under the animal protocol number (130208061).WT, Gas8GT/WT, and Gas8GT mouse embryonic fibroblasts using Trizol reagent according to the manufacturer’s protocol . cDNA was generated using Verso cDNA kit . 5’ Gas8 RT-PCR was performed using the following primers spanning exons 3 and 4: 5’-GAATCGAAGAATACCACCATC-3’ and 5’-CTGAGAAGATGGCTATGTAG-3’. 3’ Gas8 RT-PCR was performed with primers spanning exons 9 and 10: 5’-CTGGACCCCACAGCATTAAC-3’ and 5’-CTTGATGGTGGTATTCTTCG-3’. Actin control primers: 5’-ATGGGTCAGAAGGACTCCTA-3’ and 5’-GGTGTAAAACGCAGCTCA-3’ were used in all samples.RNA was isolated from Gas8Animals were anesthetized by a 0.1 ml per 10 g of body weight intraperitoneal injection of 2.5% tribromoethanol , killed by cardiac puncture, and perfused with PBS followed by 4% paraformaldehyde . The brains were further fixed in 4% paraformaldehyde 1h at room temperature followed by successive dehydration through 1 hour alcohol incubations at 30% and 50% and placed finally in 70% overnight. Tissues were further dehydrated through 1 hour alcohol incubations at 80%, 95%, and finally 100%. Tissues were placed in xylenes for 1 hour and then placed in a 50/50 xylenes/paraffin mix for 1 hour at 60°C under vacuum followed by a final paraffin penetration in paraffin at 60°C under vacuum for 1 hour and then paraffin embedded. The brains were sectioned at 10μm and stained with Cresyl Violet stain as previously described .WT, Gas8GT/WT, and Gas8GT mice. Samples were submerged in ice cold RIPA  mixed with one cOmplete Protease Inhibitor tablet  per 10mL at 300uL per 5mg of tissue. Tissues were sonicated 3x for 10 seconds each. After sonication, tissues were placed on a rotary agitator for 2 hours at 4°C and then spun for 20 minutes at 12,000rpm at 4°C. Supernatant was removed and protein levels were assayed using a BioRad DC protein assay kit . Approximately 20μg per sample was used for SDS-PAGE with a 12% Tris-Glycine gel . Proteins were transferred overnight to nitrocellulose. The membrane was blocked for 45 minutes in 5% milk in PBS and incubated with primary antibody in 5% milk in PBS with 0.02% Tween-20 overnight at 4°C. Primary rabbit anti-Gas8/DRC4 antibody was used at 1:20000 [Fresh tracheas were extracted from p21 Gas8Mouse embryonic fibroblasts (MEFs) were generated from E14.5 embryos and cultured in DMEM growth medium with High Glucose, 0.05mg/ml Penicillin/Streptomycin, 2mM L-Glutamine, 0.2mM β-mercaptoethanol, and 20% Fetal Bovine Serum (FBS). Prior to immunolabeling, MEFs were cultured in reduced serum medium containing 0.5% FBS for 48 hours to induce primary cilia formation as previously described .Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS with 2% donkey serum, 0.02% sodium azide and 10 mg/ml bovine serum albumin (BSA). Cells were labeled with anti-acetylated α-tubulin, 1:1000 , anti-SmoN, 1:1000 . Sections from E10.5 neural tubes were immunolabeled with the following antibodies from Developmental Studies Hybridoma Bank : anti-ShhN 1:1000 (5E1), anti-FoxA2 1:1000 (74.5a5), anti-Mnr2 1:1000 (81.5C10), anti-Pax7 1:1000 (Pax7), and anti-Msx1+2 1:1000 (4G1) as previously described . TracheaDIC images of p14 trachea prepared for IF were used for length analysis. Images were captured with a 40x objective . Length was measured manually by drawing a line from the tip of the cilium to the base using Volocity v6.3.Gas8WT and Gas8GT MEFs were grown to confluency on 0.17mm coverslips and serum starved for 48 hours to induce ciliation. Cells were treated with 150nM Smoothened agonist (SAG)  for 2 hours in low serum media to induce Hedgehog pathway activation and Smoothened translocation. Cells were fixed and stained as described in the immunofluorescence section and imaged by spinning disk confocal. Amount of Smoothened per cilia volume was measured using Volocity v6.3 software.Postnatal day 14  after setting the average angle to 0°. The frequency of angles in each image was measured and plotted.2PO4, 2mM CaCl2, 1mM MgCl2, 25mM NaHCO3, 25mM Glucose, pH 7.35). Brains were placed on a Zeiss Axioskop microscope and imaged with a 5x objective  and a 10x objective  using a Photometrics CoolSnap HQ CCD camera at 30fps. Red fluorescent latex beads  were diluted 1:100 from stock and 10μL of diluted beads was added to the ventricles. Bead tracking analysis was performed using the MTrack2 plugin in FIJI.Brains of experimental mice were extracted, sliced in half to expose the ependymal of the lateral ventricles and placed in pre-warmed, pre-oxygenated artificial cerebrospinal fluid  before imaging with Differential Interference Contrast (DIC). All high speed video was captured at 240fps using a modified Casio Exilim EX-ZR100 attached to a Nikon TE-200 using a 60x water objective (Plan-Apo WI NA = 1.2). Videos saved as quick time files were then extracted into individual frames using VirtualDub 1.10.4 software and all analysis was performed in ImageJ.Mice trachea were dissected out into fresh PBS and cut lengthwise into strips. Trachea were kept in warm media  to generate the plasmid pf2-Y1-A. This plasmid was further digested with KpnI and SpeI to removed repetitive DNA and subcloned into pBlueScript to generate the plasmid pf2-Y1-B. The D198K mutation was introduced into pf2-Y1-B using the primers 5’-GAAGATGCTGCGAGACaAaATGGAGCTGCGGAGAAAG-3’ and 5’-CTTCTACGACGCTCTGtTtTACCTCGACGCCTCTTTC-3’ and the QuickChange II kit (Agilent Technologies) to generate the plasmid pf2-Y1-C. After sequence verification by Genewiz, the pf2-Y1-C plasmid was digested with KpnI and SpeI and subcloned back into pf2-Y1-A to generate the plasmid pf2-Y1-D. The pf2-Y1-D plasmid was digested with BclI and ClaI to release the 1483 bp fragment now carrying the D198K mutation. This fragment was subcloned back into the original DRC4-GFP plasmid by Genewiz. The completed plasmid, DRC4-D198K-GFP, was linearized with EcoRI for transformation into the pf2-4 strain [To make the D198K mutation in  plasmid  was usedGGG-3’ (reverse strand), CRISPR2: 5’-GGTGCTGGCCGCCTCCAACC TGG-3’ (forward strand), CRISPR3: 5’-GACACAAGCGTTAATGCTGTGGG-3’ (reverse strand). Pronuclear injections were performed with Cas9 mRNA (100 ng/ul), CRISPR3/sgRNA (50 ng/ul) and ssODN (200 ng/ul). Efficiency of nuclease activity was assessed using a blastocyst assay. In brief, injected zygotes were cultured to the blastocyst stage and lysed to obtain genomic DNA. Genomic DNA was used in PCR and the amplicons (215 bp) were resolved by heteroduplex mobility assay (HMA). CRISPR3 was found to be most efficient and was used to generate the SNP edited mouse (C57Bl/6 background). Injected zygotes were cultured to 2-cell stage in KSOM mixed with the NHEJ inhibitor SCR7 at a final concentration of 10 mM. The 2-cell stage embryos were transferred to psuedopregnant recipient female mice, which gave birth to 13 pups. The SNP was introduced with the help of a 154 nt single stranded oligo DNA (ssODN) HDR template. Since the PAM sequence (CCC>Pro) could not be modified without changing the amino acid, multiple silent changes were made in the protospacer (sgRNA binding) sequence . These changes were made to eliminate the chances of the sgRNA binding to the repaired allele. The SNP change introduced a restriction enzyme recognition enzyme site (BsmBI/Esp3I) and the silent changes introduced two new restriction enzyme recognition sites (BtgI and HaeII). HaeII sites were used to distinguish the wildtype and the modified alleles. Specific primers were also designed that can preferentially amplify the modified allele. HDR template (ssODN) 5’-GGCCCTGAACGCTGCTGTGGAGAAGAGAGAGGTTCAGTTCAATGAGGTGCTGGCTCTCCAACCCGTGGACCCCACgGCgcTgACGtTgGTGTCCCGCAAACTTGAGGTAGGTGCCCTCCTGTCCTGTGCTGTGGTACGCCTTCTTGGGTGGCAC-3’. After the initial characterization of the F0 litter by PCR, the 215 bp amplicons were cloned, and selected individual clones were subjected to Sanger sequencing. Sequence analysis of the 13 pups revealed that 2 had complete knock-in of the edited/repair sequence, 1 pup had incorporated the silent changes but did not have the desired SNP change, and 1 pup had indels. F0 animals were bred with wildtype C57Bl/6 mice to test germline transmission of the desired alleles. All alleles were successfully transmitted through the germline, and the positive F1 animals were used to create homozygous and compound heterozygous F2 animals.CRISPR/sgRNA target sequences were queried using the MIT CRISPR server. Three sites most proximal to the desired SNP change were selected to test nuclease efficiency. CRISPR1: 5’- CTTCTCCACAGCAGCGTTCA Cilia length analysis, bead flow tracking, cilia orientation, and cilia beat frequency were tested with Student’s t-test and graphed in Microsoft Excel. Smoothened trafficking assay and Chlamydomonas swim speeds were tested by ANOVA followed by Student’s t-test with a Bonferroni correction and graphed in Microsoft Excel. All error is represented in Standard Error of Means (SEM).S1 FigChlamydomonas flagellar axonemes showing that pf2 cells transformed with DRC4-DK-GFP (DK) have proper localization of the protein product. The DRC4-GFP fusion proteins are detected by a DRC4 antibody (top panel) and a GFP antibody (bottom panel). An IC69 antibody against an outer arm dynein intermediate chain serves as a loading control for the blot (bottom panel).Western Blot of (TIF)Click here for additional data file.S1 Movie(AVI)Click here for additional data file.S2 Movie(AVI)Click here for additional data file.


Ku80 is encoded by the XRCC5 gene that contains a variable number tandem repeat (VNTR) insertion in its promoter region. Different VNTR genotypes can alter XRCC5 expression and affect Ku80 production, thereby affecting NHEJ and HR pathways. VNTR polymorphism is associated with multiple types of sporadic cancer. In this study, we investigated its potential association with familial breast cancer at the germline level. Using PCR, PAGE, Sanger sequencing, and statistical analyses, we compared VNTR genotypes in the XRCC5 promoter between healthy individuals and three types of familial breast cancer cases: mutated BRCA1 (BRCA1+), mutated BRCA2 (BRCA2+), and wild-type BRCA1/BRCA2 (BRCAx). We observed significant differences of VNTR genotypes between control and BRCA1+ group (P < 0.0001) and BRCA2+ group (P = 0.0042) but not BRCAx group (P = 0.2185), and the differences were significant between control and cancer-affected BRCA1+ cases (P < 0.0001) and BRCA2+ cases (P = 0.0092) but not cancer-affected BRCAx cases (P = 0.4251). Further analysis indicated that 2R/2R  and 2R/1R  were associated with increased risk but 1R/1R  and 1R/0R  were associated with decreased risk in cancer-affected BRCA1+ group; 2R/1R  was associated with increased risk in cancer-affected BRCA2+ group. No correlation was observed for the altered risk between cancer-affected or -unaffected carriers and between different age of cancer diagnosis in cancer-affected carriers. The frequently observed VNTR association with in BRCA1+ and BRCA2+ breast cancer group indicates that VNTR polymorphism in the XRCC5 promoter is associated with altered risk of breast cancer in BRCA1+ and BRCA2+ carriers.Ku80 is a subunit of the Ku heterodimer that binds to DNA double-strand break ends as part of the non-homologous end joining (NHEJ) pathway. Ku80 is also involved in homologous recombination (HR)  BRCA1 and BRCA2 confirmed the presence of genetic predisposition for familial breast cancer  hosting a putative Sp1-binding site  are tandem repeat DNA sequences often located in gene regulatory regions that can influence gene expression –25. VNTRing site . Studiesing site , 23. Exppression –29 . The results showed that certain genotypes of VNTR polymorphisms are associated with the risk of familial breast cancer in BRCA1+ and BRCA2+ carriers.Given the transmission pattern of VNTR, the uncertainty regarding the role of NHEJ in familial breast cancer, the presence of VNTR polymorphisms in the BRCA1 mutation (BRCA1+), familial breast cancer with BRCA2 mutation (BRCA2+), and familial breast cancer without BRCA1 or BRCA2 mutations (BRCAx). Samples were obtained from the Hereditary Cancer Center at Creighton University . Healthy control samples of age- and gender-matched, de-identified Caucasian individuals were obtained from the Nebraska Biobank of the University of Nebraska Medical Center and The Nebraska Medical Center. The use of patient samples for this study was approved by the Institutional Review Board of Creighton University School of Medicine (00-12265) and of the University of Nebraska Medical Center (718-11-EP). Written and informed consent to participate in the study and to publicate the results was obtained from all subjects.The familial breast cancer cases used in this study included three subtypes: familial breast cancer with XRCC5 promoter of each patient. PCR primer sequences were based on a previously published study  with 2R/2R, 2R/1R, and 1R/1R as the major genotypes, but a significant difference was seen in the genotypes between local Caucasian and Chinese populations (P < 0.0001), and Iranian Caucasian and Chinese (P < 0.0001), whose genotypes included 2R/2R, 2R/1R, 2R/0R, 1R/1R, 1R/0R, and 0R/0R .Data from Iranian and Chinese healthy populations showed that VNTR genotypes in the c groups , 28. To c groups . The res0R Table . BecauseP-values were computed by using exact methods to keep consistency .Fisher’s exact test was applied to determine the differences of VNTR polymorphism between the groups of familial breast cancer populations and control population, each type of breast cancer and cancer-affected and -unaffected subgroups within each type of cancer. Both odds ratios and their 95% confidence intervals and sistency . Benjamisistency . AnalyseBRCA1+ carrier refers to the women who tested positive for a pathogenic BRCA1 mutation; BRCA2+ refers to the women who tested positive for a pathogenic BRCA2 mutation; and BRCAx refers to the women who tested negative for the mutations in BRCA1, BRCA2, and p53, with two or more first or second degree relatives affected with primary in situ or invasive breast, ovarian, fallopian tube, or peritoneal cancer, and at least one person must have negative test result. Under each group, the cases were further divided into breast cancer (ovarian cancer)-affected and -unaffected carriers. The average ages at breast cancer diagnosis among the groups were 41.4 (BRCA1+), 43.6 (BRCA2+), and 47.7 (BRCAx). The age distributions are consistent with existing data that BRCA1 and BRCA2 mutation carriers tend to suffer cancer at earlier age. Most of the breast cancers were ductal type and ER-positive; all, but one, of the cases of ovarian cancer were invasive at diagnosis (Table XRCC5 promoter consist of three 21-bp (TGCGCATGCTCGGCGGGAATC) tandem repeats (3R), two 21-bp repeats (2R), one 21-bp repeat (1R), or without repeat (0R). The combination of PCR, PAGE, and Sanger sequencing methods provided an effective means to determine VNTR genotypes formed by the four alleles. Figure The four VNTR alleles in the XRCC5 promoter between three types of familial breast cancer: BRCA1+, BRCA2+, and BRCAx . The results show that the BRCA1+ and BRCA2+ groups differed significantly from the control group , but no difference was observed between the BRCAx groups and control group (P = 0.1308)  and BRCA2+ , but not in BRCAx   and 2R/1R  were associated with increased risk of breast cancer in cancer-affected BRCA1+ group, and 1R/1R  and 1R/0R  were associated with the decreased risk in cancer-affected BRCA1+ group;BRCA2+ group. 2R/1R  was associated with increased risk in cancer-affected BRCA2+ group. 2R/2R, 1R/1R, and 1R/0R had no association with the risk in cancer-affected BRCA2+ group;BRCAx group. 2R/2R, 2R/1R, 1R/1R, and 1R/0R had no association with the risk of breast cancer in breast cancer-affected BRCAx group.Through comparing between control, breast cancer-affected and breast cancer-unaffected groups, we tested odds ratio to identify specific genotypes associated with risk of breast cancer Table . ConsideXRCC5 promoter is associated with risk of familial breast cancer with BRCA1+ and BRCA2+ predisposition. Data from sporadic breast cancer showed that 2R/1R was not associated , but 0R/0R was associated with the disease   . The difBRCA1+ and BRCA2+ groups, the results can be explained by synergistic roles between Ku80 and BRCA1/BRCA2 in maintaining genome stability through the NHRJ and HR pathways (BRCA mutation carriers. The results also suggest that genotype 1R/1R and 1R/0R can reduce the risk of breast cancer in BRCA1+ carriers. Based on current knowledge, it is difficult to relate VNTR polymorphisms to BRCAx familial breast cancer, as genetic predisposition in this heterogeneous group of familial breast cancer remains to be determined. We did not observe a relationship between age at onset of disease, cancer status, and VNTR polymorphism. This could be due to the weaker influence by the VNTR polymorphism compared with that of the BRCA mutation predisposition. Alternatively, it could be due to the limited sample size used in the study, which restricts the statistical power to detect the potential significance. Further studies with larger sample size will help to address the issue.For the pathways –39. AlteBRCA1+ breast cancer, whereas 2R/1R was significantly associated with the increased risk of BRCA2+ breast cancer.In summary, our study indicates that 2R/2R and 2R/1R were significantly associated with increased risk, and 1R/1R and 1R/0R were significantly associated with the decreased risk of JC and BD performed the genotyping and Sanger sequencing experiments; YK analyzed the genotyping data; JC and JL performed statistical analyses; CS, DB, and HL recruited breast cancer cases and extracted genomic DNA; and SW designed the study, interpreted the data, and drafted the manuscript. All authors read and approved the final manuscript.The authors declared that the research was conducted in the absence of any commercial and financial relationships that could be constructed as a potential conflict of interest.


Since the impact of these Fel d 1 structure modifications on the protein dynamics, physiology and pathology are not well established, the present work employed computational biology techniques to tackle these issues. While conformational effects brought upon by glycosylation were identified, potentially involved in cavity volume regulation, our results indicate that only the central Ca2+ ion remains coordinated to Fel d 1 in biological solutions, impairing its proposed role in modulating phospholipase A2 activity. As these results increase our understanding of Fel d 1 structural biology, they may offer new support for understanding its physiological role and impact into cat-promoted allergy.The major cat allergen, Fel d 1, is a structurally complex protein with two N-glycosylation sites that may be filled by different glycoforms. In addition, the protein contains three putative Ca Felis domesticus), which are present in up to 50% of homes  and truncated [(-Asn-) GlcNAc2Man3GlcNAc3Gal2] forms of the glycosidic chains  previously identified [Each dimer of Fel d 1 has one N-glycosylation site, at Asn103 , in a toentified  to buildentified , 38. Theentified , 40. Sucentified , 42. Theentified , 19 and We submitted the Fel d 1 systems to MD simulations with the GROMACS 4.5 suite  for 200 Cavities were measured with Mole2 , 50. Ess2+ ions to semiempirical quantum chemistry calculations using MOPAC [2+ ions. We used the obtained minimum energy conformation was employed to evaluate the bond orders and distances to estimate the possible coordination between amino acid residues and Ca2+ ions.In order to add information on metal coordination to molecular mechanics calculations we submitted Fel d 1 with all four crystallographic Cang MOPAC  with theng MOPAC  and MOZYng MOPAC . The folng MOPAC , were emng MOPAC , and witS1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig2+ ions (black), simulations with Ca2+ ions (red).Color coding: Simulations without Ca(PDF)Click here for additional data file.S4 Fig2+; (B) Fel d 1 with Ca2+; (C) Fel d 1 with minimal glycosylation; (D) Fel d 1 with full glycosylation.(A) Fel d 1 without Ca(PDF)Click here for additional data file.S5 Fig1 in black, region A2 in red, region B1 in blue, region B2 in green.(A) Calcium-free system; (B) Calcium-bound system; (C) Minimally glycosylated system; (D) Fully glycosylated system. Region A(PDF)Click here for additional data file.S6 Fig(A) initial cavity volume, (B) final cavity volume (cavity volumes shown as green surfaces); (C) cavity volume measurements for each Fel d 1 chain in four simulations.(PDF)Click here for additional data file.S7 FigThe distance between each original chain in the native Fel d 1  is shown. The oscillation may indicate an opening-closing behavior. .(PDF)Click here for additional data file.S1 TableThese encompass atom types, atomic partial charges, charge-group definition, bond stretching, bond-angle bending, improper dihedral deformation and torsional potential dihedral.(PDF)Click here for additional data file.S2 TableNodes that appear more than once are grouped by color. Interactions detected between pairs by PDBsum for the crystallographic structure are in parentheses.(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Wcor15-2A, Wcor15-2B and Wcor15-2D in 23 diploid, 10 tetraploid and 106 hexaploid wheat varieties and analyzed their molecular evolution to reveal the origin of the A, B and D genome in Triticum aestivum. Comparative analyses of sequences in diploid, tetraploid and hexaploid wheats suggest that T. urartu, Ae. speltoides and Ae. tauschii subsp. strangulata are most likely the donors of the Wcor15-2A, Wcor15-2B and Wcor15-2D locus in common wheat, respectively. The Wcor15 genes from subgenomes A and D were very conservative without insertion and deletion of bases during evolution of diploid, tetraploid and hexaploid. Non-coding region of Wcor15-2B gene from B genome might mutate during the first polyploidization from Ae. speltoides to tetraploid wheat, however, no change has occurred for this gene during the second allopolyploidization from tetraploid to hexaploid. Comparison of the Wcor15 gene shed light on understanding of the origin of the A, B and D genome of common wheat.The allohexaploid bread wheat originally derived from three closely related species with A, B and D genome. Although numerous studies were performed to elucidate its origin and phylogeny, no consensus conclusion has reached. In this study, we cloned and sequenced the genes  Triticum aestivum L.) is an annual species in the tribe Triticeae of the grass family Poaceae. It is the most widely cultivated food crop followed by rice and maize, and is the primary cereal in the temperate region, serving as a staple food for about 40% of the world’s population (http://faostat.fao.org)2Wheat (Triticum aestivum (AABBDD) as a good example of allopolyploid is derived from the three homologous genomes, A, B, and D, each of which contributes 7 pairs of chromosomes to the wheat’s total genome (2n = 6x = 42)1112Triticum aestivum L.) involved two sequential allopolyploidization events1416Aegilops tauschii Coss) and the tetraploid (AABB) emmer wheat  led to the formation of common wheat (AABBDD) approximately 8,000 years ago16Polyploidization played an important role in the evolution of eukaryotes, and is one of the important mechanisms for creating genetic variation, and major evolutionary factor affecting genome size and gene copy number4568Triticum urartu Thum ex Gand (genome Au)Triticum monococcum Linn (genome Am) including two subspecies: the wild T. monococcum subsp. boeoticum Boiss. (T. m. boeoticum)T. monococcum subsp. monococcum (T. m. monococcum)u and Am genomes have similar genome size and gene contentT. urartu, the wild diploid wheat from the Fertile Crescent region, has long been considered as the A-genome donor to tetraploid and hexaploid wheat species23Sitopsis section2526Ae. bicornis , Ae. longissima , Ae. sharonensis , Ae. searsii  and Ae. speltoides 28303132Ae. speltoides is phylogenetically distinct from the other species in the Sitopsis section. Ae. speltoides (S genome) has been suggested as the most likely progenitor of the B genome26et al.Sitopsis species they investigated is a close relative of the B genome in T. aestivum, and concluded that the B genome donor remains unknown. There has been little debate on Ae. tauschii Coss (genome DD) as the D genome progenitor of T. aestivumThe progenitor of the A genome of the tetraploid and hexaploid wheat species contains There has been great interest in the determination of ancestral diploid genome donors of hexaploid wheat102035Wcor15 (GenBank: AB095006), a member of the wheat cold-responsive gene family, which could encode the chloroplast-targeted protein when exposed to low temperature, plays an important role in the cold hardiness of wheatWcor15 gene was very conservative in the hexaploid wheat, not only the coding region but also the 5′-upstream non-coding region. In this study, we cloned and sequenced the Wcor15 gene from diploid, tetraploid and hexaploid wheats to reveal the origin of A, B and D genome in common wheat, and compared their evolution among diploid, tetraploid and hexaploid wheats.Wcor15 sequences were identified using the ORF sequence  of Wcor15 gene (GenBank: AB095006) as probe to screen the nucleotides databases of EBI Wcor15 sequences which contained intact ORFs were designed, based on the highly variation region of accession CBTL0110083500 (2AL), CBTL0111257031 (2BL) and CBTL0110522649 (2DL).The three homoeologous Wcor15-2A (KT264885), Wcor15-2B (KT264957) and Wcor15-2D (KT265022) respectively, which contained the 5′ upstream region, two exons, one intron and 3′ downstream region. Further analysis demonstrated that these three sequences are very similar with a few nucleotide insertions, deletions, and substitutions  is the same gene. After RT-PCR using RNA templates from Annong 0822, all of the three homoeologous Wcor15 genes were specifically induced by low temperature (data not shown), suggesting the three homoeologous Wcor15 genes are the cold-responsive gene.The primer pairs were used to amplify genomic DNA of hexaploid wheat cultivar Annong 0822. Each primer pair generated single-band amplicon with the expected size. The genes were designated as itutions . The WcoWcor15-2B was found in the lines except nullisomic 2B–tetrasomic 2D (N2B–T2D). This indicates that the Wcor15-2B is located on chromosome 2B. In turn, Wcor15-2A and Wcor15-2D were assigned to chromosome 2A, and 2D, respectively    are exacd wheats , suggestWcor15-2B sequences from tetraploid wheat showed a number of single nucleotide substitutions among these sequences whose situation was the same to Wcor15-2B in the 54 hexaploid varieties  surveyed, the primer Wcor15B and Wcor15D did not generate any amplification products  showed 100% identity with the Wcor15-2A sequences from tetraploid and hexaploid wheats . Three Wcor15 sequences were obtained  . Eleven Wcor15 sequences were obtained , Ae. longissima (Q03-004), Ae. sharonensis , and Ae. searsii  showed 100% identity with each other, nevertheless, besides the difference of base G indel mentioned above, there were still many base differences compared with the gene from Ae. speltoides, 2 located in the first exon, 7 in the intron, and 5 in the second exon   surveyedides SS) . In ordeobtained . Sequence intron . The genond exon . These rAe. tauschii , six Wcor15-2D were cloned with the primer Wcor15D (Wcor15-2D sequences were divided into two types: (I) As 2386, As 2387 and As 2388 with 100% identity, (II) As 80, As 77 and As 2392 with only a base substitution in the upstream non-coding regions. However, the Wcor15-2D from As 2386, As 2387 and As 2388 which belong to Ae. tauschii subsp. strangulata showed 100% identity with the Wcor15-2D from hexaploid wheat varieties (Ae. bicornis (Q03-021), Ae. longissima (Q03-004), Ae. sharonensis , and Ae. searsii  are same to the sequences from As 80, As 77 and As 2392 of Ae. tauschii subsp. tauschii. The primer Wcor15A and Wcor15B failed to amplify a product from these species  geneWcor15-2A, in addition to this gene, we cloned the other two homoeologous Wcor15 sequences (Wcor15-2B and Wcor15-2D) from the wheat genome 2BL and 2DL, respectively. Gene characterization analyzing showed that the three homoeologous Wcor15 genes may belong to the chloroplast-targeted LEA3 protein, which is consistent with previous studies about characterization of Wcor15-2A37Wcor15-2A, Wcor15-2B and Wcor15-2D genes are a conserved gene or not, the Wcor15-2A, Wcor15-2B and Wcor15-2D genes were cloned from 106 hexaploid wheat varieties from different geographical areas, 10 tetraploid species and 23 diploid species. Comparative analyses indicated that the Wcor15-2A  to tetraploid (AABB)  to hexaploid (AABBDD)  , and frod (AABB) . Wcor15-(AABBDD) . CompariT. urartu (genome Au), T. monococcum (genome Am) and T. boeoticum (genome Am). To investigate the evolutionary relationships of Wcor15-2A genes between diploid and polyploid wheats, the sequences from T. urartu, T. monococcum, T. boeoticum, tetraploid and hexaploid wheats were compared. The six genes in diploid wheats (genome AA) were classified into two types  were type I (Wcor15-2A1). The two T. monococcum (Mo4 and TL) and one T. boeoticum (Bo8) were type II (Wcor15-2A2). Compared to the Wcor15-2A2 sequence, the Wcor15-2A1 sequence showed much higher identity (100%) with the Wcor15-2A sequences from tetraploid and hexaploid wheats, suggesting that the T. urartu might be the direct donor of the Wcor15-2A in common wheat and that Wcor15-2A gene from A genome has no mutation during two sequential allopolyploidization events from T. urartu to tetraploid and hexaploid wheats. The result is consistency with the previous studies2324T. monococcum and T. boeoticum when using Wcor15A primer, it suggested that non-coding regions of Wcor15-2A1 were obviously different from Wcor15-2A2. Coding regions alignments also revealed variation between Wcor15-2A2 and Wcor15-2A1 from T. urartu  Ae. speltoides , and (II) Ae. bicornis (Q03-021), Ae. longissima (Q03-004), Ae. sharonensis , and Ae. searsii   . Our resWcor15-2B sequences from different tetraploid and hexaploid wheats were divided into two groups by the insertion and deletion of a nucleotide G in the intron. All three Ae. speltoides sequences shared 100% identity, are different from tetraploid and hexaploid wheats with only a G deletion in the intron. On the other hand, no amplicon obtained from Ae. speltoides when using Wcor15B primer, suggested that non-coding regions of Wcor15-2B might be obvious differences between Ae. speltoides and tetraploid and hexaploid wheats. Our results suggested that Ae. speltoides might be the direct donor of the Wcor15-2B in tetraploid and hexaploid wheat varieties, non-coding region of Wcor15-2B gene from B genome might mutate during the first polyploidization from Ae. speltoides to tetraploid wheat, however, no change has occurred for this gene during the second allopolyploidization from tetraploid to hexaploid.In terms of coding region, Wcor15-2D sequences of D-genome were highly conservative among 106 hexaploid wheats. However, Wcor15-2D genes from six accessions of Ae. tauschii , and 23 diploid species  (One hundred and six hexaploid wheat (genome AABBDD) were used in this study, including 4 varieties from Winter wheat region of North China (WWRNC), 24 varieties from North China plain sub-region of Yellow & Huai river winter wheat region (NCPSR), 28 varieties from North Huai river plain sub-region of Yellow & Huai river winter wheat region (NHRPSR), 7 varieties from West upland sub-region of Yellow & Huai river winter wheat region (WUSR), 3 varieties from Jiaodong upland sub-region of Yellow & Huai river winter wheat region (JUSR), 11 varieties from Winter wheat region of middle and lower reaches of the Yangtze river (WWR), 7 varieties from Southwestern winter wheat region (SWWR), 13 varieties from Introduced wheat variety of foreign (IWVF) and DD) .Genomic DNA was extracted from young leaves of ten days seedlings using the Easypure plant Genomic DNA Kit . Genome-specific primers were designed for each of the homoeologous Wcor15 genes  using th2O, 10 × PCR buffer (with Mg2+) 2.0 μl, dNTPs (2.5 mM) 2.0 μl, 0.5 μl of each primer (10 mM), 2.0 μl genomic DNA and Taq DNA polymerase (5 U/μl) 0.2 μl. Amplifications were performed using a standard touchdown PCR protocol with the appropriate annealing temperature. Each PCR was done five repeats up to a total of 100 μl.PCR reaction were performed in total volumes of 20 μl, containing 12.8 μl ddHAll PCR products were directly sequenced. Each of 50 μl PCR products were sequenced by Shanghai Sangon Biological Technology Company, and the other 50 μl PCR products were sequenced by Huada Biotech Company in Beijing. To guarantee sequence accuracy, DNA sequencing was repeated three times.http://www.lynnon.com). The three homoeologous Wcor15 sequences were identified at EBI web site (http://www.ebi.ac.uk/ena/)Wcor15-2A, Wcor15-2B and Wcor15-2D were submitted to the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/)  .How to cite this article: Liu, F. et al. Molecular evolution of Wcor15 gene enhanced our understanding of the origin of A, B and D genomes in Triticum aestivum. Sci. Rep.6, 31706; doi: 10.1038/srep31706 (2016).


Ag-dependent activation of naive T cells induces dramatic changes in cellular metabolism that are essential for cell growth, division, and differentiation. In recent years, the serine/threonine kinase mechanistic target of rapamycin (mTOR) has emerged as a key integrator of signaling pathways that regulate these metabolic processes. However, the role of specific downstream effectors of mTOR function in T cells is poorly understood. Ribosomal protein S6 (rpS6) is an essential component of the ribosome and is inducibly phosphorylated following mTOR activation in eukaryotic cells. In the current work, we addressed the role of phosphorylation of rpS6 as an effector of mTOR function in T cell development, growth, proliferation, and differentiation using knockin and TCR transgenic mice. Surprisingly, we demonstrate that rpS6 phosphorylation is not required for any of these processes either in vitro or in vivo. Indeed, rpS6 knockin mice are completely sensitive to the inhibitory effects of rapamycin and an S6 kinase 1 (S6K1)–specific inhibitor on T cell activation and proliferation. These results place the mTOR complex 1-S6K1 axis as a crucial determinant of T cell activation independently of its ability to regulate rpS6 phosphorylation. Naive T cells undergo a rapid switch from quiescence to a highly metabolically active state upon recognition of cognate Ag. In recent years, it has become apparent that this metabolic reprogramming is critical not only for T cell growth and population expansion but also effector-memory differentiation during immune responses . Consequ+ Th cell differentiation  are viable  and rpS6P−/− T cells are equally sensitive to the inhibitory effects of rapamycin and S6K1-specific inhibitors, indicating a vital role for mTORC1/S6K1 in T cell activation and differentiation independent of rpS6 phosphorylation.In T cells, activation of S6K1 and entry into the cell cycle and proliferation have long been linked –25; howeP−/− mice (−/−Rag1 OT-I background (6 CFU of an attenuated (ActA mutant) ova-expressing strain of L. monocytogenes , SIITFEKL (T4) and SIIGFEKL (G4) peptides (Peptide Synthesis) were added to culture media at the concentrations stated in figure legends. In some experiments, cells were cultured in the presence of 100 nM rapamycin or 10 μM S6K1 inhibitor PF-4708671 (both Tocris Bioscience). These conditions have previously been optimized for the selective inhibition of target kinases by the drugs (Lymph node (LN) OT-I T cells were cultured in RPMI 1640 medium (Invitrogen) supplemented with 10% FCS, he drugs –30. For b dextramers (Immudex). Data were acquired using a MacsQuant flow cytometer (Miltenyi Biotec) and analyzed using FlowJo software (Tree Star).Fluorescently conjugated Abs were purchased from eBioscience, BD Pharmingen, and BioLegend. For intracellular staining of phospho-rpS6 S235/6 and S240/4 and phospho-ERK T202/Y204, cells were permeabilized using Phosflow Fixation/permeabilization buffer (BD Biosciences) and stained with phospho-specific rabbit mAb  and anti-rabbit secondary Abs. Ova-specific T cells from Lm-Ova infection experiments were identified using PE-conjugated SIINFEKL-H-2Kt tests were performed using Prism software. The Holm–Sidak correction for multiple comparisons was used where appropriate. The p values < 0.05 were considered statistically significant.Two-tailed paired or unpaired Student P−/− knockin mice (−CD4− double-negative (DN), CD8+CD4+ double-positive (DP), and CD8+CD4− and CD8−CD4+ single-positive (SP) thymocytes in WT and rpS6P−/− knockin thymi , central memory (CD44highCD62L+), and effector memory (CD44highCD62L−) CD4+ and CD8+ T cells  in rpS6P−/− as compared with WT OT-1 T cells . Differentiation in IL-2, as compared with IL-15, resulted in higher expression of CD44, CD25, granzyme B, Tbet, IRF4, and eomesodermin and lower levels of L-selectin (CD62L) by CTL  by CTL , 4B. Unduishable , 4B. FurNF+ CTLs  and leveNF+ CTLs  upon N4,P−/− mice with an ova-expressing strain of L. monocytogenes. Primary splenic CD8+ T cell responses to the immunodominant SIINFEKL peptide were monitored using MHC class I–peptide dextramers. Numbers and proportions of dextramer-positive effector CD8+ T cells were comparable in WT and rpS6P−/− mice as measured at 7 d postinfection (+) CD8+CD44hi dextramer-negative and CD4+ cells were comparable in WT and rpS6P−/− mice, suggesting that the overall T cell response to Lm-Ova infection was similar in both groups  or via S6K1-dependent rpS6 phosphorylation–independent mechanisms. For example, S6K1 can modulate gene expression via the phosphorylation of additional substrates such as elongation factor 2 kinase . To disc of CD44 . As expe of CD44 . TreatmemTOR signaling pathways are central to the regulation of T cell growth, metabolism, differentiation, and memory. An understanding of the effector mechanisms that regulate these processes is essential for the development of more sophisticated and targeted therapies to manipulate mTOR activation and T cell responses in the clinic. We sought to determine the role of downstream effectors of mTORC1 and, surprisingly, found that phosphorylation of the canonical target of the S6K1 signaling axis, rpS6, was dispensable for T cell development, activation, differentiation, and effector function both in vitro and in vivo.P−/− mice demonstrated that rpS6 phosphorylation is dispensable for the translation of 5′-terminal oligopyrimidine tract mRNAs and plays only a minor regulatory role in global protein synthesis (P−/− MEFs were smaller than WT counterparts and their size was not further reduced by rapamycin (P−/− and WT T cells are similar in cell size both under basal conditions and following Ag-induced stimulation. Furthermore, whereas rpS6P−/− MEFs have an elevated rate of cell cycle progression and proliferation compared with WT MEFs, rpS6P−/− and WT T cells have comparable proliferative responses to TCR stimulation and mitogenic cytokines such as IL-2. Indeed, we found that, in all parameters of TCR-induced activation, differentiation, and effector function assessed in the current work, rpS6P−/− and WT T cell responses were comparable.More than 20 y ago, it was hypothesized that rpS6 phosphorylation is an important regulator of the translation of mRNAs containing a 5′-terminal oligopyrimidine tract . Howeverynthesis . Nonetheynthesis , 22, 37.apamycin , indicat+ T cell memory differentiation in a similar manner to rapamycin treatment. Furthermore, expression of a constitutively active form of S6K1 in CD4+ T cells counteracted the inhibitory effects of rapamycin on Th17 differentiation (These results raise the question of what are the important downstream effectors of mTORC1 if rpS6 phosphorylation is dispensable for T cell activation? Araki et al.  reportedntiation . In the ntiation , initiatntiation , and prontiation  that regntiation . Furtherntiation . Interesntiation , whereasntiation . Thus, tntiation . Interesntiation , suggestIn conclusion, our data show that mTORC1 and S6K1 influence T cell activation and differentiation independently of their capacity to mediate rpS6 phosphorylation. Further analysis of the role of additional downstream effectors of this pathway in T cells may provide insight into the targets of this pathway that are important for T cell function.Data Supplement


MYD88 mutations in conjunction with cytogenetic study in 22 consecutive Korean WM patients. Conventional G-banding and interphase fluorescence in situ hybridization (FISH) were performed at regions including 6q21 using bone marrow (BM) aspirates. Sixteen patients were subjected to Sanger sequencing-based MYD88 mutation study. Five patients (28%) showed cytogenetic aberrations in G-banding. The incidence of 6q21 deletion was 17% by conventional G-banding and 37% by FISH. Ten patients (45%) showed cytogenetic aberrations using FISH: 6q deletion in eight (37%) and IGH rearrangement in four (18%). Two patients had both the 6q deletion and IGH rearrangement, and two had only the IGH rearrangement. Eleven patients (69%) presented with the MYD88 L265P mutation. MYD88 mutations were significantly associated with the presence of 6q deletions (P = 0.037). Six patients with the 6q deletion for whom sequencing was possible were found to harbor MYD88 mutations. The MYD88 L265P mutation was also associated with increased lymphocyte burden in BM biopsy. This is the first report of high frequency MYD88 L265P mutations in Korean WM patients.Waldenström macroglobulinemia (WM) is a malignant lymphoplasma-proliferative disorder with IgM monoclonal gammopathy. A recent whole-genome study identified MYD88 L265P as the key mutation in WM. We investigated  Waldenström macroglobulinemia (WM) is a malignant lymphoplasma-proliferative disorder characterized by bone marrow infiltration with lymphoplasmacytic lymphoma (LPL) and the secretion of monoclonal immunoglobulin M (IgM) –3. DiffeMYD88) in WM /46,XY[4] and 46,XX[14] 46,XX,−1,der(1)add(1)(q42)dup(1)(q21q32)?dup(1)(q32q12),+3,add (14)(q32)[1]/46,XX[19].Conventional cytogenetic analysis using G-banding was performed in 18 patients. Five patients (28%) exhibited structural abnormalities: three presented with 6q deletions 46,XY,14pstk+[18]/46,XY,idem,i(6)(p10)[2], 46,XY,1qh+,del(6)(q23)[2]/46,XY,1qh+[22], and 45,X,−Y,1qh+,del(6)(q21),inc[3]/45,idem,add(3)(q26.2),der(3)add(3)(p?23)add(3)(q26.2)[13]/46,XY,1qh+[5] as abnormal karyotypes, which were also detected using FISH. Two patients identified as having theIGH rearrangement was observed in four patients (18%), amongst whom two patients had both 6q deletion and IGH rearrangement, and two patients had only the IGH rearrangement. TP53 deletion was observed in one patient who also had a 6q deletion, and 1q amplification was detected in one patient with an IGH rearrangement. The CDKN2A (9p21) and RB1 (13q14) FISH probes did not reveal any abnormalities. Among the ten patients with FISH abnormalities, eight had G-banding data available. Of the six patients with 6q deletions revealed using FISH, three (50%) presented normal karyotypes, whereas other three exhibited concurrent abnormalities by G-banding analysis. Two patients with both the 6q deletion and IGH rearrangement by FISH did not exhibit abnormalities at the 14q21 locus by G-banding, and two patients with only the IGH rearrangement by FISH presented with concordant abnormalities by G-banding of t and add(14)(q32), respectively.Among the 22 patients, ten (45%) exhibited cytogenetic aberrations . Of thes MYD88 gene was possible, 11 (69%) carried the L265P mutation .Among the 16 patients for whom the sequencing formutation . When MYo groups . In addi MYD88 mutation was found in all six patients with 6q deletion in whom sequencing was possible (P = 0.037). There was no significant difference in the presence of the IGH rearrangement between mutation-positive and mutation-negative patients (P = 0.350). However, neither of the two patients with IGH rearrangement without 6q deletion carried a MYD88 mutation. When the prognosis was compared according to the presence of the MYD88 L265P mutation and 6q deletion, there was no significant difference between patients with and without MYD88 mutations or 6q deletions , which might be more consistent with the involvement of BM in diffuse large B cell lymphoma after the review of lymph node biopsies.When we retrospectively reviewed fiveThis is the first report describing the MYD88 L265P mutation status in Korean WM patients. MYD88 L265P mutation has been commonly reported in recent studies of WM, with frequencies of 70–100%, depending on the method and tissues used for genetic analyses. The MYD88 L265P mutation can also be found in patients with IgM monoclonal gammopathy of unknown significance (IgM MGUS) at a lower frequency than WM, ranging from 10 to 87% , 19. BecIn this study, the frequency of the MYD88 L265P mutation was 69% in Korean patients with WM assessed by PCR and Sanger sequencing in unsorted BM cells, which is comparable with a previous study performed in Caucasian patients . Future  IGH rearrangement, which was identified in 18% of patients. Half of the patients with the IGH rearrangement presented with both 6q deletion and IGH rearrangement, whereas the other half presented with only the IGH rearrangement.We also investigated the correlation of cytogenetic aberrations with the MYD88 L265P mutation. The 6q deletion was the most frequent abnormality and was found in 37% of Korean patients. Previous studies performed in Western patients with WM reported the frequency of 6q deletions to be 32–54%, identified using FISH –24. Prev IGH rearrangement did not present with the MYD88 L265P mutation. The deletion of 6q is the most frequent chromosomal abnormality in WM. Candidate tumor suppressor genes identified in this region are B lymphocyte-induced maturation protein 1 (BLIMP1) andα-induced protein 3  tumor necrosis factor (TNFAIP3 or A20) [BLIMP1 is a transcriptional repressor that plays a pivotal role in the differentiation of B cells into plasma cells [ BLIMP1 would block the differentiation of B cells into plasma cells. TNFAIP3 participates in terminating NF-κB signaling, and its loss of function by deletion might enhance inflammatory, autoimmune, and malignant human diseases, including WM [ MYD88 is a key player in the activation of the canonical NF-κB pathway, which is downstream of Toll-like receptor and interleukin-1 receptor signaling [ BLIMP1 or TNFAIP3 enhance inflammatory reactions that contribute to the pathogenesis of WM.Interestingly, the 6q deletion was significantly associated with the presence of the MYD88 L265P mutation. All patients with 6q deletions for whom sequencing was possible harbored MYD88 L265P mutations. In contrast, patients with only the or A20) , 26. BLIma cells –29. Theruding WM , 31. Becignaling , 33, we In previous studies, Jimenez et al.  reportedThe limitations of this study are the small number of patients and the use of less sensitive Sanger sequencing to detect the MYD88 L265P mutation. Because WM is a very rare disease, an additional multicenter study should be performed to allow a more comprehensive genetic analysis of Korean WM patients. In addition, studies using different molecular methods and more sophisticated approaches for assessing mutations in minor populations of malignant cells should be performed.In conclusion, we observed a high incidence of MYD88 L265P mutation and 6q deletion in Korean WM patients. We also found a novel association between MYD88 L265P mutation and 6q deletion, though the small number of patients should be taken into consideration for interpretation of the findings of our study with caution. We suggest assessing MYD88 L265P mutation status and performing cytogenetic studies to characterize 6q deletions in WM could help with the diagnosis of WM. As such, refining the current diagnostic classification system might be attempted based on these novel findings.


Drosophila melanogaster and Drosophila simulans, the D. simulans nucleoporin-encoding simNup96 and simNup160 can cause recessive lethality if the hybrid does not also inherit the D. simulans X chromosome. In addition, simNup160 leads to recessive female sterility in the D. melanogaster genetic background. Here, we conducted carefully controlled crosses to better understandIn interspecific hybrids between simNup96 and simNup160. simNup96 did not lead to female sterility in the D. melanogaster genetic background, and double introgression of simNup96 and simNup160 did not generally lead to lethality when one was heterozygous and the other homozygous (hemizygous). It appears that introgression of additional autosomal D. simulans genes is necessary to cause lethality and that the effect of the introgression is dominant to D. melanogaster alleles. Interestingly, the genetic background affected dominance of simNup96, and double introgression carrying homozygous simNup96 and hemizygous simNup160 resulted in lethality. Thus, simNup96 and simNup160 seem to be two components of the same incompatibility.the relationship between  Drosophila encode the nuclear pore proteins (nucleoporins = Nups), which were previously thought to be functionally conserved among diverse organisms. Approximately 30 different Nups assemble to form the nuclear pore complex (NPC) and are essential for nucleocytoplasmic transport, gene regulation, and kinetochore formation  mutation of D. simulans. D. melanogaster/D. simulans hybrids carrying the simD. simulans Nup96 and simNup160 are lethal in hemizygotes (or homozygotes) if they do not inherit the D. simulans X chromosome  are components of the conserved Nup107−160 complex that has a role in the initial assembly of the NPC and functions as a stable anchoring point for other Nups—referred to as central scaffold Nups (Drosophila to address the following three questions. (1) Does simNup96 lead to female sterility in the D. melanogaster genetic background as seen with simNup160 introgression? (2) Does the simNup96 and simNup160 double introgression lead to lethality when one is heterozygous and the other homozygous (or hemizygous) in the D. melanogaster background  in the D. melanogaster background (simNup96 and simNup160 is necessary and sufficient condition for the incompatibility to the gene(s) on the D. melanogastere X chromosome. Dominance of the genes and the possible involvement of different genes to the hybrid lethality will also be discussed.We conducted interspecific crosses of ckground ? (3) Doeckground ? Based oCG10208, Nup98-96, and mbc), was amplified from DSM1-010P23, a D. simulans bacterial artificial chromosome clone established by the National BioResource Project Drosophila (BamHI-R (5′-CGCGGATCCACGCACCTGGACAATGCAAGAGGGTGATTTG-3′), RA-BamHI-F (5′-CGCGGATCCGACCAGCATGAGCATTGCCAACAGCATGCT-3′), and RA-PacI-R (5′-ACCTTAATTAATCAGCACACCGGGCATAAGGTATCCCTGCTC-3′). This fragment was subcloned into the vector attB-P[acman]-CmR by homologous recombination   and e  were used as visible markers, and Nup98-96 is at 95B1-5. To confirm that the recombinant chromosome carried the 339Nup98-96 mutation and that it was not lost by rare double recombination between e and 339Nup98-96, + Nup96sim}P{w was removed from the established chromosome by further recombination with a wild-type chromosome using the +w and e markers. The resultant chromosome again exhibited recessive lethality that was not complemented by the Nup98-96 deficiencies (Df(3R)Exel9014 and Df(3R)BSC489), thus confirming that the chromosome examined carried 339Nup98-96. A balancer chromosome, TM3, was used to isolate the recombinant chromosome in a heterozygous state, and CyO and SM1 were used as a chromosome 2 balancer. Int(2L)D+S is a chromosome 2 D. simulans introgression covering two cytological regions that include simNup160 (Int(2L)D+S introgression also carries other Nup loci (Nup107 and Nup154), but we do not believe that this could affect our overall conclusion of this study. When necessary, only simNup160 was made hemizygous by a deficiency of the Nup160 locus, Df(2L)Nup160M190  inserted at cytological position 68A4 of the same chromosome. Note that Nup98-96 is a dicistronic gene that produces the proteins Nup98 and Nup96 by autoproteolysis  is referred to as the simNup96 introgression. Both male and female simNup96 introgression homozygotes (and hemizygotes) were viable and fertile, and the strain homozygous for simNup96 could be maintained indefinitely. Although females that were homozygous for simNup96 and hemizygous over Df(3R)BSC489 exhibited lower fertility than heterozygous controls , fertility was not decreased in simNup96 hemizygotes over Df(3R)Exel9014   . TherefosimNup160 and simNup96 introgression, we produced w; Int(2L)D+S, sim/CyONup160; sim e/+Nup96 males by conventional crosses. Then, these males were crossed to females heterozygous for a balancer and a mutation (or a deficiency) of Nup160 or Nup98-96. If the introgressions were behaving similar to the F1 hybrid, then sim/Nup160(simNup160 or Df-Nup160); sim/+Nup96 is expected to be lethal; however, that is not what is observed. Instead, the simNup160 homozygotes (or hemizygotes) were viable in the simNup96 heterozygous background  is expected to be lethal; however, that is not what is observed. Instead, the simNup96 homozygotes (or hemizygotes) were viable in the simNup160 heterozygous background .Next, to examine possible synergistic and/or additive effects of ckground . If the ckground . Thus, tsimNup160 and simNup96 introgressions maintained with chromosome 2 and 3 balancers but were not successful, presumably because Int(2L)D+S can cause dominant male semisterility in some genetic backgrounds . Therefore, we could not test the viability/fertility of simNup96 and simNup160 double introgression homozygotes. Instead, we made w; Df(2L)Nup160M190/SM1; sim/TM3Nup96 females and w; Int(2L)D+S, sim/SM1Nup160; sim/+Nup96 males by conventional crosses and crossed them. Int(2L)D+S, sim/ Df(2L)Nup160M190Nup160; sim/+Nup96 flies were viable as we previously noted (simNup160 might have reduced their viability (Int(2L)D+S, sim/ Df(2L)Nup160M190Nup160; sim/TM3Nup96 was semilethal D+S, sim/ Df(2L)Nup160M190Nup160; sim/Nup96simNup96 was also absolutely lethal  for the simNup96 introgression were fertile  display karyogamy failure and female pronuclei never fuse to wild-type male pronuclei  in the previous studies. Apparently the presence of additional autosomal D. simulans genes is necessary to cause lethality, and these genes are dominant to the D. melanogaster alleles. Thus, more genes (maybe encoding other Nups) are involved in this hybrid incompatibility. Nup107 and Nup154 are excluded from the candidates because Int(2L)D+S also carries these genes from D. simulans but did not exhibit the dominant effect. One candidate for the interactant is Nup75, presumably the Drosophila homolog of Nup85. Further investigation of this system is necessary to better understand the genetic mechanisms of reproductive isolation.We found that flies with genotypes indicated in simNup96 was changed by the presence of a balancer TM3 (Lhr vs. Hmr) in the same species cross (simNup96 and hemizygous simNup160 resulted in lethality in the hybrids (simNup96 and simNup160 are two components of the same incompatibility.Interestingly, dominance of ncer TM3 . Reprodu hybrids . This is