C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved.Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR), which monitors DNA integrity, and the spindle assembly checkpoint (SAC), which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in  C. elegans proliferating germ cells and human cells to investigate the role of these checkpoints when either DNA is damaged or the spindle is perturbed. We discovered that these checkpoints function together in response to these different perturbations to ensure genome integrity. Our studies have important implications for cancer treatments, as many cancer chemotherapies target one of these checkpoint pathways without consideration for the effect on the other pathway.Checkpoints are surveillance pathways that monitor and correct cellular errors to ensure that the genome is transmitted intact through cell division; defects in checkpoints lead to human disease such as cancer. Two major checkpoint pathways that have been extensively studied are the DNA damage response and the spindle assembly checkpoint. As their names imply, they have been thought to monitor distinct chromosomal events during the cell cycle. Here, we used  Genome integrity is monitored throughout the cell cycle by surveillance mechanisms that ensure the proper order and fidelity of DNA replication and segregation through mitosis and meiosis. This is largely achieved by the actions of two checkpoint pathways: the DNA damage response (DDR) and the spindle assembly checkpoint (SAC). As its name implies, the canonical role of the DDR is to recognize DNA damage and either arrest the cell cycle and initiate DNA repair, or induce apoptosis. The DDR is composed of a large number of proteins, prominent among them are the highly conserved protein kinases, ATM, ATR, and CHK1  and mat-2(ts)] as micrmat-2(ts),24. On tmat-2(ts).zyg-1(ts) , which renders the worm incompetent for metaphase delay  mutants were competent for arrest with HU (H3S10P = 0.1±0.04) yet had accelerated mitosis following release from arrest as monitored by small (fzy-1(av15) following release from HU  worms were largely able to repair the HU-induced damage, as RAD-51 levels were equivalent to wild type   than in wild type after HU but then gradually increased throughout the time course (17% at 16 hours) , mad-3, bub-3(RNAi) had persistent RAD-51 foci  , suggest-1(av15)  and 5B. -1(av15)  and S4A.rs) Figs  and S4A.ATR Figs  and S4A.-51 foci , suggestsac mutants, fzy-1(av15) was competent for repair as monitored by RAD-51 foci during low dose HU  mutants , suggesting that SAC does not function to inhibit CDC20 when modest levels of DNA damage are induced. Defects in DNA damage repair were not limited to replication stresses in SAC mutants, as RAD-51 foci also persisted in germ cell nuclei after recovery from 30gy of IR  to determine the localization of MAD-2 and RAD-51 relative to the nuclear periphery as marked by nuclear pores (NPC). We found that approximately 90% of MAD-2 was at the nuclear periphery, juxtaposed to nuclear pore complexes . RAD-51 fzy-1(av15), indicating that SAC-dependent CENPA localization is not mediated through CDC20 inhibition  depleted animals with HU and measured the distance between the nuclear periphery and RAD-51 foci in proliferating germ cells using high resolution SIM. We found that RAD-51 foci were on average 190.8±13 nm away from nuclear pores in WT compared to 441.8±28 nm in cenpa(RNAi) and 423.1±30 nM in mad-1 (p<0.0001). We also observed that there were fewer RAD-51 foci that were significantly larger in WT compared to mad-1 and cenpa(RNAi)  = 15.1±1.9; p<0.0001; area: WT = 0.11μm2, mad-1 = 0.045μm2, cenpa(RNAi) = 0.053μm2; p<0.0001; mad-1 and cenpa(RNAi) when comparing either the distance of RAD-51 from the periphery, or the number and size of RAD-51 foci, suggesting MAD-1 and CENPA are both required to recruit damage to the periphery. Consistent with incorporation of CENPA in a subset of DNA, CENPA and DAPI did not show complete co-localization. Further, CENPA distribution was more diffuse in mad-1 mutants by SIM  under the premise that these checkpoints are independent ,84. Howesan-1/mad-3(ok1580) I, atm-1(gk186) I, fzy-1(av15) unc-4(e120) II, npp-5/nup-107(tm3039)/mIn1 [mIs14 dpy-10(e128)] II, atl-1/ATR(tm853) IV/nT1 [unc-?(n754) let-? qIs50] , mdf-1/mad-1(gk2) V/nT1 [unc-?(n754) let-? qIs50] , fog-2(q71) V, atm-1(gk186) I;atl-1/ATR(tm853) IV/nT1 [unc-?(n754) let-? qIs50] . mat-2(ax102) II, and zyg-1(b1) II were maintained at 15°C. GFP::MAD-2 was a gift from Arshad Desai (1:100)(abcam), rabbit anti-Aurora B Phospho Thr 232 (1:500)(Rockland Antibodies and Assays). Rabbit anti-MDF-1 (1:2000) , rabbit Collection of images was performed using an API Delta Vision deconvolution microscope. Images were deconvolved using Applied Precision SoftWoRx image analysis software and were subsequently processed and analyzed using Fiji (ImageJ) . All images are projections through approximately half of the germ line unless otherwise stated.Structured illumination microscopy (SIM) analysis was performed using a Nikon N-SIM super-resolution microscope and NIS-Elements 2 image processing software. Images were further processed using ImageJ.L4s were treated with 0 or 25mM HU for 16 hrs and allowed to recover for 5 hrs before dissection and staining with CENPA and Mab414 (NPC). Germ lines were imaged at the same exposure time for CENPA and the CENPA channel was not manipulated post-acquisition. To determine fluorescence intensity, a single z stack was chosen in which the middle of several nuclei were displayed. A line was drawn across a single nucleus and the RGB plot profile was collected in Image J. Intensities were binned and averaged in 10% increments of nuclear length. Measurements were taken for every arrested (enlarged) nucleus where the plane bisected the middle of the nucleus for 3 germ lines per condition and these measurements were averaged.Distances between RAD-51 and NPC were determined by obtaining fluorescence intensity plots with line scans in Image J. The number of pixels between the peaks of each signal was determined and converted to nanometers.Statistics were determined with an unpaired student’s T-test or two-way ANOVA.mat-2(ax102)and zyg-1(b1), which were propagated at 15°C. Unless otherwise noted, gravid hermaphrodites were fed RNAi-inducing HT115(DE3) bacteria strains or the same bacteria transformed with the empty feeding vector, L4440. chk-1(RNAi) was performed on L1 larvae. All feeding strains were obtained from a genomic RNAi feeding library [RNAi experiments were performed using the feeding method  at 20°C, library . Culturecyb-3 RNAi for 24 hrs. Worms were dissected and embryos were placed on a 3% agarose pad and imaged by DIC at 40X on a Ziess compound microscope to monitor arrest.Following depletion of SAC, L4 worms were fed Young adult worms were irradiated with 30Gy (3000 rad) from a Cs-137 source. Worms were dissected 8 hrs post irradiation for MAD-2 and CENPA localization studies and 24 hrs post irradiation for recovery experiments.chk-1 and atr RNAi efficiency. For low dose HU experiments, staged young adults were exposed to 5mM HU for 2 hrs before being moved to a—HU plate for dissection, recovery, or progeny viability assays. HU was allowed to dissipate into plates for at least 3 hrs before worms were introduced. For low dose HU exposure, cell cycle kinetics were assayed by counting H3S10P.For high dose experiments, L4s were placed on NGM plates containing 25mM hydroxyurea (HU) (Sigma Aldrich) for 16 hrs before either dissection, transfer for recovery, or progeny viability assays. Cell cycle arrest was assayed by counting DAPI stained nuclei for mat-2(ax102) and zyg-1(b1) L4s were transferred to the restrictive temperature of 25°C for 16 or 48 hrs before dissection, respectively. To determine metaphase delay/arrest, zyg-1(b1) and mat-2(ts) L4s were transferred to the restrictive temperature of 25°C for 24 hrs before dissection and staining with H3S10P.For antibody staining after MAT-2 or ZYG-1 inactivation, fog-2(q71) worms to eliminate contribution from embryos and were resolved on 12% SDS-PAGE gels and transferred to Millipore Immobilon-P PVDF membranes. Membranes were blocked with 5% BSA and probed with rabbit anti-CENPA , and anti-Mortalin/Grp75 mouse monoclonal antibody N52A/42  as loading control., followed by IRDye680LT- and IRDye800-conjugated anti-rabbit and anti-mouse IgG secondary antibodies obtained from LI-COR Bioscience . 0.01% SDS was added to anti-rabbit secondary.Worm lysates were generated from unmated 2.Human U2OS osteosarcoma cells and COS monkey kidney cells were, obtained from the ATCC. U2OS cells were grown in McCoy’s 5A modified medium, COS cells were grown in DMEM and both were supplemented with 10% fetal bovine serum and were cultured at 37°C in 5% COCells were grown on glass coverslips and treated with 5mM HU for 24 hrs or 1μg/mL colchicine (Sigma) for 16 hrs. Cells were fixed with 4% paraformaldehyde and 0.1% triton, then blocked with 5% BSA for 1 hr before primary antibodies were added and incubated at room temperature overnight. Secondary antibodies were incubated for 2 hrs at room temperature.To determine fluorescence intensity, integrated density was identified for 2 equal areas in both the nucleus and the cytoplasm. The ratio of nuclear to cytoplasmic signal was calculated dividing the averages of the 2 measurements. For each condition, n≥50 cells.Primary antibodies were used at the following dilutions: rabbit anti-H3S10P (1:500) (Millipore), rabbit anti-MAD2L1 (1:500) and mouse anti-nuclear pore complex (MAb414) (1:500)(Abcam), mouse anti-CENPA (1:100)(GeneTex), mouse anti-MAD1L1 IgG2b (1:250) and human CREST antiserum (1:500) (a generous gift from N. Hunter ). The foS1 Figmat-2(ts) worms stained for either CENPA (red) or both CENPA and MAD-1 (red) and co-stained with α-tubulin (green) and DAPI (blue). The average width of CENPA staining is 308±12nM, the average width of CENPA/MAD-1 staining is 316±14nM, p = 0.87 (B) Nucleus with a monopolar spindle from a zyg-1(ts) worm stained with NDC-80 (red) α-tubulin (green) and DAPI (blue). Scale bars = 2mM. (C) Wild-type and GFP::MAD-2 embryos either arrested at 1-cell stage by cyb-3(RNAi) or not arrested and stained for either MAD-1 or MAD-2 (red), α-tubulin (green) and DAPI (blue). Arrows indicate MAD-1 or MAD-2 staining. (D) DAPI stained germ lines of wild type and chk-1(RNAi) worms with and without HU. Scale bars = 10μM.A) Metaphase arrested nuclei from (TIF)Click here for additional data file.S2 Figatr(tm853), chk-1(RNAi), mad-1(RNAi) and mad-3(RNAi) and mat-2(ts);mad-2(RNAi) treated worms at 25° stained with H3S10P (red), α-tubulin (green) and DAPI (blue). (B) Percentage of tubulin arrays in proliferative zones of the above genotypes at 25° (n≥10). (C) Efficiency of SAC RNAi as measured by failure to arrest in the early embryo following cyb-3(RNAi). Percentage of cell numbers in the early embryo of the given genotypes (n≥100).(A) Dissected germ lines from (TIF)Click here for additional data file.S3 Fignup-107. In the presence of HU, MAD-1 (red) and MAD-2 (red) fail to localize to the nuclear periphery  in nup-107(tm3039). (C) MAD-1(green) and DAPI (magenta) in the proliferative zones of wild-type and atr(tm853) germ lines in the presence and absence of HU. Scale bar = 10μm.(A) MAD-2 is not enriched at the nuclear periphery after cell cycle disruption. Depletion of CDK-2 or CYE-1by RNAi does not induce MAD-2 (red) enrichment to the nuclear periphery , however germ lines are competent for MAD-2 relocalization if treated with HU. (B) MAD-2 is not enriched at the nuclear periphery in (TIF)Click here for additional data file.S4 Figmad-1(gk2), atr(tm853), and fzy-1(av15) worms (n>10). (B) Cartoon of the germ line showing approximately how long it takes for nuclei to travel through the germ line to form embryos. (C) Progeny inviability after 5mM HU separated into embryos laid in the 1st 24 hrs and the last 48 hrs for wild type and mad-3(ok1580) (n>13) (D) Dissected WT germ line showing MAD-2 (green) accumulation in nuclei after release from 5mM HU treatment, DAPI (magenta). Scale bar = 10μm. (E) Percent of nuclei with RAD-51 in wild type fzy-1(av15), mad-3(ok1580), mad-1(gk2), and atr(tm853) germ lines after recovery from 30 gy of gamma irradiation. *p<0.05, ***p<0.0001 (two-way ANOVA).(A) Percent of nuclei that contain at least 1 RAD-51 focus—HU or after 0, 2, 4, 6, or 16 hours of 5mM HU recovery for wild-type, (TIF)Click here for additional data file.S5 Figfog-2(q71) worms with and without HU treatment and in worms depleted for CENPA. Mortalin was used as a loading control. (C) NDC-80 is not enriched in the nucleus after HU. Wild-type germ lines stained with NDC-80 (red) and DAPI (blue) in the presence and absence of HU. (D) Partial depletion of CENPA by cenpa(RNAi). Germ line stained with CENPA (red) and DAPI (blue). (E) atr(tm853) worms are still competent for loading CENPA during metaphase. atr(tm853) germ line stained for CENPA (red) and DAPI (blue). Arrows indicate CENPA staining. Scale bars = 10μm. (F) P-AIR-2 localization is not disrupted after depletion of DDR or SAC in metaphase arrested nuclei. P-AIR-2(red), α-tubulin (green) and DAPI (blue) staining in mat-2(ts), mat-2(ts);atr(RNAi), and mat-2(ts);mad-1(RNAi) germ lines. (G) SIM images of nuclei from wild type and worms treated with HU and stained for CENPA(cyan), DAPI(magenta), and NPC(yellow). Scale bar 2 μm. (H) CENPA is not enriched in meiotic nuclei. Germ line from an HU-treated wild-type worm stained with CENPA (red) and DAPI (blue). Arrows indicate pachytene nuclei. Scale bar = 10μm.(A) Proliferative zones of wild-type worms after IR or in the absence of damage stained with CENPA (red) and DAPI (blue). (B) CENPA steady state levels are not up-regulated after HU. Western blot showing CENPA in (TIF)Click here for additional data file.S6 Fig(A) COS cells stained with MAD2L1 (red) or MAD1 (green) and counterstained with DAPI (blue) in untreated cells, with colchicine or HU. (B) Graph shows the average ratio of nucleoplasmic MAD2L1 fluorescence to cytoplasmic signal in the presence and absence of HU; Error bars indicate SEM. Scale bar = 2μm.(TIF)Click here for additional data file.


Vibrio parahaemolyticus is an important pathogen that causes food-borne gastroenteritis in humans. The type III secretion system encoded on chromosome 2 (T3SS2) plays a critical role in the enterotoxic activity of V. parahaemolyticus. Previous studies have demonstrated that T3SS2 induces actin stress fibers in various epithelial cell lines during infection. This stress fiber formation is strongly related to pathogenicity, but the mechanisms that underlie T3SS2-dependent actin stress fiber formation and the main effector have not been elucidated. In this study, we identified VopO as a critical T3SS2 effector protein that activates the RhoA-ROCK pathway, which is an essential pathway for the induction of the T3SS2-dependent stress fiber formation. We also determined that GEF-H1, a RhoA guanine nucleotide exchange factor (GEF), directly binds VopO and is necessary for T3SS2-dependent stress fiber formation. The GEF-H1-binding activity of VopO via an alpha helix region correlated well with its stress fiber-inducing capacity. Furthermore, we showed that VopO is involved in the T3SS2-dependent disruption of the epithelial barrier. Thus, VopO hijacks the RhoA-ROCK pathway in a different manner compared with previously reported bacterial toxins and effectors that modulate the Rho GTPase signaling pathway. Vibrio parahaemolyticus induced robust actin stress fiber formation in infected host cells. Furthermore, this actin rearrangement induced barrier disruption in a colon epithelial cell line. Although many types of effector proteins have been reported, VopO does not share homology with previously reported effector proteins, and no putative functional motifs could be identified. Finally, we determined that the direct binding of VopO to a RhoA guanine nucleotide exchange factor (GEF) is a key step in the induction of stress fiber formation. These findings indicate that VopO plays a unique role in the pathogenicity of V. parahaemolyticus.Many bacterial pathogens manipulate the actin cytoskeleton of mammalian cells to establish pathogenesis via invasion, to evade killing by phagocytes, to disrupt a barrier function, and to induce inflammation caused by translocation type III secretion (T3S) effector proteins. We demonstrated that the T3S effector protein (VopO) of the enteric pathogen  Vibrio parahaemolyticus is a Gram-negative halophilic bacterium that causes acute gastroenteritis in humans after the consumption of contaminated raw or undercooked seafood. The emergence of pandemic strains poses a worldwide health threat  containing an EDTA-free protease inhibitor cocktail . After agitation for 15 min at 4°C, the lysates were harvested with a cell scraper and centrifuged for 15 min at 10,000 × The samples used for western blotting were separated by SDS-PAGE. After electrotransfer, the polyvinylidene fluoride (PVDF) membranes  were probed with anti-GEF-H1, p(Thr18/Ser19)-MLC, MLC, anti-GST , anti-LARG, anti-Ect2, anti-RhoA, anti-β-actin, anti-α-tubulin, or anti-FLAG  antibodies, followed by horseradish-peroxidase-conjugated goat anti-rabbit or rabbit anti-mouse antibodies . The blots were developed using an ECL Western Blotting Kit .5 Caco-2 cells were plated into Transwell chambers  with a pore size of 0.4 μm (6.5 mm diameter) and cultured for 12–14 days. The medium was changed every 2 days until a steady-state TER (450–550 Ωcm2) was achieved. The cells were infected with V. parahaemolyticus strains, and the TER was measured using an epithelial voltmeter  at the indicated time. The FITC-dextran leakage assay was conducted as described previously [A total of 2 x 10eviously . MixtureSpecific siRNAs for RhoA, GEF-H1, LARG, or Ect2 were purchased from Applied Biosystems. Silencer Select Negative Control #1 siRNA  was used as a negative control. The siRNAs were transfected using siPORT NeoFX Transfection Agent . At 24 h after transfection, the cells were subjected to western blot analysis to confirm the knockdown efficiency and used subsequently in the infection studies.t tests that assumed unequal variances were used for the statistical analyses. P < 0.05 was considered significant.All of the data are expressed as the mean and standard error based on at least three determinations per experimental condition. Student’s S1 FigV. parahaemolyticus mutant strains at a MOI of 10 for 3 h. Then, the cells were fixed and stained to detect F-actin (green) and cellular and bacterial DNA (blue). Bars = 20 μm.Caco-2 cells (A) or HeLa cells (B) were infected with isogenic (TIF)Click here for additional data file.S2 FigtdhAS-deficient strain), POR-2 (T3SS1-deficient strain derived from POR-1), POR-3 (T3SS2-deficient strain derived from POR-1), POR-2∆vcrD2 (T3SS1- and T3SS2-deficient strain derived from POR-1), and POR-2∆vopO (vopO-deficient strain derived from POR-2). The membranes were probed with anti-VopO, anti-VopB1 (translocon of T3SS1), anti-VopB2 (translocon of T3SS2), anti-VopD2 (translocon of T3SS2), anti-VopL (T3SS2 effector), or anti-VopC (T3SS2 effector) antibodies. (B) Effect of vopO deletion on T3SS2-dependent cytotoxicity against Caco-2 cells. Caco-2 cells were infected with isogenic mutants of POR-2 at a MOI of 10. At 6 h after infection, cytotoxic activity was evaluated by determining the amount of lactate dehydrogenase released. The asterisks indicate results that differ significantly from those obtained using the wild-type strain (*p < 0.01). The error bars indicate the standard errors for experiments performed in triplicate. n.s. = not significant. (C) Effect of vopO deletion on T3SS2-dependent enterotoxicity. The enterotoxicity of various isogenic V. parahaemolyticus mutant strains was evaluated using the rabbit ileal loop test. The fluid accumulation (FA) ratio in each loop was measured 18 h after infection. FA is the amount of accumulated fluid (in ml) per length (in cm) of ligated rabbit small intestine. The asterisks indicate results that differ significantly from those obtained using the wild-type strain (*p < 0.01). The error bars represent the standard errors. n.s. = not significant. (D) Effect of vopO deletion on T3SS2-mediated cell invasion. HeLa cells were infected with isogenic mutant strains of POR-2 at an MOI of 2 for 2 h. Frequencies of invasion are expressed relative to the invasion of POR-2, which was set at 1.0. The error bars indicate the standard errors for experiments performed in triplicate. The asterisks indicate results that differ significantly from those obtained using the POR-2 strain (*p < 0.01).(A) Secreted protein profiles of POR-1 ((TIF)Click here for additional data file.S3 FigFLAG-tagged DN-RhoA (pMEPyori-RhoN19) or mock vector (pMEPyori) was transfected into HeLa cells. At 24 h after transfection, the cells were infected with a POR-2 strain at a MOI of 10 for 3 h. The cells were stained to detect transiently expressed DN-RhoA (red), F-actin (green), and cellular and bacterial DNA (blue).(TIF)Click here for additional data file.S4 Fighttp://cib.cf.ocha.ac.jp/bitool/MIX/).The secondary structure of the VopO protein was predicted using the Chou-Fasman secondary structure prediction program ((TIF)Click here for additional data file.S5 Fig(A) Visualization of F-actin (green), DsRed (red), and cellular DNA (blue) in HeLa cells transfected with a VopO expression construct (DsRed-vopO) or empty DsRed vector (DsRed). (B) Effect of cellular VopO expression on the microtubule network. Cells transfected with a VopO-expression construct (DsRed-vopO) or empty DsRed vector (DsRed) were stained to detect tubulin (green) and cellular and bacterial DNA (blue). (C) Effect of cellular VopO expression on association of GEF-H1 with microtubules. A VopO expression construct (DsRed-vopO) or empty DsRed vector (DsRed) were co-transfected with GFP-GEF-H1 expressing vector (green). After staining to detect tubulin (cyan), the cells were examined by confocal microscopy. (D) Effect of V. parahaemolyticus infection on microtubule dynamics. HeLa cells were infected with a vopO mutant (POR-2∆vopO) and its complemented strain (POR-2∆vopO/pvopO) for 3 h. The cells were stained to detect tubulin (green), cellular and bacterial DNA (blue), and F-actin (red).(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Sc).This study aimed to evaluate the potential of radioiodinated flavonoid derivativesfor single photon emission computed tomography (SPECT) imaging ofPrPSc. In vitro binding assays using recombinant mouse PrP(rMoPrP) aggregates revealed that the 4-dimethylamino-substituted styrylchromonederivative (SC-NMe2) had higher in vitro binding affinity(Kd = 24.5 nM) andcapacity(Bmax = 36.3 pmol/nmolprotein) than three other flavonoid derivatives .Fluorescent imaging using brain sections from mouse-adapted bovine spongiformencephalopathy (mBSE)-infected mice demonstrated that SC-NMe2 clearlylabelled PrPSc-positive prion deposits in the mice brain. Two methoxySC derivatives, SC-OMe and SC-(OMe)2, also showed high binding affinityfor rMoPrP aggregates with Ki values of 20.8 and26.6 nM, respectively. In vitro fluorescence and autoradiographyexperiments demonstrated high accumulation of [125I]SC-OMe and[125I]SC-(OMe)2 in prion deposit-rich regions of themBSE-infected mouse brain. SPECT/computed tomography (CT) imaging and ex vivoautoradiography demonstrated that [123I]SC-OMe showed consistentbrain distribution with the presence of PrPSc deposits in themBSE-infected mice brain. In conclusion, [123I]SC-OMe appears apromising SPECT radioligand for monitoring prion deposit levels in the livingbrain.Prion diseases are fatal neurodegenerative diseases characterised by deposition ofamyloid plaques containing abnormal prion protein aggregates (PrP C) to abnormal PrP aggregates (PrPSc). Thehuman prion diseases, including Creutzfeldt–Jakob disease (CJD), variant CJD(vCJD), Gerstmann–Sträussler–Scheinker (GSS)disease, kuru and fatal familial insomnia are histopathologically typified by neuronalloss, astrocytosis, appearance of spongiform, and the presence of PrPScdeposits in the brain1Sc at anearly stage is considered important for the effective treatment against prion diseasesbecause PrPSc has been found in the brain prior to the appearance ofextensive clinical symptoms4Sc is still needed for definitiveconfirmation of prion diseases6et al.developed an ultrasensitive detection method for PrPSc from CSF calledreal-time quaking-induced conversion (RT-QUIC)8antemortem diagnosis method for prion diseases. However, further clinical studies oflarge numbers of patients may be needed to establish the RT-QUIC as a standarddefinitive diagnosis method. On the other hand, nuclear medicine imaging such as singlephoton emission computed tomography (SPECT) and positron emission tomography (PET) mayallow the direct visualisation of prion deposits composed of PrPSc in theliving brain of prion disease patients. Hence, specific in vivo imaging agentsfor PrPSc deposits may be useful for monitoring the progression of thesediseases and evaluating the efficacy of therapeutic interventions. Prion disease andAlzheimer’s disease have common histological features of insoluble amyloidformation from amyloid beta (Aβ) and PrPSc,respectivelyβ imaging agents forSPECT and PETβ have beenapplied for imaging of prion deposits. [125I]IMPY has shown differentialin vitro and in vivo brain distribution between scrapie-infected miceand age-matched control mice, but high background binding was observedSc plaques in the brain of scrapie-infected mice invivo1411C]2-(2-[2-dimethylaminothiazol-5-yl]ethenyl)-6-(2-[fluoro]ethoxy)benzoxazole([11C]BF-227) demonstrated significant retention in cortical andsubcortical brain regions, which are known as PrPSc-rich areas, althoughfurther investigations may be necessaryβ imaging agents may be useful for diagnosing prion diseases. Wehave developed radiolabelled flavonoid-related compounds, such as flavones (FLs)17192123β plaques  and brain slicesfrom mouse-adapted bovine spongiform encephalopathy (mBSE)-infected mice as priondisease models, followed by SPECT/CT studies in the mBSE-infected mice. We discoveredthat SPECT/CT imaging with a methoxy SC derivative [123I]SC-OMesuccessfully visualised the PrPSc–positive regions in thebrain of the prion disease mouse model.We considered that these flavonoid derivatives have potential as diagnostic agents forprion diseases. Herein, we aimed to explore the feasibility of the flavonoid derivativesas imaging probes for detecting PrPSc model according toprevious reports8in vitro binding assays offlavonoid derivatives to PrPSc. Conversion of rMoPrP toβ-sheet rich rMoPrP aggregates was confirmed by anincrease in the fluorescence intensity of ThT (data not shown). We previouslyreported that flavonoid derivatives with a 4-dimethylamino group in a benzenering showed the highest levels of binding affinity for Aβaggregates among these series171921125I]FL-NMe2, achalcone derivative [125I]CL-NMe2, an auronederivative [125I]AR-NMe2 and a styrylchromonederivative [125I]SC-NMe2, for rMoPrP aggregates wereevaluated to discover lead scaffolds of PrPSc imaging probes. Asshown in 125I-labelled flavonoid derivatives to the rMoPrP aggregatesdemonstrated sigmoidal saturation curves and linear Scatchard plots that werefitted to single binding site models. [125I]FL-NMe2< [125I]CL-NMe2 (16.7) <[125I]AR-NMe2 (34.9) <[125I]SC-NMe2 (36.3). These data indicate that[125I]SC-NMe2 has the highest binding affinity andcapacity for rMoPrP aggregates among the four flavonoid derivatives. To evaluatethe binding properties of flavonoid derivatives for PrPSc inbrain tissue, the mBSE-infected mice were prepared as a mouse model of priondiseases252,CL-NMe2, AR-NMe2 andSC-NMe2) were detectedin brain sections from mock-infected mice  of the SCs for rMoPrP aggregates varied from17.0 to 221 nM 2 had a slightly lower binding affinity(Ki = 26.6 nM).Replacing the 4-methoxy group of SC-OMe with a hydroxyl group  led to aslight decrease in binding affinity. The ethyleneoxy derivative SC-OEtOH showedan approximately six-fold lower binding affinity(Ki = 221 nM) than SC-OH.The primary amine derivative SC-NH2 exhibited a 2.8-fold lowerbinding affinity than SC-OMe, whereas the methylamino derivative SC-NHMe hadcomparable affinity(Ki = 17.0 nM) withSC-OMe. Because three SC derivatives, including SC-OMe, SC-(OMe)2 andSC-NHMe, showed high affinity for rMoPrP aggregates and had preferablelipophilicities 2 and [125I]SC-NHMe)demonstrated the homogeneous distribution of radioactivity in the brain sectionsof mock-infected mice (125I]SC-OMe and [125I]SC-(OMe)2exhibited high signals in the right corpus callosum region of mBSE-infected mice than that in the same concentration of native rMoPrP (3.3%). We further evaluated in vitro autoradiographs of[125I]SC-OMe in the brain sections of patients with AD, whichdemonstrated an inconsistent accumulation of [125I]SC-OMe withthe existent Aβ plaques  wereobserved and only 17% of the unchanged compound was detected. On the other hand,most of the parent compound remained unchanged (85%) in the brain homogenates compared withthose in the contralateral site  and the PBS-injected ipsilateral hemisphere of mock-infected mice.At the late period, the [123I]SC-OMe binding in the righthemisphere of mBSE-infected mice (%SUV = 7.5) was stillsignificantly higher than those in the contralateral siteand the ipsilateral site of mock-infected mice. There wSc in the living brain may be usefulfor monitoring the progression of these diseases and for the evaluation of theefficacy of therapeutic interventions at an early stage. There have been severalreports on Aβ imaging agents being applied to prion imaging12131415et al. reported onthe consistent in vitro autoradiograms of [18F]BF-227 with PrPdeposits in GSS brain sections. They also showed high [11C]BF-227retention in PrPSc-rich brain tissue from GSS patients using PETstudiesin vitroevaluations of flavonoid derivatives using rMoPrP aggregates as aPrPSc model25in vivo potential using SPECT/CT imaging andex vivo autoradiography of mBSE-infected mice. To our knowledge, thisstudy is the first to describe in vivo imaging of PrPSc in arodent model of prion diseases using small-animal nuclear medicine imaging systems.We have demonstrated that SC derivatives can be applied to in vivo imagingprobes for the detection of prion deposits in the brain. It should be noted thatSPECT/CT studies with [123I]SC-OMe successfully visualisedPrPSc-positive regions in the mBSE-infected mouse brain.Nuclear medicine imaging of PrPrams of 1FBF-227 wIn vitro binding studies suggested that SC derivatives may be the mostpromising candidate imaging probes for PrPSc among the four flavonoidderivatives  all showed highbinding affinities for Aβ aggregates and clearly stained amyloidplaques in AD mouse model (Tg 2576 mice) brains1719212 bound with the highest affinity to rMoPrPaggregates and prion deposits while other compounds had unsatisfactory bindingproperties for PrPSc. Considering that styrylbenzoazole derivativesalso showed binding affinity for prion depositsSc deposits were onlydetected close to the mBSE infection site in the mouse brain  fibrils of 34.3 pmol/nmol125I]SC-NMe2 exhibited a Kdvalue of 24.5 nM and a Bmax for rMoPrP aggregates of36.3 pmol/nmol. Although the amyloid models differ, the results of ourin vitro experiments of SC derivatives could provide one of the criteriafor the development of in vivo imaging probes for PrPSc. Amongthe SC derivatives, the methoxy derivatives (SC-OMe and SC-(OMe)2) andSC-NHMe showed relatively high affinity for rMoPrP aggregates, suggesting thatelectron-donating and lipophilic substituents in the 4-position of the 2-styrylgroup may be important for binding interaction with rMoPrP aggregates (125I]SC-OMe and[125I]SC-(OMe)2 labelled prion deposits in the brainsections from mBSE-inoculated mice by fluorescence microscopy . It should be taken intoconsideration that overall radioactivity levels in the brain of mBSE-infected micewere higher than those of mock-infected mice, indicating that theblood–brain barrier (BBB) of mBSE-infected mice was altered. In fact,several reports suggested that prion infection is related to BBB disruption31123I]SC-OMe can be helpful in distinguishingPrPSc-positive regions from PrPSc-negativeregions. Although further preclinical SPECT imaging studies of[123I]SC-OMe using various animal models of prion diseases arenecessary, [123I]SC-OMe has exhibited higher selectivity forPrPSc than other previously reported amyloid imaging probes, andmay be a prospective SPECT imaging probe for prion deposits. Such a probe can beused for further investigations into the mechanisms of prion diseases as well asdevelopment of therapeutic agents for these diseases both in basic investigationsand clinical studies.ivatives , 3, 4, 5del Tg 256 mice brgregates . In partcroscopy  and in viography .Moreoveted mice . Importainvivo imaging agents of PrPSc. We discovered theradioiodinated SC-OMe exhibited high affinity for rMoPrP aggregates and highaccumulation in PrPSc positive regions of the mBSE-infected mousebrain. Notably, [123I]SC-OMe allowed prion deposit regions inmBSE-infected mice to be visualised by small animal SPECT/CT imaging systems.Overall, we demonstrate that [123I]SC-OMe could be a potential SPECTimaging probe for visualisation of PrPSc in the living brain.In conclusion, we found that a SC backbone can be used as a scaffold for 125I]NaI was obtained by MPBiomedicals . [123I]NaI was supplied byFUJIFILM RI Pharma Co., Ltd. . High-performance liquidchromatography (HPLC) analysis was performed on a Shimadzu HPLC system . An automated gammacounter with a NaI(Tl) detector (2470 WIZARD2) and[125I]FL-NMe2 were prepared according to theliteratureE)-3-(4-(Dimethylamino)phenyl)-1-(4-iodophenyl) prop-2-en-1-one(CL-NMe2) and [125I]CL-NMe2 wereprepared as described previously2) and [125I]AR-NMe2 were preparedin accordance with another studyE)-6-Tributylstannyl-2-(4-methoxystyryl)-chromone (1),(E)-6-Iodo-2-(4-methoxystyryl) chromone (SC-OMe),(E)-6-Iodo-2- chromone {SC-(OMe)2},(E)-6-Iodo-2-(4-hydroxylstyryl)-chromone (SC-OH),(E)-6-Iodo-2-(4- hydroxyethoxystyryl)-chromone (SC-OEtOH),[125I]SC-OMe and [125I]SC-(OMe)2were prepared as described previouslyE)-6-Iodo-2-(4-aminostyryl)-chromone (SC-NH2),(E)-6-Iodo-2-(4-(methylamino)styryl)-chromone (SC-NHMe),(E)-6-Iodo- 2-(4-(dimethylamino)styryl)-chromone (SC-NMe2),[125I]SC-NHMe and [125I]SC-NMe2were prepared as described previouslyAll reagents were commercial products and used without further purificationunless otherwise indicated. 15INaI was, USA). 1INaI was 123I]SC-OMe was prepared using a similar procedure for[125I]SC-OMe2O2 (100 μL) was added to amixture of corresponding tributyltin derivative(1.0 mg/400 μL-EtOH),[123I]NaI , and 1 M HCl (100 μL) in asealed vial. The reaction was allowed to proceed at room temperature for10 min and terminated by addition of saturated NaHSO3aq(0.5 mL). After alkalisation with 0.5 mL of saturatedNaHCO3aq and extraction with ethyl acetate, the extract wasevaporated to dryness. The crude products were purified by HPLC on a CosmosilC18 column  with an isocraticsolvent of CH3CN/H2O (7:3) at a flow rate of4.0 mL/min.The ligand was calculated. Thedissociation constant (Kd) and binding capacity(Bmax) of compounds were estimated by Scatchard analysisusing PRISM4 . For competitive binding assays,the mixture contained [125I]SC-NMe2(0.02 nM), test compound(8.0 pM–12.5 μM), and rMoPrPaggregates (100 nM) in NaCl/HEPES buffer (pH 7.5) containing 20%(v/v) DMSO. After incubation for 2 h at room temperature, themixture was filtered and the filters were measured using the gamma counter.Nonspecific binding was defined in the presence of 10 μMfor nonradioactive SC-NMe2. Values for the half maximal inhibitoryconcentration (IC50) were determined from displacement curves ofthree independent experiments using PRISM4, and those for the inhibitionconstant (Ki) were calculated using theCheng–Prusoff equation.The saturation assays were performed by mixing an appropriate concentration ofAll animals were supplied by Kyudo Co., Ltd. . Experiments usinganimals were conducted in accordance with our institutional guidelines and wereapproved by the Nagasaki University Animal Care Committee.in vitro studies at 22–25 weeks post-infection. Theanimals for in vitro experiments were exsanguinated by transcardialperfusion with saline under ether anaesthesia, and their brains weresubsequently removed. Sacrificed brain tissues were fixated in 10% (v/v)buffered formalin for 1 week, and then each sample was embedded in paraffin andcut into 3-μm-thick sections.The mBSE-infectious animal experiments were conducted under biosafety level 3(BSL3) containment in accordance with institutional guidelines. mBSE-infectedmice were prepared as reported previously252O2for 30 min, normal goat serum (1:20) was added for30 min. The tissues were incubated overnight with SAF32 anti-PrPantibody (1:20). Following washing with Tris-HCl buffer including 0.05% (v/v)Tween 20, the slices were incubated with secondary anti-mouse biotinylatedantibody for 1.5 h. The signal was visualised by a reaction withhydrogen peroxidase-activated diaminobenzidine.The sections from mBSE-infected and mock-infected mice were dewaxed and incubatedwith a 50% (v/v) EtOH solution containing the test compound(100 μM) for 1 h. The sections were washedin 50% (v/v) EtOH for 2 min, two times. The fluorescence images werecollected by an Eclipse 80i microscope  using a V-2Afilter set  or a B-2A filter set. After fluorescentimaging analysis, the tissues were washed with 50% (v/v) EtOH and autoclaved in1.2 mM of HCl at 121 °C for10 min and then the sections were treated with formic acid for15 min. After blocking with 0.3% (v/v) H125I]ligand  for1 h. The slices were rinsed for 5 min, two times each,with 70% (v/v) DMSO solution, and subsequently dipped into cold water for30 s. The sections were dried under a steam of cold air and placedin contact with imaging plates  for24 h. Distribution of radioactivity on the plates were analysedusing the Fluoro Image Analyzer . Thereafter, serialsections were also analysed by immunohistochemical staining ofPrPSc deposition as described above.Each brain section was incubated in 40% (v/v) DMSO solution containing[123I]SC-OMe(32.0–43.4 MBq) via tail vein injection. Immediatelyafter injection, the mice were anaesthetised with 1.5% (v/v) isoflurane. SPECTimage acquisitions were performed with a four-head γ-camera equippedwith five pinhole collimators . SPECT data were acquired for 33 min  starting at 15 or50 min after intravenous injection. The SPECT imaging was followedby CT image acquisition , withthe animal in exactly the same position. The SPECT data were reconstructed usinga 3D-ordered subset expectation maximisation (3D-OSEM) algorithm in FLEX SPECTsoftware. The semiquantitative values obtained from the SPECT images areexpressed as the percent standardised uptake values (%SUV), which was calculatedas follows:SPECT/CT imaging studies of mBSE-infected mice  ormock-infected mice  wereperformed using Triumph combined PET/SPECT/CT systems . Each mouse was administered [Sc and theautoradiograms of radioactivity in the brain sections were obtained using thesame methods as described above.After SPECT/CT imaging, each mouse was sacrificed and the whole brain was frozenon dry ice/ethanol baths, followed by preparation of coronal sections(10 μm) using a cryostat microtome. Thereafter, theimages of immunohistochemical staining of PrPP value <0.05 was considered statistically significant.One-way analysis of variance followed by the post hoc tests usingBonferroni’s correction were used for analysis of significantdifferences for the %SUV values of SPECT images in the mouse brain tissues. AHow to cite this article: Fuchigami, T. et al. Characterisation ofradioiodinated flavonoid derivatives for SPECT imaging of cerebral prion deposits.Sci. Rep. 5, 18440; doi: 10.1038/srep18440 (2015).


The p53 tumor suppressor protein is a transcription factor that initiates transcriptional programs aimed at inhibiting carcinogenesis. p53 represses metabolic pathways that support tumor development (such as glycolysis and the pentose phosphate pathway (PPP)) and enhances metabolic pathways that are considered counter-tumorigenic such as fatty acid oxidation.de novo synthesis of glucose). Examining glucogenic gene expression revealed a p53-dependent induction of genes involved in both gluconeogenesis  and in supplying glucogenic precursors (glycerol kinase (GK), aquaporin 3 (AQP3), aquaporin 9 (AQP9) and glutamic-oxaloacetic transaminase 1 (GOT1)). Accordingly, p53 augmented hepatic glucose production (HGP) in both human liver cells and primary mouse hepatocytes.In an attempt to comprehensively define metabolic pathways regulated by p53, we performed two consecutive high-throughput analyses in human liver-derived cells with varying p53 statuses. A gene expression microarray screen followed by constraint-based modeling (CBM) predicting metabolic changes imposed by the transcriptomic changes suggested a role for p53 in enhancing gluconeogenesis (These findings portray p53 as a novel regulator of glucose production. By facilitating glucose export, p53 may prevent it from being shunted to pro-cancerous pathways such as glycolysis and the PPP. Thus, our findings suggest a metabolic pathway through which p53 may inhibit tumorigenesis. Recentlsh-p53) while the control group expressed a non-relevant short hairpin RNA (termed HepG2sh-con). Second, we treated the cells with Nutlin-3a, a p53-activating agent, resulting in accumulation of p53 protein levels and induction of p53-dependent transcriptional programs  with α = 0.05), while no enrichment was found for these pathways in the Nutlin-treated HepG2sh-p53 cells . Gluconeogenesis is activated in hepatocytes when blood glucose levels diminish (due to fasting or vigorous exercise), the glucose is then secreted to the bloodstream thereby restoring glucose homeostasis. Upon a hormonal stimulus the induction of gluconeogenesis is achieved through transcriptional, post-translational and allosteric alterations in four main reactions of gluconeogenesis. These reactions are catalyzed by the following enzymes: pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-bisphosphatase  and glucose-6-phosphatase (G6Pase). Transcriptional regulation of the genes encoding these enzymes is crucial for an effective induction of gluconeogenesis [Our analyses pointed to a role for p53 in regulating gluconeogenesis  were predicted to carry higher metabolic flux in cells expressing either basal or activated p53 than in any of the other states  and PCK2  were significantly elevated in Nutlin-treated HepG2sh-con cells but not in Nutlin-treated HepG2sh-p53 cells , aquaporin 9 (AQP9), glycerol kinase (GK) and glutamic-oxaloacetic transaminase 1 (GOT1)). Notably, most of these genes participate in the uptake and metabolism of glycerol into the glucogenic pathway following lypolysis . The mRNA levels of in silico predictions and with the p53-dependent changes in glucogenic gene expression, we found that HepG2 cells exhibit increased hepatic glucose production (HGP) following Nutlin-3a treatment  . The p53In accordance with the computational analysis and the gene expression data, we found that p53 enhances glucose production in both human and mouse hepatocytes. Our findings are strongly supported by several published data. First, p53 induces the expression of TP53-induced glycolysis and apoptosis regulator (TIGAR), which lowers the levels of fructose-2,6-bisphosphate , an inhibitor of gluconeogenesis . Second,G6PC expression and enhances HGP adds another layer for the metabolic regulation exerted by p53 in an effort to curtail carcinogenesis.Reduced G6Pase levels are correlated to the development of hepatocellular carcinoma . It has The microarray results were deposited in GEO Accession Number GSE30137.AQP3: Aquaporin 3; AQP9: Aquaporin 9; CBM: Constraint-based modeling; ChIP-seq: Chromatin immunoprecipitation-sequencing; DHAP: Dihydroxyacetone phosphate; F1,6BPase: Fructose-1,6-bisphosphatase; F2,6BP: Fructose-2,6-bisphosphate; FDR: False discovery rate; G3P: Glyceraldehyde 3-phosphate; G6P: Glucose-6-phosphate; G6Pase: Glucose-6-phosphatase; GCPC: Glucose-6-phosphatase, catalytic subunit; G6PD: Glucose-6-phosphate dehydrogenase; GK: Glycerol kinase; GOT1: Glutamic-oxaloacetic transaminase 1; GR: Glucocorticoid receptor; sh-con: Non-relevant short hairpin RNA; sh-p53: Short hairpin RNA targeting p53; HGP: Hepatic glucose production; iMAT: Integrative metabolic analysis tool; OAA: Oxaloacetate; PC: Pyruvate carboxylase; PCK2: Phosphoenolpyruvate carboxykinase 2; PEP: Phosphoenolpyruvate; PEPCK: Phosphoenolpyruvate carboxykinase; PGC1α: Peroxisome proliferator-activated receptor gamma coactivator 1 alpha; PPP: Pentose phosphate pathway; qPCR: Quantitative PCR; TIGAR: TP53-induced glycolysis and apoptosis regulator; TSS: Transcription start site.The authors declare that they have no competing interests.IG conceived the study, designed and performed the experiments and drafted the manuscript. KY performed the computational analysis. SM and NG designed and performed the experiments. ER performed the computational analysis and drafted the manuscript. VR designed the experiments and drafted the manuscript. All authors read and approved the final version of the manuscript.Detailed methods.Click here for fileiMAT analysis.Click here for fileFigure S1 Hepatocytes were isolated and incubated in gluconeogenic medium [c medium  for 4 hoClick here for file


In this regard, patients with pemphigus were compared to patients with myasthenia gravis (MG), an unrelated auto-ab–mediated autoimmune disease, and healthy controls. In pemphigus and MG, the plasma concentrations of the APC-derived immunomodulatory cytokine IL-27 were highly increased. Strikingly, IL-27 strongly correlated with Dsg-specific IgG auto-ab titers. T helper (Th) 17 cells were augmented in both pemphigus and MG patients while T follicular helper (Tfh) cells, which are essential in providing B cell help, were increased only in pemphigus along with increasing plasma concentrations of IL-21, a cytokine produced by Th17 and Tfh cells. Moreover, we could detect Dsg3-specific autoreactive T cells producing IL-21 upon ex vivo stimulation with Dsg3. These findings suggest that IL-27 and IL-21-producing T cells, are involved in the pathogenesis of pemphigus. The further characterization of IL-21-producing T cells and of the role of IL-27 will lead to a more defined understanding of the auto-ab response in pemphigus.Pemphigus is an autoimmune disease in which IgG auto-antibodies (auto-ab) against the desmosomal cadherins desmoglein (Dsg) 3 and Dsg1 cause loss of epidermal keratinocyte adhesion. Aim of this study was to investigate cytokines derived from antigen-presenting cells (APC) and their relation to CD4 Differences between the groups were analyzed using Mann-Whitney-U-Test with p values indicated as *p<0.05, **p 0.01 and ***p 0.001.(TIF)Click here for additional data file.S2 Fig(A) Serum anti-AChR IgG titers did not correlate with plasma IL-27 in MG (n = 12). (B) Auto-ab titers in MG did not correlate with the plasma levels of the pro-inflammatory cytokine IL-6 (C) A negative correlation between TNF-α and the auto-ab titers was found in MG. Spearman’s rank correlation coefficients (rs) are shown with p values indicated as *p<0.05.(TIF)Click here for additional data file.S3 Fig(A) Lymphocytes were gated on size and granularity. Th cells were defined as CD3+CD8- T cells and intracellular cytokine expression was detected. (B) Gates for cytokine detection were defined according to the respective isotype control.PBMC stimulated with PMA, ionomycin and monensin were used for the analysis (TIF)Click here for additional data file.S4 Fig(A) Lymphocytes were gated on size and granularity. cTfh cells were identified as CD4+ T cells expressing CXCR5 and further characterized by measuring determining PD-1 and ICOS expression. (B) Gates were defined according to the respective isotype controls.ACK-lysed blood cells were used for analysis. (TIF)Click here for additional data file.S5 Fig(A) Lymphocytes were gated on size and granularity. T cells were gated on CD4 expression and intracellular IL-21 and IL-17 expression was detected. (B) The CD4-gate was defined according to the respective isotype control. Gates for IL-21 and IL-17 were defined by staining non-stimulated PBMC with the IL-21 or IL-17 antibody.PBMC stimulated with PMA, ionomycin and monensin were used for the analysis (TIF)Click here for additional data file.S6 FigA) IL-27 plasma levels do not correlate with CD4+CXCR5+ T cells (B) and IL-21 plasma levels. Spearman’s rank correlation coefficients (rs) are shown.((TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Chronic liver disease leads to neurocognitive impairment (NCI) and more advanced liver fibrosis is associated with greater deficits. Further, cognitive performances do not differ significantly among patients affected by diverse types of chronic liver diseases. Thus, it would be useful to have a clinical tool associated with early cognitive change applicable to the HIV-infected population with high HCV prevalence. Aim of the analysis was to assess the association between NCI and aspartate aminotransferase-platelet ratio index (APRI) or Fibrosis-4, which are non-invasive scores used to assess liver fibrosis.Single-centre, retrospective, cross-sectional analysis of cART-treated HIV-infected patients undergoing neuropsychological assessment (NPA) by a set of 14 standardized and comprehensive tests on five different domains: concentration and speed of mental processing; mental flexibility; memory, fine motor functioning; visual-spatial and constructional abilities. NPA obtained from the same patient were included, if collected while receiving different cART. Patients were classified as having NCI, if they scored >1 SD below the normative mean in at least two tests, or below >2 SD in one test. HIV-associated neurocognitive disorders (HAND) were classified according to Frascati's criteria, controlling for confounding comorbidities. Univariable analysis and multivariable logistic regression models were carried out.3; median current CD4 501/mm3; undetectable HIV-RNA in 368 (67%); and HCV-positivity in 150 (29%). Frequency among score levels was for FIB-4: min-1.44=404 (73%), 1.45–3.25=118 (21%), 3.26-max=28 (5%); for APRI: min-0.5=414 (75%), 0.5–1.5=112 (20%), 1.5-max=24 (4%). Median FIB-4 and APRI were 0.98 and 0.27 in HIV+/HCV- and 1.40 and 0.50 in HIV+/HCV+ individuals, respectively. HAND was found in 176 (32%): 91 ANI, 73 MND, 12 HAD. Association of variables with NCI are shown in A total of 556 HIV-infected cART-treated patients from 2006 to 2013 were included: male 78%; IVDUs 14%; CDC stage C 31%; median CD4 nadir 200/mmIn this large population, HAND was not associated with commonly used non-invasive liver fibrosis scores. As aetiology of cognitive dysfunction in HIV mono- and HCV co-infected patients is multifactorial and partially unknown, our results support the hypothesis of a direct or indirect effect on cognitive function of the viruses, rather than the consequence of alterations residing outside the brain.


Hepatitis C virus (HCV) infection causes chronic liver diseases leading to hepatocellular carcinoma (HCC) and liver failure. We have previously shown that HCV sensitizes hepatocytes to mitochondrial apoptosis via the TRAIL death receptors DR4 and DR5. Although TRAIL and its receptors are selective targets for cancer therapy, their potential against HCC with chronic HCV infection has not been explored yet. Here we show that HCV induces DR4/DR5-dependent activation of caspase-8 leading to elevation of apoptotic signaling in infected cells and also present TRAIL effect in HCV-induced apoptotic signaling. HCV induced proteolytic cleavage of caspase-9 by stimulating DR4 and DR5, resulting in subsequent cleavage of caspase-3. Further, HCV-induced proteolytic cleavage in caspase-8, caspase-9, and caspase-3 was enhanced in the presence of recombinant TRAIL. HCV-induced cleavage in caspase-9 and increase in caspase-3/7 activity was completely suppressed by silencing of either DR4 or DR5. Perturbing DR4/DR5-caspase-8 signaling complex by silencing DR4 and DR5 or by chemical inhibitor specific to caspase-8 led to decrease of HCV-induced cleavage of poly(ADP-ribose) polymerase (PARP), a substrate for caspase-3 during apoptosis, indicating the functional role of caspase-8 in HCV-induced apoptotic signaling network. Furthermore, TRAIL enhanced PARP cleavage in apoptotic response induced by HCV infection, indicating the effect of TRAIL for the induction of selective apoptosis of HCC cells infected with HCV. Given the importance of apoptosis in HCC development, our data suggest that HCV-induced DR4 and DR5 may be considered as an attractive target for TRAIL therapy against HCC with chronic HCV infection. Hepatitis C virus (HCV) infects about 170 million people worldwide in vivoRegulation of apoptosis is complex and mainly triggered by the activation of so-called death receptors In addition to inducing apoptosis, these death receptors initiate independent intracellular signaling cascades through mitochondrial dysfunction. Mitochondrial dysfunction plays a critical role in integrating death receptor-initiated apoptosis into a final common pathway by activating a caspase-dependent apoptosis We recently demonstrated that co-infection with HCV and HIV stimulates DR4 and DR5 gene expression and subsequently activates caspase-3 signaling pathway, resulting in induction of mitochondrial apoptosis of human hepatocytes The human hepatocellular carcinoma Huh7.5.1 cells used in this study were grown in high-glucose Dulbecco's Modified Eagle's Medieum  supplemented with 10% fetal bovine serum , 100 U/ml penicillin and 0.1 mg/ml streptomycin . Cell culture-derived HCV JFH-1 (HCVcc) used in this study was propagated and prepared, as described previously Recombinant human TRAIL, pan-caspase inhibitor Z-VAD-FMK, caspase-8 specific inhibitor Z-IETD-FMK, and caspase-9 specific inhibitor Z-LEHD-FMK used in this study were purchased from R&D systems (Abingdon). TRAIL and caspase inhibitors were reconstituted according to the manufacturer's instructions. Primary antibodies used in this study include the following: rabbit monoclonal anti-cleaved PARP ; mouse monoclonal anti-DR4 (Abcam); rabbit monoclonal anti-DR5 ; rabbit monoclonal anti-cleaved caspase-3 ; rabbit monoclonal anti-cleaved caspase-8 ; rabbit monoclonal anti-cleaved caspase-9 ; rabbit monoclonal anti-cleaved Bid (Invitrogen); mouse monoclonal anti-HCV core (Thermo Fisher Scientific); mouse monoclonal anti-α-tubulin (Sigma); mouse monoclonal anti-β-actin (Sigma). The secondary antibodies used for Western blot analysis were HRP-conjugated anti-mouse IgG (Santa Cruz Biotechnology) and HRP-conjugated anti-rabbit IgG (Santa Cruz Biotechnology).siGENOME non-targeting siRNA pool #1 and ON-TARGETplus SMARTpool small interfering RNA (siRNA) for DR4 (NM_003844) and DR5 (NM_003842) were purchased from Dharmacon. Huh7.5.1 cells were transfected with siRNAs (50 nM) using Lipofectamine RNAiMAX transfection reagent according to the manufacturer's instructions (Invitrogen) prior to HCVcc infection.5′-CTGAGCAACGCAGACTCGCTGTCCAC; DR4 reverse, 5′-TCAAAGGACACGGCAGAGCCTGTGCCA; DR5 forward, 5′-GGGAGCCGCTCATGAGGAAGTTGG; DR5 reverse, 5′-GGCAAGTCTCTCTCCCAGCGTCTC; GNB2L1 forward, 5′-GACCATCATCATGTGGAAACTGA; GNB2L1 reverse, 5′-CCGTTGTGAGATCCCAGAGG. GNB2L1 was used as an internal control. Real-time qPCR was conducted by using a StepOnePlus Real-Time PCR System (Applied Biosystems).To analyze the expression levels of DR4 and DR5, total cellular RNA was extracted from Huh7.5.1 cells using RNeasy Mini kit (Qiagen) and subsequently, complementary DNA was synthesized by using GeneAmp RNA PCR kit (Applied Biosystems) according to the manufacturer's instructions. Power SYBR Green PCR Master Mix (Applied Biosystems) was used to quantify the cellular RNA levels. The following primer sets were used for RT-PCR: DR4 forward, Cells were resuspended in lysis buffer  supplemented with protease inhibitor cocktail (Roche). Whole cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose membrane (Millipore), and Western blot analyzed with antibodies against the indicated proteins.Caspase-3/7 activity in HCV-infected cells was measured using the Caspase-Glo 3/7 assay kit according to the manufacturer's instruction (Promega). Briefly, Huh7.5.1 cells transfected with non-targeting (NT) or specific-targeting siRNA pools for DR4 and DR5 were infected with HCVcc. At 3 days post-infection, caspase-3/7 specific luminescence activity was measured using a Synergy 2 Multi-Mode Microplate Reader (BioTek).Statistical analysis was performed using a paired student's t-test. Results were expressed as means ± standard deviation of at least three sample replicates.We have recently shown that HCV infection activates DR4 and DR5-mediated intrinsic apoptotic signaling in human hepatocytes TRAIL induces the recruitment of Fas-associated death domain and procaspase-8 to DR4 and DR5, leading to activation of caspase-8 and subsequent caspase-9/-3-dependent apoptotic signaling pathway PARP cleavage by caspase activation is a hallmark of apoptosis Apoptosis, or programmed cell death, is a process in which cells activate intracellular death pathways to terminate themselves systematically in response to a wide variety of stimuli A therapeutic potential of TRAIL for liver cancer with persistent HCV infection has been overlooked. TRAIL receptors including DR4 and DR5 are very attractive targets for cancer therapy et al have shown that HCV infection upregulates DR4 and DR5 expression levels and triggers TRAIL-mediated apoptosis in human hepatocellular carcinoma LH86 cells It is commonly assumed that virus clearance requires the destruction of infected cells by virus-specific cytotoxic T-lymphocytes via the CD95 receptor, TNF-receptor-1 and TRAIL receptors DR4 and DR5. If death receptor-mediated apoptosis works adequately, it is beneficial for the organism and may lead to elimination of virus-infected cells. However, dysregulation of this system might result in liver damage. Therefore, TRAIL receptors, which selectively kill virus-infected cells while leaving uninfected cells intact, have evolved as a prime physiological mechanism in order to selectively eliminate virus-infected cells et al have shown that HCV infection induces TRAIL-mediated mitochondrial apoptosis via caspase-9 activation A number of liver diseases are characterized by elevated caspase activation and apoptosis. Caspase is a cysteine protease which plays a critical role in initiating and executing the apoptotic signaling by degrading several cellular components, resulting in the apoptotic phenotype


Electrocardiograms (ECGs) obtained within High-field MRIs are distorted due to the Magneto-hydrodynamic (MHD) effect. Blood plasma electrolytes ejected into the aorta during early systole interact with the strong magnetic field of the MR scanner to produce a MHD-induced voltage (VMHD) . The VMH12-lead ECG data was recorded using a prototype MRI-conditional 12-lead ECG recorder  from 2 PTable The 3DQRS method represented a higher sensitivity in QRS detection than the VCG based approach, which resulted in decreased error levels in high field MRI.NIH U41-RR019703, NIH R03 EB013873-01A1, SBIR-1 R43 HL110427-01.


Hap1-null and wild-type mouse brains. Breakpoint cluster region protein (Bcr), a Rho GTPase regulator, was identified as a Hap1-interacting partner. Bcr was co-immunoprecipitated with Hap1 from transfected neuro-2a cells and co-localized with Hap1A isoform more in the differentiated than in the nondifferentiated cells. The Bcr downstream effectors, namely ERK and p38, were significantly less activated in Hap1-null than in wild-type mouse hypothalamus. In conclusion, Hap1 interacts with Bcr on microtubules to regulate neuronal differentiation.Alterations in microtubule-dependent trafficking and certain signaling pathways in neuronal cells represent critical pathogenesis in neurodegenerative diseases. Huntingtin (Htt)-associated protein-1 (Hap1) is a brain-enriched protein and plays a key role in the trafficking of neuronal surviving and differentiating cargos. Lack of Hap1 reduces signaling through tropomyosin-related kinases including extracellular signal regulated kinase (ERK), resulting in inhibition of neurite outgrowth, hypothalamic dysfunction and postnatal lethality in mice. To examine how Hap1 is involved in microtubule-dependent trafficking and neuronal differentiation, we performed a proteomic analysis using taxol-precipitated microtubules from  The prevalence of neurodegenerative diseases is increasing due to an increase in aging population, but the molecular mechanism remains elusive. Neurodegenerative diseases such as Alzheimer’s disease (AD) and Huntington’s disease (HD) are characterized by progressive neuropsychiatric dysfunction and loss of specific subtypes of neurons. Although there are distinct neuropathology and clinical profiles among these diseases, many similarities in pathological pathways were reported . One of Glued (p150)  arginine and [13C6] lysine before pregnancy. Its offspring kept the same diet to adulthood. Brain lysates from the isotope-labeled wild-type (WT) adult mouse and a non-labeled newborn mouse at P1, with either WT or Hap1-null genotype, were mixed at 1:1 ratio in a buffer containing 50 mM PIPES pH 6.8, 1 mM EGTA, 1 mM MgSO4 and complete protease inhibitor cocktail (Roche). Microtubule-associated proteins were polymerized by taxol (Sigma) as described previously . Th. Th38]. S1 FigA) Micrographs (phase images in the upper panel and fluorescent images in the lower panel) showing neuro-2a cells expressing GFP, GFP-Hap1A or GFP-Hap1B after 48 h of transfection. Note that cells expressing GFP-Hap1A or GFP-Hap-1B display long neurites. (B) Statistical analysis indicating the percentage of differentiated cells with process extension more than two-fold their cell bodies. The number of cells examined was at least 100 in each group in three independent experiments. *** p < 0.001.((TIF)Click here for additional data file.S2 FigThe cells were transfected with GFP, GFP-Hap1A or GFP-Hap1B (green) with Bcr-myc (red). Scale bar, 10 μm.(TIF)Click here for additional data file.S3 FigA) Western blotting analysis of neuro-2a cells transfected with GFP, GFP-Hap1A, or GFP-Hap1B. After 48 hours of transfection, cells were harvested and homogenized for the analysis. Antibodies against Bcr and downstream signaling molecules including p38, ERK1/2, JNK, PAK, and their phosphorylated forms were used. (B) Quantitative and statistical analysis of the changes ERK1 in the transfected cells. Three independent experiments were performed for statistical analysis. * p < 0.05((TIF)Click here for additional data file.


C. elegans sensory neuron PVD. In rab-10 mutants, PVD dendritic branches are reduced in the posterior region of the cell but are excessive in the distal anterior region of the cell. We also demonstrate that the dendritic branch distribution within PVD depends on the balance between the molecular motors kinesin-1/UNC-116 and dynein, and we propose that RAB-10 regulates dendrite morphology by balancing the activity of these motors to appropriately distribute branching factors, including the transmembrane receptor DMA-1.The construction of a large dendritic arbor requires robust growth and the precise delivery of membrane and protein cargoes to specific subcellular regions of the developing dendrite. How the microtubule-based vesicular trafficking and sorting systems are regulated to distribute these dendritic development factors throughout the dendrite is not well understood. Here we identify the small GTPase RAB-10 and the exocyst complex as critical regulators of dendrite morphogenesis and patterning in the  Building a complex dendritic arbor requires tremendous cellular growth, and how membrane and protein components are transported to support a rapidly growing, polarized dendrite remains unclear. We have identified the small GTPase RAB-10 as a key regulator of this process, providing insight into both dendritic development and the control of trafficking by small GTPases. There is great diversity in the structure and complexity of dendritic arbors across neuron types, and establishing the correct dendritic morphology is critical for the proper connectivity and function of neural circuits. A developing dendritic arbor must target a specific receptive field, adopt the appropriate neuron-specific architecture, and avoid overlapping in connectivity with itself and neighboring dendrites. A number of extrinsic cues and intrinsic mechanisms help orchestrate the formation of these complex neuronal morphologies, including transcriptional programs, extracellular guidance cues, and contact-dependent repulsive molecules that mediate self-avoidance –5.dar) genes have been identified which are required for dendritic arbor outgrowth but not axon growth. These dar genes are important for ER-to-Golgi transport . In. Inser2p:myrGFP) . To undebranches . wy787 m 17 ± 10 . In addibranches . To quanbranches . In wy78 region. . The sev region. . The incwy787 showed no significant difference in the number of secondary filopodia  failed to form 3° branches (dma-1 , RB1928 exoc-8 (ok2523), and VS1026 rab-10 (ok1494) were obtained from the Caenorhabditis Genetics Center.Worms were raised on OP50  at 20°C . The wilrab-10 locus was identified with SNP mapping, fosmid rescue, and sequencing.The wy787 allele was isolated from an F2 semiclonal screen of 3,000 haploid genomes in the PVD::myrGFP strain. Worms were mutagenized with 50mM ethyl methanesulfonate. The ser2prom3 and pdes2short [phlh-1 was used for muscle-specific expression. The integrated wyIs592 (ser2prom3::myrGFP) and the extrachromosomal array ser2prom3::myrmCherry were used to visualize PVD. wyEx4286 (ser2prom3::dma-1::GFP) was used to visualize DMA-1 localization. wyEx4985 (pdes2::ebp-2::GFP) was used to visualize plus-end microtubules. Transgenes generated for this work include: wyEx7698 (ser2prom3::rab-10), wyEx7701 (pdes2short::rab-10::GFP), wyEx7700 (pdes2short::rab-10(GTP)::GFP), wyEx7772 (pdes2short::rab-10(GDP)::GFP), wyEx7763 (ser2prom3::exoc-8), wyEx7699 (hlh-1::rab-10), rab-10::rab-10, wyEx7069 (PVD::mcd8.3), ser2prom3::rab-10::mCherry; wyEx7137 (ser2prom3::rab-7::mCherry), wyEx50081 , and Phsp16.2::Cas-9;PU6::denn-4-sgRNA. Podr-1::RFP and Punc-122::RFP were used as coinjection markers.Two PVD-specific promoters, es2short , were usImages were captured in live animals using a Plan-Apochromat 63x/1.4 objective on a Zeiss LSM710 confocal microscope or using a Plan-Apochromat 63x/1.4 objective on a Zeiss Axio Observer Z1 microscope equipped with a Yokagawa spinning disk head. For still images, worms were immobilized using 10 mM Levamisole (Sigma-Aldrich) in M9 on 2% agarose pads. For FRAP imaging of DMA-1::GFP and time-lapse imaging of EBP-2::GFP, worms were soaked in 0.1% tricane/0.01% levamisole in M9 for 20 minutes and were then mounted on 5% agarose pads with 0.05μg of polysterene microspheres (Polysceinces). Video for EBP-2::GFP comet analysis was acquired at 8 frames per second.Images were straightened with the ImageJ software  using the primary dendrite as a reference. In L4 animals, the PVD dendrite was divided into four segments  and the number of secondary and quaternary branches per segment was counted. Quaternary branches were assigned to a segment based upon the location of the secondary branch forming the base of the menorah from which the quaternary branch originated. Any protrusion from the primary dendrite was classified as a secondary branch, and any correctly-oriented protrusion from the tertiary dendrite was classified as a quaternary branch. A “branch complexity” index, defined in Phsp16.2, a heat-shock promoter expressed in many tissues, was used to drive Cas9 expression. Worms were synchronized via bleaching and L1 plates were heat shocked at 33°C for two hours. The efficiency of the CRISPR-Cas9 editing was determined by observing >30 animals of each genotype. For the denn-4 experiment, which had ~20% efficiency, animals with the CRISPR-Cas9 array and an observed PVD phenotype were selected for imaging and quantification, in comparison to wild-type controls without the array from the same plate. For the dhc-1 experiment, all animals with the CRISPR-Cas9 array were included for imaging and quantification, without pre-screening for neuronal phenotype, because the dhc-1 sgRNA had a much higher efficiency (~60%). Animals from multiple replicates of each CRISPR experiment were pooled for the quantification.CRISPR-Cas9 constructs were designed according to previously established methods . Phsp16.FRAP experiments were performed using the Zeiss Axio Observer Z1 microscope equipped with a Yokagawa spinning disk head. A small region of primary dendrite just anterior to the cell body was bleached for 1000ms. Recovery images were taken every second for five minutes.wy787 were assessed using multiple t-tests.Branch complexity numbers per segment were compared across all genotypes using the 2-way ANOVA with post-hoc Tukey’s multiple comparisons test. Similarly, branch complexity numbers were compared across all genotypes within each somatic CRISPR experiment using the 2-way ANOVA with post-hoc Tukey’s multiple comparisons test. FRAP data were compared using 2-way ANOVA. Developmental differences in secondary branches between wild-type and S1 Figrab-10(ok1494) with expression of rab-10 in muscle (B) or rab-10 genomic DNA under its own promoter (C). (D-E) Representatives images of PVD::GFP in wild-type expressing an inactive, dominant negative GDP-bound RAB-10 in PVD, demonstrating the range of branch loss caused by DN RAB-10. Scale bars represent 20 μm.(A) Schematic of conservation of the RAB-10 GTPase site, with homology across species and conservation between various Rabs. (B-C) Representative images of PVD::GFP in (TIF)Click here for additional data file.S2 Figsec-5 (pk2358). (B) Quantification of subcellular distribution of branch complexity in the sec-5(pk2358) mutant animals. Scale bars represent 20 μm. Error bars represent SEM. * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by 2-way ANOVA with post-hoc Tukey’s multiple comparisons test. N≥3 for all genotypes.(A) Representative image of PVD::GFP in (TIF)Click here for additional data file.S3 Figrab-10(ok1494), dma-1(wy686), and dma-1(wy6860);rab-10(ok1494) double mutant animals. (D) Quantification of DMA-1::GFP puncta size in rab-10(ok1494) and wild-type. (E-F) Representative image of mcd8.3::GFP in wild-type (E) and rab-10(ok1494) (F). Zoom-in images indicate mcd8.3::GFP localization in the +1 and +2 regions. Arrows indicate mcd8.3::GFP puncta, arrowheads indicate abnormal accumulations, and asterisks indicate regions from which diffuse staining is lost. The primary dendrite is indicated on each zoom-in image. Scale bars represent 20 μm. Error bars represent SEM. * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by 2-way ANOVA with Tukey’s multiple comparisons test. N≥3 for all genotypes. Arrowheads indicate diffuse staining; arrows indicate puncta.(A-B) Representative images of PVD::GFP in various genotypes, as indicated on micrographs. (C) Quantification of subcellular distribution of branch complexity in the (TIF)Click here for additional data file.S4 Figrab-10(ok1494) (B) animals. (C) Representative images of DMA-1::GFP and mCherry::RAB-10 in the anterior primary dendrite of wild-type animals.(A-B) Representative images of DMA-1::GFP and mCherry::RAB-7 in the anterior primary dendrite of wild-type (A) and (TIF)Click here for additional data file.S1 TableThe average number of branches per subcellular compartment and the total number of branches are reported for each genotype. Wild-type is listed first, with other genotypes listed in order of increasing total quaternary branches.(XLSX)Click here for additional data file.


Glioblastoma multiforme (GBM) is a uniformly fatal brain tumor. The GBM microenvironment includes abundant tumor-associated macrophages (TAMs) that predominantly assume a pro-tumor “M2” phenotype rather than a pro-inflammatory “M1” phenotype. TAMs suppress T cell activation, produce VEGF, secrete proliferative factors, and encourage tumor invasion and metastasis. The inhibitory p50 subunit of the NF-kB transcription factor exhibits markedly increased nuclear expression in TAMs and M2-polarized macrophages [1], and p50 knockdown or deletion suppresses expression of M2-associated factors [1-3]. We hypothesize that absence of TAM p50 will convert TAMs to a pro-inflammatory M1 phenotype that will reduce tumor growth and prolong survival.in vitro with wild-type T cells, they do not suppress T cell proliferation to the same extent as wild-type BMM. These data suggest that NF-kB p50 is an important modulator of the suppressive TAM phenotype in GBM and that deletion of the gene encoding p50 promotes conversion to a pro-inflammatory phenotype that is less tumor-permissive. We anticipate that targeting p50 in combination with immune checkpoint inhibition might prove synergistic in facilitating anti-tumor immunity and tumor regression.The murine glioma cell line GL261-Luc was intracranially implanted into wild-type and p50(-/-) mice. Tumors grew 6-fold slower in p50(-/-) compared with wild-type mice, and p50(-/-) mice exhibited significantly increased survival. RT-qPCR analysis of CD11b+ myeloid cells isolated from the brains of tumor-bearing mice revealed decreased M2 marker expression and increased M1 marker expression in the absence of NF-kB p50. Evaluation of tumor-infiltrating T cells indicated that p50(-/-) mice possess decreased Treg cells, and that more p50(-/-) CD4 T cells induce IFNg expression after PMA/Ionomycin stimulation than wild-type CD4 T cells. When M2-polarized p50(-/-) bone marrow-derived macrophages (BMM) are co-cultured


C. elegans sgk-1 null mutants, GFP-tagged MIG-14/Wntless, the sorting receptor of Wnt, failed to localize to the basolateral membrane of intestinal cells; instead, it was mis-sorted to lysosomes. This effect can be explained in part by altered sphingolipid levels, because reducing glucosylceramide biosynthesis restored the localization of MIG-14::GFP. Membrane traffic was not perturbed in general, as no obvious morphological defects were detected for early endosomes, the Golgi apparatus, and the endoplasmic reticulum (ER) in sgk-1 null animals. The recycling of MIG-14/Wntless through the Golgi might be partially responsible for the observed phenotype because the subcellular distribution of two plasma membrane cargoes that do not recycle through the trans-Golgi network (TGN) was affected to a lesser degree. Consistently, knockdown of the ArfGEF gbf-1 altered the distribution of SGK-1 at the basolateral membrane of intestinal cells. In addition, we found that sgk-1(RNAi)  induced unfolded protein response in the ER, suggesting at least an indirect role of SGK-1 early in the secretory pathway. We propose that SGK-1 function is required for lipid homeostasis and that it acts at different intracellular trafficking steps.The mammalian serum- and glucocorticoid-inducible kinase SGK1 regulates the endocytosis of ion channels. Here we report that in  Mammalian serum- and glucocorticoid-inducible kinase 1 (SGK1) is an AGC kinase that was cloned as a gene whose transcription was stimulated by serum and glucocorticoids in rat mammary tumor cells . AlthougHow SGK1 regulates these ion channels and transporters is mostly unknown. Recently, it was suggested that SGK1 is involved in the endocytosis of membrane proteins . Cystic Endocytosis is a key process by which cells internalize molecules . ThroughIn yeast, Ypk1—a homologue of SGK-1—activates serine palmitoyl-CoA acyltransferase (SPT) and promotes the biosynthesis of ceramide and sphingolipid , 26. CerC. elegans, sgk-1 encodes the sole ortholog of mammalian SGK1. Compared to the wild type (WT), loss-of-function (lf) mutants of sgk-1 are abnormal in egg laying, development, stress response, and lifespan /[CGCAACGTATGATGGAGTGATTCT], primers #2 [TTCCGTGCTACATTGAAGCCGGTT]/[GCTTCGTCAGGGTTGATTCCACGA], primers #3 [GGACTTGTTCCGTGCTACATTGAAG]/[GCTTCGTCAGGGTTGATTCCACGA] and pmp-3F [GAATGGAATTGTTTCACGGAATGC]/ pmp-3R [CTCTTCGTGAAGTTCCATAACACGATG] for pmp-3 as the internal standard. qPCR was carried out on an ABI 7500 Fast real-time PCR system using a Takara real-time PCR kit (SYBR Premix Ex TaqTM II).Total RNA was extracted from synchronized L4 worms using TRIZOL (Invitrogen), followed by the removal of contaminant DNA using DNase I. cDNAs were synthesized from the total RNA templates using a reverse transcription kit (Takara). Primers used for qPCR of 2) OP50 mixed with an equal volume of myriocin or solvent control (methanol). Precipitation of myriocin occurred at 25.2 μM and 50.4 μM. The first batch of L4 larvae of the next generation were imaged.A 1 mg/ml myriocin (Sigma) stock solution was made in methanol. Gravid adults (five per plate) were transferred to plates containing UV-irradiated  and GLUT1 (glucose transporter 1) are plasma membrane receptors internalized via clathrin-independent endocytosis and recycled back to the plasma membrane through the endocytic recycling compartment (ERC) and RME-1-positive basolateral recycling endosomes [sgk-1(ok538) mutant, the pool of hTAC::GFP on the basolateral membrane decreased slightly. However, strikingly, large hTAC::GFP structures, possibly aggregated vesicles, accumulated in the cytoplasm (sgk-1(mg455) mutant, similar cytoplasmic accumulation of hTAC::GFP was observed, as well as a slight reduction of hTAC::GFP on the basal membrane (sgk-1(ok538) mutant worms. In wild type worms, only a small portion of the cytoplasmic hTAC::GFP puncta co-localized with early endosomes labeled by RFP::RAB-5 (sgk-1(ok538) mutant worms, most of the hTAC::GFP labeled large, aggregated structures that are also labeled by RFP::RAB-5  worms with 4.2 μM, 25.2 μM and 50.4 μM of Myriocin, but did not detect any obvious growth or morphological effect in either strain. Also, we did not see any effect of Myriocin on the localization pattern of either hTAC::GFP or MIG-14::GFP in wild type animals  backgrounds (sgk-1(ok538) mutant that were positive for RFP::RAB-5 and hTAC::GFP (sgk-1(ok538) had no effect on GFP::RAB-5, they were most likely protein aggregates that contained both hTAC::GFP and RFP::RAB-5. Alternatively, loss of sgk-1 may affect early endosomes only in a sensitized background such as when the trafficking system is overloaded with cargoes. Similarly, loss of sgk-1 did not change the morphology of the Golgi compartment labeled by mCHERRY::MANS (mannosidase), which appeared as dispersed punctate structures in the intestinal cells (sgk-1 mutant (sgk-1 induced unfolded protein response in the ER (UPR-ER) as indicated by an elevated expression of hsp-4pr::GFP (hsp-4 mRNA levels in sgk-1(ok538) L4 mutant worms . After subtracting background binding proteins and non-specific interacting proteins that co-immunoprecipitated with GFP alone or a number of unrelated GFP fusion proteins [To identify potential SGK-1 binding proteins on membranes, we performed co-immunoprecipitation experiments. Using GFP binding protein (GBP), which is a high-affinity single-chain antibody of GFP , we immuproteins , we founproteins –52. For proteins , 49. By proteins , 52. In proteins .arl-1, gbf-1, mon-2 and unc-11. We found that gbf-1(RNAi) caused an accumulation of hTAC::GFP in the cytoplasm  abolished specifically the lateral membrane localization of MIG-14::GFP along the circumferential  had no effect on steady-state GBF-1 localization (data not shown). In contrast, gbf-1(RNAi) affected the localization of SGK-1. SGK-1::GFP displayed a smooth, continuous distribution along the cortex of basolateral membrane of the intestinal cells. Upon gbf-1 RNAi, SGK-1::GFP at the cortex of basolateral membrane changed to small punctate structures  exacerbated the developmental phenotype of cgt-1/cgt-3 RNAi  animals , which catalyzes the first and the key step of ceramide , 26. Recs d17iso . It is ss d17iso . InactivL1 stage  or causeL1 stage . Our obst-3 RNAi  is consials Figs  and 5 by did not  Figs. PoS1 Figsgk-1(ok538) mutant animals  in red channel (A’ B’), green channel  and blue channel . Scale bars: 5 μm.Confocal images of intestinal cells of wild type worms  and (EPS)Click here for additional data file.S2 Figsgk-1(ok538) mutant animals (A’ to I’) in red channel , green channel  and blue channel  in “low” condition , “middle” condition  and “high” condition . Low, middle, high denote three different settings of pinhole size and detector gain. High means larger number and low means smaller number. Scale bars: 5 μm.Confocal images of intestinal cells of wild type worms (A to I) and (EPS)Click here for additional data file.S3 FigConfocal images of intestinal cells of wild type worms in green channel showing MIG-14::GFP  and blue channel  indicating the autofluorescence. The basolateral and apical membranes are indicated by arrows and arrowheads, respectively. The autofluorescent spots were different in shape and size from the MIG-14::GFP positive structures and the GFP signals in (EPS)Click here for additional data file.S4 Figsgk-1(ok538) mutant worms in green channel showing MIG-14::GFP  and blue channel  indicating the autofluorescence. The autofluorescent spots were different in shape and size from the MIG-14::GFP positive structures and the GFP signals in Confocal images of intestinal cells of (EPS)Click here for additional data file.S5 Figsgk-1(mg455) mutant worms in green channel showing MIG-14::GFP  and blue channel  indicating the autofluorescence. The autofluorescent spots were different in shape and size from the MIG-14::GFP positive structures and the GFP signals in Confocal images of intestinal cells of (EPS)Click here for additional data file.S6 FigConfocal images of intestinal cells of wild type worms in green channel showing hTAC::GFP  and blue channel  indicating the autofluorescence. The autofluorescent spots were different in shape and size from the hTAC::GFP positive structures and the GFP signals in (EPS)Click here for additional data file.S7 Figsgk-1(ok538) mutant worms in green channel showing hTAC::GFP  and blue channel  indicating the autofluorescence. The autofluorescent spots were different in shape and size from the hTAC::GFP positive structures and the GFP signals in Confocal images of intestinal cells of (EPS)Click here for additional data file.S8 Figsgk-1(mg455) mutant worms in green channel showing hTAC::GFP  and blue channel  indicating the autofluorescence. The autofluorescent spots were different in shape and size from the hTAC::GFP positive structures and the GFP signals in Confocal images of intestinal cells of (EPS)Click here for additional data file.S9 Figsgk-1(null)  intestinal cells expressing hTAC::GFP  or RFP::RAB-5 . Insets show magnified areas (× 2.5). The overlapped and non-overlapped regions are indicated by arrows and arrowheads, respectively. Scale bars: 5 μm.Confocal images of the wild type  and (EPS)Click here for additional data file.S10 Figsgk-1(ok538) (B) worms expressing GLUT1::GFP. The basolateral membranes are indicated by arrows. Scale bars: 5 μm.Confocal images of intestinal cells of young adult wild type (A) and (EPS)Click here for additional data file.S11 FigConfocal images of wild-type intestinal cells treated with control , 4.2 μM of myriocin , 25.2 μM of myriocin  and 50.4 μM of myriocin  expressing hTAC::GFP. The basolateral membranes were indicated by arrows. Scale bars: 5 μm.(EPS)Click here for additional data file.S12 FigConfocal images of wild-type intestinal cells treated with control , 4.2 μM of myriocin , 25.2 μM of myriocin  and 50.4 μM of myriocin  expressing MIG-14::GFP. The basolateral membranes were indicated by arrows. Scale bars: 5 μm.(EPS)Click here for additional data file.S13 FigSGK-1::GFP expression is seen in embryos, all larval stages and throughout adulthood. SGK-1::GFP is highly expressed in the intestine and the head and tail neurons, as described previously . The api(EPS)Click here for additional data file.S14 FigS. pombe [C. elegans proteins. MON-2N: 1–800 aa; MON-2C: 801–1648 aa; GBF-1N: 1–1147 aa; GBF-1C: 1148–1975 aa.Yeast cells were co-transformed with a GAL4BD-bait plasmid and a GAL4AD-prey plasmid as indicated. Among the double transformants  only two (AD-SGK-1/BD-COGC-3 and AD-SGK-1/BD-MON-2N) displayed positive interactions as they grew in the absence of Leu, Trp and His (–LWH). The apparent positive interaction of AD-SGK-1/BD-UNC-11 was false because BD-UNC-11 showed self-activating activity independent of the prey (AD-SGK-1 or AD-Xrc4). Xrc4 is a negative control; it is a nonhomologous end joining factor from S. pombe  and is n(EPS)Click here for additional data file.S15 Figarl-1 RNAi , gbf-1 RNAi , mon-2 RNAi , and unc-11 RNAi  and sgk-1(ok538) mutants  expressing hTAC::GFP. Scale bars: 5 μm.Confocal images of intestinal cells of wild type worms treated with control RNAi , (EPS)Click here for additional data file.S16 Figarl-1 RNAi , gbf-1 RNAi , mon-2 RNAi , and unc-11 RNAi  and sgk-1(ok538) mutants  expressing MIG-14::GFP. The basolateral membranes are indicated by arrows. Scale bars: 5 μm.Confocal images of intestinal cells of wild type worms treated with control RNAi , (EPS)Click here for additional data file.S17 Figgbf-1 RNAi treatment. * P value <0.05 (Student’s t-test).The overall fluorescence intensity of SGK-1::GFP was unaffected by (EPS)Click here for additional data file.S1 File(AVI)Click here for additional data file.S2 File(AVI)Click here for additional data file.S3 File(AVI)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.