Here, we develop a fast and genetically tractable assay to test the efficiency and dynamics of fluospheres (FS) using microinjection into Drosophila oocytes coupled with time-lapse microscopy. We designed dynein motor driven FS using a collection of dynein light chain 8 (LC8) peptide binding motifs as molecular linkers and characterized in real time the efficiency of the FS movement according to its linker’s sequence. Results show that the conserved LC8 binding motif allows fast perinuclear nanoparticle's accumulation in a microtubule and dynein dependent mechanism. These data reveal the Drosophila oocyte as a new valuable tool for the design of motor driven nanovectors.Molecular motors transport various cargoes including vesicles, proteins and mRNAs, to distinct intracellular compartments. A significant challenge in the field of nanotechnology is to improve drug nuclear delivery by engineering nanocarriers transported by cytoskeletal motors. However, suitable  Dynein is a large multi protein complex, containing a motor domain composed of two heavy chains (HC), and a cargo binding domain, made of intermediate chains (IC), light intermediate chains (LIC) and dimers of light chains including LC8 [Drosophila and human [in vivo and in vitro studies identified a short consensus LC8 binding sequence, (KXTQT) [Drosophila, the LC8 binding sequence is also present in proteins involved in dynein dependent mRNA transport such as Swallow (Swa) [Drosophila oocyte microinjection combined with genetic, pharmacological and videomicroscopy. We designed dynein motor driven fluorospheres using a collection of dynein light chain 8 (LC8) peptide binding motifs as molecular linkers and demonstrate their ability to move toward the nucleus of Drosophila oocytes. We further showed that conserved LC8 binding motifs allow the rapid perinuclear accumulation of nanoparticles in a microtubule and dynein dependent mechanism. This new approach represents a valuable tool allowing the identification of new motor-cargoes biolinkers and the genetic characterization of their intracellular transport efficiency.Active transport in cells is largely driven by the kinesin and dynein motor families that move toward the plus and minus end of microtubules (MT), respectively . Variousding LC8 . Dynein  (KXTQT) -12, foun (KXTQT) -15. In Dow (Swa)  and Egalow (Swa) . Here, wDrosophila oocytes  with various peptides and tested their capacity to travel in the cytoplasm after injection into  oocytes , 2b. Pep oocytes . To extecterized ,19. Morecterized -27  could be considered as being among the best LC8 ligands. We tested LC8 binding motifs identified in three known LC8 binding proteins: dynein intermediate chain (DIC), adenovirus-associated protein BS69 [Drosophila and can be used as universal molecular linkers. First, injected control FSs tagged with PEG only (FS-PEG-OMe) (see Methods) spread randomly in the oocyte cytoplasm and showed no specific accumulation  Movie S. FS's inein BS69  and Egl ein BS69 . FSs carein BS69 . AltogetY-2 predicted to have slightly improved interaction with LC8 homodimer  (G-1 (T-G), Mut2G0 (Q-G) and Mut3G+1 (T-G) predicted to have a low binding affinity for LC8  failed to localize and were instead evenly distributed within the oocyte   (Movie SPEG-DIC) . We also for LC8 . As expee oocyte   or Dhc (Dhc6‑6/Dhc6‑12). FS's localization is strongly affected in both mutant conditions, with 60% of injected oocytes showing random distribution of the FSs  and FS-PEG-BS69 (68%) move anteriorly toward the MT minus ends. In Dhc hypomorphic mutant, the percentage of FS-PEG-WTY-2 showing directed motion is decreased, which could explain the decreased localization index observed  peptides but not with Mut2G0  accumulate around the nucleus forming aggregates (size > 1000 μm2) adjacent to the nuclear envelope suggesting that LC8 binding peptides could facilitate nuclear delivery  and high resolution time lapse imaging. Movies were taken 5 to 10 min post injection near the nucleus and during 1 min. The mean square displacement (MSD) was calculated for each individual particle and plotted versus Δt. FS's movements were classified into three groups of motility based on theoretical previous studies –31: statransport . A majorobserved , 3f. Findelivery  . The BS69 sequence is of viral origin and it has already been shown that viruses are able to efficiently hijack the cellular transport machinery [Y-2 affinities may induce transitory interaction and frequent dissociation of the complex. While nuclear accumulation is apparent with all peptides it could generate differences in pulldown efficiency. Finally, the tight binding of BS69 to LC8 might also be responsible for the large FS-PEG-BS69 aggregates observed around the nucleus.In this study, we found that G0 mutant peptide, consistently failed to localize fluospheres and to bind LC8. Interestingly, while BS69, DIC, Egl and WTachinery . We thusDrosophila oocytes provide a suitable and powerful system to characterize and develop nanovectors able to transport therapeutic cargoes to specific intracellular compartments including the nucleus. Direct injection in the cytoplasm of the large oocyte was shown to allow for fast, efficient, non-cytotoxic testing of heterologous materials circumventing the cell entrance step. As shown in this study, Drosophila oocytes represent a unique system combining genetic, pharmacology and real time videomicroscopy analysis in a single package. Our approach can be adapted to test and improve various kinds of carriers including gold and magnetic particles combined with a large spectrum of motor biolinkers.In conclusion, G0-GFP, UASp-WTY-2-GFP, UASp –BS69-GFP were cloned KpnI, NotI. LC8 DIIA82 and LC8ins1 mutants were from K. Ray’s lab. Dhc64c6 , Dhc64c6-12 were from T. Hays’ lab.Flies were raised on standard cornmeal-agar medium at 25°C. The following strains were used: nlsGFP, tauGFP, ctp-GFP, Mat-tub-gal4 were from the Bloomington stock center (BL# 7062). Coding sequence for GFP tagged peptides were cloned in pUASp vector using KpnI-NotI cloning site. UASp-GFP-LC8, UASp-Egl-GFP, UASp- Mut2For injection, ovaries were dissected in halocarbon oil . Injecti2; 1% triton X100) before running the blots. Westerns blots were performed on protein extracts from pools of three different strains for a same construct. Extracts protein concentrations’ were evaluated using Bio-Rad protein assay method based on the Bradford assay (Bio-Rad). We used the same total amount of protein per extract for each experiment (approximately 750 μg). IP were performed overnight at 4°C. A mix 1:1 of protein A sepharose: protein G sepharose was used (2 hours at RT). Beads were washed 3 times 10 min with the assay buffer  conjugate carrying the protected DIC, Egl, BS69, WTY-2, Mut1G-1, Mut2G0, or Mut3G+1 peptide sequence  was dissolved into a mixture of MES  and DMF (200 μL). 543/620 or 488/645 carboxylated fluospheres from Molecular Probes  were then added. After 15 min of stirring, EDC (4 mg) was added and the mixture was incubated for 18 h. Remaining unreacted carboxylated functions were capped with glycine (100 mM) for 30 min. The functionalized fluospheres FS-PEG-Peptide (P) were collected after 5 cycles of centrifugation/resuspension (sonication)  with ultrapure water (1 mL). After the last cycle, supernatant was discarded and freshly prepared HCl 6N solution (400 μL) was added to FS-PEG-Peptide(P) for the removal of the various aminoacid side chain protecting groups. The mixture was vortexed for 96-120 h. Deprotected FS-PEG-Peptide beads were then purified through 10 cycles of centrifugation/resuspension (sonication) with ultrapure water (1.5 mL) till conductivity reaches ultrapure water one’s. As for the FSs carrying both the PEG-Peptide and PEG-OMe, they were prepared according to the same two-step procedure described above but starting with a 90/10 or a 50/50 mol% mixture of NH2-PEG-OMe (2 kDa) and NH2-PEG-Peptide (P). Conjugation of the peptides onto the FS's surface was confirmed by IR spectroscopy, which indicated the presence of NH vibrations . For aggregates quantifications, oocytes have been injected, fixed and collected 45 min after injection. Oocytes were stained for Lamin C in order to visualise the nuclear envelope together with the fluospheres. Statistical significance was assessed using a Chi-2 test of FSPEGBS69 versus FSPEGPeptide  *, p<0.05; **, p<0.005. Aggregates characterization was made using Image J, the data presented correspond to the maximum surface of each object obtained using 3D object counter. Imaging was performed on a confocal microscope Zeiss LSM 510 or on a widefield DeltaVision microscope  and deconvolved using SoftWorx software. 100x, fast imaging was performed on a Spinning disk microscope (OlympusAndor technology). Particle tracking was made manually using Image J. The speed of particles was calculated in micrometers per second according to the distance travelled in each time interval. The mean square displacement (MSD) was calculated according to the published formula ,36 for eFigure S1DIC peptide-protein complex structure. Peptide ribbon is drawn in cyan and dynein light chain is in gray.(DOCX)Click here for additional data file.Figure S2Functionalized FSs localize at the minus ends of MTs.(a) βGal staining of a Nod-LacZ oocyte showing the minus ends of MTs. (b) Localized FS-PEG-WTY-2 colocalized with βgal staining (arrow head).(TIF)Click here for additional data file.Materials S1Molecular Modeling of Dynein peptides.(DOCX)Click here for additional data file.Movie S1FS-PEG-OMe move randomly through the oocyte cytoplasm.FS-PEG-OMe injection in a NLS-GFP oocyte. Movie starts 30 min +/- 5 mins after injection in this and all subsequent movies unless otherwise mentioned. The frame rate is 1 image every 5 min in this movie and all subsequent movies unless otherwise mentioned.(AVI)Click here for additional data file.Movie S2FS-PEG-DIC localize specifically close to the oocyte nucleus.FS-PEG-DIC injection in a NLS-GFP oocyte. (AVI)Click here for additional data file.Movie S3Co injected FS-PEG-DIC (red) and FS-PEG-OMe (green) show different behaviour in the oocyte cytoplasm.FS-PEG-DIC (red) and FS-PEG-OMe (green) co-injection in a W118 oocyte. (AVI)Click here for additional data file.Movie S4Y-2 localize specifically close to the oocyte nucleus.FS-PEG-WTFS-PEG-WTY-2 injection into a nls-GFP oocyte. (AVI)Click here for additional data file.Movie S5G+1 move randomly through the oocyte cytoplasm.FS-PEG-Mut3FS-PEG-Mut3G+1 injection in a nls-GFP oocyte.(AVI)Click here for additional data file.Movie S6Colcemid inhibits FS-PEG-DIC specific localization.Colcemid and FS-PEG-DIC co-injection in a Tau-GFP oocyte. Movie starts 20 min after injection. (AVI)Click here for additional data file.Movie S7P1H4 inhibits FS-PEG-DIC specific localization.P1H4 and FS-PEG-DIC co-injection in a Tau-GFP oocyte. (AVI)Click here for additional data file.Movie S8Y-2 compete for LC8 binding.FS-PEG-WTLeft panel, FS-PEG-WTY-2 localization is strongly decreased in oocytes expressing WTY-2-GFP. Right panel, FS-PEG-WTY-2 efficiently localize in Mut2G0-GFP expressing oocyte. (AVI)Click here for additional data file.Movie S9FS-PEG-BS69 movement isdirected toward the anterior.FS-PEG-BS69 move anteriorly. Colored lines follow FS showing directed motion toward the MT minus ends. The frame rate is 2 images/s. Movie starts 5 min after injection.(AVI)Click here for additional data file.Movie S10FS-PEG-BS69 form aggregates embedded into the nuclear envelope.3D reconstruction and rotation around the nucleus of an oocyte injected with FS-PEG-BS69 and stained for Lamin C (blue) 45 min after injection. This oocyte is the same as in (AVI)Click here for additional data file.Table S1Binding free energy differences of mutant sequences related to DIC peptide according to various computational methods.(DOCX)Click here for additional data file.


Salinarchaeum sp. strain HArcht-Bsk1T, was determined using next-generation sequencing. The genome comprises a 3,255,260-bp circular chromosome with a G+C content of 66.7%. Automatic annotation of the genome revealed a single rRNA operon, 45 tRNAs, and 3,013 protein-coding gene sequences.The complete genome sequence of a novel halophilic archaeon,  Euryarchaeota growing optimally at salt-saturating conditions. Strain HArcht-Bsk1T was isolated from the top aerobic sediment layer of the hypersaline chloride-sulfate Lake Baskunchak (south Russia). Cells of strain HArcht-Bsk1T were occasionally motile pleomorphic flat rods during the exponential phase of growth, becoming irregularly shaped coccoids and discoids at the later stages. Strain HArcht-Bsk1T grew optimally at 40°С (рН 7.5) and 3.5 M of NaCl. It grew organotrophically using various sugars as substrates. A BLAST search  and long mate pair  approaches were used. The fragment library was sequenced with two bar-coded runs of the Illumina MiSeq system, resulting in 5,919,667 101-bp and 222,683 250-bp paired-end reads. Reads were corrected with the Quake sequencing error correction tool . The chromosome contains a single rRNA operon, 45 tRNA genes for all 20 standard amino acids, and 3,013 protein-coding sequencesThe Salinarchaeum sp. strain HArcht-Bsk1T has been deposited in NCBI GenBank under the accession number CP005962.The genome sequence of


Acinetobacter sp. strain MCC2139. This bacterium, isolated from dairy sludge and with optimum growth at 37°C, has a genome size of 2,967,280 bp with a G+C content of 42.3%.We report the draft genome sequence of an ammonia-producing, esculin-hydrolyzing, catalase-positive, gram-negative bacterium,  It accounts for 1.2% of the global primary energy demand  under ambient conditions. The strain is available at the Microbial Culture Collection at the National Centre for Cell Sciences, Pune, India, with the accession number MCC2139.Sludge from a dairy effluent treatment plant was used for the isolation of a bacterial strain, The genome sequencing was carried out on an Ion Torrent PGM instrument using a 316 chip. The total assembled reads (using MIRA Assembler version 3.4.0) were 1,435,352 bp, with 69.01× coverage. There were 141 contigs. A total of 29.67 Mb of data was sequenced, with the largest contig size of 163,183 bp. All contigs generated were submitted to the RAST (Rapid Annotation using Subsystem Technology) ; phosphoserine phosphatize; 23S rRNA; Na(+) H(+) antiporter subunits; type IV fimbrial assembly (AOTJ01000001); copper chaperone as well as lead-, cadmium-, zinc- and mercury-transporting ATPase; Cu(I)-responsive transcriptional regulator (AOTJ0100009); ferric iron ATP-binding cassette (ABC) transporter; aromatic amino acid transport protein AroP(AOTJ0100008); cobalt-zinc-cadmium resistance protein CzcD (AOTJ0100076); chromosome (plasmid) partitioning proteins ParB and ParA (AOTJ0100068); chromate transport protein ChrA/resistant protein ChrB (AOTJ0100061); phage-related protein (AOTJ0100053); superoxide dismutase [Fe] (AOTJ0100056); rod-shape-determining protein MreB/MreC (AOTJ0100057); ABC1 family protein (AOTJ0100048); exonuclease SbcC/SbcD (AOTJ0100049); transcriptional regulator (LuxR family); Kup system potassium uptake protein (AOTJ0100047); Fe-S oxidoreductase (AOTJ0100045); inorganic pyrophosphatase; phosphate transport system regulatory protein PhoU; ADP-ribose pyrophosphatase; ammonium transporter; and nitrogen regulatory protein P–II (AOTJ0100042).AOTJ00000000. The version described in this paper is the first version, AOTJ01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number


Herpes Simplex virus thymidine kinase were eliminated by ganciclovir treatment, indicating that the HAC safeguard system functioned in iPS cells. Thus, the HAC vector could generate uniform, integration-free iPS cells with a built-in safeguard system.Human artificial chromosomes (HACs) have unique characteristics as gene-delivery vectors, including episomal transmission and transfer of multiple, large transgenes. Here, we demonstrate the advantages of HAC vectors for reprogramming mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. Two HAC vectors (iHAC1 and iHAC2) were constructed. Both carried four reprogramming factors, and iHAC2 also encoded a p53-knockdown cassette. iHAC1 partially reprogrammed MEFs, and iHAC2 efficiently reprogrammed MEFs. Global gene expression patterns showed that the iHACs, unlike other vectors, generated relatively uniform iPS cells. Under non-selecting conditions, we established iHAC-free iPS cells by isolating cells that spontaneously lost iHAC2. Analyses of pluripotent markers, teratomas and chimeras confirmed that these iHAC-free iPS cells were pluripotent. Moreover, iHAC-free iPS cells with a re-introduced HAC encoding  Reprogramming somatic cells to become induced pluripotent stem (iPS) cells is important in making regenerative medicine a reality With respect to the generation of iPS cells, human artificial chromosomes (HACs) have two important and unique characteristics as gene-delivery vectors; effectively unlimited carrying capacity for transgenic material and autonomous maintenance through cell division that is independent of host chromosomes. We have created several HAC vectors from human chromosome 21 using a top down method Herpes Simplex virus thymidine kinase (HSV-TK), and we confirmed that iPS cells and/or their differentiated derivatives carrying our HAC can be killed by ganciclovir (GCV), providing a safeguard system if unexpected events  occur.Here, we have taken advantage of these features of HAC vectors to generate vector-free and transgene-free iPS cells. Recent attempts to generate iPS cells using polycistronic vectors to express multiple proteins demonstrated that a significant portion of the iPS clones carried more than two copies of the polycistronic vector All animal experiments were approved by the Institutional Animal Care and Use Committee of Tottori University (the permit number: 08-Y-69).We constructed individual expression cassettes for each reprogramming factor in pBSII (Stratagene) and combined all cassettes into a pPAC4 backbone as follows. The pBSII multiple cloning site was replaced with either KpnI-XhoI-AscI-BsiWI-NheI-ClaI-SalI-PstI-AvrII-PmeI-FseI-XbaI-SpeI-SacI or KpnI-XhoI-AscI-BsiWI-NheI-ClaI-SalI-MluI-SphI-SnaBI-NotI-SacII-BamHI-AvrII-PmeI-FseI-XbaI-SpeI-SacI, resulting in the vectors pB3 and pB4, respectively. Two different 1.2 kb fragments of the chicken HS4 insulator were excised by either SacI or XbaI digestion of pCJ5-4 , blunted by KOD polymerase (Toyobo), and cloned into the SmaI site or the blunted HindIII site of pBSII, respectively. The resulting vector, harboring 2 copies of HS4, was called pBSI-I. A ClaI-BamHI fragment of pBSI-I was cloned into (1) a blunted ClaI site of pB3, (2) a blunted ClaI site of pB4, or (3) blunted ClaI and PmeI sites of pB4, resulting in (1) pinsB3, (2) pinsB4 and (3) pB4ins2, all of which retained the BamHI site immediately downstream of the HS4 dimer. All subcloned HS4 insulators had the same orientation.Mouse Klf-4, c-Myc, Sox2 and Oct4 were PCR-amplified and individually cloned into the EcoRI site of pCAGGS , resulting in pCX-Klf4, pCX-c-Myc, pCX-Sox2 and pCX-Oct3/4, respectively. SalI-BamHI fragments of pCX-Klf4, pCX-c-Myc and pCX-Sox2 were blunted and cloned into blunted BamHI sites of pinsB4, resulting in pB4K, pB4M and pB4S, respectively; an SspI-BamHI fragment of pCX-Oct3/4 was cloned into a SnaBI site of pB4ins2, resulting in pB4O. To combine four factors in a single vector, AscI-AvrII fragments from pB4K and pB4S were inserted into the AscI-NheI sites of pB4M and pB4O, resulting in pB4KM and pB4SO, respectively. Finally, an AscI-AvrII fragment of pB4KM and an NheI-FseI fragment of pB4SO were ligated into the AscI and FseI sites of pPH3-9, which was generated by modifying pPAC4; specifically we exchanged the region between the pUC link and the CMV promoter with HPRT ex3-ex9 and added an FseI site immediately downstream of HPRT ex9. The resulting vector was designated pPAC-KMSO. This KMSO reprogramming cassette was duplicated by the same strategy, resulting in pPAC-2CAG-KMSO. A fragment of the duplicated pB4O was cloned into the AscI-NheI site of pPAC-2CAG-KMSO, resulting in pPAC-2CAG-O2.GTACATGTGTAATAGCTCC and cloning the product into the BglII-XbaI sites of pENTR4-H1 , resulting in pENTR4-H1-mp53sh. A SalI-XbaI fragment of pENTR4-H1-mp53sh was inserted into the SalI-AvrII site of pinsB3, resulting in pinsB3mp53sh. Finally, an AscI-SpeI fragment of pinsB3mp53sh was inserted into the AscI-NheI site of pPAC-2CAG-O2, resulting in pPAC-2CAG-O2mp53sh.A mouse p53-knockdown construct was generated by annealing two complementary synthetic oligonucleotides with the target sequence Hprt-deficient Chinese hamster ovary cells  each bearing a HAC vector, (CHO(21HAC2), CHO/iHAC1/E15 and CHO/iHAC2/mp25) were maintained at 37°C in Ham’s F-12 nutrient mixture (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 8 µ/ml Blasticidin S (Funakoshi). Mouse embryonic fibroblasts (MEFs), isolated from 13.5 day post-coitum (d.p.c.) wild-type embryos (C57BL/6-J), were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma) plus 10% FBS. The mouse ES cell lines, TT2  6 cells in PBS) were mixed with 10 µg of the Cre expression plasmid (pBS185) and 20 µg of pPAC-2CAG-KMSO. Electroporation was performed at 450 V and 500 µF using a GenePulser Xcell (Bio-Rad). pBS185 (1 µg) and pPAC-2CAG-O2mp53sh (2 µg) were transfected into semi-confluent CHO(21HAC2) cells in a 60 mm dish using Lipofectamine2000 (Invitrogen) according to the manufacturer’s instructions. These cells were treated with HAT (Sigma) and 8 µg/ml Blasticidin S two days after transfection. After 2-3 weeks, drug-resistant colonies with a functional HPRT allele were identified by genomic PCR and isolated.The reprogramming cassettes were introduced into 21HAC2 vectors using the Cre-loxP system. Suspensions of CHO(21HAC2) cells  human COT-1 DNA (Invitrogen) and biotin-labeled pPAC-KMSO, essentially as described previously Procedures for the denaturation of metaphase chromosomes and mFISH probes (MetaSystems), hybridization, post-hybridization washes and fluorescent staining were performed in accordance with the manufacturer's instructions. Metaphase images were captured digitally with a cooled CCD camera equipped with an ISIS mFISH software program (MetaSystems), processed and stored for subsequent analysis.Total RNA was extracted with TriZol (Invitrogen) and cDNA was synthesized using an oligo(dT) primer and ReverTra Ace (Toyobo). Quantitative RT-PCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems) on an ABI7900HT (Applied Biosystems). Primer sequences are listed in Cells were fixed with 4% paraformaldehyde in PBS at 4°C overnight and permeabilized and blocked in 0.1% Triton X-100, 5% normal goat serum (Millipore) and 5% skim milk in PBS for 15 min at room temperature. Primary antibodies used were rabbit anti-Nanog , mouse monoclonal anti-Oct4 , rabbit anti-α-fetoprotein , mouse monoclonal anti-α-smooth muscle actin  and rabbit anti-βIII-tubulin . Secondary antibodies used were Alexa 594-conjugated goat anti-rabbit IgG  and Alexa 594-conjugated goat anti-mouse IgG . Samples were counterstained with 1 µ/ml DAPI. A Leukocyte Alkaline Phosphatase Kit (Sigma) was used to detect alkaline phosphatase activity.Total RNA was prepared with the RNeasy midi Kit (Qiagen). RNA (500 ng) was labeled with Cy3 using the Quick Amp Labeling Kit (Agilent) according to the manufacturer’s instructions. cRNA was column-purified using the RNeasy Kit (Qiagen) and hybridized to the Agilent Whole Mouse Genome Oligo Microarray (G4122A). The data were normalized and analyzed using GeneSpringGX11 (Agilent). Genes with a 4-fold change in expression between MEFs and ESCs were selected and hierarchically clustered using the centroid linkage rule.The microarray data is MIAME compliant and the raw data have been submitted to the NCBI GEO database under the accession number GSE29441.Cell suspensions were prepared by trypsinization and 500 cells were aggregated in hanging drops of ES medium without LIF. After 3 days, embryoid bodies (EBs) were seeded onto gelatin-coated culture dishes for 4 days. Spontaneous germ-layer differentiation of EBs was assayed using markers for endoderm (AFP), mesoderm (α-SMA), and ectoderm (βIII-tubulin).6 cells were transplanted subcutaneously into dorsal flanks of CD-1 (ICR)-nu mice (Charles River). Tumors were isolated after 5-6 weeks and subjected to hematoxylin-eosin staining. Histological examination confirmed differentiation into all three germ layers.Approximately 1 × 10Chimera production was performed as described previously To introduce four reprogramming factors into recipient cells without the risk of integrating foreign genetic material into host chromosomes, we constructed HAC-based cell-reprogramming vectors. An expression cassette consisting of CAG promoter-driven cDNAs of Klf4, c-Myc, Sox2 and Oct4, each surrounded by copies of the HS4 insulator, was introduced into a modified pPAC4 vector to generate pPAC-KMSO . To achi6 MEFs via MMCT. A schematic of this process is shown in To assess the reprogramming ability of iHAC1 and iHAC2, each was transferred from a CHO donor cell line into approximately 10-5). An EGFP-negative fraction from one iHAC1-iPS line (clone 1-D6) (up to 105 cells) was harvested by FACS , plated onto a feeder layer, and re-cloned based on morphological criteria. EGFP-negative colonies (n = 4) were selected randomly and assayed for the presence or absence of the iHAC1 vector. FISH and genomic PCR analyses revealed that all 4 EGFP-negative clones  retained the iHAC1 vector  and in vivo (teratoma) assays. Furthermore, 4 of 4 iHAC-free iPS clones tested gave rise to live chimeras  HSV-TK, and a full-length Dystrophin locus. GCV, administered consecutively over 32 days, selectively repressed advancement of teratomas from the mdx-iPS(DYS-HAC) cells, but not of control mdx-iPS cells, and administration of PBS had no effect on either cell line , suggesting that the re-introduced 21HAC2 vector did not affect the pluripotency of iPS cells.Our 21HAC vectors carry a suicide gene, ell line . Next, w-5. To overcome this drawback, our protocol was enhanced with two procedures. First, to sustain high expression levels of individual reprogramming factors, each factor was surrounded with insulators. Second, to potentiate the reprogrammed state, miR294 cluster mimics, which promote induced pluripotency We demonstrate that a HAC vector containing expression cassettes for four reprogramming factors and a p53-knockdown construct efficiently reprogrammed somatic cells to pluripotency. In addition, we established integration-free iPS cells derived from these reprogrammed cells. Inserting all expression constructs into a defined cloning site on the HAC vector, which was maintained stably and independently of host chromosomes in cells, resulted in homogenous expression levels of the transgenes in HAC donor and recipient cells. Once configuration of an expression cassette in the HAC vector is optimized for the generation of iPS cells, the resulting uniformity of transgene expression in target cells is an advantage in promoting reprogramming efficiency and reducing clonal variation in the resulting iPS cells. Thus, the HAC-based reprogramming strategies are expected to be more effective in establishing homogenous iPS clones than other methods, including DNA transfection and viral transduction, which are both unable to regulate the quantity of xeno-products in modified cells. Nonetheless, the transfer rate of HAC vectors via MMCT is relatively low, i.e., 10All iHAC1-iPS cells satisfied some criteria of pluripotency ; however, the iHAC1-iPS cells expressed only low levels of various pluripotent markers, indicating only partial reprogramming. In contrast, expression of pluripotent markers in most of the iHAC2-iPS cells was significantly upregulated and close to that in ES cells. These results were consistent with previous studies, which demonstrated that increasing Oct4 expression relative to the other three reprogramming factors Removal of potential obstacles, such as persistent exogenous genes or chemicals, the maintenance of normal cellular functions and the preservation of genome integrity are fundamental to the application of iPS cells in regenerative medicine. Here, we demonstrated that a HAC vector can mediate somatic cell reprogramming and that transgene-free, vector-free iPS cells can be obtained from iHAC2-iPS cells using simple FACS sorting; in contrast, other systems require prolonged culture, vector excision and drug selection to obtain integration-free iPS cells. Furthermore, the iHAC-free iPS cells generated using iHAC2 did not exhibit chromosomal aberrations, although the present version of iHAC2 contained a p53-knockdown construct. Thus, the strategy of using HAC vectors to generate integration-free iPS cells might be valuable. Moreover, the HAC vector itself was safe because it was maintained independently of host chromosomes, and importantly, chimeric mice were produced from mouse ES/iPS cells harboring a HAC vector Figure S1Transgene expression in CHO donor cells. Expression of the four reprogramming factors contained in the iHAC vectors was confirmed by RT-PCR. In qRT-PCR assays, transcript levels of transgenes were standardized to Gapdh. Transcript levels in CHO/iHAC2/mp25 were compared to levels in CHO/iHAC1/E15.(EPS)Click here for additional data file.Figure S2Characterization of iHAC1-iPS cells. (A) Alkaline phosphatase staining of iHAC1-iPS 1-A10, 1-C3, 1-D10 and mouse ES TT2 cells.  Immunostaining of pluripotent markers. iPS cells with the iHAC1 vector and that were positive for EGFP (green) were stained for Nanog (B) and Oct4 (C).(EPS)Click here for additional data file.Figure S3In vitro differentiation of iHAC1-iPS cells. (A) EB formation. (B) Phase-contrast images showing differentiated cells derived from EBs. (C) Immunostaining confirming that iHAC1-iPS cells differentiated into each germ layer in vitro.(EPS)Click here for additional data file.Figure S4Expression levels of pluripotent markers, Nanog and Rex1. Pluripotency of iHAC2-iPS cells generated without miR-294/295 was compared to that of iHAC2-iPS cells with miR-294/295 by quantitative RT-PCR analyses of Nanog and Rex1 expression. Transcript levels were standardized to Nat1 and normalized to B6ES cells.(EPS)Click here for additional data file.Figure S5Anti-proliferative effect of GCV on HSV-TK-containing mdx-iPS(DYS-HAC) cells in vivo. Parental mdx-iPS cells and mdx-iPS(DYS-HAC) cells were subcutaneously transplanted into nude mice, then GCV was administered intraperitoneally. Tumor volume was calculated using the formula [width (mm)]×[length (mm)]×[depth (mm)]. Administration of PBS was used as a control.(EPS)Click here for additional data file.Figure S6Expression levels of p53, Ink4a, Arf and Cip1. Quantitative RT-PCR analyses of p53, Ink4a, Arf and Cip1 levels in iHAC1- and iHAC2-iPS cells were compared with levels in parental MEFs and mouse ES cells. Transcript levels were standardized to Nat1 and normalized to B6ES cells.(EPS)Click here for additional data file.Table S1Primers used for PCR analyses.(XLS)Click here for additional data file.


Bone mineralization disturbances are common in children with juvenile idiopathic arthritis (JIA) and depend on inflammation, medications, lack of motion and genetic factors.To evaluate the role of genetic markers in bone metabolism and mineralization in JIA children.1-L4. Bone biochemical markers were osteocalcine, C-terminal telopeptides (CTT), parathyroid hormone (PTH), Ca, Ca++, P, total alkaline phosphatase (TAP) activity. We have detected Sp1 (rs1800012) and -1997G/T (rs1107946) polymorphisms in type I alpha-1 chain collagen gene (COL1A1).We included 196 JIA children, 81 boys and 115 girls. Bone mineralization parameters were detected by dual-energy X-ray absorptiometry of lumbar spine LWe revealed gender differences in Sp1 genotype distribution in children with low bone mineral density (LBMD): boys had GG genotype in 89.5% and girls in 54.2% (p=0.03). In boys GG genotype presence increased LBMD – OR=2.96 (95%CI: 0.59-14.9) compare in girls in which GG presence decreased LBMD – OR=0.56 (95%CI: 0.36-2.7). Also, children carried T allele (GT and TT genotypes) despite on higher inflammatory parameters had better mineralization dates. In total group children with GG genotype had higher osteocalcine  and CTT levels . In children, who have not been treated with steroids GG genotype was associated with lower BMD-Zscore in boys  and lower height in girls . In children with Tanner stage I GG genotype was associated with more rare LBMD  and with frequent LBMD in children with Tanner stage II-III .++ , inorganic phosphate  and osteocalcine level  in children with Tanner stage II-III and lower BMD  and lower BMD-Zscore GG genotype of -1997G/T polymorphism was associated with lower CaWe have revealed different changes in mineralization and metabolism, associated with sex, Tanner stage and treatment due to COL1A1 gene polymorphisms in children.


In vitro RNA-pull down assays indicated that miR-20b targets numerous tumor suppressors, including PTEN and BRCA1, which were downregulated in HCC1806. Conversely, suppression of miR-20b increased PTEN and BRCA1 levels. Moreover, immunohistochemical and FISH analyses showed that the miR-20b expression correlated significantly with EGR1 levels in breast cancer tissues. Our findings thus demonstrate for the first time that EGR1 is a key player in the transcriptional control of miR-20b, and miR-20b may in turn function as an oncogene by contributing to breast tumorigenesis via tumor suppressor targeting.Transcriptional regulation of miRNAs that control the pathogenesis of breast cancer remains largely unknown. Here, we showed that ionizing radiation, a known breast carcinogen, triggered the differential expression of miR-20b in mammary tissues. We identified several GC-rich consensus binding motifs for the zinc finger transcription factor early growth response-1 (EGR1) in miR-20b promoter. miR-20b was upregulated by IR and its upregulation correlated with EGR1 expression in the breast cancer cell line HCC1806. Therefore, we used HCC1806 cells as a model system to explore the role of EGR1 in miR-20b transcription. siRNA knockdown of EGR1 attenuated miR-20b expression. Luciferase assays showed that whereas EGR1 stimulated luciferase activity driven by the wild-type miR-20b promoter, this induction was abolished in the mutant miR-20 promoter construct. We noted significant enrichment of EGR1 at miR-20b promoter in HCC1806 cells compared with normal human mammary epithelial cells. Suppression of miR-20b significantly inhibited HCC1806 cell proliferation and migration, and led to G 0/G 1 and S phase arrest.  Breast cancer is the most common malignancy in women worldwide and the second leading cause of cancer-related deaths among North American women. Most brIn recent years, one of the key advances in our understanding of the fundamental mechanisms of gene regulation has been the discovery of microRNAs (miRNAs/miRs). miRNAs are small noncoding RNA molecules that regulate gene expression either through translational repression or mRNA degradation, as determined by the degree of complementarity to the 3' untranslated regions of cognate mRNAs, 7. ApprmiRNA also partake in genotoxic stress responses. Many genotoxic carcinogens affect miRNA patterns in the exposed tissues and organs. Amongst those, IR profoundly affects tissue miRNAome. Recently we have shown that IR exposure affects mammary gland tissue and causes profound deregulation of miRNA expression. Amongst miRNAs, miR-20b was significantly affected.miRNA-20b (miR-20b) is encoded by the miR-106a-363 cluster which, together with the miR-17-92 and miR-106b-25 clusters, forms a large family of highly similar miRNAs called the miR-17 family. Members0/G1 and S phase arrest. Furthermore, we provide the key evidence that miR-20b targets tumor suppressors BRCA1 and PTEN. Finally, immunohistochemical and FISH analyses indicate that miR-20b is elevated in 30% of breast cancer and 50% of metastatic breast cancer specimens examined, and this upregulation correlates significantly with EGR1 levels.Transcriptional regulation of miR-20b in human cancers remains poorly understood to date. We therefore explored the transcription factor(s) involved in miR-20b expression and the role of miR-20b in breast tumorigenesis. The data presented in this paper indicate that IR induces miR-20b expression in rat mammary gland tissues in a dose- and time-dependent manner. We also show that miR-20b is upregulated in HCC1806 breast cancer cells, and this upregulation correlates with EGR1 expression. We provide evidence that EGR1 controls miR-20b transcription via putative EGR1 binding motifs present in miR-20b promoter. Suppression of miR-20b inhibits HCC1806 proliferation and migration, resulting in GOur previous studies demonstrated that IR triggered a significant and sex-specific deregulation of the microRNAome, as well as altered levels of Dicer and components of the RNA-induced silencing complex in the spleen of C57BL/6 mice . To undeTo explore our hypothesis, we determined the expression of EGR1 and miR-20b, as well as the contribution of EGR1 to miR-20b transcription in breast cancer cells. qRT-PCR showed an aberrant expression of miR-20b in the breast cancer cell lines examined Fig. , which c0/G1 and S phase cell cycle arrest  have been implicated in prostate tumorigenesis [EGR1 is a zinc finger transcription factor that plays a crucial role in controlling cell growth, proliferation, differentiation and apoptosis -29. EGR1igenesis . Knockdoigenesis . Furtherigenesis  althoughigenesis , 41.Although this is the first report regarding EGR1 in the transcriptional regulation of miR-20b, EGR1 was previously reported to regulate mir-106a expression. This reThe absence of ERα; in breast carcinomas has been known for years to be associated with a less-differentiated phenotype and with resistance to endocrine therapies, thus presenting poor prognosis. A recent study identified a new modulator of ERα;, miR-20b, which downregulated ERα; in MCF7 breast cancer cells . However0/G1 and S phase arrest in cell cycle that clearly indicated the key role of miR-20b in the development of this disease. To globally identify miR-20b targets associated with cell proliferation, migration, and cell cycle control, in vitro RNA pulldown and deep RNA sequencing analyses were performed. Our results indicated that miR-20b could target many tumor suppressors ; Group 2: 80 kVp X-ray, 2.5 Gy (High dose/high energy); and, Group 3: sham-treated controls. Ten rats per group were sacrificed at 6 hours, 96 hours, 4 weeks or 24 weeks after irradiation. Mammary gland specimens were frozen immediately and stored at −80°C, or fixed in 10% neutral buffered formalin and embedded in paraffin. Handling and care of animals were in accordance with the recommendations of the Canadian Council for Animal Care and Use. Procedures were approved by the University of Lethbridge Animal Welfare Committee. Animals were housed in a virus-free facility and given food and water Total RNA was isolated from mammary gland tissue of different group IR-exposed rats using TRIzol reagent (Invitrogen) according to the manufacturer's instruction. MicroRNA profiling, clustering and data analysis were carried out by LC Sciences.2.Human mammary epithelial cells (HMEC) purchased from Invitrogen were cultured in HuMEC Basal Serum-Free Medium (Invitrogen) containing HuMEC Supplement (Invitrogen); breast cancer cell lines ZR75-1, HCC1419, and HCC1806 obtained from ATCC were grown in ATCC-formulated RPMI-1640 Medium (ATCC) containing 10% FBS; MCF7 cells were cultured in DMEM/F-12 (HyClone) containing 10% FBS; HEK293 cells were grown in DMEM/High Glucose (Thermo Scientific Limited) containing 10% FBS at 37°C in a humidified atmosphere of 5% COTotal RNAs isolated from IR-exposed rat mammary gland tissue, HMEC, MCF7, ZR75-1, HCC1419, and HCC1806 cells were subjected to real-time RT-PCR using primer sets for either rno-miR-20b-5p or hsa-miR-20b (SABiosciences) according to the manufacturer's instruction. Rat and human RNU6-2 served as a loading control.RPP38) was calculated by the comparative threshold cycle (Ct) method, and results are shown as fold induction.Genomic DNAs extracted from IR-exposed HMEC cells using a kit for purification of total DNA from animal blood or cells (QIAGEN) were subjected to real-time PCR using SsoFast EvaGreen Spermix (Bio-Rad) with the following primers; 20bCopyNo-F: 5'-TGC AGG TAG TTT TGG CAT GA-3', 20bCopyNo-R: 5'-TCA ACA AGA GAT TTG TTA TCC AAG AG-3'; RPP38-F: 5'-TGG TTG TGA AGA CGT CGT TGA-3', RPP38-R: 5'-TGC ATA TCC TCG CTC TCC AGA-3'. The copy number level relative to the internal control (RNase P/55 Fluorescence was observed under 400x on an inverted microscope (ZEISS).HMEC cells grown on coverslips were exposed to either 30 kVp/0.1 Gy, 80 kVp/2.5 Gy X-ray or left sham-treatment as a control. 96 hours after irradiation, immunofluorescence staining was performed using rabbit monoclonal antibody against EGR1  as described previously.Total RNA isolated from HCC1806 transiently transfected with 10 nM or 50 nM of siEGR1 (QIAGEN) or 50 nM of AllStar negative control (QIAGEN) for 96 hours was subjected to real-time RT-PCR using EGR1 primers  and SsoFast EvaGreen Supermix (BIO-RAD) according to manufacturer's instruction.Kpn I and Hind III, and subcloned into pGL3-Basic vector (Promega); sequence identity was confirmed by automatic sequencing; primers used here for amplifying miR-20b promoters are as follows, 20b WT-Prom F: 5'-ATT GGT ACC GTT TTC GCT TTG-3', 20b mt-Prom F: 5'-TTG GTA CCG AGA CTG CGC T-3', 20b Prom R: 5'-ATA AGC TTG CCC CAA CGA AG-3'. To generate luciferase miR-20b target reporters, oligos corresponding to portions of the 3'UTRs of either PTEN or BRCA1 were synthesized, annealed and cloned into downstream of the luciferase gene in the pGL3-Basic vector between Xba I and EcoR I (a linker introduced by Dr James Meservy); the sequence identity was confirmed by automatic sequencing. Oligo sequences were as follows. PTEN 3'UTR-1: 5'-/5Phos/CTA GAA GAT GGC ACT TTC ACT GCT TGT TGT TTG CGC ATT TTT G-3', PTEN 3'UTR-2: 5'-/5Phos/AAT TCA AAA ATG CGC AAA CAA CAA GCA GTG AAA GTG CCA TCT T-3'; BRCA1 3'UTR-1: 5'-/5Phos/CTA GAT CAC GCC TGT AAT CCC AGC ACT TTG GGA G-3', BRCA1 3'UTR-2: 5'-/5Phos/AAT TCT CCC AAA GTG CTG GGA TTA CAG GCG TGA T-3'.Wild-type and mutant fragments of the hsa-miR-20b promoter amplified by PCR using genomic DNA were cloned into pGEM-T easy vector (Promega), released by digestion with The transcription start site of hsa-miR-20b was predicted using Promoter 2.0 Prediction Server. Common transcription factor binding sites at hsa-miR-20b promoter were analysed using Genomatix. Potential hsa-miR-20b targets were predicted by both MIRANDA and RNAhybrid softwares. A network of predicted hsa-miR-20b targets were generated by STRING 9.0.HCC1806 cells grown to 90% confluency  were transiently transfected with either miRCURY LNA hsa-miR-20b power inhibitor or miRCURY LNA microRNA power inhibitor negative control A (Exiqon). 96 hours after transfection, the cells were harvested for cell cycle and apoptosis analyses that were performed with a BD FACSCanto II Flow Cytometer (BD Biosciences) using propidium iodide staining solution and FITC Annexin V Apoptosis Detection Kit II (BD Biosciences) according to manufacturer's instruction.™-P, GE Healthcare) at 4°C for 1.5 hours. Blots were incubated for 1 hour with 5% nonfat dry milk to block nonspecific binding sites and then incubated with polyclonal/monoclonal antibodies against PTEN, BRCA1 (Santa Cruz Biotechnology) or EGR1  at 4°C overnight. The immunoreactivity was detected using peroxidase-conjugated antibody and visualized by ECL Plus Western Blotting Detection System . The blots were stripped before reprobing with antibodies to GAPDH or actin (Santa Cruz Biotechnology).HMEC, MCF7, ZR75-1, HCC1419, and HCC1806 cells grown to 90% confluency were rinsed twice with ice-cold PBS and scraped off the plate in radioimmunoprecipitation assay buffer (RIPA). 30-100 μg of protein per sample was electrophoresed on 6% or 10% SDS-PAGE and electrophoretically transferred to PVDF membrane -2,5-diphenyltetrazolium bromide assays were carried out using the Cell Proliferation Kit I (Roche Diagnostics GmbH) as described by the manufacturer. The spectrophotometric absorbance of samples was measured at 595 nm using a microtiter plate reader .24 hours after transfection with miR-20b inhibitor (Exiqon), 3.0 × 102 for another 24 hours. The cells were treated with 10 μg/ml mitomycin C (Sigma) for 2 hours before injury. The wound healing assay was carried out as described previously[24 hours after transfection with miR-20b inhibitor (Exiqon), HCC1806 cells were replated in 6-well plates and incubated at 37°C in a humidified atmosphere of 5% COreviously.Firefly luciferase data normalized to Renilla luciferase.HEK293 cells grown to 90% confluence in 6-well plates in antibiotic-free DMEM/High Glucose (Thermo Scientific Limited) containing 10% FBS were transiently cotransfected with either 0.5 μg WT-miR20b promoter or MT-miR20b promoter reporter, 0.2 μg or 1 μg pCB6-Egr1, 5 ng pRL-TK, and left empty vector pCB6 as a control; or contransfected with 0.4 μg reporter plasmid (either pGL3-PTEN or pGL3-BRCA1), 5 ng pRL-TK plasmid, 10 nM or 50 nM hsa-miR-20b mimic  and mirVana miRNA mimic negative control #1 (Ambion) using Lipofectamine 2000  as per manufacturer's instruction. 24 hours after transfection, cells were harvested, the relative luciferase activity was measured by the Dual-Luciferase Reporter Assay System (Promega) using a luminometer  and with HMEC and HCC1806 cells grown to 90% confluence were subjected to quantitative ChIP assays as detailed elsewhere, 58. BriHCC1806 cells grown to 90% confluency, nuclear extracts were prepared using NE-PER Nulcear and Cytoplasmic Extraction Reagents (Thermo Scientific Limited), and EMSA was performed using Lightshift Chemilluminescent EMSA Kit (Thermo Scientific Limited) according to the manufacturer's instruction. 20 μl of binding reaction contained 1 × EGR1 binding buffer, 50 ng/μl Poly(dI/dC), 0.05% NP-40, 800 nM cold probe , 2 nM Biotin probe , 2 μl nuclear extract, 1 μg/μl BSA, 2 μl EGR1 antibody or 2 μl normal rabbit IgG .Hsa-miR-20b expression in breast cancer specimens  was determined by FISH as detailed elsewhere . BrieflyTotal RNA isolated from HCC1806 cells using TRIzol Reagent (Invitrogen) and treated RNase-free DNase I (Fermentas) was subjected to RNA pulldown assay using μMACS Streptavidin Kit (Miltenyi Biotec Inc.) as described previously  and accoThe expression of EGR1, PTEN and BRCA1 in breast cancer specimens  was determined by immunohistochemical staining using antibodies to EGR1 , PTEN and BRCA1  as described by manufacturers. Stained tissue sections were analyzed independently by a pathologist and scientists in blind manner.t test was used for statistical significance of differences in miR-20b expression, EGR1 expression, luciferase activity, enrichment of EGR1 at miR-20b promoter, cell growth, and cell migration between groups. Pearson Correlation was used for statistical significance between miR-20b expression and the exression of EGR1 or PTEN or BRCA1 in breast cancer specimens examined. P < 0.05 was considered significant.The Student's No potential conflicts of interest were disclosed.


Patients with HTLV-1 associated myelopathy (HAM/TSP) tend to present higher HTLV-1 proviral load (PVL) than asymptomatic carriers (AC). PVL remains the only marker consistently associated with HAM/TSP across several populations. We report a candidate gene study to identify associations between host genetic markers and PVL.170 HTLV-1 positive individuals were analyzed (56 HAM/TSP and 114 AC). Six HLA alleles and 140 SNPs belonging to 40 candidate genes were genotyped, with 36 ancestry informative markers included into the analysis to correct by population stratification. PVL was determined by a SYBR Green-based realtime quantitative PCR using ERV-3 as reference gene.The effect of each SNP was evaluated with linear regression analysis. Disease status, age, gender, and the first three principal components were included as covariates.Due to the exploratory nature of the study, no correction for multiple testing was performed; a P value<0.05 was considered significant.After multivariate linear regression analysis, two SNPs: IL6–174 (Interleukine 6) (P=0.0013) and ICAM1-R241G (Intercellular Adhesion Molecule 1) (P=0.0084), and HLA-A*02 (P=0.0306) showed association with PVL.These results suggest a possible implication of IL6–174, ICAM1-R241G and HLA-A*02 allele in the PVL among Peruvian HTLV-1 infected individuals. IL-6 promotes T cell activation, growth, and differentiation of cytotoxic T lymphocytes; ICAM-1 was reported as cofactor for HTLV-1-induced cell fusion and is upregulated in HTLV-1 infected cells; a protective effect associated with TAX has been suggested for HLA-A*02. A replication study in an independent population is needed to confirm our findings.


Survivin is a unique member of the inhibitor of apoptosis (IAP) proteins that is overexpressed in numerous cancers through poorly defined mechanisms. One such mechanism may be through constitutive activation of the insulin-like growth factor-I (IGF-I) signaling pathway, implicated in the development and progression of prostate cancer. Using the pre-neoplastic NRP-152 rat prostate cell line as a model, we showed that IGF-I induces Survivin expression, and that silencing Survivin by lentiviral-mediated small hairpin RNA (shRNA) represses IGF-I-stimulated cell growth, implicating Survivin as a mediator of this growth response. Moreover, our data support that the induction of Survivin by IGF-I occurs through a transcriptional mechanism that is mediated in part by the PI3K/Akt/mTORC1 pathway. Use of various Survivin promoter-luciferase constructs revealed that the CDE and CHR response elements in the proximal region of the Survivin promoter are involved in this IGF-I response. Transforming growth factor (TGF-β) signaling antagonists similarly activated the Surivin promoter and rendered cells refractory to further promoter activation by IGF-I. IGF-I suppressed levels of phospho-Smads 2 and 3 with kinetics similar to that of Survivin induction. Suppression of TGF-β signaling, either by TGF-β receptor kinase inhibitors or by silencing Smads 2 and 3, induced Survivin expression and promoted cell growth similar to that induced by IGF-I. TGF-β receptor antagonists also rescued cells from down-regulation of Survivin expression and growth suppression by pharmacological inhibitors of PI3K, Akt, MEK and mTOR. Sh-RNA gene silencing studies suggest that mTORC1 induces while mTORC2 represses the expression of Survivin by IGF-I. Taken together, these results suggest that IGF-I signaling through a PI3K/Akt/mTORC1 mechanism elevates expression of Survivin and promotes growth of prostate epithelial cells by suppressing Smad-dependent autocrine TGF-β signaling. As a regulator of Survivin TGF-β receptor type II (TβRII) and TβRI, which upon TGF-β ligand binding form a receptor tetrameric complex. TβRI , which is activated through phosphorylation by TβRII kinase, recruits and phosphorylates the two C-terminal serines of Smads 2 and 3. Such phosphorylation exposes their nuclear import sequence, promoting their nuclear localization where they engage in transcriptional control of numerous targets An important negative regulator of Survivin is transforming growth factor-beta (TGF-β cell cycle-dependent element (CDE) and a cell cycle genes homology region (CHR) TGF-β is well recognized to function as a tumor suppressor of the prostate Sources were: Recombinant human TGF-β1 and anti-Survivin (#AF886) (R&D Systems); anti-P-Smad3 , anti-P-Smad2 , and P-Smad1/5/8 , anti-mTOR (#2972), anti-Raptor (#2114), anti-Rictor (#4978), anti-P-Rb , Akt1 (#2967), Akt , anti-P-S6  antibodies ; anti-Survivin (sc-10811) and anti-Smad3 (sc-8332) antibodies (Santa Cruz); anti-β-actin antibody (Sigma); anti-Smad2 (#S66220) antibody (Transduction laboratories); anti-XIAP ; anti-P-Smad3 (Ser423/425) was generous gift obtained from Dr. Dr. Ed Leof; U0126 and rapamycin (LC laboratories), perifosine, Ku-0063794 (Selleck Chem); SB431542 (Tocris Bioscience), SB202190, SP600125, LY294002, HTS-466284 and ALK5 inhibitor-II (EMD Millipore), MK2206 (ChemieTek), DMEM/F12 (Invitrogen), characterized fetal bovine serum (FBS) (HyClone). The rat Survivin promoter-luciferase reporter, sh-Survivin, sh-mTOR, sh-Raptor, and sh-Rictor constructs were developed previously NRP-152 prostatic epithelial cell line 6 cells/10 ml/100 mm dish with 5% FBS+DMEM/F12 medium and transfected with MD2G, PCMV-dr2.74, and PLV-tTR-KREB-Red, using Lipofectamine Plus. Lentiviral supernatants, collected between 24 to 48 h after transfection, were passed through a 0.22 µpore filter, and used to transduce NRP-152 cells. The resulting NRP-152-tTR cells were then infected with lentiviruses harboring sh-LacZ or sh-Survivin. The ensuing inducible silenced cell lines were maintained in GM2.1 without doxycycline, and experiments with these cell lines were performed in GM3 supplemented with 0.1 µg/ml of doxycycline.Doxycycline-inducible silencing cell lines were developed as previously described Immunoblotting methods are as described previously 5′-GAGTGACATGCCACGGCTAA-3′ (forward) and 5′-CCAGGCATGGAAACATCAAG-3′ (reverse). Quantitative (Q) PCR was performed using the Bio-Rad CFX Connect Real-Time Detection System and Invitogen SYBR Green Real-Time PCR Master Mix using the above primers and conditions.RT was performed as described Cells were transfected using polyethylenimine method as before Adenovirus shuttle vectors (pDC515) that direct the expression of WT-Akt1 (AdMax-Myc-Akt1WT), Active-Akt1 (AdMax-Myc-Akt1Myr), KD-Akt1 (AdMax-Myc-Akt1K179M), DN-P85 (AdMax-Myc-p85αΔSH2N), and CA-P110α (AdMax-Myc-p110αCAAX) were constructed using the AdMax system (Microbix Biosystems) and high-titer adenoviruses were prepared and titered as described previously 50  values for reliable quantification of viral titers.NRP-152 cells were plated at a density of 50,000 cells/2 ml GM2.1/well in six-well plates and the next day infected with lentiviruses (MOI = 7) expressing sh-LacZ, sh-mTOR, sh-Rictor and sh-Raptor, using protamine sulfate (4 µg/mL) to facilitate infection. The viral supernatant was replaced 24 h later with GM2.1±200 nM TKDI, and 72 h later cells were harvested for Western blot and cell growth analysis. Viral titers were measured by Flow Cytometry of GFP-positive cells, interpolating the ID3 cells/1 ml/well in 12 well plates with GM3, and the following day treated with various indicated agents 2 h prior to addition of LR3-IGF-I (2 nM) or vehicle. Cell growth was assessed either enumerating single cells (following trypsinization) with a Coulter Electronics counter or by staining adherent cells in wells with crystal violet. For the latter assay, adherent cells were washed with PBS, fixed in 2% formalin/PBS  and stained with 0.2 mg/ml Crystal Violet in PBS . Stained cells were washed twice with PBS, and the dye was eluted with 1% Triton/PBS . One hundred µl of the eluted dye was transferred to a 96-well plate for assessing optical adsorption at 550 nm with a Tecan microplate spectrophotometer.Unless indicated, cells were plated at a density of 5×103-IGF-I, which has similar affinity for IGF-IR but binds poorly to IGF-I binding proteins 3-IGF-I in GM3 medium decreased the doubling time of NRP-152 cells to<24 h after a two day lag -negative DU145, and the immortalized non-tumorigenic RWPE-1  derived from the preneoplastic prostate of a Lobund Wistar rat  day lag . Under t by 16 h , and Sur by 16 h , consist by 16 h . We alsoc RWPE-1 .To examine whether the induction of Survivin expression by LR3-IGF-I is necessary for its ability to promote growth of prostate epithelial cells, we stably silenced expression of Survivin in NRP-152 cells using a doxycycline-inducible shRNA lentiviral transduction system 3-IGF-I, NRP-152 cells were first transduced with adenoviruses carrying constitutively-active (CA) and dominant-negative (DN) PI3K and Akt. Cells then received 2 nM LR3-IGF-I and their Survivin levels were assessed 24 h later  gave the lowest basal activity, it conferred a similar fold induction by LR3-IGF-I relative to the other promoter constructs  inhibitor SP600125 partially blunted promoter activation by LR3-IGF-I. As SB202190 partially antagonizes the TβRI kinase, it is likely that activation of this promoter by SB202190 is primarily through inactivation of TβRI. These results suggest that IGF-I induces Survivin expression principally by blocking endogenous TGF-β.We next assessed the effect of various small chemical inhibitors on the ability of IGF-I to activate the Survivin promoter using the second smallest (−370/−4) construct. The PI3K inhibitor LY294002 effectively and fully repressed basal and IGF-I-induced activity of the Survivin promoter, respectively . Rapamyc3-IGF-I  in association with E2F4 bind to CDE and CHR response elements of the Survivin promoter and repress promoter activity 3-IGF-I , suggestR3-IGF-I . The mRNPrevious studies from our group indicated that IGF-I suppresses the ability of TGF-β to activate Smad3 We next examined whether the ability of IGF-I to stimulate growth of NRP-152 cells was through suppressing autocrine activity of TGF-β. For this, NRP-152 cells were plated overnight in GM3 medium, treated with various TβRI kinase inhibitors and changes in cell growth was assessed after 5 to 6 days by counting total cell numbers and by crystal violet staining of fixed cells. Each of these TβRI kinase inhibitors enhanced cell growth between 4- to 10-fold . The mosThe above results support our hypothesis that IGF-I promotes the growth of NRP-152 cells and their expression of Survivin through inactivating autocrine TGF-β/Smad activity. We next explored the impact of the signaling pathway activated by IGF-I on cell growth and Survivin expression by autocrine TGF-β. When cultured in GM3, NRP-152 cells undergo increased cell death/growth arrest by rapamycin [40]. ThiSer235/236), mTORC2 (by P-AktSer473), Smad2 (P-Smad2Ser465/467), Smad3 (P-Smad3Ser423/425) and Rb (P-RbSer807/811) were assessed by Western blot analysis , indicating that mTORC1 represses or/and mTORC2 activates TGF-β signaling; and this also opens up the likelihood that an mTORC2-independent function of Rictor represses Smad activation. The anti-P-Smad3Ser423/425 IgG used recognizes P-Smad3  as well as P-Smad1/5/8 . TKDI, treatment or silencing mTOR, Rictor, and Raptor each alone enhanced P-Smad1/5/8, with silencing of Rictor blunting the induction by TKDI.Despite their differential effects on the regulation of Survivin expression, sh-mTOR, sh-Raptor and sh-Rictor (compared with sh-LacZ) each activated TβRI  by which TGF-β may induce loss of Survivin expression, namely through a proteosomal mechanism involving Smad3-dependent activation of protein kinase A (PKA), which phosphorylates Survivin at SerIntriguingly, we show that suppression of TGF-β signaling by a highly-specific TGF-β receptor kinase inhibitor (TKDI)Functional loss of PTEN, which is a hallmark of most prostate cancers, robustly contributes to cell survival through the PI3K/Akt/mTOR pathway Ser473 by sh-TOR but not by sh-Raptor (Another fascinating observation is that TKDI, sh-mTOR and sh-Raptor but not sh-Rictor increased levels of P-Smad1/5/8  & 8B. Thh-Raptor , suggesth-Raptor . ExploriIn summary, we provide the first evidence using a pre-neoplastic model of prostate cancer that an autocrine TGF-β loop serves as a critical barrier between the IGF-I/PI3K/Akt/mTORC1 signaling network and the induction of cell growth/survival associated with inactivation of the Rb pocket protein and induction of Survivin. As such, functional inactivation of TGF-β signaling, particularly loss of TGF-β-induced apoptosis or growth arrest, which is a common occurrence during prostate carcinogenesis, serves as a driver of malignant transformation through inactivation of Rb and induction of Survivin. As we and others have demonstrated that activation of the AR can directly antagonize TGF-β signaling


K-RAS mutation poses a particularly difficult problem for cancer therapy. Activating mutations in K-RAS are common in cancers of the lung, pancreas, and colon and are associated with poor response to therapy. As such, targeted therapies that abrogate K-RAS-induced oncogenicity would be of tremendous value.We searched for small molecule kinase inhibitors that preferentially affect the growth of colorectal cancer cells expressing mutant K-RAS. The mechanism of action of one inhibitor was explored using chemical and genetic approaches.We identified BAY61-3606 as an inhibitor of proliferation in colorectal cancer cells expressing mutant forms of K-RAS, but not in isogenic cells expressing wild-type K-RAS. In addition to its anti-proliferative effects in mutant cells, BAY61-3606 exhibited a distinct biological property in wild-type cells in that it conferred sensitivity to inhibition of RAF. In this context, BAY61-3606 acted by inhibiting MAP4K2 (GCK), which normally activates NFκβ signaling in wild-type cells in response to inhibition of RAF. As a result of MAP4K2 inhibition, wild-type cells became sensitive to AZ-628, a RAF inhibitor, when also treated with BAY61-3606.These studies indicate that BAY61-3606 exerts distinct biological activities in different genetic contexts. RAS family GTPases act as binary switches that undergo a conformational change upon binding to GTP, allowing them to engage a host of downstream signaling effectors including RAF, PI3K, and RALGDS The main challenge of counteracting the oncogenic effects of activated K-RAS is the inability to directly inhibit the mutant protein. Because the signaling properties of K-RAS are enhanced via inactivation of its GTPase activity, direct pharmacologic inhibition of RAS is not a viable therapeutic strategy. An alternate strategy to counteract mutant K-RAS is to inhibit its downstream effectors, for example, the RAF-MEK-ERK (MAPK) pathway. MEK inhibitors have received attention due their allosteric mechanism of action, which confers extreme specificity, and their demonstrated efficacy in melanomas and colon cancers expressing activated B-RAF bona fide therapeutic targets for K-RAS mutant cancers. Whereas understanding the mechanisms by which K-RAS signals through these targets is central to the design of effective drugs, a less studied, and often overlooked, question is why wild-type cells, which also express these targets, tolerate loss of function of these enzymes. This issue is equally important for drug design because the advantage of targeted therapies  is their potential selectivity for malignant cells.In recent years, large-scale functional genomic approaches have been employed to discover kinase targets that when knocked down are “synthetic lethal” with mutant RAS. Potential therapeutic targets that have been identified include TBK1 In this study, we have characterized the activity of BAY61-3606 in the context of colorectal cancer, providing insight into (1) potential therapeutic targets for cancers expressing mutant K-RAS and (2) pathways that regulate the response of non-mutant cells to targeted inhibitors. BAY61-3606 was originally identified as an orally available, ATP-competitive inhibitor of Spleen Tyrosine Kinase (SYK) All colon cancer cell lines were maintained in DMEM supplemented with penicillin (100 units/mL), streptomycin (100 μg/mL), and 10% fetal bovine serum (FBS). The rectal cancer cell line (Car1) was maintained in DMEM/F12 supplemented with penicillin (100 units/mL)/streptomycin (50 μg/mL), and 5% FBS. Knockdowns were achieved with pSICOR or pLKO lentiviral vectors Cell cycle analysis was performed via FACS-based propidium iodide quantification, using standard methods. To measure cell viability, cells were grown in 96-well plates in the presence or absence of drug for 72 hours, fixed with 4% paraformaldehyde, and then stained with Syto60 (Invitrogen). Plates were imaged and quantified using the LiCor Odyssey system (LiCor).The Bio-Plex assay system was used to measure signaling in drug-treated cells. Briefly, cells were incubated in the presence of drug for various amounts of time and then lysed in Bio-Rad cell lysis buffer (Bio-Rad). Protein quantification was performed using BCA assay (Pierce) and 5 µg of protein from each sample was used for Bio-Plex analysis. Phospho-signaling assays were performed using available phospho-signaling assay kits and quantified on a Bio-Plex 200 system (Bio-Rad): p-Iκβα (Ser32/Ser36), p-JNK (Thr183/Tyr185), p-MEK1 (Ser217/Ser221), p-ERK1/2 , p-p90RSK (Thr359/Ser363), p-p38 (Thr180/Tyr182), p-c-JUN (Ser63), p-ATF2 (Thr71), p-AKT (Ser473), p-S6 (Ser235/Ser236), p-STAT3 (Ser727), p-STAT3 (Tyr705), and p-GSK3α/β (Ser21/Ser9). Bio-Plex assay for total MEK1 was also performed as a loading control. All signals were normalized to a common control cell line lysate in order for assays between plates to be comparable.in vitro IC50s for the compounds against specific kinases.The biochemical activity of BAY61-3606 and derivatives were measured in two ways. First, we used Ambit's KINOMEscan™ technology to identify those kinases that are inhibited for substrate binding by the compounds, all assayed at 1 μM. Second, we used Invitrogen's SelectScreen® Biochemical Kinase Profiling Service to determine the Details on the synthesis of BAY derivatives, and the structures of those derivatives, can be found in Figure S5.In an effort to identify novel therapeutic targets for colorectal cancers expressing mutant K-RAS, we performed a screen for small molecule kinase inhibitors that affect viability in a genotype-specific manner. In these studies, we utilized a set of isogenic colon cancer cell lines that differ only in their K-RAS mutation status. The parental cell lines, HCT-116 and DLD-1, carry a heterozygous activating mutation in K-RAS (G13D/+). The derivative cell lines, HKe-3 and DKs-8, retain the wild-type allele, but have lost the mutant allele of K-RAS by virtue of gene targeting To determine how BAY61-3606 affected viability of cells expressing mutant K-RAS, we analyzed the cell cycle of cells treated with the drug. We found that BAY61-3606 did not alter the cell cycle profile of DLD-1 cells, nor did it induce apoptosis . In contTo test the second part of this hypothesis directly, we measured the activation state of MEK and ERK in cells that were treated with BAY61-3606. We found that inhibition of RAF with AZ-628 suppressed MEK and ERK phosphorylation, but that BAY61-3606 was unable to do so . Thus, wGiven that BAY61-3606 was originally developed as an ATP-competitive inhibitor of SYK in vitro IC50 values for BAY61-3606 against many of these candidate targets. This study revealed MAP4K2, a STE-20 family kinase, as the kinase most sensitive to BAY61-3606, with an in vitro IC50 of 11.3 nM  . For theSecondary to our genetic analysis, we took a chemical approach to studying BAY61-3606. In this study, we generated 30 structurally related derivatives of BAY61-3606 and assayed their ability to selectively affect viability of cells expressing mutant K-RAS . Of thesin vitro activity assays revealed a loss of kinase inhibition activity for the two derivatives tested . Based on these observations, we explored whether MAP4K2 plays a role in the sensitivity of wild-type and K-RAS mutant cells to BAY61-3606. HKe-3 and Dks-8 cells lacking MAP4K2 were hypersensitive to AZ-628, while MAP4K2 knockout had no effect on the response of HCT-116 or DLD-1 cells , JNK (Thr183/Tyr185), MEK1 (Ser217/Ser221), ERK1/2 , RSK (Thr359/Ser363), p38 (Thr180/Tyr182), c-JUN (Ser63), ATF2 (Thr71), AKT (Ser473), S6 (Ser235/Ser236), STAT3 (Ser727), STAT3 (Tyr705), and GSK3α/β (Ser21/Ser9) . We found two differences between HKe-3 and HCT-116 that we posited might be related to the function of MAP4K2. First, we found that the basal level of Iκβα phosphorylation was significantly lower in HCT-116 than in HKe-3 cells, suggesting that NFκβ signaling is suppressed in cells expressing activated K-RAS . Iκβα phosphorylation was further induced in HKe-3 cells after treatment with AZ-628 and this induction was dependent upon MAP4K2 . This obSince NFκβ was appeared to hyper-activated in wild-type cells in a MAP4K2-dependent manner, we surmised that inhibition of the NFκβ pathway would have the same effect as BAY61-3606 or MAP4K2 knockdown. That is, we expected that inhibition of NFκβ would increase the sensitivity of K-RAS wild-type cells to AZ-628 without affecting the sensitivity of K-RAS mutant cells. As predicted, inhibition of NFκβ increased the sensitivity of HKe-3 and Dks-8 cells to AZ-628, essentially phenocopying MAP4K2 knockdown . By contK-RAS is mutationally activated in approximately 40% of colorectal cancers In our study, we characterized the activity of a small molecule, BAY61-3606, that preferentially affected viability in colorectal cancer cells expressing mutant K-RAS compared to isogenic cells expressing only wild-type K-RAS . Since BAlthough BAY61-3606 was initially characterized as an ATP competitive kinase inhibitor, its selectivity for decreasing the viability of K-RAS mutant cells may not require this activity. Our studies of BAY61-3606 derivatives demonstrate that those lacking the ability to affect active site binding can still maintain their ability to selectively affect cell viability. One possible explanation for this observation is that the relevant target of BAY61-3606 and its biologically active derivatives is a non-kinase protein. Aside from inhibiting kinases, ATP analogs can affect biology through other processes, including nucleic acids synthesis In addition to its activity in cells expressing mutant K-RAS or B-RAF, we also identified a secondary biological effect of BAY61-3606; it conferred upon wild-type cells, which are normally resistant to AZ-628, sensitivity to RAF inhibition . Using aInhibition of RAF by AZ-628 leads to MAP4K2-dependent activation of NFκβ signaling in wild-type cells, presumably to ensure cell survival . To mainWhile our studies specifically address the function of the small molecule kinase inhibitor BAY61-3606 in colorectal cancer cells, they more generally address the varied activities that kinase inhibitors can have in cancer cells. Although inhibitor promiscuity is often viewed in a negative light, the ability of a given small molecule to target multiple kinases may, in fact, be beneficial for targeting diverse genotypic classes of cancer.Figure S1Genetic analysis of BAY61-3606 response in DLD-1 cells. (a) Cell viability quantified by Syto60 after 72 hours of BAY61-3606 treatment in DLD-1  or DKs-8  cell lines. Relative cell viability was normalized to DMSO vehicle treated control for each cell line. Error bars represent SEM for 3 independent experiments. The differential response in the two cell lines is statistically significant (p = 0.019 at 1 μM). (b) Cell viability quantified by Syto60 after 72 hours of BAY derivative 6 treatment in DLD-1  or DKs-8  cell lines. Relative cell viability was normalized to DMSO vehicle treated control for each cell line. Error bars represent SEM for 3 independent experiments. (c) Validation of shRNAs. Relative gene expression of shRNA-mediated knockdowns of potential BAY61-3606 targets in DLD-1 (red) and DKs-8 (blue) cells. Gene expression is measured via Taqman assay and calculated using standard methods in reference to the housekeeping gene TBP. Error bars represent SEM for 3 independent experiments.(PDF)Click here for additional data file.Figure S2Chemical derivation of BAY61-3606 derivatives. (a) Synthesis of BAY derivative 6. To a stirred solution of 5,7-dichloroimidazo  pyrimidine  in DMF (5.0 mL) was added 2-methoxybenzenamine . After 1 h heating at 70°, the mixture was purified on silica gel column with methylene chloride and methanol (10∶1) as eluent to give of 7-chloro-N-(2-methoxyphenyl)imidazo  pyrimidin-5-amine. To a solution of 7-chloro-N-(2-methoxyphenyl)imidazo  pyrimidin-5-amine  and 3,4-dimethoxyphenylboronic acid  in 5.0 mL 1,4-dioxane was added Bis(triphenylphosphine) palladium(II) dichloride  as catalyst and saturated potassium carbonate aqueous solution (2.0 mL) as base. The mixture was heated for 2 h at 80° and then was diluted with chloroform and 2-propanol . The organic layer was washed with water, brine and was dried with sodium sulfate. After removal of solvent, the crude was purified by column with methylene chloride and methanol (10∶1) to give BAY derivative 6 . 1H NMR (DMSO-d6) 9.05 , 8.22 , 7.23 , 7,61-7.53 , 7.26 , 7.16 , 7.04 , 6.94 , 3.80 , 3.75 , 3.72 . (b) Chemical structures of all BAY derivatives.(PDF)Click here for additional data file.Figure S3Evaluation of BAY derivative 6 activity in colorectal cancer cell lines. Cell viability quantified by Syto60 after 72 hours of BAY derivative 6 treatment in 5 cell lines expressing mutant K-RAS and in 3 cell lines expressing wild-type K-RAS. Relative cell viability was normalized to an untreated control for each cell line. Error bars represent SEM for 3 independent experiments. With the exception of GP5d (highlight in bold red), all of the cell lines expressing mutant K-RAS respond to BAY derivative 6.(PDF)Click here for additional data file.Figure S4BAY61-3606 derivatives lose ATP competitive activity. TREEspot images for five different derivatives of BAY61-3606. Both inhibitors that retained selectivity for K-RAS mutant cells (e.g. 6 and 8) and those that lost selectivity , failed to effectively inhibit ATP binding by the majority of kinases that were assayed.(PDF)Click here for additional data file.Figure S5Kinase inhibition profiles of BAY61-3606 and its derivatives. Inhibitor activity was measured using Invitrogen's SelectScreen® Biochemical Kinase Profiling Service.(PDF)Click here for additional data file.Figure S6Evaluation of MAP4K2 in the BAY61-3606 response. (a) Cell viability quantified by Syto60 after 72 hours of AZ-628 treatment in DLD-1 or DKs-8 cell lines with MAP4K2 knockdown. Loss of MAP4K2 does not affect AZ-628 response in cells expressing mutant K-RAS, but enhances the effect of AZ-628 in cells expressing wild-type K-RAS. (b) Time course of phospho-JNK (Thr183/Tyr185) after 1 µM AZ-628 treatment in HCT-116 (red lines) or HKe-3 (blue line) cells with MAP4K2 knock down, as measured by Bio-Plex. Relative signal was normalized to a master control lysate. Error bars represent SEM for 3 independent experiments. JNK signaling was enhanced in HKe-3 cells but was independent of MAP4K2. (c) Cell viability quantified by Syto60 after 72 hours of combinatorial treatment with IKK inhibitor VII and 1 µM AZ-628. Relative cell viability was normalized to DMSO vehicle treated control for each cell line. Like BAY61-3606, IKK inhibitor VII enhanced the effect of AZ-628 specifically in K-RAS wild-type cells. (d) Cell viability quantified by Syto60 after 72 hours of combinatorial treatment with the JNK inhibitor SP600125 and 1 µM AZ-628. Relative cell viability was normalized to DMSO vehicle treated control for each cell line. Unlike BAY61-3606, SP600125 did not affect AZ-628 sensitivity in K-RAS wild-type cells.(PDF)Click here for additional data file.Figure S7Signaling pathway activity in response to AZ-628. Phospho-protein measurements were made after 45 minutes of exposure to 1 μM AZ-628 or DMSO vehicle control treatment in HCT-116 (red squares) or HKe-3 (blue dots) cells. X's represent measurements from cells with MAP4K2 knock down. All measurements were quantified by Bio-Plex signaling assays. Relative signal was normalized to a master control lysate.(PDF)Click here for additional data file.Table S1Active site binding inhibition data.(PDF)Click here for additional data file.Table S2shRNAs used in this study.(PDF)Click here for additional data file.


Recent research highlight that newly synthesized HIV-1 capsid protein is required to induce dendritic cell activation through a pathway involving interaction with peptidyl-prolyl cis-trans isomerase cyclophilin A (CYPA),which subsequently induce an antiviral type I interferon response and activation of T cells.Previous studies also revealed HIV Gag-CYPA interaction inhibited recognition by host restriction factors. In another perspective, the inhibition effect might be benefit for HIV Gag immunogenicity.Consequently, we assume CYPA might be a potential genetic adjuvant co-inoculation with HIV-1 Gag DNA vaccine, which not only circumvent the restriction of host factors, but also activate dendritic cell and enhance adaptive immune response.In this study, 6-8-week-old female BALB/C mice were administrated thrice intramuscularly with pDRVI4.0-Gag HIV-1 DNA vaccine co-formulated with pDRVI4.0-CYPA plasmid at two weeks interval. IFN-γ ELISPOT, Gag-specific ELISA, IgG isotype ELISA detected cellular and humoral immunological response.The regimen of HIV-1 Gag DNA premixed with CYPA DNA induced robust immune responses in mouse model. IFN-γ ELISPOT result demonstrated Gag DNA alone only generated 180 ± 63(mean ± SD) SFCs/million. However, Gag DNA combined with CYPA DNA strategy produced 353 ± 80(mean ± SD) SFCs/million. In humoral immune response, Gag DNA co-inoculation with CYPA DNA showed high level antibody titer (GMT=22800),whereas Gag DNA alone induced slight antibody response (GMT=8000). IgG isotype results confirmed that co-vaccination with CYPA DNA induced Th1-bias immune response, however, Gag DNA alone activated Th1/Th2 response in balance.This is the first report demonstrating that mixture of CYPA DNA and HIV-1 gag DNA vaccine could induce robust cellular and humoral immune response in the mouse model. Ongoing studies are focusing on construction of dual expression cassette DNA vaccine, which including both Gag and CYPA gene. Further research will also construct others HIV DNA vaccine involving human or non-human primates CYPA.


To map and identify the genetic defect underlying autosomal dominant cataract segregating in a 5-generation Caucasian American family.Genomic DNA was prepared from blood leukocytes, genotyping was performed using microsatellite markers, and logarithm of the odds (LOD) scores were calculated using the LINKAGE programs. Mutation profiling was performed using direct exon cycle-sequencing and restriction fragment analysis. Protein function effects were evaluated using in silico prediction algorithms.max]=3.67; maximum recombination fraction [θmax]=0.04) and D13S1316 . Haplotyping indicated that the disease lay in the ~170 Kb physical interval between D13S1316 and D13S175, which contained the gene for gap-junction protein alpha-3 (GJA3) or connexin-46. Sequencing of GJA3 detected a heterozygous transition (c.130G>A) in exon-2 that resulted in gain of an Hsp92 II restriction site. Allele-specific PCR amplification and restriction analysis confirmed that the novel Hsp92 II site co-segregated with cataract in the family but was not detected in 192 normal unrelated individuals. The c.130G>A transition was predicted to result in a non-conservative substitution of valine-to-methionine at codon 44 (p.V44M) with damaging effects on protein function.Significant evidence of linkage was obtained at marker D13S175 (maximum LOD score [ZGJA3 as one of the most frequently mutated genes that underlie autosomal dominant cataract in humans, and further emphasize the importance of connexin function in maintaining lens transparency.These data confirm  Inherited forms of cataract(s) constitute a clinically heterogeneous disorder of the ocular lens that usually present with an early-onset ranging from birth  through infancy into the fourth decade . Congenital and infantile forms of cataract that cause blurring of images on the immature retina are a clinically important cause of impaired form vision development (deprivation amblyopia), and pediatric cataract surgery is associated with increased risk of aphakic glaucoma and lifelong visual impairment -3.In addition to being found as a secondary feature of many genetic syndromes and metabolic disorders involving other ocular and/or systemic abnormalities (OMIM), cataract may be inherited as a primary or isolated lens phenotype ,5. All tCRYAA), alphaB-crystallin (CRYAB), betaB1-crystallin (CRYBB1), betaB2-crystallin (CRYBB2), betaB3-crystallin (CRYBB3), betaA1-crystallin (CRYBA1), bataA4-crystallin (CRYBA4), gammaC-crystallin (CRYGC), gammaD-crystallin (CRYGD), and gammaS-crystallin (CRYGS) that encode the major “refractive” proteins of the lens [% of known mutations have been detected in two genes encoding gap-junction protein alpha 3 and alpha 8  [HSF) [MIP) [LIM2) [TMEM114) [BFSP1, BFSP2) [CHMP4B) [EPHA2) [TDRD7) [FYCO1) [Approximately 55% of the known mutations underlying inherited forms of primary cataract have been detected in ten crystallin genes; alphaA-crystallin  ,17. The 8) [HSF) , lens maF) [MIP) , lens in) [LIM2) , transmeTMEM114) , beaded , BFSP2) ,23, chro[CHMP4B) , Eph-rec [EPHA2) , Tudor d [TDRD7) , and FYV [FYCO1) . Here weA 5-generation Caucasian pedigree (family Sh) from the midwestern United States was ascertained through ophthalmic records in the Department of Ophthalmology and Visual Sciences at Washington University School of Medicine, St. Louis MO. Blood samples were obtained from 22 family members including 11 affected individuals. Leukocyte genomic DNA was purified using the Gentra Puregene Blood kit , and quantified by absorbance at 260 nm . Ethical approval for this study was obtained from the Washington University Human Research Protection Office, and written informed consent was provided by all participants before enrollment in accordance with the tenets of the Declaration of Helsinki, and Health Insurance Portability and Accountability Act (HIPAA) regulations.NCBI) combined Généthon, Marshfield, and deCODE genetic linkage maps were genotyped by means of a 4200 DNA analyzer running Gene ImagIR software  as described [LINKAGE (5.1) package of programs [Microsatellite markers from the National Center for Biotechnology Information  probe database or custom designed (IDT Primer Quest). Genomic DNA , was amplified (35–40 cycles) in a GeneAmp 9700 thermal cycler using AmpliTaq polymerase  and gene-specific primers (10 pmol). Resulting PCR amplicons were either enzyme-purified with ExoSAP-IT  or gel-purified with the QIAquick gel-extraction kit (Qiagen). Purified amplicons were direct cycle-sequenced in both directions with BigDye Terminator Ready Reaction Mix (version 3.1) containing M13 forward or reverse sequencing primers then ethanol precipitated and detected by capillary electrophoresis on a 3130xl Genetic Analyzer running Sequence Analysis (version 5.2) software (Applied Biosystems), and Chromas (version 2.23) software .Genomic sequence for  primers  were selAllele-specific restriction fragment length analysis was performed on gel-purified PCR amplicons, amplified with primers GJA3-Ex2F1 and GJA3-Ex2R1  using HsPSSM), Sorting Intolerant From Tolerant substitutions (SIFT) [PolyPhen-2) [Entrez protein database, and aligned by means of the ClustalW multiple sequence alignment web server [HMMTOP transmembrane topology prediction server [CDD) [Missense mutations in GJA3 were evaluated for pathogenicity using three in silico prediction algorithms: Position-Specific Scoring Matrix analysis (s (SIFT) , and PolyPhen-2) . GJA3 amb server . The hydn server , and strer [CDD) .GJA8), 2q , 3q (BFSP2), 11q (CRYAB), 12q (MIP), 13q (GJA3), 16q (HSF4), 17q (CRYBA1), 19q (LIM2), 21q (CRYAA), and 22q . Following exclusion of 10 of these loci , we obtained significant evidence of linkage  and D13S1316  on 13q11-q12. Haplotyping of the pedigree (GJA3.We studied a 5-generation Caucasian American pedigree (family Sh) segregating autosomal dominant cataract in the absence of other ocular or systemic defects. Autosomal dominant inheritance was supported by the absence of gender bias or skipping of generations. Ophthalmic records described the cataract as congenital in at least four affected individuals ; however, no slit-lamp images of the lens opacities pre-surgery were available. Twenty-two members of the family , includi linkage  for markpedigree  detectedGJA3 (GeneID: 2700) comprises two exons with exon-2 containing the entire coding region for a 435-amino-acid protein. Sequencing of exon-2 including flanking 5′-intron and 3′-UTR boundaries in two affected relatives detected a heterozygous G-to-A transition . Finally we excluded the c.130G>A transition as a single nucleotide polymorphism (SNP) in a panel of 192 normal unrelated control individuals (384 chromosomes) using allele-specific restriction analysis described in ansition  located ansition . Moreoveansition  we obtaiGTG>ATG) and was predicted to result in the missense substitution of valine-to-mehionine (p.V44M) at the level of protein translation. The predicted p.V44M substitution represented a relatively conservative amino acid change, with the small non-polar side-group of valine (CH3-CH-CH3) replaced by the larger non-polar side-group of methionine (CH2-CH2-S-CH3). However, cross-species alignment of GJA3 amino-acid sequences revealed that p.V44 is phylogenetically conserved from Zebrafish to man . SIFT analysis gave a score of 0.00 consistent with an “intolerant” amino-acid change, and PolyPhen-2 analysis gave a score of 1.00 consistent with a “probably damaging” change, further raising the likelihood of GJA3 dysfunction.Based on the hydrophobicity profile of GJA3, the p.V44M substitution is likely located in the first extracellular (EC-1) loop close to the boundary with the first transmembrane (TM-1) domain . To evalgorithms . PSSM anGJA3 co-segregating with autosomal dominant cataract linked to chromosome 13q in a Caucasian American family. This missense mutation was predicted to result in a conservative p.V44M substitution in the first extracellular domain of GJA3 with damaging effects on protein function. Recently, the same GJA3 mutation was detected by candidate-gene sequencing in a Han Chinese family segregating autosomal dominant cataract described as central nuclear with punctate cortical opacities [Here we have identified a heterozygous transition (c.130G>A) in exon-2 of pacities . HoweverGJA3  and the gap-junction channel protein cysteine-rich or conexin_CCC domain (CCD: pfam10582). Five of these missense substitutions, including p.V44M identified here, are believed to be located in the first extracellular (EC-1) domain of GJA3  in rat Gja3 underlies a spontaneous form of autosomal recessive nuclear cataract in the SHRSPwch1.9Cat strain [Gja3 does not elicit cataract in mice.So far no mutations in the mouse ution p.EK in rat t strain ,44. HoweXenopus oocytes revealed that it exhibited impaired hemi-channel activity in single oocytes, and failed to elicit gap-junction coupling in paired oocytes [GJA3 mutations with cataract.Mouse Gja3 has been proposed to function in gap-junction coupling of lens fiber cells ; the pri oocytes . While p