Stress granules (SGs) are cytoplasmic granular aggregations that are induced by cellular stress, including viral infection. SGs have opposing antiviral and proviral roles, which depend on virus species. The exact function of SGs during viral infection is not fully understood. Here, we showed that mumps virus (MuV) induced SGs depending on activation of protein kinase R (PKR). MuV infection strongly induced interferon (IFN)-λ1, 2 and 3, and IFN-β through activation of IFN regulatory factor 3 (IRF3) via retinoic acid inducible gene-I (RIG-I) and the mitochondrial antiviral signaling (MAVS) pathway. MuV-induced IFNs were strongly upregulated in PKR-knockdown cells. MuV-induced SG formation was suppressed by knockdown of PKR and SG marker proteins, Ras-GTPase-activating protein SH3-domain-binding protein 1 and T-cell-restricted intracellular antigen-1, and significantly increased the levels of MuV-induced IFN-λ1. However, viral titer was not altered by suppression of SG formation. PKR was required for induction of SGs by MuV infection and regulated type III IFN (IFN-λ1) mRNA stability. MuV-induced SGs partly suppressed type III IFN production by MuV; however, the limited suppression was not sufficient to inhibit MuV replication in cell culture. Our results provide insight into the relationship between SGs and IFN production induced by MuV infection. Mumps is an infectious disease caused by mumps virus (MuV) and is characterized by swelling of the parotid gland . Mumps hThe innate immune response is known to be one of the most important defense mechanisms against pathogenic bacteria, viruses, and foreign antigens. The innate immune sensors in host cells, called pattern recognition receptors (PRRs), detect pathogen-associated molecular patterns and initiate antimicrobial immune responses . PRRs coCellular stress, such as heat shock, hypoxia, and viral infection, induces formation of cytoplasmic granules called stress granules (SGs) . SGs areIn the present study, we demonstrated that MuV-induced SG formation was dependent on PKR. The PKR-dependent SGs partly suppressed production of IFN, especially IFN-λ; however, this did not affect viral replication. We discuss the relationship between SG formation and MuV-induced IFNs.Rabbit monoclonal antibodies (mAbs) against phospho-(P-)IRF3(Ser96) (4D4G), eIF2α(D7D3) and P-eIF2α(Ser51) (D9D8), and rabbit polyclonal antibody (PcAb) against PKR (3072) were purchased from Cell Signaling Technology . Rabbit mAb against P-PKR(Thr451) EPR2152Y) (ab81303) and mouse mAb against G3BP1 (ab56574) were purchased from Abcam . Rabbit PcAbs against IRF3 (309033), RIG-I (PAB12973), melanoma differentiation-associated gene 5 (MDA5) (29020), Hu-antigen R (HuR), and insulin-like growth factor 2 binding protein (IGF2BP)1 were purchased from Active Motif , Abnova , Immuno-Biological Laboratories , Medical and Biological Laboratory  and Abgent , respectively. Mouse mAbs against MAVS (E-3) and β-actin (AC-74) were purchased from Santa Cruz Biotechnology  and Sigma–Aldrich , respectively. Rabbit PcAb against mumps P/V protein (T61), and mouse mAb against mumps F protein (44C) were generated as previously described . Experimental methods and preparation of human respiratory syncytial virus (RSV) were as described previously  octane (Sigma–Aldrich) and 50% glycerol in PBS. The specimens were examined and photographed using confocal microscopy .Cytokines in cell culture supernatants were determined using an enzyme-linked immunosorbent assay (ELISA). Human IFN-λ1, 2 and 3 were measured using respective DuoSet ELISA Development Systems . Human IFN-β ELISA kit was purchased from Kamakura Techno-Science .Silencer selected small-interfering RNAs (siRNAs) were obtained from Thermo Fisher Scientific. Targeting siRNA was transfected using Lipofectamine RNAimax Transfection Reagent (Thermo Fisher Scientific).5′-cggtctaatggagggtcaga-3′ and reverse 5′-cagtgctacaaatcgcctca-3′; MuV P/V forward, 5′-cattcagggaaccaactcgt-3′ and reverse 5′-aaattctgttaccggcgttg-3′. cDNA was synthesized using SuperScript III First-Strand Synthesis Kit (Thermo Fisher Scientific). Primer sets of IFN-λ1 and Mx1 were purchased from Thermo Fisher Scientific. Hypoxanthine phosphoribosyltransferase (HPRT)1 and IFN-β primer sets for 5′ nuclease assay were synthesized by Integrated DNA Technologies . Primer set and probe sequence of IFN-β were: 5′-/56-FAM/CAGACAAGA/ZEN/TTCATCTAGCACTGGCTGG/3lABkFQ/-3′, forward, 5′-GCCGCATTGACCATCTATG-3′, and reverse, 5′-GCCAGGAGGTTCTCAACAATAG-3′. G3BP1 and TIA-1 mRNA was quantified using specific primers: G3BP1 forward 5′-GCAGATGCAGTCTACGGACA-3′, reverse 5′-TCACCTGGACTACCACACCA-3′ and TIA-1 forward 5′-AAGCTGCTTTTGCACCATTT-3′ and reverse 5′-TTCAGCATCCCATTTGTTGA-3′. Real-time PCR was performed using Thunderbird Probe qPCR Mix or KOD SYBR qPCR mix  by LightCycler480 . mRNA was normalized with HPRT1 and relative quantitation was calculated by ΔΔCt methods.Total RNA was purified using an RNeasy Plus Mini Kit . Reverse transcription polymerase chain reactions (RT-PCRs) were performed using the Onestep RT-PCR kit (Qiagen) and specific primers as below: MuV F forward, Reporter plasmids pIFN-βP125-Luc and pPRDIII-I-Luc were constructed as described previously . pGL3-ELg for 5 min, protein concentration in supernatant was determined using the bicinchoninic acid method (Thermo Fisher Scientific). Sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting were performed as described previously , IFN-λ2 (IL-28A) and IFN-λ3 (IL-28B) were discovered by two independent groups in 2003 , 32. IFNWe found that protein and mRNA levels of IFN-λ1 induced by MuV were markedly higher than those of IFN-β . IFN-λ1 There are three types of SG formation depending on virus species: stable formation, transient formation, and no formation . MuV infMuV infection induced strong PKR phosphorylation and weak eIF2α phosphorylation. Although production of IFN, especially IFN-λ, was potentially suppressed by SGs induced by MuV infection in a PKR-dependent manner, the SGs did not promote viral replication. There are two possible reasons why SG-dependent suppression of IFNs had no effect on viral replication. (i) PKR induces SG formation in viral infection, and has antiviral activity, as an ISG , 52. ForIn conclusion, MuV induces SGs in a PKR-dependent manner and partly suppresses MuV-induced IFN production. MuV infection induces type I and III IFNs, and RIG-I and MAVS are important molecules for IFN production. Our results promote understanding of the relationship between virus-induced SGs and the innate immune response; however, further studies are needed to understand the role of SGs and innate immune responses against viral infection, including mumps.


Salinomycin is an antibiotic that has been shown to target cancer stem cells (CSC); however, the mechanisms of action involved have not been well characterized. The objective of the present study was to investigate the effect of salinomycin on cell death, migration, and invasion, as well as CSC-like properties in MDA-MB-231 breast cancer cells. Salinomycin significantly induced anoikis-sensitivity, accompanied by caspase-3 and caspase-8 activation and PARP cleavage, during anchorage-independent growth. Salinomycin treatment also caused a marked suppression of cell migration and invasion with concomitant downregulation of MMP-9 and MMP-2 mRNA levels. Notably, salinomycin inhibited the formation of mammospheres and effectively reduced the CD44+/CD24- stem-like population during anchorage-independent growth. These observations were associated with the inhibition of STAT3 phosphorylation (Tyr705). Furthermore, interleukin-6 (IL-6)-induced STAT3 activation was strongly suppressed by salinomycin challenge. These findings support the notion that salinomycin may be potentially efficacious for targeting breast cancer stem-like cells through the inhibition of STAT3 activation.Triple-negative breast cancer (TNBC) is an aggressive tumor subtype with an enriched CD44 The quantity of formazan product was determined by measuring absorbance at 490 nm using a Spectramax Plus384 microplate analyzer .Cells were solubilized in lysis buffer  containing phosphatase and protease inhibitor cocktail tablets. Supernatant was collected after centrifugation  and protein concentrations were measured with a Bradford protein assay kit (Bio-Rad Laboratories). Equal quantities of protein (30 μg) were subjected to SDS-PAGE and electrotransferred onto a nitrocellulose membrane . The membranes were incubated overnight at 4°C with primary antibodies diluted in 5% BSA , followed by incubation with horseradish peroxidase (HRP)-conjugated anti-rabbit and mouse IgG . Signal intensity was detected using an Enhanced Chemiluminescence Kit  and X-ray film  and quantitated using AlphaEaseFC software .Cells were harvested and fixed with 95% ethanol containing 0.5% Tween-20 for 24 h, washed with PBS, and incubated with propidium iodide  and RNase (50 μg/ml) for 30 min. Sub-G1 analysis was conducted by flow cytometry using a Beckman Coulter Expo.+/CD24- staining analysis, cells were incubated for 30 min at 4°C with FITC- and PE-conjugated anti-mouse IgG or FITC-conjugated anti-CD24 and PE-conjugated anti-CD44 antibodies (BD Biosciences). Cells were analyzed by flow cytometry using a Beckman Coulter Expo.Cells were stained using a FITC-conjugated Annexin V apoptosis detection kit  according to the manufacturer’s protocol. For CD44Cells were plated in ultralow attachment dishes  and cultured in HuMEC basal serum-free medium (Gibco), supplemented with B27 , 20 ng/mL basic fibroblast growth factor , 20 ng/mL human epidermal growth factor , 4 μg/ml heparin, 1% antibiotic-antimycotic agent, and 15 μg/mL gentamycin. The number and volume of mammospheres were determined under an Olympus IX 71 inverted microscope and photos were acquired with Olympus DP capture software.Cells on 8-well chamber slides (BD Biosciences) were fixed with 4% PFA, washed with PBS, and permeabilized with 0.2% Triton X-100 for 10 min. Cells were incubated with primary antibodies in antibody diluent  overnight at 4°C and then with Alexa Fluor-488 goat anti-rabbit and mouse IgG (Invitrogen) secondary antibodies. Cells were mounted with ProLong Gold Antifade Reagent with DAPI . Images were acquired using a Carl Zeiss confocal microscope .5’-TCT CCT GAC ATT GAC CTT GGC-3’, reverse 5’-CAA GGT GCT GGC TGA GTA GAT C-3’; MMP-9, forward 5’-TTG ACA GCG ACA AGA AGT GG-3’, reverse 5’-CCC TCA GTG AAG CGG TAC AT-3’; cyclin D1, forward 5’-ACA CGG ACT ACA GGG GAG TTT-3’, reverse 5’-GGA AGC GGT CCA GGT AGT TC-3’; actin, forward 5’-ACC CAG ATC ATG TTT GAG AC-3’, reverse 5’-GGA GTT GAA GGT AGT TTC GT-3’. The PCR products were separated on 1.2% agarose gels and visualized using a Gel Doc™ XR+ System (Bio-Rad Laboratories).Total RNA was extracted using the RNeasy mini kit , according to the manufacturer’s instructions. Transcript amplification was achieved by reverse transcriptase-polymerase chain reaction (RT-PCR) using 1 μg/μl total RNA, Molony Murine Leukemia Virus reverse transcriptase , and oligo-d(T)15 primer (Roche Applied Sciences). PCR amplification was performed using the following primers: MMP-2, forward 2. Invaded cells were fixed and stained with Diff-Quik  and counted under a BX51 microscope .Invasion chambers were coated with Matrigel matrix (BD Biosciences) according to the manufacturer’s recommendations. Cells were treated with salinomycin and then trypsinized, washed, and transferred to the upper chambers in serum-free media. Conditioned media was added to the lower chambers. The chambers were incubated for 48 h at 37°C in a humidified atmosphere of 5% CO™ ZOOM® Kinetic Imaging System. The relative wound density was analyzed using the IncuCyte™ Scratch Wound Cell Migration Software Module.For kinetic migration analysis, cells were seeded to ~90% confluency in 96-well plates . Wound areas were made with a 96-pin Wound Maker device and washed with PBS to prevent reattachment of dislodged cells. Cells were treated with salinomycin immediately after wound scratching, and images of the scratched fields were automatically acquired and registered every hour for 24 h with an IncuCytepost hoc test. Statistical significance was defined at p < 0.05 (*).All data were analyzed using GraphPad Prism 5.0 statistical software . The results are presented as mean ± SEM of at least three independent experiments. Data were analyzed by Student’s t test and one- or two-way ANOVA, as appropriate. A two-way ANOVA was used to assess the effects and interactions of two variables and multiple comparisons were performed using the Bonferroni p<0.05, p = 0.5639, Recent reports have demonstrated that salinomycin exerts anticancer effects by inhibiting cell growth, inducing apoptosis, and overcoming ABC transporter-mediated multidrug resistance in several cancers –32. To eWe therefore speculated that salinomycin might be sensitizing the TNBC cells to anoikis. Cells undergo anoikis in the absence of cell-ECM interaction via intrinsic and extrinsic apoptotic pathways ,17. We cRecent reports have shown that STAT3 activation is associated with anoikis and that phospho-STAT3 (Tyr705) levels are elevated in anoikis-resistant melanoma and pancreatic cancer cells ,19,33. WWe next focused on the effect of salinomycin on STAT3 downstream target genes such as cyclin D1 and the matrix metalloproteinases MMP-2 and MMP-9 ,37,38. Ckip1 E3 ligase) in ovarian cancer cells in vitro. Furthermore, salinomycin induces destabilization of Skp2 via ubiquitin-mediated proteasomal degradation, which leads to cyclin-dependent kinase inhibitor p27kip1 upregulation, thereby contributing to G1 phase arrest and induction of apoptosis [Similar to these findings, salinomycin has been shown to induce STAT3 inactivation and subsequently reduce expression levels of its downstream target genes such as cyclin D1, survivin, and Skp2  , and inh in vivo .+/CD24- levels in several breast cancer cell lines. The results revealed that MDA-MB-231 cells harbored the highest CD44+/CD24- stem-like population, at approximately 98%  has emerged as a CSC marker in breast cancer ,45, promin vitro . Therefoin vitro . Althoug31 cells .+/CD24- stem-like population, whereas neither salinomycin nor STAT3 inhibitors alone could induce this effect . (B) Effect of salinomycin  on activation of JAK2 and STAT3 in anchorage-dependent and -independent cells. The graphs represent the ratios of phospho-JAK2/JAK2 and phospho-STAT3/STAT3 . The results are presented as mean ± SEM (n = 3 independent experiments) and were analyzed by one- or two-way ANOVA as appropriate, followed by Bonferroni’s post hoc test.(A) After exposure to salinomycin (1–5 μM) or DMSO for 48 h, protein levels of JAK2, phospho-JAK2 (Tyr1007/1008), STAT3, and phospho-STAT3 (Tyr705) were determined using Western blot analysis. Quantitative graphs of phospho-JAK2/JAK2 ratio and phospho-STAT/STAT3 ratio are shown Click here for additional data file.S2 Figp<0.001). (B) Effect of salinomycin on mRNA expression of Nanog and Sox2. Quantitative graphs represent mRNA abundance of Nanog and Sox2 . The p values are displayed. All experiments were independently repeated 3 times (n = 3).(A-B) MDA-MB-231 cells were treated with salinomycin (2 μM) or DMSO for 48 h in anchorage-independent conditions. (A) Effect of salinomycin on ALDH1 activity. Cells were incubated with the ALDH protein substrate BAAA in the presence or absence of DEAB (a specific inhibitor of ALDH) to define the ALDEFLUOR-positive cell population. Quantification of ALDEFLUOR-positive population Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


We found that MSC osteogenic differentiation was inhibited in the presenceof the activated immune response and that overexpression of CTLA4 inallo-MSCs suppressed the immune response and promoted osteogenic differentiation. Ourresults support the application of CTLA4-overexpressing allo-MSCs inbone tissue engineering.Allogeneic mesenchymal stem cells  have recently garnered increasinginterest for their broad clinical therapy applications. Despite this, many studieshave shown that allo-MSCs are associated with a high rate of graft rejection unlessimmunosuppressive therapy is administered to control allo-immune responses. CytotoxicT-lymphocyte-associated protein 4 (CTLA4) is a co-inhibitory molecule expressed on Tcells that mediates the inhibition of T-cell function. Here, we investigated theosteogenic differentiation potency of allo-MSCs in an activated immune system thatmimics the  Bone marrow-derived mesenchymal stem cells (MSCs) are a type of pluripotent stem cellthat possesses the capacity for self-renewal and differentiation into multiplefunctional cell types . Althougin vitro and in vivo(The low immunogenicity of MSCs is attributable to their expression of majorhistocompatibility complex (MHC) class I and adhesion molecules including vascular celladhesion molecule 1 (VCAM-1), intracellular adhesion molecule 1 (ICAM-1), and lymphocytefunction-associated antigen 3 (LFA-3) and their lack of expression of MHC class IImolecules  . Indeed, in vivo,8. Despi in vivo–13. For in vivo found th in vivo. In this in vivo. AccordiCTLA4-Igdifferentiate normally into osteoblasts in vitro and form bone tissuein xenotransplantation models in the absence of general immunosuppression (invitroimmune activation microenvironment and to characterize the mechanismunderlyingCTLA4 promotion of MSC osteogenic differentiation.Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) is an important co-inhibitorymolecule expressed on activated T cells that mediates T-cell anergy, apoptosis, andclone deletion and is associated with increased immune tolerance . The mecpression . Accordig for 20 min at20°C. Nucleated cells were collected from the interface, diluted with 20 mL PBS, andcentrifuged at 900g for 10 min at 20°C. Cells were resuspended inDulbecco's modified Eagle's medium (DMEM) containing F12  and 10% fetalbovine serum . Cells were cultured at a density of7.5×106 cells per 37.5 cm2 flask in F12-DMEM containing 10%FBS at 37°C and 5% CO2. When subconfluent, cells were detached using 0.05%trypsin-EDTA (Gibco) and subcultured at a density of 1.8×105 cells per37.5-cm2 flask. After the third passage, cells were harvested fordetection of surface antigens by flow cytometry. Cells (5×105) wereincubated for 30 min with fluorescein isothiocyanate (FITC)-labeled anti-CD105monoclonal antibody  and phycoerythrin (PE)-labeled anti-CD34 monoclonalantibody (Sigma). FITC-labeled anti-IgG2b monoclonal antibody (Sigma) and PE-labeledanti-IgG1 monoclonal antibody (Sigma) were used as isotype controls. Cells werewashed with PBS and resuspended in PBS containing 1% formalin and 0.1% bovine serumalbumin . Data were acquired on a FACSCalibur platform , and CD105-positive/CD34-negative populations were designated as BMMSCs.MSCs were isolated and cultured using a previously described method . BrieflyCTLA4-Ig(pAdEasy-CTLA4-EGFP) and EGFP(pAdEasy-EGFP) were gifts from Dr. Jun Wu . Viral solutions at a titer of 3×106 colony formingunits (CFUs)/mL were used to infect MSCs at 70% confluence. Green fluorescent protein(GFP)-positive cells were detected by fluorescence microscopy, andCTLA4 expression was characterized by flow cytometry and Westernblot (see respective sections).Recombinant adenoviruses expressing genes encoding humanPeripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradientcentrifugation of heparinized peripheral blood collected from healthy humanvolunteers. PBMCs were cultured in RPMI 1640 medium (Gibco) supplemented with 100U/mL penicillin and 100 U/mL streptomycin, 2 mM glutamine, and 10% FBS (Gibco). PBMCswere stimulated with 2.5 μg/mL phytohemagglutinin  for 72 h to establishthe immune activation microenvironment.5) were stimulated with 2.5 μg/mL PHA for 24 h, after whichcells were harvested, washed, and stained for 30 min with FITC-labeled anti-CD80monoclonal antibody (BD Biosciences) and PE-labeled anti-CD86 monoclonal antibody (BDBiosciences). FITC-labeled anti-IgG2b monoclonal antibody (BD Biosciences) andPE-labeled anti-IgG2a (BD Biosciences) were used as isotype controls, respectively.Cells were washed with PBS and resuspended in PBS containing 1% formalin and 0.1%BSA. Data were acquired on a FACSCalibur platform (BD Biosciences) and analyzed usingthe FlowJo software .PBMCs (5×105) were cocultured with 1×105 or 5×104γ-irradiated (30 Gy) MSCs orCTLA4-infected MSCs (MSCs-CTLA4) in aU-bottom 96-well plate for 3 days in the presence or absence of 2.5 μg/mL PHA. Cellproliferation was measured using a Cell Counting Kit-8 assay according to the manufacturer's instructions. Levels of interleukin (IL)-2 andinterferon (IFN)-γ in supernatants were measured by enzyme-linked immunosorbent assay(ELISA) in accordance with the manufacturer's instructions .PBMCs (5×10In vitro osteogenic differentiation of MSCs was performed asdescribed previously (eviously . BrieflyRUNX2 (125 bp): 5′-AGATGATGACACTGCCACCTCTG-3′(F), 5′-GGGATGAAATGCTTGGGAACTGC-3′ (R); ALP(162 bp):5′-ACCATTCCCACGTCTTCACATTTG-3′ (F), 5′-AGACATTCTCTCGTTCACCGCC-3′ (R);Collagen 1 (147 bp): 5′-CCTGGAAAGAATGGAGATGATG-3′ (F),5′-ATCCAAACCACTGAAACCTCTG-3′(R);GAPDH (159 bp):5′-ACCCATCACCATCTTCCAGGAG-3′ (F), 5′-GAAGGGGCGGAGATGATGAC-3′(R). Relative expressionlevels were calculated by normalizing to the expression of the housekeeping geneglyceraldehyde 3-phosphate dehydrogenase (GAPDH).Total RNA was extracted using an RNA Pure Separate Extraction kit according to the manufacturer's instructions. cDNA was synthesized using First StrandcDNA Synthesis Kit according to the manufacturer's instructions . Real-time PCR was performed on an ABI7500 real-time PCR system using SYBR Premix EX Taq™ II . The primersused were as follows:Total protein was extracted with radioimmunoprecipitation assay (RIPA) lysis buffer and separated by 8% sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE), then transferred onto polyvinylidene fluoride membranes. After blocking nonspecific protein binding with 5% BSA, blots wereincubated with primary antibodies against CTLA4 ,Collagen 1 , Runx2  andGAPDH . After extensive washing with PBS containing 0.1%Triton X-100, membranes were incubated with horseradish peroxidase (HRP)-conjugatedsecondary antibody  for 30 min at room temperature. Thesignals were visualized by ChemiDoc XRS  using an EnhancedChemiluminescence kit  and analyzed using the ImageJ2xsoftware (USA).t-tests.Analyses were performed using the GraphPad Prism 5.0 statistical software package. P<0.05 was considered to be statisticallysignificant.Results are reported as means±SD. The statistical significance of differences betweengroups was analyzed using two-tailed independent Student's CTLA4-Ig in MSCs wasdetermined by flow cytometry (Fluorescent-activated cell sorting (FACS) analysis demonstrated expression of CD105and a lack of expression of CD34 in third-passage MSCs, indicating their successfulexpansion (data not shown). Expression ofytometry  andfluoytometry , whichsytometry .invitro.Next, to investigate the effect of allo-MSCs on T lymphocyte proliferation,γ-irradiated MSCs-CTLA4 or MSCs were cocultured with PBMCs at a ratio of 1:5 or 1:10in the presence or absence of 2.5 μg/mL PHA. We found that lymphocyte proliferationwas inhibited by allo-MSCs-CTLA4 at both ratios in the presence or absence of PHA,but not by allo-MSCs , confirmConsistent with this, levels of IL-2 and IFN-γ in the supernatant of MSCs-CTLA4 weresignificantly lower than in the supernatants of MSCs and PHA-positive control cellsand E.CTLA4 expression on MSC osteogenicdifferentiation in an immune activation state. MSCs-CTLA4 and MSCs exhibitedcomparable rates of osteogenic differentiation when cultured in osteogenic mediumalone. However, when cells were cocultured with PHA-stimulated PBMCs in osteogenicmedium, expression of Runx2, Collagen 1, and ALP were significantly lower at both theprotein , and NK cellshas been shown to result in immune rejection  signaltransduction by binding to TCRζ and inhibiting tyrosine phosphorylation after T cellactivation (CTLA4 has beenshown to suppress T cell activation and function via recruitment of the phosphatases SH2domain-containing tyrosine phosphatase 1 (SHP1), SHP2, and serine/threonine proteinphosphatase 2A (PP2A). These phosphatases dephosphorylate several of the major signalingnodes such as nuclear factor of activated T cells (NF-AT), NF-κB, mammalian target ofrapamycin (mTOR) and activator protein 1 (AP-1), which are essential for costimulationof T cells . Indeed, although grafted allo-MSCs have been shownto be capable of prolonged survival, they fail to successfully differentiate into bonetissue in vivo (CTLA4 promoted the osteogenic differentiation ofMSCsin vitro, whether it has similar effects invivo will require further study. Secondly, although adenoviruses achievemore efficient gene transfer than retroviruses and do not integrate the target gene intothe host cell genome, their potential tumorigenicity restricts their clinicalapplications (CTLA4 overexpression inallo-MSCs in vivo.Our study has a number of limitations. The intricate nature of bone reconstructioninvolves the interactions of variety of cells  in vivo .Consequications . ConsequCTLA4 reversed this inhibition. Our study holds promise for thefuture success of allo-MSC engraftment in bone engineering and holds out the possibilitythat CTLA4-expressing MSCs can give rise to bone tissue invivo, even in the setting of immune-mediated rejection.In conclusion, our results demonstrated that the osteogenic differentiation of allo-MSCsis compromised in an activated immune environment and that expression in these cells ofClick here toview [pdf]


Staphylococcus aureus infection. Expression of cytokine genes induced by Staphylococcus aureus was not inhibited by a caspase-1 inhibitor, and did not occur through an autocrine mechanism in response to newly synthesized cytokines. We also demonstrated that NLRP3 could activate NF-κB and induce cytokines in response to sterile signals, monosodium urate crystals and aluminum adjuvant. Thus, NLRP3 mediates NF-κB activation in both sterile and microbially induced inflammation. Our findings show that not only does NLRP3 activate caspase-1 post-translationally, but it also induces multiple cytokine genes in the innate immune system.Nucleotide-binding domain and leucine-rich repeat-containing family, pyrin domain containing 3 (NLRP3) has recently emerged as a central regulator of innate immunity and inflammation in response to both sterile inflammatory and microbial invasion signals. Although its ability to drive proteolytic procaspase-1 processing has drawn more attention, NLPR3 can also activate NF-κB. To clarify the physiological relevance of this latter function, we examined the effect of NLRP3 on NF-κB activation and cytokine induction in RNA-interference-based NLRP3-knockdown cell lines generated from the human monocytic cell line THP-1. Knocking down NLRP3 reduced NF-κB activation and cytokine induction in the early stages of  Nucleotide-binding domain and leucine-rich repeat-containing family, pyrin domain containing 3 , such as bacterial and viral RNA and muramyl dipeptide ; damage-NLRP3−/− or ASC−/− mice [For macrophages to produce mature IL-1β, two signals are required. The first  activates NF-κB and induces pro-IL-1β synthesis, and the second  activates caspase-1 and induces the proteolytic maturation of pro-IL-1β. Since NF-κB activation in response to PAMP stimulation and bacterial infection is normal in macrophages from −/− mice , 13, NLRPorphyromonas gingivalis infection [Genetic reconstitution experiments using HEK293 cells have shown that human NLRP3 can activate NF-κB , 24 in cnfection . HoweverTo address human NLRP3's role in the NF-κB signaling pathway, we generated THP-1-derived NLRP3-knockdown cells and demonstrated that NLRP3 mediates NF-κB activation and cytokine gene induction in a caspase-1 independent manner. More importantly, NLRP3 could activate NF-κB and induce cytokines following stimulation by monosodium urate (MSU) crystals and aluminum adjuvant, sterile activators for NLRP3. These results suggest that NLRP3 is important for NF-κB activation, especially in sterile inflammatory diseases.Doxycycline (Dox) was purchased from AppliChem , recombinant human IL-1Ra from Genzyme , recombinant human IL-1β from PeproTech , Ac-YVAD-CMK from Bachem , bafilomycin A1 from Fermenteck , and CA-074Me from Merck . Diphenyleneiodonium (DPI) was purchased from Enzo . LPS from Escherichia coli K235 was purchased from Sigma-Aldrich . ATP was purchased from YAMASA CORPORATION . Cycloheximide (CHX) and MSU crystals were purchased from Wako Pure Chemical Industries . Aluminum adjuvant (Imject Alum) was purchased from Pierce . Ficoll-Paque PLUS was purchased from GE Healthcare Japan .Expression cassettes for artificial micro RNAs (miRNAs) targeting NLRP3, ASC, and MyD88 and for negative control miRNAs were generated using the BLOCK-iT Pol II miR RNAi Expression Vector Kit  and the following target sequences: for NLRP3, 5′-TCCTGAATCAGACTGAAGGCT-3′; for ASC, 5′-TAGGTCTCCAGGTAGAAGCTG-3′; and for MyD88, 5′-TCTCCAAGTACTCAAAGTCCA-3′. The DNA fragment containing the microRNA expression cassette was cloned into a Tet-on lentiviral vector pLVCT-tTRKRAB  and transferred into HEK293FT packaging cells using the packaging vectors pMD2G and psPAX2. Recombinant lentiviruses collected from the culture supernatant were used to transduce THP-1 cells. We used vector-coded GFP to select THP-1 cells that expressed pLVCT-tTRKRAB-derived miRNA. The collected cells were cultured without Dox or treated with Dox for 5 d to induce miRNA expression.The target sequence for NLRP3 (5′-GCTGGAATTGTTCTACTGTTT-3′) and a negative control sequence (5′-CCTAAGGTTAAGTCGCCCTCG-3′) were used to generate oligonucleotide pairs that were inserted into the pLKO.1 TRC cloning vector (Addgene) to produce lentiviral vectors that were used to transduce THP-1 cells. THP-1 cells stably expressing shRNA were selected by puromycin resistance for 2 weeks, and these cells were used for experiments.5 cells were primed with LPS (20 ng/ml) for 12 h and then with ATP (3 mM) for 1 h. Unprimed THP-1 and THP-derived cells were used for bacterial-infection and MSU-stimulation experiments. For bacterial-infection experiments, S. aureus  was grown in Luria broth until OD 1.9–2.2 at 600 nm, then collected and washed in RPMI 1640 with 10% FCS.The THP-1 cell line (ATCC TIB-202) and its derivatives expressing miRNA or shRNA were cultured in RPMI 1640 medium supplemented with 10% FCS. To induce IL-1β release, 1x1050 ml of fresh blood from a healthy donor was fractionated using 1.074 g/ml density barrier solution as described . A monocThe NLRP3-targeted (HSS132811), MyD88-targeted (#428431 and #42832), ASC-targeted (HSS147064), and control siRNA (12395-115 and 12395-113) were purchased from Life Technologies. THP-1 and primary human monocyte were transfected with siRNA (20 nM) using the Neon Transfection System (Life Technologies).Immunoblotting was performed as described previously . A hybriThe amount of human TNF-α, IL-1β, and IL-8 in culture supernatants was determined using the OptEIA ELISA kit  according to the manufacturer’s protocol.EMSA was performed as described previously .Real-time PCR was performed with primers for TNF-α , IL-1β , and MyD88 . Semiquantitative RT-PCR was performed as described previously  with priP-values are indicated in the text and figures above the two groups. P <0.05 was considered statistically significant.All experiments were done in duplicate or triplicate and repeated at least three times. Error bars indicate standard deviations. Statistical significance of difference between two experimental groups was accessed by two-tailed Student’s t-test. To determine NLRP3's role in activating NF-κB in human monocytes, we generated Dox-inducible, artificial miRNA-based NLRP3- or ASC-knockdown cell lines  by transducing human monocytic THP-1 cells with Tet-on-regulated lentiviral vectors expressing miRNA against NLRP3 or ASC. Before Dox treatment, the NLRP3 and ASC protein levels in miNLRP3 and miASC cells, respectively, were the same or slightly lower than those in cell lines transduced with negative-control miRNA (miCtrl) . After DS. aureus infection [S. aureus infection  were activated early in is study . HoweverTogether, these results indicate that NLRP3 plays an important role in activating NF-κB and inducing cytokine genes in THP-1 cells in response to microbial infection.S. aureus infection in the Dox-treated MyD88-knockdown cells, TNF-α release evaluated by ELISA  cells even before Dox treatment, and was reduced further by Dox treatment . Followiby ELISA  and NF-κTo further investigate the pathophysiological significance of the NLRP3-mediated NF-κB activation, we examined the effect of NLRP3 knockdown on uric acid-induced inflammatory responses. MSU-induced TNF-α and IL-1β mRNA was reduced in the NLRP3-knockdown (shNLRP3) cells compared to control (shCtrl) cells . ConsistTo further confirm the role of NLRP3 in sterile inflammation, we next examined the effect of NLRP3 knockdown on aluminum adjuvant-induced inflammatory responses. For this purpose, PMA-primed shCtrl and shNLRP3 cells were stimulated with aluminum adjuvant. In shNLRP3 cells, the extent of aluminum adjuvant-induced IL-1β mRNA induction was significantly lower than those in shCtrl cells . SimilarS. aureus infection  has been unclear. It has been proposed that TLRs might mediate signal 1 by sensing DAMPs such as high mobility group box-1, hyaluronic acid, and biglycan, which are released from dead cells or extracellular matrix upon tissue injury , 31–35. S. aureus-induced activation of NF-κB but not MAPKs, indicating that ASC mediates the NLRP3-induced NF-κB activation, but NLRP3 and ASC are not involved in MAPK activation under these conditions. The different results in terms of the involvement of ASC in MAPK activation may be due to different infectious agents between this and the previous study.Genetic reconstitution experiments with HEK293 cells have demonstrated that ASC can mediate NF-κB activation. ASC has also been reported to mediate NF-κB and MAPK activation following bacterial infection in THP-1 cells , 36. HowS. aureus-induced NF-κB activation and cytokine gene expression in THP-1 cells (data not shown). Thus, at least one more mechanism exists for ASC-mediated NF-κB activation under these conditions. In this context, it should be note that NLRP3 and ASC have been reported to interact with MAVS and RIPK2, respectively [Our previous genetic reconstitution experiments using HEK293 cells demonstrated that caspase-8 is involved in ASC-mediated NF-κB activation , 37. Howectively –40, thatAlthough NLRP3 has been linked to inflammasome in many microbially and sterile inflammatory responses, inflammasome-independent role for NLRP3 has also been reported –43. In oIL1B.In summary, our results indicate that NLRP3 mediates the NF-κB activation in sterile inflammatory and microbially induced immune responses. So far, NLRP3 has been implicated in procaspase-1 activation through inflammasome formation. Our findings significantly broaden NLRP3's physiological importance in innate immunity: NLRP3 not only activates caspase-1 post-translationally, but also induces multiple cytokine genes, including S1 FileS. aureus infection was dampened in Dox-treated NLRP3- and ASC-knockdown cells. Fig. B, NLRP3 and ASC was dispensable for TNF-α and IL-8 production at 180 min after S. aureus infection. Fig. C, Establishment of shRNA-based knockdown cells, and evaluation of TNF-α and IL-1β induction following S. aureus infection. Fig. D, The involvement of NLRP3 and ASC in MSU-induced TNF-α and IL-1β expression. Fig. E, Caspase-1 inhibitor Ac-YVAD-CMK did not inhibit aluminium adjuvant-induced TNF-α and IL-1β mRNA expression in primary human monocytes.Fig. A, IL-8 secretion following (PDF)Click here for additional data file.


This protein was recently found to regulate lipolysis, whole body adiposity, and glucose homeostasis in vivo. Here we identify VPS13C, a member of the VPS13 family of vacuolar protein sorting-associated proteins highly conserved throughout eukaryotic evolution, as a major galectin-12-binding protein. VPS13C is upregulated during adipocyte differentiation, and is required for galectin-12 protein stability. Knockdown of Vps13c markedly reduces the steady-state levels of galectin-12 by promoting its degradation through primarily the lysosomal pathway, and impairs adipocyte differentiation. Our studies also suggest that VPS13C may have a broader role in protein quality control. The regulation of galectin-12 stability by VPS13C could potentially be exploited for therapeutic intervention of obesity and related metabolic diseases.Galectin-12, a member of the galectin family of β-galactoside-binding animal lectins, is preferentially expressed in adipocytes and required for adipocyte differentiation  Galectins are a family of β-galactoside-binding animal lectins with one or two conserved carbohydrate-recognition domains (CRDs) ,2. Thesein vitro , and have recently found that the protein is associated with adipocyte lipid droplets to regulate lipolysis [Galectin-12 has two CRDs separated by a linker sequence and is preferentially expressed in adipocytes , whose dipolysis ,17. Abla2, 37°C.Polyclonal galectin-12 antibodies were generated in galectin-12 knockout mice . Mouse aVPS13C cDNA plasmid as a template, and sub-cloned into pOPINF vector as previously described [E. Coli bacteria transformed with pF-C3 were grown on Overnight ExpressTM Instant TB medium (Novagen), and processed as previously described [A cDNA fragment encoding positions 1582–1882 of human VPS13C isoform 2A (UniProtKB Q709C8-1)  was amplified with primers C3F (5’-AAGTTCTGTTTCAGGGCCCGATCGCTGTCAAAGCTGTATCC-3’) and C3R (5’-ATGGTCTAGAAAGCTTTACAAAATTTTCATTAAAACTGTCAAG-3’), using Deep Vent DNA polymerase (NEB) and a full-length escribed . The oveVPS13C cDNA fragments amplified by RT-PCR [HindIII (blunt) and XbaI sites in pcDNA4-TO-mycHis (Invitrogen) to generate plasmid pcD13C6. This insert contains SNPs rs3784634, rs2303405, rs11629598, rs12907567, rs10851704 and rs765205705, at positions 2866, 3850, 4428, 6909, 8683 and 9968, respectively. We used a retro-lentiviral system for conditional gene knockdown through doxycycline-regulated expression of artificial amiRs [DNA inserts encoding full-length galectin-12 protein or individual CRDs with three copies of FLAG tag (3xFLAG) were generated by PCR using the high-fidelity DNA polymerase PicoMaxx (Stratagene), and cloned into the pSC-A-amp/kan vector using the StrataClone PCR Cloning Kit (Stratagene). After confirmation by sequencing, the inserts were excised and cloned into the pMSCVpuro retroviral vector (Clontech). The resultant constructs encode 3xFlag-tagged versions of LC3 (3F-LC3), full-length galectin-12 , C-CRD-deleted galectin-12 , and N-CRD-deleted galectin-12 . For mammalian over-expression of a C-terminally myc-His-tagged human VPS13C protein, several overlapping y RT-PCR  and clonal amiRs . For eacal amiRs . The insWe produced viruses by packaging viral particles into 293T cells. For retrovirus production, we co-transfected cells with the transfer vector, the packaging plasmid pUMVC, and the envelope plasmid pMD2.G. Lentiviruses were produced similarly, except that pPax2 was used as the packaging plasmid. Transient transfection of 293T cells was performed using FuGENE 6 Transfection Reagent (Promega). Viral supernatants were harvested and pooled 2 and 3 days after transfection.7 3T3-L1 adipocytes transduced with control retrovirus or retrovirus expressing 3xFLAG-galectin-12 were rinsed with PBS and then lyzed in 1 ml of ice-cold buffer containing 50 mM Tris (pH 8), 120 mM NaCl, 0.5% NP-40, and Protease Inhibitor Cocktail (Sigma). After centrifugation, supernatants were incubated with 30 μl anti-FLAG M2 agarose beads (Sigma) at 4°C for 2 h. Beads were then spun down, washed 6 times in lysis buffer, and eluted by boiling 5 min in 30 μl 2x SDS-sample buffer. Proteins in eluted samples were then separated on a SDS-PAGE gradient acrylamide gel  and visualized by silver staining. Protein bands of interest were excised and submitted to UC Davis Proteomics Core for in-gel protein digestion and identification by LC-MS/MS. Mass spectrometry results were searched using X!tandem and search results were returned using the Scaffold software. Samples from four independent IP experiments were analyzed, either as total immunoprecipitates or isolated gel bands. All came back with highly consistent results.We transduced 3T3-L1 cells by incubating cells with pMSCVpuro-3xFLAGm12  retroviruses and 8 μg/ml polybrene. Cells were selected one day later in medium containing 1 μg/ml puromycin for 5 days. The whole puromycin-resistant cell population was expanded and then cryopreserved or used for experiments. To identify galectin-12-binding proteins, these cells were induced to differentiate into adipocytes . A totalLipid droplets were isolated from 3T3-L1 adipocytes by density gradient centrifugation, as described ,23.Cells were fixed with paraformaldehyde, permeabilized with digitonin, and further processed for immunostaining of cellular proteins with specific primary antibodies and appropriate corresponding fluorescence-labeled secondary antibodies, as described . Where iLacZ (control), galectin-12, or Vps13c genes, using the same method. After 5 days of selection in medium containing 1 μg/ml puromycin, the surviving cell population was either used for experiments or cryopreserved. To induce gene knockdown, these co-transduced cells were cultured in the presence of 1 μg/ml doxycycline for 3 days, and subjected to gene expression analysis by real-time RT-PCR and immunoblot, as described [We generated conditional knockdown stable cell lines by sequential transduction. Cells were first transduced with the pMA2641 retrovirus by incubation with viral supernatant and 8 μg/ml polybrene. One day later, cells were switched to fresh medium containing 10 μg/ml Blasticidin S and selected in this medium for 5 days. The whole blasticidin-resistant cell population was then transduced with the pMA2867 lentiviruses targeting escribed . Primer Data are presented as means ± standard error (s.e.). Measurements in control and experimental groups were compared by unpaired two-tailed Student’s t-tests using Prism 5 . Results were considered statistically significant at p < 0.05.We chose a system to stably express 3xFLAG-tagged galectin-12 in adipocytes as the bait, followed by purification of galectin-12-containing protein complexes by immunoprecipitation with an anti-FLAG tag antibody, and then identification of the binding proteins by mass spectrometry (LC-MS/MS). Because endogenous galectin-12 is preferentially expressed in adipocytes, this method subjects the bait protein to a relevant cellular context, increasing the likelihood of identifying bona fide protein interactions. Similar approaches have been successfully used to show that the zinc finger protein PRDM16 (PR domain containing 16) specifies the brown fat lineage from a progenitor by binding to the transcription factors PPARγ  and C/EBGalectin-12 consists of two carbohydrate-recognition domains connected by a linker sequence . To deteVPS13 genes are ubiquitously expressed in many tissues and cell types examined; however, their expression in adipocytes has not been investigated [Vps13c during adipocyte differentiation of 3T3-L1 cells induced with an adipogenic cocktail [Vps13c mRNA was upregulated 13-fold (relative to subconfluent cells) and its levels plateaued by day 6 (Previous studies have shown that human stigated . We examcocktail  and founby day 6 . In compby day 6 . These rby day 6 .LacZ (control), galectin-12, or Vps13c, respectively, we induced adipocyte differentiation in the absence or presence of doxycycline, and determined adipocyte differentiation and the expression of galectin-12 and Vps13c genes 10 days after induction  depletion of the galectin-12 protein, respectively  conditions [Vps13c knockdown cells under these conditions P localization and levels . WhetherVPS13C are associated with a variety of disorders, including glucose homeostasis [We have previously shown that galectin-12 is a protein preferentially expressed in adipocytes and plays roles in adipocyte differentiation and lipolysis important for the regulation of whole-body adiposity and glucose homeostasis. In this paper, we further identify VPS13C as a galectin-12-binding protein required for galectin-12 protein stability and show that VPS13C depletion also impairs adipogenesis. The fact that VPS13C-depleted cells express lower levels of galectin-12 than galectin-12 knockdown cells yet exhibit lesser adipogenesis suppression is somewhat surprising and probably suggests that VPSC13depletion sets off other pathways with opposing effects on adipocyte differentiation. Genetic studies have found that variants of eostasis –54, canceostasis ,56, as weostasis ,44. This


Concerns were raised regarding errors in panel B of S2 FigTotal RNA from indicated tissues—dry pollen, 0.5 h PT, 4 h PT, 8- and 21-day-old seedlings (DS)—was used as templates to perform oligo-dT primed reverse transcription reactions followed by cDNA synthesis. RT-PCR was performed with cDNAs from indicated tissues and gel images of PCR products amplified are shown. (A) RT-PCR analysis of pollen-enriched and pollen-expressed genes. (B) RT-PCR analysis of genes that are significantly altered in SIV PT compared to 4 h PT. (C) RT-PCR analysis of pistil-dependent gene expression changes in vivo. Samples analyzed were dry pollen, unpollinated ms1 pistils (virgin pistil), ms1 pistils pollinated for one minute (1 m pollinated pistil) and ms1 pistils pollinated for two hours (2 h pollinated pistil).(TIF)Click here for additional data file.


In vivo, pressure overload by transverse aorta binding (AB) induced a significant cardiac hypertrophic response as well as re-expression of ANP and BNP in mice heart, which were markedly reduced by AG-490 and TSA. Significantly, AG-490, the Jak2 inhibitor, largely suppressed pressure overload-/Ang-II-induced HDAC2 nuclear exportation in vivo and in vitro. Meanwhile, TSA or HDAC2 siRNA knockdown reduced Ang-II-induced ANP/BNP expression in Jak2 over-expressed H9c2 cardiomyocytes. Together, these results suggest that HDAC2 might be a downstream effector of Jak2 to mediate cardiac hypertrophic response by pressure overload or Ang-II.Pressure overload induces cardiac hypertrophy through activation of Janus kinase 2 (Jak2), however, the underlying mechanisms remain largely unknown. In the current study, we tested whether histone deacetylase 2 (HDAC2) was involved in the process. We found that angiotensin II (Ang-II)-induced re-expression of fetal genes  and brain natriuretic peptide (BNP)) in cultured cardiomyocytes was prevented by the Jak2 inhibitor AG-490 and HDAC2 inhibitor Trichostatin-A (TSA), or by Jak2/HDAC2 siRNA knockdown. On the other hand, myocardial cells with Jak2 or HDAC2 over-expression were hyper-sensitive to Ang-II.  Pressure overload-induced cardiac hypertrophy is mediated partly by activation of Janus kinase 2 (Jak2) ,2. UnderRecent studies show that transverse aorta binding (AB)-induced cardiac hypertrophy is associated with increased expression/activity of histone deacetylases (HDACs) . HDACs aANP and BNP  leucine (1 μCi/mL) DMEM.  Cardiomyocytes were treated with 5% Trichloroacetic acid . The protein precipitates were dissolved in 1 mL of 100 mM/L sodium hydroxide, and radio activities were determined with a liquid scintillation counter.Cultured neonatal mouse cardiomyocytes were incubated with [t-test (Excel 2007). A value of p < 0.05 was considered statistically significant.Data were expressed as means ± SEM, multiple group comparison was performed by the Bonferroni method . Comparison between two groups under identical conditions was performed by the two-tailed Student’s In summary, these results suggest that pressure overload-induced cardiac hypertrophy response requires Jak2-HDAC2 signaling.


The cardiovascular magnetic resonance (CMR) is usually complements echocardiography, provides a non- ionizing radiation evaluation of congenital heart disease(CHD). The congenital air way malformation occur significantly more patients with CHD than patients without CHD. Echocardiography can not show air way well and CT can show air way very well. MRI has emerged as a powerful approach to imaging CHD in children. The question is if MRI is good enough for showing air way malformation.We review our 428 MR cases of CHD in 2014. CMR was performed using a 1.5T unit. The range of age in 428 patients was 10 days to 12 years (mean 3.2 years). Imaging sequences included ECG-triggered 2D balanced steady state free precession (b-SSFP) Cine, phase contrast sequences cine (PC Cine), ECG-triggered 2D Spin echo black blood, Contrast-enhanced 3D magnetic resonance angiography (CE-MRA), Navigator-gated, ECG-triggered 3D balanced steady state free precession (SSFP) MRA and Navigator-gated, ECG-triggered 3D T1W TFE sequences. Minimum-intensity (MinIP) reconstruction was used to evaluate the tracheobronchial tree in CE-MRA, 3D SSFP and 3D T1W TFE sequences.The tracheobronchial tree can be visualized clearly in 82.7% (286/348) of CE-MRA sequences, in 92.6% (224/242) of 3D SSFP sequences and in 98.7% (155/157) of 3D T1W TFE sequences.In 36 of the 428(8.4%)cases with CHD, congenital air way malformation observed in MRI. The congenital air way malformation include tracheal bronchus 18, asplenia syndrome 7 , polysplenia syndrome 3 , congenital tracheal stenosis 3, bridging bronchus 3 and bilateral tracheal bronchi with asplenia syndrome 2 cases. The bilateral tracheal bronchi with asplenia syndrome is very rare congenital air way malformation.This data shows that congenital air way malformation is common in children with CHD. Our data also means that MRI also can show tracheobronchial tree and navigator-gated, ECG-triggered 3D T1W TFE sequences and MinIP reconstruction is the best sequences for air way.


As the world's population ages, the number of patients with Alzheimer's disease (AD) is predicted to increase rapidly. The presence of neurofibrillary tangles (NFTs), composed of hyperphosphorylated tau protein, is one of the neuropathological hallmarks of AD brain. Since the presence of NFTs is well correlated with neurodegeneration and cognitive decline in AD, imaging of tau using positron emission tomography (PET) and single-photon emission computed tomography (SPECT) is useful for presymptomatic diagnosis and monitoring of the progression of AD. Therefore, novel PET/SPECT probes for the imaging of tau have been developed. More recently, several probes were tested clinically and evaluated for their utility. This paper reviews the current state of research on the development and evaluation of PET/SPECT probes for the imaging of tau in AD brain. On the basis of these results, [18F]THK-523 was tested clinically [18F]THK-523 might not reach the necessary optimal levels for PET probes.First, Fodero-Tavoletti and coworkers developed 6- , 31 and ]BF-227) , which ainically . However18F]-fluoro-2-hydroxy)propoxy]-2-(4-dimethylaminophenyl)quinoline ([18F]THK-5105) and 6-[(3-[18F]-fluoro-2-hydroxy)propoxy]-2-(4-methylaminophenyl)quinoline ([18F]THK-5117) for use as novel tau imaging PET probes -N-(2-(2-(2-fluoroethoxy)ethoxy)ethyl)aniline (T557) was identified as having good tau binding properties. However, imaging studies of [18F]T557 in rodents showed poor brain uptake. Therefore, they developed 2-(4-(2-[18F]-fluoroethyl)piperidin-1-yl)benzoimidazopyrimidine ([18F]T808) as a novel imaging probe for tau. By in vitro autoradiography in AD brain sections, this probe localized in the tau-rich sections with considerable selectivity and specificity. Compared with [18F]T557, [18F]T808 rapidly crossed the blood brain barrier, followed by rapid clearance, suggesting little nonspecific binding in mice.Zhang and coworkers designed and synthesized a novel class of compounds, benzoimidazopyrimidines, for in vivo imaging of tau in brain  24]. Am. Am24]. β aggregates -4-((6-iodobenzo[d]thiazol-2-yl)diazenyl)-N,N-dimethylaniline (PDB-3) showed high and selective binding affinity for tau aggregates in vitro. However, [125I]PDB-3 showed poor pharmacokinetics in normal mouse brain. Therefore, we developed (E)-4-((6-(2-(2-(2-[18F]-fluoroethoxy)ethoxy)ethoxy)benzo[d]thiazol-2-yl)diazenyl)-N,N-dimethylaniline ([18F]FPPDB), which is a novel probe for the imaging of tau, to improve the in vivo pharmacokinetics of PDB-3 -4-(6-(methylamino)pyridin-3-yl)buta-1,3-dienyl)benzo[d]thiazol-6-ol (PBB3) clearly stained tau aggregates in tau model mouse brain sections and human AD brain sections. By ex vivo autoradiography, PBB3 selectively labeled the brain stem and spinal cord of tau model mice harboring neuronal tau inclusions. These accumulations could be detected with a micro-PET system in vivo. In addition, by in vitro autoradiography in AD hippocampus, a notable difference in the distribution between [11C]PBB3 and [11C]PIB was observed in the tau-rich sections (11C]PBB3 is a useful PET probe for the imaging of tau, they conducted an exploratory clinical PET study for patients with probable AD and healthy controls (11C]PBB3 for clarifying correlations between the distribution of tau deposition and the symptomatic progression of AD.More recently, Maruyama and coworkers developed novel tau imaging probes, phenyl/pyridinyl-butadienyl-benzothiazole/benzothiazolium derivatives, for the imaging of tau in AD brain  28]. Am. Am28]. sections . Since tcontrols . The res18F]N-methyl lansoprazole is the lead compound for progression into clinical trials because this probe showed rapid brain uptake in nonhuman primates, favorable kinetics, low white matter binding, and selectivity for binding to tau aggregates over Aβ aggregates. In addition, some compounds were also developed as novel PET and SPECT probes for the imaging of tau THK-523, [18F]THK-5105, [18F]T807, [18F]T808, and [11C]PBB3 were tested clinically. The results showed their feasibility for imaging tau aggregates for the diagnosis of AD. Therefore, we hope that further studies of these probes should lead to novel methods for presymptomatic diagnosis and monitoring of the progression of AD.In the past decade, novel PET and SPECT probes for the imaging of tau were developed and evaluated for their utility. Among them, several compounds including [


Sc, an aggregated conformer of the cellular prion protein PrPC. Antibody-derived ligands to the globular domain of PrPC (collectively termed GDL) are also neurotoxic. Here we show that GDL and prion infections activate the same pathways. Firstly, both GDL and prion infection of cerebellar organotypic cultured slices (COCS) induced the production of reactive oxygen species (ROS). Accordingly, ROS scavenging, which counteracts GDL toxicity in vitro and in vivo, prolonged the lifespan of prion-infected mice and protected prion-infected COCS from neurodegeneration. Instead, neither glutamate receptor antagonists nor inhibitors of endoplasmic reticulum calcium channels abolished neurotoxicity in either model. Secondly, antibodies against the flexible tail (FT) of PrPC reduced neurotoxicity in both GDL-exposed and prion-infected COCS, suggesting that the FT executes toxicity in both paradigms. Thirdly, the PERK pathway of the unfolded protein response was activated in both models. Finally, 80% of transcriptionally downregulated genes overlapped between prion-infected and GDL-treated COCS. We conclude that GDL mimic the interaction of PrPSc with PrPC, thereby triggering the downstream events characteristic of prion infection.Prions induce lethal neurodegeneration and consist of PrP Prion diseases are a group of infectious, invariably fatal neurodegenerative diseases. Progress in developing therapeutics is slow, partly because animal models of prion diseases require stringent biosafety and are very slow. We recently found that treatment of cerebellar slices with antibodies targeting the globular domain (GD ligands) of the prion protein (PrP) is neurotoxic. Here we compared this model to prion infection, and describe striking similarities. Both models involved the production of reactive oxygen species, and antioxidants could reverse the toxicity in cerebellar slices and even prolong the survival time of prion-infected mice. Antibodies targeting the flexible tail of PrP that prevent toxicity of GD ligands reduced the toxicity induced by prions. Endoplasmic reticulum stress, which is involved in prion toxicity, is also found in GD-ligand induced neurotoxicity. Finally, changes of gene expression were similar in both models. We conclude that prion infection and GD ligands use converging neurotoxic pathways. Because GD ligands induce toxicity within days rather than months and do not pose biosafety hazards, they may represent a powerful tool for furthering our understanding of prion pathogenesis and also for the discovery of antiprion drugs. C) into a pathological conformer, the scrapie-associated prion protein (PrPSc)  cyclohepten-5,10-imine maleate , 6-cyano-7-nitroquinoxaline-2,3-dione , cyclosporine A (1 μM), ascorbate (1.5 mM), isoascorbate (1.5mM), N-acetamide , MnTBAP (100 μM), benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone , diphenyleneiodonium chloride , N-([3-(Aminomethyl)phenyl]methyl)- ethanimidamide dihydrochloride , N-benzyloxycarbonyl-L-leucylnorleucinal , N-acetylcystein , -trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester , 1-[(5-(p-Nitrophenyl)furfurylidene)amino]-hydantoin sodium salt . A summary table of the used tool compounds and their biological targets are reported in -1, generously provided by Bene Pharmachem), Congo red (1 mg ml-1), and amphotericin B (4.5 mg ml-1).For antibody experiments, drug treatment was initiated at the time of antibody addition (10–14 days post-culturing), whereas for prion experiments, drug treatment was started at 21 dpi when PrP1 antibody raised against PrPC , anti-α-fodrin , anti-GAPDH , anti-actin  and anti-calnexin . Secondary antibodies were horseradish peroxidase (HRP)-conjugated rabbit anti–mouse IgG1 , and goat anti–rabbit IgG1 . The following rabbit monoclonal antibodies were used: anti-phospho PERK , anti-PERK , anti-phospho eIf2α , anti-eIf2α , and anti-ATF4 .Inserts containing the slices were transferred to new plates containing PBS for washing (twice) and tissue was then scraped off the membrane using 10 μl per slice of lysis buffer (0.5% sodium deoxycholate (DOC), 0.5% Nonidet P-40 (NP-40) supplemented with complete mini protease inhibitor cocktail (Roche) and PhosphoStop (Roche) in PBS). The harvested tissue was homogenized by trituration using a 30G syringe and protein concentrations were measured using the bicinchoninic acid assay (Pierce). Samples were mixed with loading buffer  and heated at 95°C for 5 min. Equal volumes were loaded (10 μg proteins per lane) and separated on a 12% Bis-Tris polyacrylamide gel or for higher molecular weight proteins, on a 4–12% gradient gel , and transferred onto a nitrocellulose membrane. These membranes were blocked with 5% w/vol Topblock (Fluka) in TBS-T ) for 1 h and incubated with primary antibodies diluted in 1% Topblock in TBS-T at 4°C overnight. After 4 washes of 15 minutes each with TBS-T, membranes were incubated with secondary antibody diluted in 1% Top Block in TBS-T for 1 h at RT. Primary mouse monoclonal antibodies used were: POM1 mouse IgGSc, 20 μg of protein were digested with 25 μg ml-1 proteinase K in 20 μl final volume of digestion buffer (0.5% wt/vol sodium deoxycholate and 0.5% vol/vol Nonidet P-40 in PBS) for 30 min at 37°C  an antga20 (TIF)Click here for additional data file.S5 Fig(A) Autophagy, (B) CCL2 signaling, (C) ER stress response, (D) ERK inhibition and (E) TNF-ROS-Caspase 3 cascade.Scatter plots comparing the expression level of genes that are involved in specific signaling pathways found in RML-infected and POM1-exposed COCS. The 45 days RML time point (x-axis) and the different time points of POM1 exposure (y-axis) are represented. The correlation coefficient (cor) is indicated above each graph. These five signaling pathways have been described to be activated upon prion infection. (TIF)Click here for additional data file.S6 FigDownregulated (blue) and upregulated (red) genes (log2ratio) are shown for RML vs NBH  and POM1 vs IgG . Four replicates are depicted for each condition. The specific pattern of upregulated and downregulated genes observed at 45 days post RML infection can also be found with increasing exposure time of POM1.(TIF)Click here for additional data file.S7 FigAll investigated parameters are congruent in both models, except for the time line of toxicity (which is explained by the large differences in the concentration of the active principle).(TIF)Click here for additional data file.S8 Figtga20 mice infected with 22L and optionally treated with AcHyT were digested with proteinase K and probed with the antibody POM1 for PrPSc. Negative control: non-infectious brain homogenate (NBH).Brain homogenates from (TIF)Click here for additional data file.S1 Tablep value < 0.01 vs NBH-exposed COCS.Differential expression was defined as fold change ≥ 2; (XLSX)Click here for additional data file.S2 Tablep value < 0.01 vs IgG exposed COCS.Differential expression was defined as fold change ≥ 2; (XLSX)Click here for additional data file.S3 TableGenes upregulated and downregulated at 45dpi (RML), 3 dpe and 7 dpe (POM1).(XLSX)Click here for additional data file.S4 TableLists of the upregulated and downregulated genes at 45dpi for RML, and 3dpe and 7dpe for POM1 were uploaded. The top 40 pathways for POM1 at 3dpe and top 50 for POM1 at 7dpe were computed. Nine of the top 10 of upregulated pathways at 45dpi for RML were either activated at 3dpe or 7dpe POM1.(XLS)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.


Group A rotaviruses (RVAs) are the most important etiological agent of acute gastroenteritis in children <5 years of age worldwide. The monovalent rotavirus vaccine Rotarix was introduced into the national Expanded Programme on Immunization (EPI) in Ghana in May 2012. However, there is a paucity of genetic and phylogenetic data on the complete genomes of human RVAs in circulation pre-vaccine introduction. The common bovine rotavirus VP7 genotype G8 has been sporadically detected in Ghanaian children, usually in combination with the VP4 genotype P[6]. To investigate the genomic constellations and phylogeny of RVA strains in circulation prior to vaccine introduction, the full genomes of two unusual G8P[6] strains, GH018-08 and GH019-08, detected during burden of disease surveillance, were characterized by Illumina MiSeq sequencing. The Ghanaian isolates, GH018-08 and GH019-08, exhibited the unusual, previously unreported genotype constellation G8-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H3. Phylogenetic analyses confirmed that 10 out of the 11 genes of GH018-08 and GH019-08 were identical/nearly identical, with significant variation detected only in their VP1 genes, and clearly established the occurrence of multiple independent interspecies transmission and reassortment events between co-circulating bovine/ovine/caprine rotaviruses and human DS-1-like RVA strains. These findings highlight the contribution of reassortment and interspecies transmission events to the high rotavirus diversity in this region of Africa, and justify the need for simultaneous monitoring of animal and human rotavirus strains. Rotavirus within the family Reoviridae, characteristically possess a genome consisting of 11 double-stranded RNA segments that encode 6 viral structural proteins  and 6 non-structural proteins (NSP1-NSP6) Group A rotaviruses (RVAs) are the most important etiological agent of acute gastroenteritis in children <5 years of age worldwide, and are estimated to be responsible for most of the 453,000 rotavirus-associated deaths annually, of which the majority occur in countries of sub-Saharan Africa and South East Asia Based on complete RVA genome sequence comparisons, three human genotype constellations of the non-G, non-P genes have been described: Wa-like (genotype 1), DS-1-like (genotype 2), and AU-1-like (genotype 3). This classification agrees with the RVA genogroups established by RNA-RNA hybridization assays, represented by prototype human RVA strains Wa, DS-1 and AU-1 The G8 genotype is common in bovine rotaviruses Ghana introduced the monovalent rotavirus vaccine Rotarix into the national Expanded Programme on Immunization (EPI) in May 2012, and has set in place a surveillance system to monitor rotavirus strains post-vaccine introduction, as well as the emergence of new strains. However, there is a paucity of genetic and phylogenetic data on the complete genomes of human RVA in circulation in Ghana; information that will allow the comparison of strains pre- and post-vaccine introduction. In this study, we describe the complete genome characterization of two unusual Ghanaian G8P[6] RVA strains.This study involved the further characterization of stool samples collected as part of WHO rotavirus burden of disease surveillance studies in Ghanaian children carried out per the WHO generic protocol GH018-08 and GH019-08 were isolated in 2008 from children <5 years old with diarrhoea seeking medical attention at the Navrongo War Memorial Hospital in the Kassena-Nankana district of the Upper East Region of Ghana. They were part of 35 randomly selected, partially characterized, single infection RVA strains of various G- and P-genotype combinations from samples collected from the study site between 2008 and 2010. GH019-08 was associated with gastroenteritis severe enough to warrant hospitalization. GH018-08 and GH019-08 had been screened for RVA by Enzyme Immunoassay (EIA) using the commercial IDEIA enzyme immunoassay kit , and polyacrylamide gel electrophoresis (PAGE) of RNA as previously described Viral RNA was extracted from 10∼20% faecal suspensions in PBS using the Direct-zol RNA MiniPrep kit  according to manufacturer's instructions. To assess the integrity of RVA dsRNA, polyacrylamide gel electrophoresis (PAGE) was carried out at constant current (30 mA per gel) on a precast 10% ePAGEL . RVA dsRNA bands were visualized by SYBR Gold staining.Using the Qubit RNA Assay, sample RNA concentrations were determined on a Qubit 2.0 Fluorometer , and starting concentrations normalized to 10∼100 ng for library construction. A 200 bp fragment library was constructed for each sample using the NEBNext Ultra RNA Library Prep Kit for Illumina v1.2  according to manufacturer's instructions. Samples were bar-coded for multiplexing using NEBNext Multiplex Oligos for Illumina, Index Primer Sets 1 and 2 . Library purification was done using Agencourt AMPure XP magnetic beads  as recommended in the NEBNext protocol. The quality of the purified libraries was assessed on a MultiNA MCE-202 bioanalyzer  and the concentrations determined on a Qubit 2.0 flourometer using the Qubit HS DNA Assay .de novo assembly. Subsequently, assembled contig sequences were used to query the non-redundant nucleotide database in GenBank employing the Basic Local Alignment Search Tool [BLAST] (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Genotypes of confirmed RVA sequences were then determined according to the recommendations of the Rotavirus Classification Working Group using the automated genotyping tool for Group A rotaviruses, RotaC v2.2b A 151-cycle paired-end read sequencing run was carried out on a MiSeq desktop sequencer  using the MiSeq Reagent Kit v2 (300 cycles). Following preliminary analysis, the MiSeq reporter programme was used to generate FASTQ formatted sequence data for each sample. Sequence data was analyzed using CLC Genomics Workbench Software v6.5.1 . Contigs were assembled from obtained sequence reads by The nucleotide sequence data for the complete genomes of strains GH018-08 (RVA/Human-wt/GHA/GH018-08/2008/G8P[6]) and GH019-08 (RVA/Human-wt/GHA/GH019-08/2008/G8P[6]) have been deposited in GenBank under the accession numbers KJ748465 – KJ748475 and KJ748476–KJ748486 respectively.An assessment of the integrity of dsRNA genomes of the Ghanaian G8P[6] RVA strains GH018-08 and GH019-08 by PAGE revealed identical, unusual, long RNA migration patterns .Full-length or nearly full-length sequences of all 11 genomic segments of GH018-08 and GH019-08 were successfully determined by Illumina MiSeq sequencing. The lengths of nucleotide and deduced amino acid sequences of genomic segments for GH018-08 and GH019-08, with related sequence read data is summarized in To further investigate the genetic relationships among GH018-08, GH019-08 and other RVA strains, phylogenetic analyses were conducted based on nucleotide sequences of the entire open reading frames of all 11 gene segments. Compared to commonly detected G2P[4] and G3P[6] Ghanaian RVA strains VP7 genes of GH018-08 and GH019-08 were identical, forming a monophyletic cluster closely related to (95.3–97.7% nucleotide sequence identity), but distinct from human G8 strains, especially those of African origin  to porcine (RVA/Porcine-wt/IND/HP113/XXXX/G6P[13] and RVA/Porcine-wt/IND/HP140/XXXX/G6P[13] The VP1 genes of GH018-08 and GH019-08 were distantly related (91.1% nucleotide sequence identity), clustering separately in different lineages within the R2 genotype , forming a monophyletic cluster within the C2 genotype. They were closely related to the Indian bovine strain RVA/Cow/IND/M1/UKD/2010/GXP[X], with 97.2% and 96.9%% nucleotide sequence identity respectively , and formed a monophyletic cluster within the M2 genotype. They were distantly related (90.1% nucleotide sequence identity) to their closest neighbour, the African bovine strain RVA/Bovine-tc/ZAF/‘O’;Agent/1965/G8P[1], as well as other strains of African origin - RVA/Human-tc/KEN/B12/1987/G8P[1] The NSP1 genes of GH018-08 and GH019-08 were identical, and formed a monophyletic cluster closely related (94.3-99.3% nucleotide sequence identity) to contemporary human strains within the A2 genotype  with their closest neighbour, the human-artiodactyl reassortant strain RVA/Human-wt/HUN/BP1062/2004/G8P[14] , forming a monophyletic cluster closely related (99.3–99.4% nucleotide sequence identity) to the human strains RVA/Human-wt/ZAF/2371WC/2008/G9P[8] The NSP4 genes of GH018-08 and GH019-08 were identical, forming a monophyletic cluster within the E2 genotype. They revealed a fairly close relationship (93.6–94.1% nucleotide sequence identity) with the human-artiodactyl reassortants RVA/Human-wt/BEL/B10925/1997/G6P[14], RVA/Human-wt/ITA/111-05-27/2005/G6P[14] The NSP5 genes of GH018-08 and GH019-08 were identical and formed a monophyletic cluster within the H3 genotype. They were closely related to bovine, guanaco, ovine and caprine strains , as well as human-artiodactyl and multiple bovine-feline/canine-human reassortants (95.3–96.7% nucleotide sequence identity), clustering within the same lineage  backbone The unusually high prevalence of the common bovine genotype G8 in humans in Africa has been speculated to be the result of interspecies transmission of rotaviruses between cattle and humans The P[6] genotype is epidemiologically important in Ghana, detected quite commonly in association with a wide variety of G-types Despite bearing the same genotype constellation, not all genome segments of GH018-08 and GH019-08 shared close phylogenetic relationships. Of the 11 gene segments of GH018-08 and GH019-08, 10  possessed identical or nearly identical (99.6–100% nucleotide sequence identity) open reading frames, forming monophyletic clusters within their respective genotypes. This suggests the presence of a recent common ancestor. However, the VP1 genes exhibited significant variation (91.1% nucleotide identity), clustering separately into different lineages within the R2 genotype. GH018-08, while forming a monophyletic cluster with the human-artiodactyl interspecies reassortant strain RVA/Human-wt/BEL/B1711/2002/G6P[6] (96.1% nucleotide sequence identity), was distantly related to other contemporary human strains, the caprine strain RVA/Goat-tc/BGD/GO34/1999/G6P[1], as well as the recently reported camel strain RVA/Camel-wt/SDN/MRC-DPRU447/2002/G8P[11] , and clustered separately. On the contrary, GH019-08 was closely related to contemporary human strains, RVA/Camel-wt/SDN/MRC-DPRU447/2002/G8P[11] and RVA/Goat-tc/BGD/GO34/1999/G6P[1] , and clustered together in the same lineage. Considering that the VP1 gene of GH019-08 clustered within a major lineage of co-circulating DS-1-like Ghanaian strains while GH018-08 formed a unique lineage, these observations provide strong evidence of at least one recent intragenogroup reassortment event with an established locally circulating DS-1-like strain carrying a phylogenetically different VP1 gene.Excluding VP7, VP4 and VP1, the 8 remaining genome segments forming the I2-C2-M2-A2-N2-T2-E2-H3 backbone of GH018-08 and GH019-08 could be divided into three groups based on their phylogeny – (i) segments closely related to bovine/ovine/caprine RVA strains , (ii) segments distantly related to human RVA strains (VP3 and NSP2), and (iii) segments closely related to human RVA strains (NSP1and NSP3).In the Upper East region, where the samples were collected, the human population live in close proximity with, and share common water supply with domestic animals such as goats, sheep and cattle. This highlights the potential for the occurrence of gene reassortment While NSP1 and NSP3, along with the VP4 genes of GH018-08 and GH019-08 were closely related to contemporary human strains, the VP6 and VP2 genes were closely related to recently isolated bovine strains, and the NSP4 and NSP5 genes were closely related to bovine/ovine/caprine strains. The NSP1- and NSP3-encoded proteins, which interact extensively with host cell factors but are not known to bind to any other viral proteins, reassort more freely than those encoding the inner capsid proteins, VP1-VP3 Though the VP3 genes of GH018-08 and GH019-08 were distantly related to cognate rotavirus genotypes, their closest neighbour was bovine. The apparent lack of a common ancestor among the segments related to bovine, ovine or caprine strains  challenges our speculation on the direction of interspecies transmission, especially since the two G8P[6] strains investigated most likely represent only a small part of a complex series of events. The availability of sequence information from local bovine species should clarify this.Taken together, these findings suggest multiple independent interspecies transmission and reassortment events between co-circulating bovine/ovine/caprine rotaviruses and human DS-1-like RVA strains. Considering that G8 strains are only sporadically detected in Ghana, and that 10 out of the 11 genome segments of GH018-09 and GH019-08 had identical or nearly identical open reading frame nucleotide sequences, it is reasonable to suggest that while these strains might have a recent common ancestor, they are unlikely to be well adapted to humans for efficient transmission at present. These findings highlight the need for simultaneous monitoring of animal and human rotavirus strains, especially in the era of rapid spread of rotavirus vaccines in Africa.Figure S1Electrophoretic migration pattern of genome segments of G8P[6] RVA strains GH018-08 and GH019-08, with representative Ghanaian Wa-like and DS-1-like strains.(PDF)Click here for additional data file.Table S1Sequence data for genome segments of GH018-08 and GH019-08 generated by de novo sequence assembly.(DOCX)Click here for additional data file.Table S2GenBank accession numbers for all rotavirus strains used for phylogenetic analyses.(DOCX)Click here for additional data file.Table S3Nucleotide sequence identities (%) of the full-length ORFs of all 11 gene segments of GH018-08 to selected human and animal reference rotavirus strains.(DOCX)Click here for additional data file.Table S4Nucleotide sequence identities (%) of the full-length ORFs of all 11 gene segments of GH018-08 to selected, relevant ‘non-reference’ human and animal rotavirus strains.(DOCX)Click here for additional data file.


Ventricular fibrosis in adult congenital heart disease (ACHD) has been shown to relate to adverse clinical markers., 2 NovelTen adult repaired Tetralogy of Fallot patients aged ≥16 years with glomerular filtration rate>30ml/min completed clinical CMR scans acquired by a single operator on a 1.5T Siemens Avanto for volumetric and functional biventricular assessment. LGE imaging was performed 10 minutes after 0.1mmol/kg intravenous gadobutrol administration .o (BH) and 50o (FB), pixel bandwidth 140Hz (BH) 977Hz (FB) with acquisition window 166ms (BH) 196ms (FB) were set. Inversion times were adjusted for myocardial nulling throughout, and kept constant between both sequences when acquiring identical slice locations in long and short-axis views. When inversion time drift was minimal, transverse navigator-gated 3D LGE imaging was performed as per published protocol as a reference standard.4Interspersed breath-hold (BH) segmented gradient echo and free-breathing (FB) motion-corrected balanced steady state free precession phase-sensitive inversion recovery (PSIR) pulse sequences were acquired with identical typical imaging parameters of: field of view 380x320mm, matrix 256x144, 6mm slice thickness with 4mm gap for short-axis stacks, and acceleration factor (GRAPPA) 2. Repetition time/echo time of 8.3/3.8ms (BH) 2.7/1.7ms (FB), flip angle 255 image confidence scores and semi-quantitative RV LGE scoring.1Blood-myocardium and Gd-myocardium contrast-to-noise ratios (CNRs) were also compared.Anonymised, blinded image analysis was carried out by two experienced observers assessing RV LGE image quality,RV LGE was present in all patients and LV LGE in 7 patients . There was reasonable interobserver agreement when scoring the extent of RV LGE (α=0.605) with good overall agreement in quantifying the extent of RVOT and VSD patch scar Figures  and 2. IFree-breathing LGE provides comparable, diagnostic image quality to conventional 2D BH LGE imaging and may be practical for clinical use, especially for patients unable to breath-hold or with arrhythmias that would otherwise compromise image quality.British Heart Foundation, NIHR Cardiovascular Biomedical Research Unit of Royal Brompton & Harefield NHS Foundation Trust and Imperial College London.


Lin28b RNA-binding protein and a conditional knockout of the MirLet7c-2/Mirlet7b locus. This ablation of Let-7 triggers the development of intestinal adenocarcinomas concomitant with reduced survival. Analysis of both mouse and human intestinal cancer specimens reveals that stem cell markers were significantly associated with loss of Let-7 miRNA expression, and that a number of Let-7 targets were elevated, including Hmga1 and Hmga2. Functional studies in 3-D enteroids revealed that Hmga2 is necessary and sufficient to mediate many characteristics of Let-7 depletion, namely accelerating cell cycle progression and enhancing a stem cell phenotype. In addition, inactivation of a single Hmga2 allele in the mouse intestine epithelium significantly represses tumorigenesis driven by Lin28b. In aggregate, we conclude that Let-7 depletion drives a stem cell phenotype and the development of intestinal cancer, primarily via Hmga2.Let-7 miRNAs comprise one of the largest and most highly expressed family of miRNAs among vertebrates, and is critical for promoting differentiation, regulating metabolism, inhibiting cellular proliferation, and repressing carcinogenesis in a variety of tissues. The large size of the Let-7 family of miRNAs has complicated the development of mutant animal models. Here we describe the comprehensive repression of all Let-7 miRNAs in the intestinal epithelium via low-level tissue-specific expression of the  MirLet7c-2/Mirlet7b. Mice with this comprehensive depletion of Let-7 develop intestinal cancers that resemble human colon cancers. Our further analysis identified another gene, HMGA2, downstream of this pathway that is critical to this outcome.Cancer develops following multiple genetic mutations (i.e. in tumor suppressors and oncogenes), and mutations that cooperate or synergize are often advantageous to cancer cell growth. To study how multiple genes might cooperate, it is usually informative to generate candidate mutations in cells or in mice. Large gene families, such as the Let-7 family, are difficult to silence or mutate because of the large amount of redundancy that exists between similar copies of the same gene; the mutation of one will often be masked or compensated by the continued function of others. In the mouse intestine we have achieved comprehensive depletion of all Let-7 miRNAs in this large multi-genic family through use of an inhibitory protein, called LIN28B, that specifically represses Let-7, and genetic inactivation of another gene cluster called  Dicer1, an enzyme needed for miRNA processing, in which haplo-insufficiency of Dicer1 and global reduction of miRNA levels significantly accelerates tumorigenesis  XP Rabbit mAb , rabbit anti-HIF3A antibody , anti-HMGA1 antibody [EPR7839] , and anti-HMGA2 [D1A7] rabbit mAb .https://genome-cancer.ucsc.edu/proj/site/hgHeatmap/) . For examination of Let-7 miRNA expression and expression relative to candidate target genes we examined a cohort of 199 CRC patients from the TCGA Pan-Cancer analysis project visualized using the starbase miRNA CLIP-seq portal (http://starbase.sysu.edu.cn/) .We examined gene expression in CRC specimens from a cohort of 416 CRC patients from a TCGA dataset using the cancer genome browser at UCSC  Box-and-whisker plots for Let-7a, Let-7b, and Let-7c, demonstrating significant down-regulation in colon and rectal cancer (CRC) miRNA-seq dataset. Box plot whiskers represent 1.5x the interquartile range (IQR) above the third quartile or below the first quartile. D-I) Scatter plots of Let-7 miRNA expression vs. target mRNA levels from CRC miRNA-seq and mRNA-seq datasets. Pearson correlation coefficients and p-values are indicated on each graph.(TIF)Click here for additional data file.S2 FigA) Kaplan-Meier curve depicting survival in patients with high HMGA1 staining scores vs. low staining scores. B) Kaplan-Meier curve depicting survival in patients with high HMGA2 levels from RNA-seq data . High le(TIF)Click here for additional data file.S3 FigLin28bLo/Let7IEC-KO S.I. , Lin28bLo/Let7IEC-KO adenoma , and Lin28bLo/Let7IEC-KO adenocarcinoma . All pictures are at same magnification, with scale bar = 100 μm.Immunohistochemical staining for Arid3a (A-D) and Hif3a (E-H), in sections from WT small intestine (S.I.) , (TIF)Click here for additional data file.S4 FigGapdh) and β-Actin (Actb).A) Colony forming assay in WT mouse small intestine enteroids transduced with pTRIPz (empty vector), pTRIPz-E2f5, or pTRIPz-Imp2 (Igf2bp2). Enteroids were treated with 100 ng/ml doxycycline for 5 days, then dissociated into single cells. B-K) Q-RT-PCR for epithelial stem cell markers in WT mouse small intestine enteroids transduced with pTRIPz vectors expressing Arid3a, Hif3a, Hmga2, or Imp2 (Igf2bp2) and treated for 5 days with 100 ng/ml doxycycline. Assays were performed in triplicate. Q-RT-PCR values in B-D were normalized to Gapdh ((TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 TableIGF2BP1 did not correlate with any stem cell markers.(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S1 Methods(DOCX)Click here for additional data file.


To test this hypothesis, we compared the in vivo antitumor effects and persistence of combined CD4+ T cells (bulk or Th17-polarized) and CD8+ T cells redirected with CARs containing CD28, 4-1BB or ICOS-based costimulatory domains. Using multiple mouse tumor models, we demonstrate that the ICOS intracellular domain significantly enhanced the in vivo persistence of CAR-expressing CD4+ T cells, and that both persistence and tumor infiltration were further enhanced by culturing these cells under Th17-polarizing conditions. Importantly, Th17-polarized CD4+ T cells expressing an ICOS-based CAR significantly increased the circulatory persistence of bulk CD8+ T cells expressing either CD28- or 4-1BB-based CARs. We further demonstrate that the antitumor effect of CAR-expressing CD8+ T cells was enhanced when co-injected with ICOS-redirected Th17 cells. Collectively, our data suggest that combining Th17 CD4+ T cells redirected with an ICOS-based CAR with CD8+ CAR-T cells will enhance their persistence and antitumor efficacy.T cell persistence is likely to promote long-term anti-tumor effects after adoptive T cell transfer. We have recently shown that incorporation of the ICOS intracellular domain into chimeric antigen receptors (CARs) significantly increased Th17 cell persistence


Therefore, we aimed to test the impact of conditioning CD8+ T cells with MDSC on their differentiation potential and ACT efficacy. Our results indicate that MDSC impaired the progression of CD8+ T cells into effector populations, without altering their activation status, production of IL-2, or signaling through the T cell receptor. In addition, culture of CD8+ T cells with MDSC resulted in an increased ACT anti-tumor efficacy, which correlated with a higher frequency of the transferred T cells and elevated IFNγ production. Interestingly, activated CD62L+ CD8+ Tcells were responsible for the enhanced anti-tumor activity showed by MDSC-exposed T cells. Additional results showed a decreased protein synthesis rate and lower activity of the mammalian/mechanistic target of rapamycin (mTOR) in T cells conditioned with MDSC. Silencing of the negative mTOR regulator tuberous sclerosis complex-2 in T cells co-cultured with MDSC restored mTOR activity, but resulted in T cell apoptosis. These results indicate that conditioning of T cells with MDSC induces stress survival pathways mediated by a blunted mTOR signaling, which regulated T cell differentiation and ACT efficacy. Continuation of this research will enable the development of better strategies to increase ACT responses in cancer.The success of adoptive T cell-based immunotherapy (ACT) in cancer is limited in part by the accumulation of myeloid-derived suppressor cells (MDSC), which block several T cell functions, including T cell proliferation and the expression of various cytotoxic mediators. Paradoxically, the inhibition of CD8 The + T cells into cytolytic and cytokine-producing effector cells . All experiments using animals were approved by the GRU-IACUC and performed following GRU animal care facility guidelines.Lewis lung carcinoma (3LL) and EL-4 thymoma expressing ovalbumin (EG7) were maintained in RPMI-1640  and supplemented with 10% fetal calf serum , 25 mM Hepes , 4 mM L-glutamine , and 100 U/mL of penicillin, streptomycin . Cell lines were obtained from the American Type Culture Collection  and periodically validated to be mycoplasma-free, using an ATCC kit. C57BL/6 mice  were obtained from Harlan , C57BL/6-Tg (TcraTcrb) 1100Mjb/J (OT-1) and CD45.1+ cells within CD8+ cells.Antibodies against CD3 (clone 145-2C11), CD28 (clone 37.51), CD25 (clone PC61), CD28 (clone CD28.2), CD44 (clone IM7), and CD69 (clone H1.2F3) were obtained from Becton Dickinson Biosciences . Antibodies against Sca-1 (clone D7) CD62L (clone MEL-14), CCR7 (clone 4B12), CD122 (clone 5H4), and CD127 (clone SB/199) were obtained from eBioscience. Anti-phospho-4E-BP1 (clone 236B4), anti-4E-BP1 (clone 53H11), and anti-TSC2 (D93F12) antibodies were obtained from Cell Signaling Technology . Anti-β-actin antibody (clone AC-74) was obtained from Sigma-Aldrich . Nx-hydroxy-nor-Arginine (N-NOHA), L-NG-Monomethylarginine (L-NMMA), and Manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) were obtained from EMD Millipore . Induction of apoptosis was tested using annexin V Apoptosis Detection Kit (BD Biosciences). Results were expressed as the percentage of annexin V+ and CD3+ cells were isolated from spleens and lymph nodes of mice using negative isolation kits (Stem Cell Technologies). Cell purity ranged between 95 and 99%, as tested by flow cytometry. MDSC were isolated from tumors previously digested with DNAse and Liberase  at 37°C for 1 hour, as previously described [+ cells. iMC were isolated from spleens of mice without tumors, as we described [+ cells using magnetic-bead isolation (Stemcell technologies). T cell purity ranged from 96-100% as monitored by flow cytometry.CD8escribed . Purity escribed , followeescribed . MDSC weCellular extracts isolated from T cells cultured alone or previously co-cultured with MDSC were used for protein detection by immunoblotting. Lysates were electrophoresed in 8% TrisGlycine gels, transferred to PVDF membranes, and immunoblotted against phospho-4E-BP1, 4E-BP1, cyclin D3, cdk4, TSC2, or β-actin. Membrane-bound immune complexes were then detected using ECL western blot detection reagent , followed by exposure on X-ray films.® Labeling kit (Life Technologies). Briefly, T cells were cultured for 48 hours with or without MDSC and in the presence or the absence of specific inhibitors for arginase or iNOS, or a peroxynitrite scavenger [® L-azidohomoalanine. Protein lysates were harvested and normalized across samples followed by methanol/chloroform precipitation and solubilization in 1% SDS, 50 mM Tris-HCl, pH 8.0. Proteins were treated with Click-IT® Tetramethylrhodamine analysis detection Kit and electrophoresed in 8% TrisGlycine gels, followed by visualization through a BioRad Versadoc.T cell protein translation was measured using Click-iTcavenger . MDSC weT cells were activated with Dynabeads mouse T-Activator CD3/CD28 beads (Life Technologies) at a cell:bead ratio of 1:1, followed by addition of MDSC to the culture. Four hours later, T cells were stained with anti-CD3 and anti-phospho Zap70 (Y319) antibodies using BD Phosflow kit.T cell proliferation was measured, as we described . For the+ T cells were transfected using Accell SMART pool siRNA from GE Dharmacon. Briefly, T cells were activated and cultured in a 96 well plate overnight in 100 μL Accell media containing 1% FBS and 2 μM of mock or anti-TSC2 siRNA comprised of the following sequences: GCAAUGACUUUGUUUCUAU, GUGUCAUAUGAGAUUGUUC, CUCCUGUCUUUUGAUGAUA, CUAACAGCAUUAAUAUCUU. The following day, MDSC  were added to the cultures and the wells supplemented with complete media for a final volume of 200 μL. Transfection efficiency was monitored using control siRNA labeled with 5 carboxy-fluorescein (FAM) and was determined to be >90%.Anti-CD3/CD28-activated CD3mTOR activity was assessed using K-LISA™ mTOR Activity Kit from EMD Millipore, according to the manufacturer's instructions. Briefly, T cell lysates were prepared in a lysis buffer comprised of 50 mM Tris HCl, 100 mM NaCl, 50 mM β-glycerophosphate, 10% glycerol (w/v), 1% Tween 20 (w/v), 1 mM EDTA, 20 nM microcystin-LR, 25 mM NaF, and protease inhibitor cocktail Set III. Values were normalized to the maximum mTOR standard, while T cells treated with rapamycin (100 ng/ml) acted as the control for mTOR activity inhibition.Statistical analyses were carried in GraphPad Prism. Tests were conducted at 5% significance level. Experimental group differences of endpoints were assessed by ANOVA, whereas mean comparisons were carried out with the Tukey procedure or with the Dunnet procedure for comparisons with controls.