Arabidopsis and 67 in rice. These CCCH proteins have been shown to play diverse roles in plant developmental processes and environmental responses. However, this family has not been studied in the model tree species Populus to date.CCCH zinc finger proteins contain a typical motif of three cysteines and one histidine residues and serve regulatory functions at all stages of mRNA metabolism. In plants, CCCH type zinc finger proteins comprise a large gene family represented by 68 members in Populus was performed. Using a thorough annotation approach, a total of 91 full-length CCCH genes were identified in Populus, of which most contained more than one CCCH motif and a type of non-conventional C-X11-C-X6-C-X3-H motif was unique for Populus. All of the Populus CCCH genes were phylogeneticly clustered into 13 distinct subfamilies. In each subfamily, the gene structure and motif composition were relatively conserved. Chromosomal localization of these genes revealed that most of the CCCHs  are physically distributed on the duplicated blocks. Thirty-four paralogous pairs were identified in Populus, of which 22 pairs (64.7 %) might be created by the whole genome segment duplication, whereas 4 pairs seem to be resulted from tandem duplications. In 91 CCCH proteins, we also identified 63 putative nucleon-cytoplasm shuttling proteins and 3 typical RNA-binding proteins. The expression profiles of all Populus CCCH genes have been digitally analyzed in six tissues across different developmental stages, and under various drought stress conditions. A variety of expression patterns of CCCH genes were observed during Populus development, of which 34 genes highly express in root and 22 genes show the highest level of transcript abundance in differentiating xylem. Quantitative real-time RT-PCR (RT-qPCR) was further performed to confirm the tissue-specific expression and responses to drought stress treatment of 12 selected Populus CCCH genes.In the present study, a comprehensive analysis of the genes encoding CCCH zinc finger family in Populus CCCH proteins. Comprehensive genomic analyses suggested that segmental duplications contribute significantly to the expansion of Populus CCCH gene family. Transcriptome profiling provides first insights into the functional divergences among members of Populus CCCH gene family. Particularly, some CCCH genes may be involved in wood development while others in drought tolerance regulation. Our results presented here may provide a starting point for the functional dissection of this family of potential RNA-binding proteins.This study provides the first systematic analysis of the  Arabidopsis zinc-finger proteins, which differs from the previously identified plant zinc-finger TFs by regulating gene expression via directly binding to mRNA, was named as CCCH gene family -x -[LIVFM]-x -L-x-[LIMTKD] was written according to previous study -x -[LIVFM]-x -L-x- [LIMTKD] according to the previous method [8]. Click here for fileThe probes and microarray data of PopulusCCCH genes. Microarray data corresponds to expression analysis showed in Figure Click here for filePrimer sequences of 12 selected Populus CCCH genes for RT-qPCR analysis. The sequence, length, Tm, GC% and product sizes of primers used for RT-qPCR. Click here for file


Chinese hamster ovary (CHO) cells have become dominant host cells in the biopharmaceutical industry due to their capacity for proper protein folding, assembly and post-translational modifications. However, low specific productivity (qp) places limitations on yields obtained from mammalian host cells. MicroRNAs (miRNAs), a novel class of short, non-coding RNAs which negatively regulate target gene expression at post-transcriptional levels, have emerged as promising targets for engineering of CHO cell factories to enhance recombinant protein production. While engineering of miRNAs for enhanced cell growth and delayed cell death have been reported, miRNA targets which can enhance qp have not been identified to date.To understand the role of miRNAs in conferring high qp phenotype in CHO cells, we carried out high throughput sequencing of 4 in-house generated IgG-expressing CHO sub-clones of varying qps. Reads were mapped to miRBase and 22 miRNAs were found to be differentially expressed between the high and low producers. These miRNAs were stably transfected into an IgG-expressing sub-clone to assess their effects on growth, titer, qp and product quality attributes.Over-expression of miRs-17, 19b, 20a and 92a individually and in combination resulted in 13-27% increases in titer and 14-24% increases in qp in stably transfected pools. No significant alterations in proliferation rates were observed. 20-45 single cell clones were randomly selected from each of the 5 transfected pools for characterization. Statistical analyses showed significant differences in titer/qp between the high- and low-miRNA expressing single cell clones. The highest producing single cell clones exhibited ~100% increases in titer and qp compared to non-transfected cells. A correlation was found between increased miR-19b levels (>1.3-fold) and enhanced qp and titer. Over-expression of miR-19b does not appear to impact IgG aggregation significantly.To our knowledge, this is the first report of enhancement of recombinant protein productivity by stable miRNA over-expression. The genes and cellular pathways targeted by these miRNAs specific to enhancing protein productivity are under investigation and will be reported.


Leukemic cells from Adult T-cell leukemia (ATL) patients display elevated telomerase activity, resulting mainly from transcriptional up-regulation of the human telomerase catalytic subunit (hTERT). We have previously shown that HBZ (HTLV-1 bZip) protein cooperates with JunD transcription factor to enhance hTERT expression after JunD anchoring to Sp1 bound to Sp1 sites within the hTERT proximal promoter. In normal somatic cells, telomerase expression is negatively regulated by tumor suppressor gene products, such as Menin, encoded by the multiple endocrine neoplasia type 1 (MEN-1) gene. Interestingly, the interaction of Menin with JunD has been shown to repress its transcriptional activity.We report here that, in HBZ-expressing cells, Menin and HBZ exert opposite effects on JunD-mediated regulation of hTERT transcription. Chromatin immuno-precipitation as well as functional assays demonstrate that this antagonism is linked to the recruitment of p300 by HBZ and HDACs by Menin. Furthermore, knock-down of Menin in the ATL Tl-Om1 (only expressing HBZ) cells results in an increase of hTERT expression and of telomerase activity, whereas a knock-down of HBZ exerts opposite effects. Interestingly, primary leukemic cells isolated from ATL patients that express high amounts of hTERT transcripts, are also characterized by an elevated expression of HBZ and MEN-1 genes. Thus, in leukemic cells, HBZ behaves as a key factor impeding the Menin function and sustaining hTERT transcription. These findings underline the critical role of HBZ as a tumor-promoting protein during the development of the HTLV-1-induced leukemogenic process.


Development of a system that can mimic the in vivo responses of human cells will enable more accurate health risk assessments. A surrogate human hematopoietic lineage can be established in NOD/Shi-scid/IL-2Rγnull (NOG) mice by transplanting human hematopoietic stem/progenitor cells (Hu-NOG mice). Here, we first evaluated the toxic response of human-like hematopoietic lineage in NOG mice to a representative toxic agent, benzene. Flow cytometric analysis showed that benzene caused a significant decrease in the number of human hematopoietic stem/progenitor cells in the bone marrow and the number of human leukocytes in the peripheral blood and hematopoietic organs. Next, we established chimeric mice by transplanting C57BL/6 mouse-derived bone marrow cells into NOG mice (Mo-NOG mice). A comparison of the degree of benzene-induced hematotoxicity in donor-derived hematopoietic lineage cells within Mo-NOG mice indicated that the toxic response of Hu-NOG mice reflected interspecies differences in susceptibilities to benzene. Responses to the toxic effects of benzene were greater in lymphoid cells than in myeloid cells in Mo-NOG and Hu-NOG mice. These findings suggested that Hu-NOG mice may be a powerful in vivo tool for assessing hematotoxicity in humans, while accounting for interspecies differences.Despite recent advancements, it is still difficult to evaluate  As a result, a safe margin has been applied to health risk assessments to take into consideration the possibility of insufficient evaluation, particularly regarding interspecies differences, though such extrapolation to humans using safe margins occasionally results in overestimation of risks. However, the underestimation of risks by a small safety margin exposes humans to significant danger. Therefore, to perform more accurate health risk assessments, the development of an in vivo evaluation system that can reproduce human responses to toxic factors would be an important breakthrough.Currently, health risk assessment of various factors is evaluated based on results from epidemiologic surveys, animal testing, cytotoxicity studies, or a combination thereof. In epidemiological surveys, the influence on human health can be evaluated directly in vivo. Mice that are humanized by transplantation of human cells are able to establish networks of human cells in their bodies. The available diverse mouse models were developed by transplantation of various types of cells to immunodeficient strains of mice. In cancer research, the biology of human tumor growth, metastasis, and angiogenesis has been evaluated in these mouse models in vivo study of human hematopoietic stem cell function For many years, mouse models transgenically expressing human genes in vivo. These mice were established by transplanting NOG mice with human CD34+ cells (Hu-NOG mice). The response to benzene, a model toxicant, was measured by determining decreases in the number of leukocytes. Furthermore, we established chimeric mice by transplanting C57BL/6 mouse-derived bone marrow cells into NOG mice (Mo-NOG mice). To evaluate whether the response to benzene by Hu-NOG mice reflected interspecies differences, the degrees of benzene-induced hematotoxicities in Mo-NOG and Hu-NOG mice were compared.Hematotoxicity is evaluated according to many factors, including decreased hematopoietic cell counts, abnormal blood coagulation, aberrant myelopoiesis, and induction of leukemia, all of which can be caused by diverse risk factors + cells isolated from cord blood were purchased from Lonza  and cryopreserved in liquid nitrogen prior to use. Transplanted mouse Lin− bone marrow cells were prepared from the femurs of 6-week-old C57BL/6J mice. Bone marrow cells were collected by excising and crushing the epiphysis and metaphysis with a mortar and by pushing a needle through the diaphysis. Lin− cells were further purified using a Lineage Cell Depletion Kit . Fresh Lin− bone marrow cells were used in experiments. As hosts for cell transplantation, immunodeficient NOD/Shi-scid/IL-2Rγnull (NOG) mice  were obtained from the Central Institute for Experimental Animals . Mice were housed in a specific pathogen-free facility in autoclaved polycarbonate cages and fed sterile food and water ad libitum. In addition, NOG mice were maintained on neomycin-polymyxin B in their drinking water.Transplanted human CD34All experimental protocols involving human cells and laboratory mice were reviewed and approved by the Ethical Committee for the Study of Materials from Human Beings and for Research and Welfare of Experimental Animals at the Central Research Institute of Electric Power Industry.+ cells or mouse Lin− bone marrow cells suspended in MEM supplemented with 2% BSA (200 µL containing 4×104 cells per mouse).After a 2-week quarantine and acclimatization period, whole-body X-ray irradiation of NOG mice was performed at 2.5 Gy using an X-ray generator  operated at 300 kV and 10 mA with 1.0-mm aluminum and 0.5-mm copper filters at a dose ratio of 1.5 Gy/min and a focus surface distance of 550 mm. Three to five hours later, the irradiated mice were injected intravenously with human CD34Donor human or mouse cell-derived hematopoietic lineages were established in NOG mice by maintenance of the mice for about 3 months after transplantation. For grouping the mice, the properties of the peripheral blood leukocytes of both types of mice were analyzed using a microcavity array system On the basis of body weight, the sum of leukocyte counts, and the rates of leukocyte chimerism, Hu-NOG mice were divided into 5 groups of 9–10 mice per group without significant differences between each group. The rates of leukocyte chimerism were calculated as the percentage of donor-derived leukocytes in the total leukocyte population (the sum of donor- and host-derived leukocytes). Mo-NOG mice were divided into 4 groups of 8 mice each.3/day; absorptivity, 50%; body weight, 70 kg) Published epidemiological research regarding short-term exposure to benzene has shown that the lowest-observed adverse effect level (LOAEL) of benzene-induced hematotoxicity based on decreasing leukocyte counts in the peripheral blood, is 60 ppm After benzene administration for 2 weeks, samples from the bone marrow, spleen, thymus, and peripheral blood were harvested from each mouse. Bone marrow cells were collected as described above. The spleen and thymus were crushed between 2 glass slides. Peripheral blood was aspirated from the postcava under anticoagulation treatment. Erythrocytes that could have interfered with further evaluation were lysed using VersaLyse . Collected cells were suspended in phosphate buffered saline supplemented with 4 mM EDTA and 0.5% BSA. Mice in which edema was observed in the thymus at the time of dissection were not used for subsequent analysis. We did not observe detectable differences in the appearance of abnormalities or the amount of benzene administered.Hematopoietic cells collected from each tissue, and organs were analyzed by flow cytometry. Cells were stained with fluorophore-conjugated antibodies in BD TruCOUNT Tubes (BD Biosciences) and applied to a flow cytometer to determine cell surface markers and cell numbers simultaneously. FITC-conjugated anti-hCD45 mAbs , Lineage cocktail (BD Biosciences), PE-conjugated anti-hCD33 mAbs (BioLegend), anti-hCD38 mAbs (BD Biosciences), PerCP-conjugated anti-mCD45 mAbs (BioLegend), APC-conjugated anti-hCD4 mAbs (BioLegend), anti-hCD34 mAbs (BD Biosciences), and APC-Cy7-conjugated anti-hCD8 mAbs (BioLegend) were used to analyze Hu-NOG mice. FITC-conjugated anti-mLy6C/6G mAbs, PerCP-Cy5.5-conjugated anti-mCD45.2 mAbs, and APC-conjugated anti-mCD45.1 mAbs  were used to analyze Mo-NOG mice. Flow cytometric analysis was conducted using the FACSCanto II (BD Biosciences) system. A total of 10,000 events were analyzed for each sample. FlowJo software  was used for the analysis of flow cytometry data. Data from several samples in which the number of leukocytes exceeding 2 standard deviations of the group mean was detected were not used for analysis.5) collected from Hu-NOG mice were plated in methylcellulose-based medium . After 13 days of cultivation at 37°C in a humidified atmosphere containing 5% CO2, the numbers of colony-forming unit-granulocyte/erythroid/macrophage/megakaryocytes (CFU-GEMMs) were enumerated using visible light microscopy.Bone marrow cells . In colony-forming assays for multilineage hematopoietic progenitors, the numbers of CFU-GEMMs appearing in bone marrow cells were reduced depending on the amount of benzene administered , 3.0×108 cells/tissue (spleen), 3.1×105 cells/tissue (thymus) and 5.2×102 cells/µL .Human leukocytes were identified in the peripheral blood and hematopoietic organs of Hu-NOG mice by double staining with anti-hCD45 and anti-mCD45 antibodies. By maintenance of the mice for about 4.5 months after cell transplantation, human leukocytes were highly represented in leukocytes contained in all target tissues of Hu-NOG mice . The num+mCD45−) in the peripheral blood and hematopoietic organs of Hu-NOG mice. The numbers of human leukocytes in all samples were reduced depending on the amount of benzene administered to the same extent as human hematopoietic stem/progenitor cells in the bone marrow  than in the number of myeloid cells (hCD33+) in the bone marrow and peripheral blood .We next analyzed the population of human leukocytes in Hu-NOG mice using anti-hCD33 mAbs and found that benzene administration caused a more dramatic reduction in the number of lymphoid cells . In Mo-NOG mice, C56BL/6 mouse cells succeeded in reconstituting the hematopoietic cell population (p>0.10).In this study, NOG mice (CD45.1) with different strain-derived mouse hematopoietic lineages were established by transplanting Linpulation . After bpulation . MoreoveHere, we evaluated the toxic response of a human-like hematopoietic lineage established in NOG mice using the hematotoxicant benzene MEF2cin vivo response of human hematopoietic cells to known and suspected toxicants. Moreover, Hu-NOG mice can contribute to basic research on human hematopoietic cells, particularly with respect to internal tissues and organs. It is important to note that the LOAEL of benzene-induced hematotoxicity in Hu-NOG mice was approximately equivalent to that established for humans Human lymphoid cells showed higher sensitivity to benzene than myeloid cells in Hu-NOG mice . In a prSensitivity to benzene differs across species, and humans are more susceptible than mice MEF2c expression by benzene on the basis of the reasons stated above.The toxicity of benzene in leukocytes in the peripheral blood is induced mainly by benzene metabolites produced in organs such as the liver in vivo system for performing species-specific biochemical analyses of benzene metabolism. We think it is reasonable to assume that Hu-NOG mice will provide a powerful in vivo tool for assessing the hematotoxicity of chemical and physical agents on human hematopoietic cells. In the future, the similarities of the hematotoxic responses induced in Hu-NOG mice and humans should be evaluated more carefully by analyzing the detailed toxic response mechanism in Hu-NOG mice. Our strategy may be applicable to the study of other organs In conclusion, a human-like hematopoietic lineage established in NOG mice by transplanting human hematopoietic stem/progenitor cells exhibited human-like susceptibility to at least 1 hematotoxicant, benzene. Hu-NOG and Mo-NOG mice offer a well-defined, reproducible, and easy-to-manipulate


HIV-1 relies on the host ESCRTs for release from cells. HIV-1 Gag engages ESCRTs by directly binding TSG101 or Alix. ESCRTs also sort ubiquitinated membrane proteins through endosomes to facilitate their lysosomal degradation. The ability of ESCRTs to recognize and process ubiquitinated proteins suggests that ESCRT-dependent viral release may also be controlled by ubiquitination. Although both Gag and ESCRTs undergo some level of ubiquitination, definitive demonstration that ubiquitin is required for viral release is lacking. Here we suppress ubiquitination at viral budding sites by fusing the catalytic domain of the Herpes Simplex UL36 deubiquitinating enzyme (DUb) onto TSG101, Alix, or Gag.Expressing DUb-TSG101 suppressed Alix-independent HIV-1 release and viral particles remained tethered to the cell surface. DUb-TSG101 had no effect on budding of MoMLV or EIAV, two retroviruses that rely on the ESCRT machinery for exit. Alix-dependent virus release such as EIAV’s, and HIV-1 lacking access to TSG101, was instead dramatically blocked by co-expressing DUb-Alix. Finally, Gag-DUb was unable to support virus release and dominantly interfered with release of wild type HIV-1. Fusion of UL36 did not affect interactions with Alix, TSG101, or Gag and all of the inhibitory effects of UL36 fusion were abolished when its catalytic activity was ablated. Accordingly, Alix, TSG101 and Gag fused to inactive UL36 functionally replaced their unfused counterparts. Interestingly, coexpression of the Nedd4-2s ubiquitin ligase suppressed the ability of DUb-TSG101 to inhibit HIV-1 release while also restoring detectable Gag ubiquitination at the membrane. Similarly, incorporation of Gag-Ub fusion proteins into virions lifted DUb-ESCRT inhibitory effect. In contrast, Nedd4-2s did not suppress the inhibition mediated by Gag-DUb despite restoring robust ubiquitination of TSG101/ESCRT-I at virus budding sites.These studies provide the first demonstration of a necessary and natural role for ubiquitin in ESCRT-dependent viral release and indicate a critical role for ubiquitination of Gag rather than ubiquitination of ESCRTs themselves.


Here we review intriguing in vitro and in vivo evidence that suggest emergent roles for acidic phospholipids in regulating DnaA protein-mediated initiation of Escherichia coli chromosomal replication. In vitro acidic phospholipids in a fluid bilayer promote the conversion of inactive ADP-DnaA to replicatively proficient ATP-DnaA, yet both PG and CL also can inhibit the DNA-binding activity of DnaA protein. We discuss how cellular acidic phospholipids may positively and negatively influence the initiation activity of DnaA protein to help assure chromosomal replication occurs once, but only once, per cell-cycle. Fluorescence microscopy has revealed that PG and CL exist in domains located at the cell poles and mid-cell, and several studies link membrane curvature with sub-cellular localization of various integral and peripheral membrane proteins. E. coli DnaA itself is found at the cell membrane and forms helical structures along the longitudinal axis of the cell. We propose that there is cross-talk between acidic phospholipids in the bacterial membrane and DnaA protein as a means to help control the spatial and temporal regulation of chromosomal replication in bacteria.Anionic ( Escherichia coli inner membrane contains a mixture of phospholipids with a composition of approximately 70% phosphatidylethanolamine (PE), 25% phosphatidylglycerol (PG) and ~5% cardiolipin (CL), with a small remaining fraction of metabolic intermediates .–73.oriC,C-terminal region allow over-expression of the mutant proteins, but not the wild type protein, to bypass the arrested-growth phenotype. However, findings from a study that used an ATP fluorescent analog, 2′-(or-3′)-O-(N-methylanthraniloyl) adenosine 5′ tri-phosphate (MANT-ATP), suggest that DnaA(L366K) might require a lower concentration of acidic phospholipids to induce the exchange of ADP to ATP bound to DnaA protein iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl] nonanoyl]-sn-glycero-3-phosphocholine as a probe in acidic and neutral phospholipid bilayers provides additional evidence of a direct interaction between DnaA and acidic phospholipids. The study revealed that DnaA at the site of a putative amphipathic helix (amino acids 354–372) inserts into the hydrophobic interior of lipid bilayers only when the bilayer is enriched in acidic phospholipids and has the same degree of fluidity that promotes nucleotide exchange [Independently, crosslinking studies that utilized the photoactivable phospholipid analog 1-exchange .E. coli lacks phosphatidylinositol (PI) or sphingolipids, negatively charged PI and ganglioside GM1  are equal to PG in their capacity to stimulate the release of DnaA-bound adenine nucleotide [1 have structurally distinct polar head groups. Furthermore, in contrast to ganglioside GM1, asialo-GM1 is feeble at promoting nucleotide exchange [The requirement for the fluid bilayer to also have acidic headgroups appears to be due to the acidic head groups’ participation in the electrostatic recruitment of DnaA, a basic protein, to the lipid bilayer . Such a cleotide ,106. Notexchange ,106. Phoexchange ,106. Intexchange . TogetheE. coli DnaA is supported by the solved crystal structure for a truncated form of Aquifex aeolicus DnaA [Supporting the importance of electrostatic forces for DnaA-membrane association, stable DnaA-lipid bilayer interaction is sensitive to ionic strength, as assessed by isopycnic centrifugation and intrinsic tryptophan fluorescence measurements . Site-dicus DnaA . Indeed,cus DnaA .Examinations of additional point mutations revealed that the amino acids Arg-328, Arg-334 and Arg-342 present in another potential amphipathic helix (Lysine-327–Ile-345) also are important for DnaA-CL association . Of thesoriC . These observations reflect the possibility that the ordered assembly of DnaA protein at specific recognition sequences might depend on whether DnaA is first exposed to cognate nucleic acid binding sites or to acidic phospholipids. Notably, the negatively charged polar head groups of acidic phospholipids and the negatively charged phosphodiester backbone of DNA may interact in a similar manner with basic proteins  that lack DNA as a result of cell division occurring near the cell pole [E. coli[B. subtilis[ell pole ,118. Exa subtilis producedE. coli membranes [E. coli osmosensory transporter ProP [B. subtilis[The presence of certain lipids at regions of membrane curvature serve to target protein-lipid complexes to cell poles ,58,119. embranes . CL is ater ProP –58, and  subtilis.E. coli DnaA, visualized by confocal microscopy of an internally-tagged GFP-DnaA fusion, also forms helical structures along the longitudinal cell axis, these helical structures exist distinct from and independent of MreB filaments [Whereas some protein are found concentrated at poles, there are proteins that form helices beneath the cell membrane, extending from pole to pole along the cell’s longitudinal axis, such as the cytoskeleton protein MreB and MinCDE ,123. Altilaments . Other silaments , but thiilaments  to be moilaments . A plasmilaments . InteresE. coli chromosomal replication at oriC is influenced by acidic phospholipids present in the cell membrane. Depletion of the acidic phospholipids via repressed expression of pgsA results in under initiation of replication and arrested cell growth. The growth arrest phenotype can be relieved by abnormal initiations events at loci other than oriC or by expression of DnaA protein harboring a point mutation in its membrane-binding, both of which suggest a close link between cellular membrane composition and essential DnaA-mediated initiations at oriC. Of interest, preventing the accumulation at inner membrane of an intermediate of the murein lipoprotein (Lpp) synthesis pathway also can relieve the growth-arrest phenotype of acidic phospholipid-deficient cells, raising the question of whether the accumulated intermediate of Lpp synthesis adversely affects DnaA’s action at oriC.There is a wealth of data suggesting that the ability of DnaA to normally initiate oriC, and are changes that occur to these domains during the cell-cycle somehow linked to the onset of replication of chromosomal DNA?Acidic phospholipids are concentrated in domain-like structures within the bacterial cell membrane, which change in a cell-cycle dependent manner. The acidic phospholipids may help determine the sub-cellular localization of proteins involved in cell division. It is worth asking might these anionic phospholipid domains also help dictate functional subcellular localization of DnaA protein bound to oriC to help set the precise timing of when DNA synthesis occurs; and (iii) possibly helping define the subcellular localization of chromosomal replication components. As such, we hypothesize crosstalk between DnaA protein and acidic phospholipids.Acidic phospholipids PG and CL may participate in multiple critical cellular processes related to chromosomal replication. These include: (i) rejuvenation of ADP-DnaA to ATP-DnaA to support rounds of replication in subsequent cell-cycles; (ii) inhibition of DnaA binding to


Thus far, this scenario has only been achieved experimentally using live-attenuated SIV vaccines. As such, great interest lies in identifying correlates of protection from a successful host response to pathogenic SIV. To this end, we have used a global genomics approach, tissue analysis, and explant cultures to identify immune complex (IC) signaling as an important component of a protective host response in the female reproductive tract (FRT) of animals vaccinated with the live-attenuated virus known as SIV mac251 32H alone or SIV-specific ICs were added drop-wise to mucosal surfaces of explants and incubated for 24 hr at 37oC / 5% CO2.RNA from cervical tissue was purified for microarray analysis. Significant genes were functionally classified and protein expression determined using single-cell analytical procedures for tissue sections. FRT tissue was removed from healthy, uninfected, adult Rhesus macaques and cervix isolated/dissected into small tissue pieces for ex vivo culturing. WT SIVmac239-ΔNef-vaccinated animals, with localized expression of these anti-inflammatory mediators in the mucosal epithelium of the FRT, coinciding with dampened inflammation, limited CD4+ T cell infiltration, and stunted virus replication. Explant cultures derived from the FRT of Rhesus macaques were used as a physiological platform to identify the inhibitory Fc receptor for IgG, FcγRIIB, and carbohydrates in the Fc portion of SIV-specific ICs as centrally important in mediating this anti-inflammatory signaling program in mucosal epithelial cells.A genome-wide transcriptomics analysis revealed selective enrichment of an anti-inflammatory program upon virus exposure in SIVThese results highlight an unappreciated, non-neutralizing role for antiviral antibodies at mucosal surfaces and implicates the mucosal epithelial cell as an important host sensor that integrates external signals to elicit a host program that either promotes (inflammatory) or suppresses (anti-inflammatory) immunodeficiency virus infection.


ATP-dependent paracrine signaling, mediated via the release of ATP through plasma membrane-embedded hemichannels of the connexin family, coordinates a synchronized response between neighboring cells. Connexin 43 (Cx43) hemichannels that are present in the plasma membrane need to be tightly regulated to ensure cell viability. In monolayers of bovine corneal endothelial cells (BCEC),Cx43-mediated ATP release is strongly inhibited when the cells are treated with inflammatory mediators, in particular thrombin and histamine. In this study we investigated the involvement of RhoA activation in the inhibition of hemichannel-mediated ATP release in BCEC. We found that RhoA activation occurs rapidly and transiently upon thrombin treatment of BCEC. The RhoA activity correlated with the onset of actomyosin contractility that is involved in the inhibition of Cx43 hemichannels. RhoA activation and inhibition of Cx43-hemichannel activity were both prevented by pre-treatment of the cells with C3-toxin as well as knock down of RhoA by siRNA. These findings provide evidence that RhoA activation is a key player in thrombin-induced inhibition of Cx43-hemichannel activity. This study demonstrates that RhoA GTPase activity is involved in the acute inhibition of ATP-dependent paracrine signaling, mediated by Cx43 hemichannels, in response to the inflammatory mediator thrombin. Therefore, RhoA appears to be an important molecular switch that controls Cx43 hemichannel openings and hemichannel-mediated ATP-dependent paracrine intercellular communication under (patho)physiological conditions of stress. An important indirect manner of intercellular communication is via paracrine signaling. Unopposed Cx-based channels present in the plasma membrane, referred to as Cx “hemichannels”, are a known conduit for the release of biomolecules, including purinergic messengers. The activity of Cx hemichannels is tightly regulated and characterized by a low open probability under physiological conditions to prevent deleterious events such as excessive loss of metabolites Intercellular communication can occur through direct and indirect pathways. Direct signaling is possible We extensively characterized gap junction- and hemichannel-mediated intercellular communication in bovine corneal endothelial cells  inhibit hemichannel activity through activation of the protease activated receptor 1 (PAR-1) in BCEC In intercellular ea (AA)” . ThrombiNo significant difference was found in AA between control and C3-pretreated BCEC in the absence of thrombin (comparison of black bars in Since the hemichannel-mediated responses of BCEC in Thrombin induces rapidly the activation of RhoA (a Rho GTPase family member) in endothelial cells To examine the involvement of RhoA in thrombin-induced inhibition of Cx hemichannel activity, we performed A-duplex . These rTo demonstrate the involvement of Cx43-made channels in the downstream cascade of activated RhoA, we performed amble-1) .Since the knock-down approach with siRNA against Cx43 affects both Cx43-hemichannel- and - gap junction-mediated signaling, we performed additional experiments to investigate an eventual contribution of the gap junctional pathway, although it was previously demonstrated that the gap junctional pathway only has a limited contribution to intercellular In another set of experiments, we investigated the effect of thrombin after knock-down of RhoA when ATP-dependent paracellular communication was blocked by extracellular apyrases . A mixtugligible . These fClostridium botulinum is known to covalently modify RhoA, RhoB and RhoC GTPases by mono-ADP-ribosylation. This event inactivates these Rho GTPases by preventing nucleotide exchange i) RhoA-GTPase activity is rapidly and transiently increased within 1 minute after thrombin exposure of BCEC, ii) C3-treatment, which prevented RhoA activation, alleviated thrombin-induced inhibition of Cx43-hemichannel responses, and iii) suppressing RhoA expression using siRNA completely alleviated thrombin-induced inhibition of Cx43-channel responses. Therefore, we propose that RhoA GTPases act as a molecular switch that controls the onset of thrombin-induced actomyosin contractility causing the potent inhibition of Cx43-hemichannel activity and subsequent ATP release. Furthermore, while several studies described that thrombin-induced signaling through RhoA leads to ATP release The most important finding of this study is that RhoA is an essential component of the signaling cascade that controls inhibition of Cx43-hemichannel activity through contractility and the hemichannel-mediated ATP-dependent paracrine intercellular communication. We show that RhoA is activated upon exposure of BCEC to thrombin and that chemical inhibition of RhoA activity with C3 and genetic inhibition of RhoA expression with siRNA alleviates the inhibition of Cx43 hemichannels by increased contractility in BCEC induced by thrombin exposure. These findings are important, since the Rho-ROCK signaling axis controls the thrombin-induced activation of contractility by non-muscle myosin II. Our findings indicate that thrombin activates RhoA in BCEC cells and that C3-treatment prevents the inhibitory effect of thrombin on intercellular communication as assayed by activity  and 2. Tactivity , indicatWhile our findings suggest that possible contribution of activated RhoA in inhibiting Cx43 gap junctional coupling in thrombin-treated BCEC is likely negligible or at least difficult to detect, we do not exclude that in other cell-systems activated RhoA might be able to potently regulate gap junctional coupling. Moreover, data from various authors underpin the concept that Rho GTPases and cytoskeletal reorganization are often involved in mechanisms that influence gap junctional - or hemichannel-mediated communication Recently, we have documented possible mechanisms and relevance by which enhanced actomyosin contractility could negatively regulate Cx43 hemichannel responses Our results obtained by experiments with pharmacological inhibitors (Y-27623 and blebbistatin) In conclusion, our current findings demonstrate that besides the previously identified Rho-associated kinase 2) and were grown for three to four days to reach confluence.Primary cultures of BCEC were established as previously described Fluo-4 AM (F14217), Dulbecco’s PBS (14190-091), Dulbecco’s modified Eagle’s medium , L-glutamine , antibiotic antimycotic mixture (15240-096), EBSS , and trypsin (25300-054) were obtained from Invitrogen-Gibco. Fetal bovine serum (F-7524), a luciferin-luciferase bioluminescence assay kit (FL-AAM), ATP, thrombin (T-4648), apyrase VI (A6410), apyrase VII (A6535) and LY (L-0259) were obtained from Sigma-Aldrich . The cell-permeable Rho inhibitor exoenzyme C3-transferase (CT04), referred to as “C3”, was obtained from Cytoskeleton, Inc .TAT-Cx43L2 peptide (85% pure) was obtained from Lifetein . TAT-Cx43L2 (YGRKKRRQRRRDGANVDMHLKQIEIKKFKYGIEEHGK) was used at 100 The protocol for point mechanical stimulation of a single cell has been described in our previous studies BCEC were treated with cell-permeable C3 , was measured using the luciferin-luciferase bioluminescence technique as previously described Activation of Rho GTPase was examined by GST pull-down of GTP-bound Rho proteins by using the Rho-binding domain (RBD) of Rhothekin protein (an effector of RhoA) expressed as a GST-fusion protein and coupled to agarose beads  to capture the active (GTP-bound) Rho proteins in BCEC-lysates. Pull down samples were subjected to SDS-PAGE and Western-blotting analysis. Active and total RhoA levels were visualized after immunoblotting with a primary rabbit monoclonal anti-RhoA antibody .Screening for potent siRNA-oligonucleotides, with exclusion of off-target candidates to selectively knock-down RhoA in BCEC, was performed using the RNAi batch selector One-way ANOVA was used to compare the mean values for different treatments while unpaired tests were used to compare results of experiments with a single treatment and a single control. A p value of


Chronic myeloid leukemia (CML) is initiated from the BCR-ABL-expressing leukemia stem cells (LSCs). These LSCs are highly resistant to BCR-ABL kinase inhibitors, imatinib, dasantinib and nilotinib, and methods for eradication of LSCs are still not available. It is critical to identify genes that play roles in survival and proliferation of LSCs. We recently discovered that the tumor suppressor gene Pten is downregulated in LSCs of CML mice. By genetic deletion or overexpression of Pten, we confirmed that Pten functions as a tumor suppressor in LSCs of CML, consistent with the role of Pten in LSCs of acute myeloid leukemia (AML) and progenitor cells of T-ALL progenitors. Functional enhancement of the Pten pathway provides a therapeutic strategy for targeting LSCs. The human Philadelphia chromosome (Ph) is present in over 95% of CML cases , and ariLSCs in many types of hematologic malignancies are believed to be a cell population required for initiating and sustaining growth of the leukemia -13. In CA study showed that a quiescent cell population in LSCs (Lin-CD34+) from CML patients was resistant to imatinib , suggestPten is significantly downregulated by BCR-ABL [Pten is often deleted or inactivated in many human cancers, including glioblastoma [Pten facilitates CML development by using Pten conditional knockout mice (Ptenfl/fl) as donor mice in our retroviral transduction/transplantation mouse model of CML. In so doing, bone marrow cells of Ptenfl/fl mice were transduced with BCR-ABL-iCre-GFP retrovirus or BCR-ABL-GFP retrovirus (as a control), followed by transplantation of the transduced cells into lethal irradiated recipient mice. Mice receiving donor cells transduced with BCR-ABL-iCre-GFP developed CML much faster that mice receiving donor cells transduced with BCR-ABL-GFP, with a higher percentage of myeloid leukemia cells and more severe infiltration of leukemia cells in the lungs. To confirm the role of Pten in CML, we examined whether overexpression of Pten causes a delay of CML development. We induced CML in mice with BCR-ABL-Pten-GFP or BCR-ABL-GFP, and found that CML development was significantly slower when Pten was overexpressed. Pten overexpression caused cell cycle arrest and increased apoptosis of leukemia cells. Next, we examined whether Pten suppresses LSCs. To do so, we compared the percentages of LSCs with and without ectopically expressed Pten, and found that Pten overexpression caused a decrease in the percentage of bone marrow LSCs, suggesting that Pten has an inhibitory effect on LSCs. To support this observation, we sorted LSCs from mice with CML induced by BCR-ABL-Pten-GFP or BCR-ABL-GFP, followed by transplantation of these LSCs into secondary recipients. We found that Pten overexpression reduced the ability of LSCs to induce CML [in vitro with rapamycin, a mTOR inhibitor, and found that rapamycin induced apoptosis of these cells, suggesting that a blockade of the mTOR pathway may help to inhibit LSCs and CML development.To identify genes that are differentially regulated by BCR-ABL in LSCs, we compared the global gene expression between normal hematopoietic stem cells (HSCs) and LSCs by conducting a DNA microarray analysis . We foun BCR-ABL . Becauseblastoma , endometblastoma , and lymblastoma , we deciduce CML . Becauseduce CML , we treaAlox5 activates p-Akt through oxidation and inhibition of Pten [Although Pten is intensively studied in solid tumors and T cell-acute lymphoid leukemia (T-ALL) -32, litt of Pten . The funPten is specifically deleted in mouse hematopoietic cells, the mice develop acute myeloid leukemia (AML) and acute lymphoid leukemia, and all mice died within 4 weeks [Pten deficient mice, causing an eventual exhaustion of LT-HSC [Pten deficient mice, indicating that Pten functions as a molecular switch governing the G0-G1 transition between the quiescent and activated states of LT-HSCs to maintain normal HSCs pool [Pten deficient AML mice, a high number of cyclin D1-expressing cells were detected in bone marrow, suggesting that cyclin D1 is downstream of Pten in cell cycle regulation of HSCs. Thus, the role of cyclin D1 in cell cycle regulation of LSCs in CML requires further investigation. Furthermore, after rapamycin administration, LSCs were depleted and normal HSCs restored in Pten-deficient AML mice [Pml deficiency promoted transition of LSCs from quiescent to activated stage and Pml-/- LSCs finally failed to initiate CML disease contrary to wild type LSCs after serial transplantation. As PML is a repressor of mTOR, inhibition of mTOR with rapamycin restored Pml-/- HSCs and the long-term reconstitution functions of LSCs. Another Pten conditional deletion mouse model demonstrated that Pten partial deletion in mouse fetal liver HSCs and their differentiated progeny led to a myeloidprliferative disorder, followed by actue T-lymphoblastic leukemia (T-ALL) [Pten deficient Lin-c-KitmidCD3+ population was shown to be the T-ALL LSCs through a serial transplantation assay. Interestingly, ablation of one allele of β-catenin significantly delayed the occurrence of acute leukemia. We and others have also shown that β-catenin plays a key role in maintaining LSCs in CML [When  4 weeks , 39. LSC 4 weeks . A blockSCs pool . In addiSCs pool , maintaiAML mice . Pml def (T-ALL) . In thiss in CML , 44 and s in CML .Taken together, these results allow us to draw a picture connecting Pten with other key pathways involved in survival and proliferation of LSCs, including β-catenin, p53, Alox5, PI3K/Akt/mTOR pathways Figure . These p


The establishment and maintenance of central tolerance depends to a large extent on the ability of medullary thymic epithelial cells to express a variety of tissue-restricted antigens, the so-called promiscuous gene expression (pGE). Autoimmune regulator (Aire) is to date the best characterised transcriptional regulator known to at least partially coordinate pGE. There is accruing evidence that the expression of Aire-dependent and -independent genes is modulated by higher order chromatin configuration, epigenetic modifications and post-transcriptional control. Given the involvement of microRNAs (miRNAs) as potent post-transcriptional modulators of gene expression, we investigated their role in the regulation of pGE in purified mouse and human thymic epithelial cells (TECs). Microarray profiling of TEC subpopulations revealed evolutionarily conserved cell type and differentiation-specific miRNA signatures with a subset of miRNAs being significantly upregulated during terminal medullary thymic epithelial cell differentiation. The differential regulation of this subset of miRNAs was correlated with Aire expression and some of these miRNAs were misexpressed in the Aire knockout thymus. In turn, the specific absence of miRNAs in TECs resulted in a progressive reduction of Aire expression and pGE, affecting both Aire-dependent and -independent genes. In contrast, the absence of miR-29a only affected the Aire-dependent gene pool. These findings reveal a mutual interdependence of miRNA and Aire. Aire-binding sites in the genome largely overlap with Pol II promoter occupancy. In a fraction of these binding sites, Aire promoted the release of stalled Pol II thus enabling low-level transcription [The scope of central tolerance in the thymus is largely dictated by the ectopic expression of a plethora of tissue-restricted antigens (TRAs) by thymic epithelial cells (TECs), in particular medullary thymic epithelial cells (mTECs), a phenomenon termed promiscuous gene expression (pGE). Within the mTEC lineage, the diversity and the level of pGE increase with terminal differentiation, which is characterised by the upregulation of CD80 and MHC class II (MHCII) and the induction of the autoimmune regulator (Aire). Thus, mature CD80 factors . Aire is factors –7. Yet m factors –11. Morecription . Our precription . AltogetMicroRNAs (miRNAs) are small RNA molecules involved in the post-transcriptional control of gene expression. Mature miRNAs bind Argonaute proteins and are incorporated into the RNA-induced silencing complex, which in turn binds to the target mRNA leading to translational block and/or degradation . A primaT-cell development and function is also affected in a cell-extrinsic manner by miRNA loss in the thymic epithelium. Genetic deletion of Dicer in TECs results in premature thymic involution, progressive disorganisation of the thymic epithelium and formation of epithelial voids . The epiHere, we analysed the role of miRNAs in mTEC biology and pGE. miRNA microarray analysis of TEC subpopulations revealed several epithelium-specific miRNAs that were upregulated upon mTEC maturation in mouse and human thymus. We show that, on the one hand, Aire regulates maturation-specific miRNA expression in mTECs and, on the other hand, miRNAs regulate Aire and TRA expression in mTECs. Collectively, our data document a mutual interdependence between Aire and miRNAs in directing mTEC maturation and pGE.low and mature mTEChigh, the latter based on MHCII surface expression levels. Sets of 25 miRNAs with the highest and ten miRNAs with the lowest ratio in mTEChigh versus thymocytes of mouse and human are shown in In order to investigate the miRNA signature in thymic epithelium, we performed miRNA microarray analysis of sorted thymic cells from mouse and human samples  than in human (16). Conversely, the numbers of MHCIIlow- and MHCIIhigh-specific miRNAs were comparable across species  and 157 (human) miRNAs were upregulated (≥2-fold) in the TECs versus T cells. The analysis of TEC subtype specific miRNAs in both species showed a similar number of miRNAs expressed in all TECs (54 and 47). We found fewer miRNAs to be cortex specific (33 and 9) than medulla specific . Of the medulla-specific miRNAs, the overlap between mTEC species B. TEC-sp species C. The mi of TRAs . We choshe cells C.high/CD80high mTECs can be further divided into Aireneg and Airepos subsets  and GFPpos (Airepos) CD80high mTECs and analysed the expression of TEC-specific miRNAs within these subsets. We found that all analysed miRNAs were downregulated in Airepos mTEChigh compared with AirenegmTEChigh  alongside with their Dicerflox/flox×FoxN1wt or Dicerwt/wt×FoxN1Cre control littermates. We and others have recently shown that genetic deletion of Dicer in TECs leads to disruption of the thymic architecture in situ and premature involution [pos cells in the medulla ; (iv) Ins2 (insulin 2), Th (tyrosine hydroxylase) and Myh7 .Next, we asked whether premature involution and stroma disorganisation would affect the cell-intrinsic regulation of pGE in Dicer mutants, and whether this effect progressed with age. To this end, we isolated immature and mature mTECs from 4- and 10-week-old Dicer-deficient animals and control littermates. Because the CD80 and MHCII expression profiles were slightly shifted in the mutants, relative gates were chosen, i.e. the lower/upper 35% of cells based on the expression levels of MHCII was taken as immature/mature mTECs, respectively. Based on our previous work [As shown in high mTECs in the mutants and a shift towards lower CD80 and higher MHCII expression levels, similar to the Dicer null mutants  in the 12-week-old mutants B. The expGE in TECs is regulated at multiple levels –13. In aneg versus mature Airepos mTEC subsets. Thus, miR-129 and miR-499 expression was downregulated upon induction of Aire among mature mTECs, yet both miRNAs were also downregulated in mature mTECs in Aire null mice as compared with WT. These data argue against a direct control of miRNA expression by Aire. A role of Aire beyond the control of pGE has been previously described, e.g. in antigen processing [We identified miRNAs with TEC-specific expression in mouse and human TECs and demonstrated that there is a larger overlap in miRNA expression between immature and mature mTECs than between either of these and cTECs. Several of the mTEC-specific miRNAs showed a maturation-dependent upregulation in both mouse and human, compatible with a potential role in pGE regulation. Hence, we focused our analysis on these particular miRNAs. Interestingly, miR-124, miR-129, miR-202, miR-203, miR-302b and miR-467a were differentially regulated in immature and mature Aireocessing  and mTECocessing .In the context of a putative role of miRNA in pGE, it is noteworthy that several mRNAs, upregulated upon mTEC maturation, showed tissue-specific expression patterns, i.e. being restricted to brain (miR-124 and miR-129), heart (miR-499), testis (miR-202), skin (miR-203) or embryo (miR-467 and miR-302). Moreover, miR-124 and miR-203 have been shown to be upregulated upon terminal differentiation in neurons and keratinocytes, respectively , 34. In A possible functional relationship between pGE and miRNA has been previously addressed by analysing the impact of an Aire knockdown on the miRNome in an mTEC cell line . The miROur results indicate that miRNAs regulate Aire expression, as Aire is downregulated in mature mTECs of Dicer null mutants. Together with Aire, the expression of various Aire-dependent and -independent TRAs was affected in the Dicer null mutants. Notably, the loss of Dicer affected all four pools of TRAs, which according to a recent study are differentially regulated in a cell-autonomous or non-autonomous manner . Since oThe age-dependent decline in pGE can also not be explained by a gradual disorganisation of the thymic architecture, since it sets in before the appearance of epithelial voids in the mutant thymus. Previously described mutants with disrupted thymic architecture did not display changes in pGE when normalized to purified TEC subsets –42. ThusA first step in identifying miRNAs that cause the observed Dicer knockout thymic phenotypes was a side-by-side comparison with miR-29a null mutant mice. These mice mimic the Dicer null-associated thymic involution without presenting the gross architectural abnormalities . InteresIn conclusion, we have analysed the expression of miRNAs in the thymic epithelium and at different stages of mTEC maturation and the role of miRNA biogenesis in pGE. Our data document that particular miRNA signatures are maturation dependent and species conserved, coincide with Aire upregulation and are affected by Aire loss. Aire expression and pGE, in turn, are affected by the loss of Dicer. The comparison of Dicer and miR-29a mutants suggests that the defects in thymic involution, epithelial architecture and pGE in the Dicer null mutants are caused by the absence of miRNAs other than miR-29a. The identification of these mRNA species poses a future challenge.flox/flox mice have been provided by M. Merkenschlager [Cre transgenic mice by T. Boehm [Adig (Aire-GFP) mice on the BALB/c background by L. Klein [flox/flox×FoxN1Cre) and miR-29a knockout mouse lines were kept on the C57BL6/N background. All animal breeding and experiments were performed according to the guidelines of the German Cancer Research Center (DKFZ).When not indicated otherwise, 4- to 6-week-old WT C57BL6/N mice were used. Dicerschlager , FoxN1CrT. Boehm  and AdigL. Klein . Dicer nHuman thymi were obtained from two male children (5 and 12 months old) in the course of corrective cardiac surgery at the Department of Cardiac Surgery, Medical School of the University of Heidelberg, Germany. Human studies were approved by the Institutional Review Board of the University of Heidelberg.pos cells using CD45 MicroBeads (Miltenyi Biotec). Murine TEC-enriched single-cell suspensions were blocked with anti-FcR mAb 2.4G2 and stained for the following surface markers: PerCP-anti-CD45 , Alexa647-anti-EpCAM (clone G8.8), FITC-anti-Ly-51 , PE-anti-CD80 , biotin-anti-MHCII  and Sav-PE-Cy7 (BD Biosciences). For dead cell exclusion, PI or DAPI were used. Mouse TECs were defined as CD45neg EpCAMpos, and were further subdivided into Ly-51pos cTECs and Ly-51neg mTECs. mTECs were separated into immature (CD80low or MHCIIlow) and mature (CD80high or MHCIIhigh), representing the top and bottom 33–35% of the population. In Adig mice, mature CD80high mTECs were further separated into Aireneg and Airepos subsets based on Aire-driven GFP expression. Cell sorting was performed on a BD FACSAria cell sorter.TEC isolation was performed as previously described , 43, 44.−/loCDR2+G8.8+), immature mTECs (CD45−/loCDR2−HEA125+HLA-DRlow) and mature mTECs (CD45−/loCDR2−HEA125+HLA-DRhi) were sorted on a BD FACSAria cell sorter.After CD45 depletion, human TEC-enriched cells were blocked with mouse serum and stained with anti-CD45-PerCP , biotinylated anti-EpCAM (clone HEA125), anti-HLA-DR-Alexa647 , CDR2-Alexa488 [Total RNA was isolated using the High Pure miRNA Isolation kit (Roche) and the concentration and integrity were analysed by Eukaryote Total RNA nano Series II chip on a 2100 Bioanalyzer (Agilent). A total of 200 ng of total RNA was polyadenylated and used for cDNA synthesis. miRNA expression profiling was performed using Illumina Version 2 MicroRNA Expression BeadChip Arrays according to the manufacturer's instructions. The human v2 and the mouse v2 panels contained 1146 and 656 probes, respectively. Arrays were scanned on a BeadArray Reader and the array intensity data were imported into BeadStudio v3.2 (Illumina).Background adjustment and quantile normalisation was performed with the BeadStudio software. All sample expression values are mean values over beads and group expression values are mean values over the replicates expression.t-test, and for each quantification at least two independent experiments were performed.For RT-qPCR analysis, RNA was prepared from sorted cells by modified Trizol extraction method. The modification included precipitation with 100% Ethanol at −20°C overnight. For messenger RNA analysis, 100–500 ng total RNA was used for the first-strand synthesis with SuperScriptII and oligo-dT primer. For miRNA analysis, first-strand synthesis was performed with commercially available Taqman miRNA Assay primers according to the manufacturer's protocol. qPCR was performed in triplicates using the Applied Biosystems 7300 light cycler with Taqman probes for miRNAs and intron-spanning primers for mRNA-coding genes. The results were analysed by the ddCt method. Expression levels were quantified from three technical replicates using Student's


The cellular and molecular mechanisms of tumor angiogenesis and its prospects for anti-angiogenic cancer therapy are major issues in almost all current concepts of both cancer biology and targeted cancer therapy. Currently, (1) sprouting angiogenesis, (2) vascular co-option, (3) vascular intussusception, (4) vasculogenic mimicry, (5) bone marrow-derived vasculogenesis, (6) cancer stem-like cell-derived vasculogenesis and (7) myeloid cell-driven angiogenesis are all considered to contribute to tumor angiogenesis. Many of these processes have been described in developmental angiogenesis; however, the relative contribution and relevance of these in human brain cancer remain unclear. Preclinical tumor models support a role for sprouting angiogenesis, vascular co-option and myeloid cell-derived angiogenesis in glioma vascularization, whereas a role for the other four mechanisms remains controversial and rather enigmatic. The anti-angiogenesis drug Avastin (Bevacizumab), which targets VEGF, has become one of the most popular cancer drugs in the world. Anti-angiogenic therapy may lead to vascular normalization and as such facilitate conventional cytotoxic chemotherapy. However, preclinical and clinical studies suggest that anti-VEGF therapy using bevacizumab may also lead to a pro-migratory phenotype in therapy resistant glioblastomas and thus actively promote tumor invasion and recurrent tumor growth. This review focusses on (1) mechanisms of tumor angiogenesis in human malignant glioma that are of particular relevance for targeted therapy and (2) controversial issues in tumor angiogenesis such as cancer stem-like cell-derived vasculogenesis and bone-marrow-derived vasculogenesis. In his landmark 1971 publication, Folkman  developeCell snapshot Jain and Carmeliet listed six different principal cellular mechanisms under the heading of tumor angiogenesis. These include (1) classical sprouting angiogenesis, (2) vascular co-option, (3) vessel intussusception, (4) vasculogenic mimicry, (5) bone marrow derived vasculogenesis and (6) cancer stem-like cell derived vasculogenesis [In a recent ogenesis . Another include  classicaAngiogenesis, the sprouting of capillaries from pre-existing vessels, has since long been considered the principal mechanism of brain vascularization, both during development and in brain cancer [n cancer , 121. A n cancer , 143). Cn cancer , 31, 114Vascular co-option describes the infiltration of tumor cells into normal tissue and the adoption of the pre-existing vasculature [culature . When onVessel intussusception describes the formation of a new vessel by vascular invagination, intra-luminar pillar formation and splitting. Vascular intussusception has initially been described in physiological vascular development [elopment  but moreelopment . The molVasculogenic mimicry is defined as a process where tumor cells replace endothelial cells and form a vessel with a lumen This phenomenon was first described (mainly based on PAS staining) in a subset of aggressive uveal melanomas [kruppel-like factor2 (KLF2) and subsequent up-regulation of Ang-2 . Iniews see . CurrentSignaling pathways initiated from numerous growth factor receptors are known to play pivotal roles in developmental and tumor-mediated angiogenesis. In this review, we focus on three signaling pathways with a proven role in vascular biology and/or tumor angiogenesis that are currently being explored in clinical trials Table  or repreVEGF is the principal angiogenesis factor in both embryonic development and tumor growth (for review see ). Deletialpha and LDH are expressed in the same cell type [The Cancer Genome Atlas (TCGA) (http://cancergenome.nih.gov) suggest that in GBM the entire VEGF/VEGF receptor system is predominantly orchestrated by the up-regulation of VEGF; however, the VEGF receptors-1, -2 and -3 are also up-regulated in the tumor vasculature. The vast majority of clinical trials that target tumor angiogenesis use drugs that target the VEGF signaling pathway, either by blocking VEGF or by interfering with VEGFR-2 signaling . The consequences of blocking Tie2 signaling in vivo, as evidenced by several pre-clinical studies, appears to be a shift towards an immature vasculature, whereas Ang-1 appears to “normalize” the vasculature (see below). In addition to an impact on angiogenesis, Ang-2 was shown to mediate a pro-inflammatory phenotype. In Ang-2 null mice the inflammatory response to stimuli such as TNF-α was greatly diminished when compared to controls [Pharmacological interference of the Tie2/Angiopoietin signaling pathway has received considerable attention recently . Secontrols . Vice vecontrols . These fcontrols ). Moreovcontrols , Ang-2 ucontrols , 86, 97.The Notch signaling pathway plays an important role in organ development and recently has also received considerable attention in the vascular biology field recently  and others  switch to a pro-migratory phenotype, (2) up-regulation of other pro-angiogenic molecules  and (3)


Common variable immunodeficiency (CVID) is a heterogeneous primary immunodeficiency characterized by low serum antibody levels and recurrent infections. Cellular response to immunization in CVID has not been elucidated. In this study aimed to characterize influenza specific memory B-cell responses in patients with CVID and normal controls following influenza immunization.CVID and unaffected controls were immunized with the 2010 influenza vaccine. PBMCs were collected on the day of vaccination, and then week 8 and week 16 after vaccination. Memory B cell responses were determined by ELISPOT analysis.Both the CVID and controls showed similar induction of flu-specific IgM-secreting memory B cells after vaccination. Before vaccination, CVID subjects had significantly lower frequencies of flu-specific IgG and IgA memory B cells. Half of the CVID subjects (4/8) showed an increase in flu-specific IgG-secreting memory B cells post vaccination, whereas the other half showed none. 8/8 controls showed increased flu-specific IgG-secreting memory B cells post-vaccination. None of the CVID subjects developed flu-specific IgA memory B cells post vaccination, compared to 5/8 normal subjects.A subgroup of CVID may be capable of making IgG memory responses to protein vaccination, although, the ability to maintain these responses needs to be studied with longer follow-up. Individuals with CVID however, demonstrated severe defects in IgA memory responses to vaccination, which may have clinical relevance in terms of protection against influenza. Further work is continuing to evaluate the influenza specific T-cell responses in this patient population.


Protein production in CHO cell lines is a highly dynamic process, where the yield limiting step can shift among transcription, translation, and secretion under different cell growth stages and culture conditions. In addition, the choice of CHO cell line has been shown to affect target protein quality and quantity.Further advancements in titer and specific productivity require elimination of cellular/process bottlenecks along the generation of stable production cell lines including vector design, clone selection, genetic engineering, and media and feed optimization.To overcome these limitations, we developed a toolbox for all process phases from transfection to production. This toolbox is based on a chemically defined media platform and includes pre-adapted CHO-K1 or CHO-DG44 cell lines, optimized vectors for single and multi-chain proteins as well as fine-tuned protocols. In addition, it enables the production of antibodies with specialized glycan structures , and combined selection and cloning in 96-well plates. For a first expression analysis monoclonal primary clones were subjected to 96-well plate assay. Clones showing best performance in 96-well were expanded and taken into batch and fed-batch for productivity assessment. Best performing primary clones were subjected to a second cloning step by limiting dilution and analyzed in 96-well. Following clone scale-up highest ranking sub-clones clones were assessed in shake flask using fed-batch. Prior to master cell banking candidate lead clones were tested for fed-batch bioreactor performance in a Cellferm-pro® (DASGIP AG). Protein concentrations were determined using either an ELISA method, the Gyrolab SIA Ligand Binding Assay or an HPLC method.Protein secreted from stable cell lines was purified either by protein A affinity chromatography (Fc-protein) or Ion Exchange Chromatography using weak anion exchangers (glycoprotein) and evaluated by Size-Exclusion HPLC (SEC), Western blot and SDS-PAGE.To determine the stability of clones a continuous passage of roughly 80 population doublings with analyzing protein batch titers as well as mRNA levels and gene copy numbers (StepOnePlus™ RT-PCR System) at defined time points was performed. Cells were cultivated without selection pressure.Here we describe an innovative parallel platform cell line development for CHO-K1 and CHO-DG44 (Figure Our toolbox approach enables a fast and highly reproducible generation of high producer clones stably expressing the transgene for more than 80 generations without selection pressure and resulting in upstream yields of 4.5-13 g/L (Table


Caenorhabditis elegans must choose between reproductive development and dauer diapause. This decision is based on sensing of environmental inputs and dauer pheromone, a small molecule signal that serves to monitor population density. These signals are integrated via conserved neuroendocrine pathways that converge on steroidal ligands of the nuclear receptor DAF-12, a homolog of the mammalian vitamin D receptor and liver X receptor. DAF-12 acts as the main switch between gene expression programs that drive either reproductive development or dauer entry. Extensive studies in the past two decades demonstrated that biosynthesis of two bile acid-like DAF-12 ligands, named dafachronic acids (DA), controls developmental fate. In this issue of PLoS Biology, Wollam et al. showed that a conserved steroid-modifying enzyme, DHS-16, introduces a key feature in the structures of the DAF-12 ligands, closing a major gap in the DA biosynthesis pathway. The emerging picture of DA biosynthesis in C. elegans enables us to address a key question in the field: how are complex environmental signals integrated to enforce binary, organism-wide decisions on developmental fate? Schaedel et al. demonstrated that pheromone and DA serve as competing signals, and that a positive feedback loop based on regulation of DA biosynthesis ensures organism-wide commitment to reproductive development. Considering that many components of DA signaling are highly conserved, ongoing studies in C. elegans may reveal new aspects of bile acid function and lifespan regulation in mammals.Larvae of the nematode  C. elegans normally goes through a simple life cycle: from egg, through four larval stages, to reproductive adult. However, under adverse environmental conditions, these worms enter an alternate third larval stage termed dauer. Compared to normal third stage larvae, dauer larvae have dramatically different metabolism and physiology, and distinct morphology and behavior C. elegans exit dauer and resume reproductive development. The dauer stage is generally considered as “non-aging,” as dauers can persist for months before recovering to develop into a reproductive adult that lives the normal lifespan of a few weeks. Not surprisingly, recent findings suggest that re-activation of some of the molecular signature of dauer later in life contributes to prolonged longevity in C. elegansdaf” (dauer formation) mutants, many of which turned out to represent components of highly conserved signaling pathways. Environmental cues are perceived by a number of sensory neurons daf-2 insulin/insulin-like growth factor signaling (IIS) pathway and the daf-7 TGF-β signaling pathway The major environmental cues that regulate dauer formation include food, temperature, and a small molecule signal constitutively produced by the worms: the dauer pheromone daf-12, a nuclear hormone receptor with homology to mammalian vitamin D receptor and LXR, as the main switch on which several upstream dauer signaling pathways converge. daf-12 acts as a ligand-dependent transcription factor that locks in the final decision on reproductive versus dauer development daf-9 was found to act upstream of daf-12 antagonizing dauer entry daf-9 and (2) that these steroidal DAF-12 ligands drive reproductive development, whereas unliganded DAF-12 promotes entry into the dauer stage Genetic evidence revealed C. elegans metabolite extracts more difficult. Subsequent studies have shown that many of the dauer pheromone components also affect C. elegans behavior, often at concentrations far below those that induce dauer Until recently surprisingly little was known about the role of small molecules in these pathways. Not until 2005, more than 20 years after its initial discovery by Golden and Riddle, was the first component of the dauer pheromone identified. In the past few years, it has become apparent that the “dauer pheromone” in fact constitutes a multi-component mixture consisting of at least 10 different, but chemically and biosynthetically related compounds. All dauer pheromone components consist of the dideoxysugar ascarylose and include a variable fatty-acid derived side chain and occasionally carry other substituents  dhs-16 as an enhancer of the daf-36 dauer phenotype. Genetic epistasis experiments showed that dhs-16 acts downstream of insulin/IGF-1 and TGF-β signaling pathways, but upstream of DAF-12, indicating that DHS-16 participated in DA biosynthesis. To determine the exact role of DHS-16 in DA biosynthesis, Wollam et al. conducted a series of sterol feeding experiments, which showed that 3-keto steroids rescue the dhs-16 phenotype, whereas the corresponding 3-hydroxy steroids were without effect. These results suggested that DHS-16 has 3β-dehydrogenase activity, converting lathosterol, and perhaps other 3-hydroxy sterols, into the corresponding 3-keto derivatives (In this issue of ivatives .C. elegans development and lifespan may suggest that its mammalian homologs serve analogous roles in bile acid metabolism. If confirmed, such close homology may suggest the intriguing possibility that bile acid-like steroids regulate mammalian lifespan, which to date has not been comprehensively explored.The identification of DHS-16 as a 3β-HSD introducing the characteristic 3-keto functionality closes a major gap in the DA biosynthesis pathway. Although DHS-16 is not orthologous to known mammalian 3β-HSDs, which appear to rely on a different chemical mechanism for introducing the 3-keto functionality, there are several mammalian short-chain dehydrogenases, for example the liver-expressed SDR-O/SDR9C7, that are close homologs. The finding that DHS-16 has 3β-HSD activity and participates in the biosynthesis of bile acid-like molecules that control C. elegans larvae integrate environmental signals before committing to dauer or reproductive development. Using dauer pheromone and DA as opposing stimuli, Schaedel et al. established that the decision can be affected by DA and pheromone only during precisely defined time windows prior to ultimate commitment to life cycle fate. Furthermore, it became apparent that dauer pheromone and DA are directly competing stimuli: higher dauer pheromone concentrations require higher DA levels to prevent commitment to dauer entry. Unexpectedly, daf-9 null mutants, which are incapable of producing DA, respond differently than wild type worms to conditions that are near the dauer-inducing threshold: whereas cohorts of wild type larvae exposed to moderate dauer pheromone concentrations develop into a mixture of dauer larvae and fully developed adults, daf-9 mutants exposed to low concentrations of added DA develop into animals displaying a range of intermediate phenotypes, including worms that bypass the dauer stage yet exhibit an abnormal phenotype. That these intermediate phenotypes occur almost exclusively in daf-9 worms suggests that organism-wide commitment to either dauer or reproductive development involves direct regulation of daf-9.As a clearer picture of DA biosynthesis emerges, tools become available to address a most fascinating aspect of life history decisions: how are organism-wide developmental changes coordinated? The decision between entering the dauer diapause and reproductive development must rely on robust temporal and spatial coordination mechanisms to avoid partial phenotypes. To uncover these mechanisms, Schaedel et al. first investigated the time points at which developing daf-9 null worms expressing GFP under the control of the daf-9 promotor, Schaedel et al. showed that in the absence of DA, daf-9 transcription is turned on only in the XXX cells, whereas intermediate DA concentrations resulted in a dramatic increase in its expression in hypodermal cells during the time window the larva must decide between dauer and reproductive development (decreased hypodermal daf-9 transcription. The finding that daf-9 is expressed constitutively in the XXX cells whereas hypodermal daf-9 expression is regulated by DA suggested that DA produced by XXX may promote daf-9 expression in hypodermal cells. To test this hypothesis, Schaedel et al. laser-ablated the XXX cells in larvae expressing a DAF-9 protein tagged with GFP. They observed that almost all XXX-ablated worms enter the dauer stage, instead of developing into normal adults, and lacked DAF-9 expression in hypodermal cells. Dauer formation in these worms could be fully rescued by addition of DA, without forming any of the intermediate phenotypes seen for the daf-9 null mutants with DA. Taken together, these results suggest that the XXX cells act as a source of DA that triggers strong additional DA biosynthesis in the hypodermis, locking in organism-wide commitment to reproductive adult development. In response to favorable conditions this feedback loop maintains DA levels high enough to continue reproductive development, whereas unfavorable conditions appear to trigger reduced DA production in the XXX cells, resulting in complete shutdown of DA biosynthesis to ensure organism-wide initiation of the transition into dauer.Using transgenic elopment . Somewhadaf-9 is kept at an intermediate level, whereas under marginal conditions, when DA levels are intermediate, hypodermal daf-9 becomes highly upregulated (The study of DA biosynthesis and its regulation thus revealed an elegant self-regulatory loop enforcing a binary decision for dauer or reproductive development. However, several aspects of this regulatory network remain to be uncovered. Mechanistically it is unclear how under favorable conditions, when DA levels are high, hypodermal egulated . To addrSchaedel et al. further show that pheromone and DA act as competing signals. Presumably, higher levels of pheromone modulate upstream DAF-2 and DAF-7 signaling that eventually affects expression of DA biosynthesis genes. Similarly, the molecular mechanisms of how food-derived signals and temperature sensing contribute to the dauer decision remains to be clarified. It is likely that these factors represent environmental inputs that are sensed by upstream signaling, but it is also possible that the food signal represents a metabolic input that influences dauer decision by upregulating DAF-2/insulin signaling.7-DA biosynthesis as previously hypothesized. Nonetheless, the hsd-1 dauer phenotype C. elegans genome contains many additional HSD, DHS, and CYP450, some of which likely participate in DA biosynthesis. If additional components of DA exist, then possible tissue specificity of their biosynthesis and action will be important questions for future investigation.Lastly, the DA biosynthetic pathway is still far from complete. Wollam et al. showed that HSD-1 is not involved in ΔC. elegans, particularly in response to germline signals. Furthermore, the pheromone responsive pathways DAF-2 and DAF-7 are both key mediators of longevity C. elegans may reveal new aspects of bile acid function in mammals. Whether bile acids will turn out to be important for mammalian aging and aging pathologies remains an interesting area for future investigation.We should note that the DA biosynthetic pathway has a key role in determining longevity in


PTPN11 gene  were associated with different levels of serum lipids. The aim of this study was to explore the relationship between single nucleotide polymorphisms (SNPs) of PTPN11 and serum lipids in Northeast Chinese.Previous studies have reported that different genotypes of PTPN11 gene were genotyped using TaqMan assay method.A total of 1003 subjects, 584 males and 419 females, were included in the study and their serum lipids were determined. Five htSNPs  of All of the five SNPs were in Hardy-Weinberg equilibrium. The male subjects had higher triglyceride (TG), higher low-density lipoprotein cholesterol (LDL-C) and lower high-density lipoprotein cholesterol (HDL-C) level than females. In males, rs4767860 was found to be associated with serum TG and total cholesterol (TC) levels and rs12229892 was associated with TC level. However, these significant associations could not be observed in females. In females, rs2301756 was found to be associated with TG and rs7958372 was associated with LDL-C level. Haplotype analysis showed that the GCGTG haplotype was associated with slightly higher TG level and ATGCG with higher TC level.PTPN11 may play a role in serum lipids in a sex-specific pattern. However, more studies are needed to confirm the conclusion and explore the underlying mechanism.SNPs of  Dyslipidemia such as the increased levels of total cholesterol (TC), triglyceride (TG) or the decreased level of high-density lipoprotein cholesterol (HDL-C) has been concluded to be involved in the higher risk of cardiac-cerebral vascular disease –3] and  and 3] aPTPN11) gene, which encodes SHP2, may be associated with serum lipid levels via changing the activity of SHP2 on lipometabolism.Src homology-2 domain-containing protein tyrosine phosphatase 2 (SHP2), a ubiquitously expressed protein tyrosine phosphatase, plays an essential role in many cell signaling events such as metabolic control and transcription regulation ,8. SHP2 PTPN11 gene, rs11066320, was associated with serum low-density lipoprotein cholesterol (LDL-C) level in normal Caucasian female twins . Se. Se26]. PTPN11 gene were associated with serum lipid levels in a sex-specific pattern. Rs12229892 and rs4767860 may play an important role in lipid profile in males, and rs2301756 and rs7958372 may be related to TG and LDL-C levels in females. Further studies are needed to explore the mechanism on how PTPN11 SNPs exert their effects on lipid profile.In summary, we found that SNPs of SNPs: Single nucleotide polymorphisms; PTPN11: Protein tyrosine phosphatase, non-receptor 11; TG: Triglyceride; TC: Total cholesterol; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; SHP2: Src homology-2 domain-containing protein tyrosine phosphatase 2; BMI: Body mass index; htSNPs: Haplotype tagging SNPs; MAF: Minor allele frequency; PCR: Polymerase chain reaction.The authors declare that they have no competing interests.JJ and XYC designed the study. ZFJ, XYC, DHC and FK performed the experiments. ZFJ and JJ analyzed the data and wrote the first draft of manuscript. JJ and PK revised the manuscript. All authors read and approved the final manuscript.


Morphine and other opioid drugs produce analgesia primarily through μ opioid (MOP) receptors, which mediate beneficial but also the non-beneficial actions. Appropriate identification of novel opioid analgesics may reduce complications and improve patient compliance. It was reported that hydrazones, oximes, carbazones and semicarbazone derivatives of morphinan-6-ones, e.g. dihydromorphinone or oxymorphone, exhibit high affinity at the MOP receptor . Since mIn vitro [35S]GTPγS functional assays were performed with Chinese hamster ovary (CHO) cell membranes expressing human opioid receptors. Antinociceptive activities were assessed in mice using tail-flick, hot-plate and writhing tests.Binding affinities at opioid receptors were determined using competition binding assays in rodent brain membranes. N-methylmorphinan-6-ones leads to qualitative and quantitative differences in the interaction with opioid receptors. Consequently, we have conducted a comparison of the biological activities of the 6-cyanomorphinans to those of structurally-related opioids, oxycodone, oxymorphone and of the clinically relevant morphine. The 6-cyanomorphinans displayed high affinity and behaved as agonists at the MOP receptor. When tested in vivo, they acted as potent antinociceptive agents after subcutaneous administration, being more active than the 6-keto analogues. The presence of a 14-methoxy or a 14-cinnamyloxy group instead of a hydroxy group not only increased in vitro opioid activity at the MOP receptor, but also enhanced the antinociceptive potency.Replacement of the 6-keto group by a 6-cyano substituent in Our findings revealed that targeting position 6 in the morphinan skeleton represents a viable approach for tuning the pharmacological properties of this class of opioids. Appropriate molecular manipulations could afford ligands that, besides their scientific value as pharmacological tools, may also have the potential of emerging as novel analgesics with fewer side effects compared to currently available treatments.


Experimental findings from transgenic migraine mouse models that carry a human FHM1 gain-of-function mutation in CaV2.1 (P/Q-type) calcium channels underscore the role of neuronal hyperexcitability in migraine [Here, we aimed to set up an electrophysiology platform to study changes in cortical neuronal network activity in relation to CSD in freely behaving transgenic migraine mice.We developed an electrophysiology system for long-term recordings of DC-EEG and multi-unit-activity from the cortex of freely behaving mice. The system combines a counterbalanced 7 channel swivel with custom-built differential DC-EEG, AC-EEG and unit activity amplifiers. Stable DC-EEG recordings are obtained using AgAgCl epidural electrodes, while intracortical platinum electrodes are used for simultaneous recording of multi-unit-activity and AC-EEG. For CSD induction intracortical microdialysis was used for infusion of high KCl solution.Simultaneous recordings of multi-unit activity, DC- and AC-EEG were made from the sensorimotor and occipital cortex of wild-type and FHM1 migraine mice for up to 3 weeks. Apart from spontaneous cortical activity, visual evoked cortical responses were induced using 1 ms blue light pulses. Microdialysis with KCl solution resulted in successful induction of CSD events in the awake mice.We established a novel platform for performing longitudinal recordings of cortical neuronal activity and spreading depression in freely behaving mice carrying migraine-specific mutations. Using this platform, we aim to characterize how cortical activity is altered by modulatory factors that predispose for migraine attacks.


Thyroid cancer is one of the most common malignancies of the endocrine system with increasing incidence. The vast majority of thyroid carcinomas derive from thyroid hormone producing follicular cells. Carcinomas of follicular origin are classified as follicular (FTCs), papillary (PTCs), partially differentiated (PDTCs) or anaplastic (ATCs) thyroid carcinomas. While FTCs and PTCs can be managed effectively, ATCs are considered one of the most lethal human cancers. Despite the identification of various genetic alterations, pathogenic mechanisms promoting the progression of thyroid carcinomas are still largely elusive. Over the recent years, aberrant microRNA expression was revealed in all as yet analyzed human cancers, including thyroid carcinomas. In view of the rapidly evolving perception that deregulated microRNA expression serves a pivotal role in tumor progression, microRNAs provide powerful tools for the diagnosis of thyroid carcinomas as well as the identification of potential therapeutic targets. Here, we summarize recent findings on microRNA signatures in thyroid carcinomas of follicular origin and discuss how deregulated microRNA expression could promote cancer progression. More th2O2 is necessary for thyroid hormone synthesis) and estrogen. Historical events exhibit radiation exposure as the major risk for PTCs, since atomic bomb survivors and Chernobyl victims frequently developed these tumors [loci 9q22.33 and 14q13.3 were found to be associated with a higher risk of FTCs and PTCs [Thyroid tumors are supposed to be mainly monoclonal malignancies arising based on somatic mutations of progenitor cells. Putative risk factors for genomic instabilities are radiation exposure, active oxygen-species . These small, non-coding RNAs of 20-24 nucleotides in length are evolutionarily conserved and control gene expression at the post-transcriptional level [‘onco-miRs’ with tumor promoting effects versus ‘tumor-suppressive’ microRNAs that antagonize cancer progression. In the past years, several studies identified miRNA signatures in thyroid carcinomas aiming to reveal how they modulate thyroid cancer progression and to evaluate their potential for thyroid cancer diagnosis.In addition to genetic alterations, recent studies indicate that thyroid carcinomas like the majority of as yet analyzed tumors are characterized by aberrant expression of al level . At presal level ,20. Two In vitro, the upregulation of miR-197 and -346 was associated with elevated proliferation of HEK293T and two FTC-derived cell lines. This pro-proliferative effect was suggested to correlate with miR-controlled expression of ACVR1 (activin A receptor type 1), TSPAN3 (tetraspanin 3), CFLAR (Caspase 8 and FADD-like apoptosis regulator) and EFEMP2 (fibulin 4), although no direct regulation of these targets by miR-197 or -346 was demonstrated [in vitro, but miRNA-mediated regulation of Activin signalling remains to be shown [Malignant FTCs and benign FTAs share significant similarities at the morphological and molecular level and thus microRNAs could serve as valuable markers to distinguish these tumors. The first study addressing this aspect on the basis of a limited set of human microRNAs (235 distinct human microRNAs) revealed four microRNAs  moderately upregulated by 1.34-1.82 fold in FTCs when compared to FTAs . The autnstrated . The abunstrated . Notablybe shown . TSPAN3 be shown .In comprehensive studies aiming at the identification of microRNA signatures distinguishing thyroid carcinomas from non-transformed thyroid tissue (NT), Nikiforova and colleagues  could noIn conclusion, comprehensive miRNA signature analyses in FTCs, in particular with respect to distinct subtypes like oncocytic versus conventional FTCs, are still lacking. Thus, it remains largely elusive if FTCs can be distinguished from other thyroid carcinomas or FTAs by altered miRNA expression profiles at this point.The upregulation of the miR-221/-222 cluster and miR-181 is the most consistent finding in all studies addressing miRNA signatures in PTCs ,31,36,37In vitro analyses using thyroid cancer-derived cells confirmed an ‘oncogene connection’ for upregulated expression of miR-221 and -181b. The abundance of these microRNAs increased upon overexpression of v-raf, v-ras, RET/PTC1, RET/PTC3, E1-Abl, E1a-v-raf, middle T of polyomavirus and v-mos [Most interestingly a significant correlation of increased miRNA expression with genetic alterations was observed in PTCs . For insnd v-mos . PTC celnd v-mos ,41.Although there is substantial and consistent data on the upregulation of microRNAs in PTCs, reduced abundance was only reported for a few miRNAs. The three tumor-suppressive miRNA-families miR-26 , -30 3838 and leIn summary, PTCs appear to be well-distinguishable from NT by altered miRNA expression, in particular the upregulation of onco-miRs. However, more comprehensive analyses including FTCs are required to identify miRNAs expressed at distinct levels in these two carcinoma types.The most striking difference between ATCs and all other thyroid carcinomas of follicular origin appears to be a severely decreased expression of various miRNAs Figure  28,30,3,330,36. In conclusion, ATCs appear to be well-distinguishable from all other thyroid carcinomas by a severe decrease in the expression of various microRNAs. However, future studies have to reveal if altered miRNA profiles suffice to unambiguously distinguish ATCs from PDTCs.PDTCs are poorly defined and show morphological characteristics of both, differentiated and anaplastic thyroid carcinomas. Aiming to distinguish PDTCs from PTCs and ATCs on the molecular level, Schwertheim et al. investigated the expression of two different sets of microRNAs . Abundance of these miRNAs was analyzed in comparison to four NTs in 15 PDTCs, nine PTCs and nine ATCs . ‘Set 1’Although preliminary evidence indicates that miRNA profiles could be useful molecular markers to identify PDTCs, additional and more comprehensive studies are required.Onco-miRs are typically classified by an upregulation in cancer and the targeting of transcripts encoding effectors antagonizing tumor progression Figure , red. InKip1[Kip1[Kip2 expression [Kip1 was observed in many primary thyroid tumors as well as thyroid carcinoma-derived cell lines [The overexpression of miR-221/-222 is a hallmark of thyroid malignancies -28,30,31Kip1. This inKip1[Kip1,49. NotaKip1[Kip1. An inhipression ,51. Hencll lines .In vivo evidence for the growth promoting role of elevated miR-221/-222 expression was provided by Xenograft studies in nude mice. The constitutive overexpression of this miRNA cluster resulted in an increased gain of Xenograft weight and volume upon subcutaneous injection of tumor-derived cell lines [The expression of the c-kit tyrosine kinase receptor is suppressed or reduced in most tested FTCs and PTCs . Indicatll lines . TogetheUpregulated expression of miR-21 is observed in multiple types of cancers, such as breast, liver, brain, prostate and myometrial cancers but also in PTCs and ATCs ,60,61. MMiR-155 overexpression was observed in FTCs, PTCs and ATCs ,31. In lIn contrast to onco-miRs, tumor-suppressive microRNAs are typically downregulated during tumor progression and target transcripts encoding oncogenic factors. The regulatory role of key tumor-suppressive miRNAs in thyroid cancer is discussed in the following Figure , green.The miR-200 family  was identified as a potent suppressor of EMT -75. Redude novo synthesized in various cancers [Downregulation of the let-7 family is observed in all thyroid carcinomas of follicular origin ,28,30. L cancers . This is cancers . HMGA1 a cancers -103. Mor cancers . In concin vitro invasiveness of ATC-derived cells [in vitro invasiveness of ATC-derived cells and correlated with a downregulation of the mesenchymal marker vimentin. However, in contrast to the miR-200 family, expression of the miR-30 family is apparently controlled in a ZEB-independent manner [Significantly reduced expression of the miR-30 family was identified in ATCs. However, moderately reduced expression is also observed in FTCs, PTCs and PDTCs, with the exception of miR-30b in FTCs ,30. The t manner . Hence, The comprehensive view of distinct miRNA signatures in thyroid carcinomas of follicular origin provides novel insights in the molecular pathologies of these malignancies. However, knowledge of target mRNAs controlled by deregulated miRNAs in thyroid cancers is still sparse at present. To reveal how altered expression of microRNAs promotes or antagonizes thyroid tumor progression it is thus required to identify novel miRNA targets in future studies.In thyroid carcinomas the most striking observation is that ‘less’ aggressive FTCs and PTCs (compared to ATCs) are apparently characterized by an upregulation of ‘oncogenic’ miRNAs (e.g. miR-221/-222 or miR-146b). These miRNAs mainly act in a pro-proliferative and anti-apoptotic manner. In accord with the clonal progression hypothesis, ATCs reveal a severe reduction of tumor-suppressive miRNAs (Figure Despite their potential as diagnostic tools, miRNAs could also provide promising therapeutic targets based on onco-miR inhibition or restoring levels of tumor suppressive miRNAs. The stage for such approaches was set in rodents already in 2005 by demonstrating that the role of miR-16 as well as -122 could be antagonized by the intravenous injection of anti-miRs . Vice veA third strategy could be envisioned on the level of epigenetic silencing of tumor-suppressive miRNAs. In breast and prostate cancer cell lines methylation of the miR-200 cluster was observed and inhibition of methyl-transferases released this transcription block . MoreoveIn conclusion the here reviewed findings reveal the potency and current limitations of miRNAs in diagnosis, prognosis and potentially therapeutic strategies for the treatment of thyroid cancer. However, substantial additional work will be required to establish miRNAs in clinical practice and reveal the molecular networks via which altered miRNA expression promotes thyroid cancer progression.The authors declare that they have no competing interests.Both authors contributed equally