Heat shock factor 1 (HSF1) is a master regulator that coordinates chaperone protein expression to enhance cellular survival in the face of heat stress. In cancer cells, HSF1 drives a transcriptional program distinct from heat shock to promote metastasis and cell survival. Its strong association with the malignant phenotype implies that HSF1 antagonists may have general and effective utilities in cancer therapy. For this purpose, we had identified an avid RNA aptamer for HSF1 that is portable among different model organisms. Extending our previous work in yeast and Drosophila, here we report the activity of this aptamer in human cancer cell lines. When delivered into cells using a synthetic gene and strong promoter, this aptamer was able to prevent HSF1 from binding to its DNA regulation elements. At the cellular level, expression of this aptamer induced apoptosis and abolished the colony-forming capability of cancer cells. At the molecular level, it reduced chaperones and attenuated the activation of the MAPK signaling pathway. Collectively, these data demonstrate the advantage of aptamers in drug target validation and support the hypothesis that HSF1 DNA binding activity is a potential target for controlling oncogenic transformation and neoplastic growth. Saccharomyces cerevisiae and Drosophila melanogaster, multiple isoforms exist in mammals and plants, which appear to have specialized functions C. elegansThe Heat Shock Factor 1 (HSF1) is a transcription factor that responds to a variety of environmental stressors to activate the heat shock response in eukaryotes, a protective mechanism conserved among different kingdoms Paradoxically, under certain circumstances, HSF1's ability to promote cell survival may endanger the overall well-being of a multicellular organism. A poignant example is the function of HSF1 in cancer. It has long been noted that cancer cells can continue to proliferate in hypoxic and hostile microenvironments that are otherwise growth inhibitory to normal cells. Therefore, it is not surprising that cancer cells exhibit elevated levels of heat shock proteins (HSPs) to facilitate the folding of damaged proteins and solubilization of protein aggregates in vitro selection experiment using Drosophila HSF1 as the target, and later shown to be able to recognize HSF1 in yeast, Drosophila and humans. Deletion analysis defined a minimal binding motif of the aptamer comprised of two stems and one stem–loop joined by a 3-way junction For the purpose of studying and controlling the function of HSF1 in cells and organisms, we previously identified an RNA aptamer, AptHSF-RA1 HSF1 (“ia” stands for “inhibitory aptamer”), and delivered it into HeLa cervical carcinoma cells in the form of a synthetic gene by transfection. The anti-cancer activity of the aptamer was then investigated through three lines of studies. First, we confirmed the molecular mechanism of the aptamer action by determining the disruption of HSF1's interaction with its cognate DNA elements in vitro and in vivo. Second, we demonstrated the ability of the aptamer, when expressed in cancer cells, to promote apoptosis and inhibit colony formation in soft agar. Finally, we address the issue of specificity in cells by showing: (1) the aptamer phenotypes are suppressed by overexpression of HSF1 or HSPs, (2) aptamer expression reduces HSF1 target gene expression, and (3) aptamer expression reduces the HSF1 modulated, MAPK signaling pathway. Collectively, our results agree with previous reports that show that HSF1 is critical for the maintenance of cellular transformation. Moreover, our findings raise the interesting possibility that an aptamer that functionally inactivates HSF1 can be used to block human cancer progression.Here in the present study, we report the anti-cancer activity of this HSF1 aptamer in cultured human cells. We adopted the dimeric configuration of AptHSF-RA1 used in Drosophila HSF1 is the following (the lowercase letters represent the hammerhead ribozyme): 5′GUCGAGUGACGUUGGCAUCGCGAUACAAAAUUAAGUUGAACGCGAGUUCUUCGGAAUUCAACUGCCUUCGUCAUACUCCUUGAAUUCAACUGCCUUCGGGCAUCGCGAUACAAAAUUAAGUUGAACGCGAGUUCUUGGAGGCUCGACguc uagcgaugugguuucgcuacugaugaguccgugaggacgaaac 3′. In the process of constructing the expression vector for the dimeric aptamer, an antisense control construct, RevRA1, was generated, using primer sets that swapped the parental and lagging strands of the aptamer coding region while keeping the hammerhead unit intact. Both sense and antisense units were cloned in gateway vectors The sequence of the dimeric aptamer construct, iaRNA2. MDA-MB-231 (CRM-HTB-26) were grown in RPMI media supplemented with 10% FBS, 1X Pen/Strep in 5% CO2. The 293T cells were grown in DMEM high glucose medium (ATCC) supplemented with 10% FBS, 1X Pen/Strep in 5% CO2. Upon growth to confluency, cells were trypsinized and passed into fresh medium according to ATCC instructions. These parental cells were transfected with iaRNAHSF or RevRA1 RNA control-expressing vectors. Non-transfected cells were subsequently eliminated from the population by culturing the cells in 6 µg/ml blasticidin and washing the cells 24 hrs after transfection. Thereafter, cells were maintained and grown in (1 ug/ml) blasticidin. Only the blasticidin-resistant cells were used throughout subsequent assays. For the “rescue” experiments, constructs expressing human HSF1 (hHSF1), human Hsp90 (hHSP90), human Hsp70 (hHSP70), LacZ, or GFP were mixed with expression vectors for HSF aptamer (or control RNA) at a 20∶1 ratio to ensure proper and excess expression of the interested targets relative to the aptamers. After the plasmids were mixed, each mixture was transfected into HeLa cells in 6-well dishes. Non-transfected cells were washed away using methods described above and the remaining cells were maintained in selection medium.Cells used in this study were obtained from the ATCC and maintained according to the manufacturer instructions. HeLa (CCL-2), IMR-90 (CCL-196), 293T (CRL-3216), MCF7 (HTB-22), U87 MG (HTB-14), and BE(2)-M17 (CRL-2267) cells were grown in E-MEM low glucose medium (ATCC) supplemented with 10% FBS, 1X Pen/Strep in 5% CO32P]UTP by using a T7 in vitro transcription kit . The 10 µl binding solution contained 1X binding buffer, 1 µg carrier yeast RNA, 4 µg carrier BSA, 5 mM DTT, 10% glycerol, 6 units of SUPERase-In (RNase inhibitor), plus protein and labeled RNA aptamer. The concentration of the labeled RNA probe is below 1 nM in most experiments. The human HSF1 gene was obtained from the Thiele Lab 2 and were run at 100–150 V at 4°C for 1–2 hr.The general scheme of electrophoretic mobility shift assay (EMSA) was adopted and modified from previous work RT-PCR was performed 24 hours post transfection according to a protocol described previously using the following primers.HSF1 F:iaRNA5′-GTCGAGTGACGTTGGCATCGHSF1 R:iaRNA5′- GACGTCGAGCCTCCAAGAACRev F:iaRNA5′-GTCGAGCCTCCAAGAACTCGRev R:iaRNA5′- GACGTCGAGTGACGTTGGCAChIP assay was performed 36 hours post transfection according to a protocol described previously Samples were prepared 72 hrs post-transfection for molecular analyses unless otherwise specified. For the examination of caspase-3 samples were prepared between 72–96 hrs. In brief, cells were fed every 48 hrs, and samples were collected by centrifugating the media, pelleting the cells. Also, adherent cells were scrapped in PBS, pelleted and combined with the cells in the media. Thereupon, samples were lysed in the presence of non-ionic detergents containing protease inhibitors, and the samples were boiled in the presence of 6X-SDS. Conventional Western blot protocols were used with the following antibodies. Primary Antibodies: G6PDH was purchased from Sigma (A9521). All other antibodies were purchased from Cell Signaling Inc.: HSF1 (No. 4356), Hsp90 (No. 4875), Hsp70 (No. 4872), Hsp60 (No. 4870), Hsp40 (No. 4868), Calnexin (No. 2433), GRP78 (No. 3177), EGFR∼p (No. 2234), Erk1/2∼p (No. 9146), total Erk1/2 (No. 9102), Caspase-3 (#9662). The PARP antibody was anti PARP-DBD, a gift from Dr. W.L. Kraus, and transglulaminase (TGM2) was a gift from Dr. R. Cerione's laboratory purchased from Thermo Scientific. Secondary antibodies were used according to proper immunoreactivity. PVDF membranes were blocked using 5% BSA and membranes were incubated primary antibodies overnight at 4°C.HSF or RevRA1 RNA control cells were observed for 96 hrs post transfection. In these experiments, non-transfected iaRNAHSF was removed by culturing the cells in 6 µg/ml blasticidin and washing the cells 24 hrs after transfection. Only the blasticidin-resistant cells were used throughout subsequent assays. All statistical analyses in this study were calculated using student's t-test.Apoptosis was observed by quantifying the number of HeLa cells containing fragmented nuclei as visualized by DAPI staining under the microscope. In these experiments, parental, iaRNA3 semi-stable transfected cells were plated on 6-well dishes in appropriate medium supplemented with 3% agarose  over a warm layer of pre-solidified medium containing 6% agarose . Five days after plating the cells a new 1 cm layer of media was plated over the growing cells. HeLa cells containing 17-AGG had a final concentration of 8.8 ug/dL 17-AAG agar mixture. The ability of cells to grow on soft agar was evaluated after 14 days. Colonies were analyzed under the light microscope.6×10HSF1, contains two aptameric units for enhanced avidity and a hammerhead ribozyme in which the 5′ and 3′ ends of the RNA are protected to facilitate folding and stability. We adapted this construct by placing its coding sequence under the control of the EF-1a promoter in a mammalian expression vector. We also made a control construct, named RevRA1, in which the aptamer moiety of the iaRNAHSF1 was replaced by its antisense sequence. Before using it on cells, we produced this RNA by in vitro transcription and determined its avidity for purified human HSF1 in an electrophoretic mobility shift assay (EMSA) (HSF1 generated a shifted complex with an apparent Kd of 25 nM (HSF1 was incubated with high amounts of purified BSA (1 µM), no shifted band was observed. Together, these in vitro results demonstrated that the interaction between iaRNAHSF1 and HSF1 occurs with high affinity and is relatively selective.Previously, we developed a method for delivering RNA aptamers into the nuclei of cells as synthetic genes y (EMSA)  using puy (EMSA) . Here, tof 25 nM . In contHSF1-expressing vector into a cancer and a non-transformed cell line. We chose HeLa cervical carcinoma cells as a representative cancer cell line, because they are one of the most commonly used and well characterized human cancer cell lines HSF1 or the control RevRA1 24 hours post-transfection. Our results showed that the RNA aptamer levels were similar for both the aptamer and the control RNA in HeLa and IMR-90 cells  assays on iaRNAHSF1 or RevRA1 expressing HeLa cells  using an antibody specific to mammalian HSF1 HSF1 expression significantly compromised HSF1 binding to key heat shock promoters (Hsp90 and Hsp70) in vivo . As already demonstrated above in HSF1 in the chemically transformed human 293T kidney cell line would similarly kill the cells. We found that iaRNAHSF1 expression in the 293T cells dramatically causes the cells to round up and detach from their culture plates, similar to HeLa cells  after transfecting the cells with either iaRNA changes  or induc changes . In contLa cells . Comparef 0.0018 .HSF1-expressing cancer cells did not undergo apoptosis (approximately 30%), we sought to determine whether these cells were compromised in their ability to form colonies in anchorage-independent (soft agar) growth assays, an in vitro measurement of tumorgenicity. As expected, HeLa cells expressing a control RNA sequence (RevRA1) formed large colonies in soft agar -17-demethoxygeldanamycin (17-AAG) on colony formation in soft agar. Similarly to iaRNA expressing cells, HeLa cells grown in the presence of 150 nM 17-AAG were unable to form colonies in soft agar   .HSF1-expressing HeLa cells , Calnexin (∼50%), and Grp78 (∼25%) compared to parental HeLa or control cells (expressing RevRA1) , control RNA (Rev) and iaRNALa cells . Importa RevRA1) , n = 4. hsf1−/−) generated in Benjamin Ivor's laboratory hsf1−/− cells have relatively minor effects on the total levels of Hsp90 protein, yet displays major effects on Hsp70 under normal growth conditions when compared with wild-type MEFs , and examine the effects it had on the activation of the EGF receptor or MAPK signaling by collecting cells 10 minutes after treatment and assaying the samples by Western blotting. Importantly, these experiments were performed in a time when the cells were not actively undergoing apoptosis as visualized by the intact PARP protein (36 hrs post transfection) (hsf1−/− MEFs and find that HSF1 K.O. cells display a compromised ability to active Erk1/2 (p-Erk1/2) (G12V in hsf1−/− MEF cells.To directly investigate the ability of iaRNAfection) . Overallfection) . In contfection) . To invefection) . In a si-Erk1/2) . This atTo further assess how HSF1 modulates MAPK signaling, we measured the levels of activated EGFR (phosphorylated) or Erk1/2 protein  in aptamer-expressing cells that either over-expressed HSP90 or its co-chaperone HSP70 (36 hr post transfection). Here, over-expression of either HSP90 or HSP70 is sufficient to rescue the loss of either EGFR activation or Erk1/2 expression, resulting in an apparently normal mitogen response  A&B. We HSF1 supports highly malignant human cancers, including the most aggressive forms of breast, lung, and colon cancer The difficulty of finding small molecules to interrupt DNA-protein interaction stems from the nature of the interface. DNA binding transcription factors generally bind to an extended stretch of base pairs covering a relatively large surface area, a biophysical feature that small molecule inhibitors have a hard time competing against. In this regard, RNA aptamers have a unique advantage, as RNA molecules share quite a few common structural features with DNA. For example, several RNA aptamers have been isolated to target the DNA-binding domain of the pro-inflammatory protein NFkB One major challenge of macromolecular drugs is delivery. Nevertheless, before aptamers can be used in pre-clinical or clinical studies, their utility can be exploited as useful agents in drug target validation studies as demonstrated here. Currently, many drug target validation methods commonly use gene knock-out or RNAi technologies to gain insights of the underlying molecular mechanisms of disease. Unfortunately, not every protein is amenable to such inhibition, nor do these strategies provide sufficient information about the particular domains of the specific drug target in question. In recent years, we have generated a comprehensive set of data regarding the activity of the HSF1 aptamer described herein using three organisms from different biological kingdoms Relevant to the case of HSF1, only a small class of naturally occurring molecules have been identified that inhibits molecular chaperones, and of these, only a handful have been tested to sensitize cancer cells to pharmacological treatment Figure S1HSF1 binds avidly to human HSF1 and HSF2.iaRNA A) Bacterially expressed and His tagged full-length human HSF1 and HSF2 protein used in the in vitro binding assays used in this study. In vitro binding assays included proteins from the following bacterial preparations: HSF1a2, HSF2a6. B). Electrophoretic motility shift assay (EMSA) using radiolabeled iaRNAHSF1 (1 nM) and increasing amounts of human HSF2 protein shows that the aptamer binds to this target with an apparent affinity of 100–200 nM .(TIF)Click here for additional data file.Figure S2Heat shock protein profile in hsf1−/− mouse embryonic fibroblasts (MEF) under non-heat shock conditions (NHS). Whole cell extracts of WT and hsf1−/− MEF's show that HSF1 deletion (hsf1−/−) results in a dramatic decrease of steady state Hsp70 protein expression levels. In contrast, the observed Hsp90 levels are only is moderately affected by HSF1 deletion, partially because this gene is under the transcriptional activity of both HSF1 & HSF2 hsf1−/− MEFs. The left most three lanes are a serial dilution of parental WT extracts that provides a visual standard (n = 4).(TIF)Click here for additional data file.Figure S3HSF1 effects by human HSF1 over-expression.Suppression of iaRNA (A) Western blot analysis of HeLa cells demonstrates the time dependent and progressive loss of clinically significant oncoproteins  by iaRNAHSF1 expression. Apoptosis can be visualized by the degradation of full-length caspase-3 starting at 48–96 hrs post transfection (lane 5 &6). . (B) Differential interference contrast imaging of HeLa cells show that human HSF1 or human Hsp90 over-expression can effectively suppress many of the morphological defects induced by iaRNAHSF1 expression (96 hrs) (compare this figure with samples from (TIF)Click here for additional data file.Figure S4MAPK signaling is attenuated in hsf1−/− MEF's. In comparison of treatment of WT MEFs (lanes 1–3) with the potent mitogen, EGF, hsf1−/− cells display decreased levels of Erk1/2 activation (p-Erk1/2) (lane 4). This abnormal signaling response can be effectively suppressed by direct over-expression of constitutively activated K-RasG12V (compare lanes 4–6).(TIF)Click here for additional data file.Figure S5Differential sensitivity of human cancer cells to Hsp70 or Hsp90 inhibition. (A) Differential interference contrast imaging of HeLa, human brain glioblastoma (U87 MG), neuroblastoma (SK-N-BE(2)-M17), and triple negative  breast adenocarcinoma (MSA-231), following Hsp70 inhibition (0.32 g/dL myricetin) or Hsp90 inhibition (8.8 ug/dL 17-AAG) . (B) Quantification of nuclear condensation and DNA fragmentation (apoptotic assays) of panel (A) and estrogen responsive  breast adenocarcinoma (MCF7) (n = 4).(TIF)Click here for additional data file.


Here we define the in vivo role of the GATOR2 component Wdr24 in Drosophila. Using a combination of genetic, biochemical, and cell biological techniques we demonstrate that Wdr24 has both TORC1 dependent and independent functions in the regulation of cellular metabolism. Through the characterization of a null allele, we show that Wdr24 is a critical effector of the GATOR2 complex that promotes the robust activation of TORC1 and cellular growth in a broad array of Drosophila tissues. Additionally, epistasis analysis between wdr24 and genes that encode components of the GATOR1 complex revealed that Wdr24 has a second critical function, the TORC1 independent regulation of lysosome dynamics and autophagic flux. Notably, we find that two additional members of the GATOR2 complex, Mio and Seh1, also have a TORC1 independent role in the regulation of lysosome function. These findings represent a surprising and previously unrecognized function of GATOR2 complex components in the regulation of lysosomes. Consistent with our findings in Drosophila, through the characterization of a wdr24-/- knockout HeLa cell line we determined that Wdr24 promotes lysosome acidification and autophagic flux in mammalian cells. Taken together our data support the model that Wdr24 is a key effector of the GATOR2 complex, required for both TORC1 activation and the TORC1 independent regulation of lysosomes.TORC1 is a master regulator of metabolism in eukaryotes that responds to multiple upstream signaling pathways. The GATOR complex is a newly defined upstream regulator of TORC1 that contains two sub-complexes, GATOR1, which inhibits TORC1 activity in response to amino acid starvation and GATOR2, which opposes the activity of GATOR1. While the GATOR1 complex has been implicated in a wide array of human pathologies including cancer and hereditary forms of epilepsy, the  Drosophila. First, Wdr24 is required to oppose the activity of the GATOR1 complex, thus activating TORC1 in a broad array of Drosophila tissues. Second, Wdr24 promotes the acidification of lysosomes and thus facilitates autophagic flux. Our data support the model that Wdr24 uses both TORC1 dependent and independent pathways to regulate cellular metabolism.TORC1 is a conserved multi-protein complex that regulates metabolism and cell growth in response to many upstream inputs including nutrient availability. When amino acids are limiting, the GATOR1 complex inhibits TORC1 activation. The inhibition of TORC1 slows cellular metabolism and promotes cell survival during times of protein scarcity. A second critical response to amino acid limitation is the activation of autophagy. During autophagy cells degrade intracellular components in specialized membrane-bound organelles called autolysosomes that are formed when lysosomes fuse with autophagosomes. In times of nutrient stress, the process of autophagy allows proteins and other building blocks of the cell to be broken down and repurposed for vital cellular functions. Here we demonstrate that Wdr24, a component of the multi-protein GATOR2 complex, has a dual role in the regulation of cellular metabolism in  Target of Rapamycin (TOR). When nutrients are abundant, TORC1 activity promotes translation, ribosome biogenesis as well as other pathways associated with anabolic metabolism and cell growth. However, when nutrients or other upstream activators are limiting, TORC1 activity is inhibited triggering catabolic metabolism and autophagy  embryos. All the oligonucleotides used for cloning and screening are listed in To generate knock out escribed . pBFv-U6ed into y, vas-CasWDR24 HeLa cells, two pairs of oligonucleotide encoding the guide RNAs were cloned into px330 vector [px330 guide constructs. The third day, cells were pooled into 10 cm dishes with 200 cells each dish. After 10 days, all the clones were collected and seeded into 24 well plates. Cells were grown and expanded, and the positive colonies were identified by PCR and sequencing. All primers used for PCR amplification are listed in To generate knock out 0 vector . On day HAWDR24 pRK5 (Plasmid #46335) was obtained from Addgene. For the DQ BSA Red (Life Technology) assay, Cells were incubated for 4 h with DQ BSA (10 μg/ml) and then washed twice with HBSS. Subsequently the cells were incubated in the starvation medium  to induce autophagy. After 1 hour the DQ BSA fluorescence was detected using a Leica TCS SP5 confocal microscope.HeLa cells and knock out cells were cultured at 37°C, 5% CO2 in DMEM medium (Life Technology) supplemented with 10% fetal bovine serum , 50 U/ml penicillin and 50 μg/ml streptomycin. Plasmid transfection was performed using Lipofectamine 2000 (Life Technologies). Cells grown on coverslips were fixed in 2% (wt/vol) formaldehyde and 2% (wt/vol) glutaraldehyde in 0.1 M sodium cacodylate (pH 7.4), postfixed in 1% aqueous OsO4, and stained en bloc with 2% (wt/vol) uranyl acetate. Upon dehydration and embedding in EMBed-812 , the coverslips were removed by hydrofluoric acid, cells were thin-sectioned parallel to the glass, and sections were stained with uranyl acetate. The samples were examined on an FEI Tecnai 20 transmission electron microscope operated at 80 kV, and images were recorded on a Gatan Ultrascan CCD camera.Lysosomal pH measurements were performed as describe . In brieS1 Figwdr241 deletion. Dashed line marks the deletion position. (B) RT-PCR demonstrates that wdr24 mutants do not produce full length mRNA. Integrator subunit 11 (intS11) was used as the internal control in order to demonstrate that the deletion only affects wdr24 gene expression but not the neighboring gene intS11. (C-D) Expression of GFP-Wdr24 using the germline specific Nanos-Gal4 driver rescues the ovarian and fertility phenotypes of wdr24 mutants. (C) Dissected ovaries from control wdr241/TM6, wdr241 and Nanos-Gal4; GFP-Wdr24; wdr241 females. Size bar is 100 μm. (D) Bar graph shows the number of eggs laid by Nanos-Gal4; wdr241/TM6, Nanos-Gal4; wdr241 and Nanos-Gal4; GFP-Wdr24; wdr241 females. Error bars represent the standard deviation for three independent experiments. ** p value < 0.01.(A) Schematic map shows the (TIF)Click here for additional data file.S2 Figwdr241/TM6 (B) wdr241 (C) mio1/Df. Note that the wdr241/TM6 and wdr241 mutant egg chambers all contain ooctyes (white arrow), but multiple mio1/Df egg chambers have no apparent oocyte (white arrowhead). Size bar is 10 μm.Wdr24 is not required for the maintenance of the oocyte fate. Ovaries stained with DAPI  and Orb antibody . (A) (TIF)Click here for additional data file.S3 Figwdr241 mutant background significantly increases body weight. ** p value < 0.01. n.s. indicates not significant.Bar graph shows that overexpression of GFP-Wdr24 using the Ubi-Gal4 driver in (TIF)Click here for additional data file.S4 Figseh1Δ15/CyO-GFP (A) and seh1Δ15 (B) third instar larvae stained with LysoTracker and Hoechst. Size bar is 10 μm. (C) Quantification of body weights of seh1Δ15/SM6a and seh1Δ15/Df adult males. Error bars represent the standard deviation for three sets of experiments . n.s. indicates not significant.Fat bodies form (TIF)Click here for additional data file.S5 Figwdr241 mutant background rescues the LysoTracker accumulation phenotype. Fat bodies from GFP-Wdr24; wdr241 /TM6 (A and A’), wdr241 (B and B’) and GFP-Wdr24; wdr241 (C and C’) third instar larvae were stained with LysoTracker and Hoechst. Size bar is 10 μm.(A-C’) Expression of GFP-Wdr24 using the Cg-Gal4 fat body driver in (TIF)Click here for additional data file.S6 Fignprl2 and nprl3 in the wdr241 mutant background results in an increased body weight. GFPRNAi; wdr241 served as a negative control. * p value < 0.05.Bar graph shows that depleting (TIF)Click here for additional data file.S7 FigGFPRNAi; wdr241 ovaries and tsc1RNAi; wdr241 were analyzed by Western blot probed with pS6K and S6K antibodies. (B) Quantification of phospho-S6K levels relative to the total S6K. Error bars represent the standard deviation for three independent experiments. ** p value < 0.01. (C-E) Depleting tsc1 fails to rescue the lysosomal phenotype in wdr241 ovaries. Ovarioles from WT (C) GFPRNAi; wdr241 (D) and tsc1RNAi; wdr241 (E) females were stained with LysoTracker and Hoechst. Size bar is 10 μm.(A) Proteins isolated from WT, (TIF)Click here for additional data file.S8 Fignprl31 allele. Dashed line marks the deletion position. N-terminal break point starts at 61th amino acid from start codon. The deletion causes frame shift and generates a new stop codon after 5 amino acids from the C-terminal break point.Schematic map shows the deletion end points of the (TIF)Click here for additional data file.S9 Figwdr241/TM6 , wdr241 , atg7d14/d77  and atg7d14/d77; wdr241  females stained with LysoTracker, anti-Atg8a and Hoechst or DAPI. Note that atg7;wdr24 double-mutant ovaries accumulate lysotracker positive puncta that are not positive for the autophagy marker ATG8a. Size bar is 10 μm.(A-D’) Ovarioles from (TIF)Click here for additional data file.S10 Figwdr24-/- deletion. (B) Western blot of wild type (WT) and wdr24-/- probed with WDR24 and actin antibodies. Note that the wdr24-/- mutant cells do not express the WDR24 protein. (C) Western blot of wild type (WT) and wdr24-/- probed with phospho-4E-BP and 4E-BP antibodies. Note that the wdr24-/- mutant cells have lower phosphor-4E-BP level suggesting a decrease of mTORC1 activity. This experiment has been done in triplicates. (D) Quantification of phospho-4E-BP levels relative to the total 4E-BP. Error bars represent the standard deviation for three independent experiments. * p value < 0.05. (E) Western blot of cell lysates from WT, wdr24-/- and wdr24-/- HA-WDR24 rescued cells probed with antibodies against pS6K and S6K. Note that the overexpression HA tagged WDR24 protein in wdr24-/- mutants increases mTORC1 activity as indicated by pS6K levels.(A) Schematic map shows the position of (TIF)Click here for additional data file.S11 Figwdr24-/- (B) HeLa cells were stained with p62 antibody and DAPI. Size bar is 10 μm.(A and B) Wild type (WT) (A) and (TIF)Click here for additional data file.S12 Figwdr24-/- (B-B”) HeLa cells were stained with Cathepsin D and LAMP1 antibodies. Size bar is 10 μm.(A–B”) Wild type (WT) (A-A”) and (TIF)Click here for additional data file.S13 Figwdr24-/- (B) cells. Autolysosomes are shown at higher magnification for both WT (A′) and wdr24-/- (B′) mutant HeLa cells. Yellow arrows mark autolysosomes. Size bar is 1 μm.(A-B’) TEM images of lysosomes or autolysosomes from WT (A) and (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


We have previously reported that RA is capable of differentiation of human trophoblast stem cells to dopamine (DA) committed progenitor cells. Intracranial implantation of such neural progenitor cells into the 6-OHDA-lesioned substantia nigra pars compacta successfully regenerates dopaminergic neurons and integrity of the nigrostriatal pathway, ameliorating the behavioral deficits in the Parkinson’s disease rat model. Here, we demonstrated a dynamic molecular network in systematic analysis by addressing spatiotemporal molecular expression, intracellular protein-protein interaction and inhibition, imaging study, and genetic expression to explore the regulatory mechanisms of RA induction in the differentiation of human trophoblast stem cells to DA committed progenitor cells. We focused on the tyrosine receptor kinase (Trk), G proteins, canonical Wnt2B/β-catenin, genomic and non-genomic RA signaling transductions with Tyrosine hydroxylase (TH) gene expression as the differentiation endpoint. We found that at the early stage, integration of TrkA and G protein signalings aims for axonogenesis and morphogenesis, involving the novel RXRα/Gαq/11 and RARβ/Gβ signaling pathways. While at the later stage, five distinct signaling pathways together with epigenetic histone modifications emerged to regulate expression of TH, a precursor of dopamine. RA induction generated DA committed progenitor cells in one day. Our results provided substantial mechanistic evidence that human trophoblast stem cell-derived neural stem cells can potentially be used for neurobiological study, drug discovery, and as an alternative source of cell-based therapy in neurodegenerative diseases like Parkinson’s disease.Understanding the mechanisms in the generation of neural stem cells from pluripotent stem cells is a fundamental step towards successful management of neurodegenerative diseases in translational medicine. Albeit  After total calcium releases, the intracellular calcium levels elevated by adding extrinsic CaCl2  and by adding extrinsic KCl . The KCl-induced calcium elevation was attenuated by pretreatment of nifedipine . Cells were stained with a Ca2+-specific dye Fura-2 in HBSS buffer for 20 min at room temperature to measure the calcium responses by real-time cell imaging microscopy  and Olympus Cell-R imaging software. The Ca2+-free medium contains EGTA  and Thapsigargin .(A-B) IP assays showed that RA  enhanced the interaction of MEF2A and NFAT1, (A) and interaction between CaMKII and Parkin as well as Parkin and MAPT (B). C as control. IgG as negative control. (C) RA (10 μM) induced [Ca(TIF)Click here for additional data file.S4 Fig(A) RA  enhanced the interaction of importin and NFAT1 by IP assay. C as control. IgG as negative control. (B) RA increased nuclear translocalization of NFAT1 by nucleocytoplasmic fractionation assay. Lamin A/C as marker of the nuclear extract. α-tubulin as marker of the cytoplasmic extract. (C-D) RA  enhanced the interaction of GSK3β -Ser21/6) and NFAT1, (C) as well as MEF2A interacted to both NFAT1 and Pitx2 bi IP assay (D). (E) Knockdown of NFAT1 reduced the expression of MEF2A by immunoblotting assay. Scramble as positive control. β-actin as loading control.(TIF)Click here for additional data file.S5 Fig(A) RA  enhanced the interaction of Akt2 and GSK3β. C as control. IgG as negative control. (B) RA induced nuclear translocalization of β-catenin by nucleocytoplasmic fractionation assay. Lamin A/C as marker of the nuclear extract. α-tubulin as marker of the cytoplasmic extract. C as control. (C) RA  enhanced the interaction of β-catenin and LEF1 by IP assay. C as control. IgG as negative control.(TIF)Click here for additional data file.S6 Fig(A) Relative expressions of components in the Wnt2B/Fzd6/Dvdl3/FRAT1/GSK3β signaling pathway assessed by DNA microarray assessment in hTS cell after treatment with RA (10 μM) for 24 hr. (B) Nuclear cytoplasmic fractionation analysis revealed the entrance of MEF2A into nucleus at 24 hr of RA induction, which was inhibited by CaN inhibitor cyclosporine . (C-D) RA induced expressions of CREB1, Pitx2 and TH at 24 hr induction by immunoblotting assay (C) and this action was inhibited by using siRNA against CREB1 (D). Scramble as positive control, β-actin as loading control.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


A and βB strands of ED3. Moreover, S15 can neutralize the infections by all four serotypes of DENVs. Our result provides a new opportunity in the development of DNA aptamers against DENVs in the future.Dengue viruses (DENVs) are members of Flaviviridae family, which are associated with human disease. The envelope (E) protein plays an important role in viral infection. However, there is no effective antibody for clinical treatment due to antibody dependent enhancement of infection. In this study, using Systematic Evolution of Ligands by Exponential Enrichment (SELEX), we demonstrated the first aptamer (S15) that can bind to DENV-2 envelop protein domain III (ED3) with a high binding affinity. S15 was found to form a parallel quadruplex based on Quadfinder prediction, gel mobility assay and circular dichroism studies. Both the quadruplex structure and the sequence on 5’-end were necessary for the binding activity of S15. NMR titration experiments indicated that S15 bound to a highly conserved loop between β Dengue viruses (DENVs) belong to the Flaviviridae family, and contain four serologically and genetically distinct viruses, termed DENV-1, DENV-2, DENV-3 and DENV-4. Like other flaviviruses such as yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV) and tick-borne encephalitis virus (TBEV), the rapid and extensive spread of DENV infection has become a major public health concern recently 0.5.HSQC spectra of ED3 (access number 2JSF) were acquired with a Bruker Avance 600 MHz spectrometer . 15N-lab5 cells per 2.5 ml MEM culture medium in each well of a 6-well plate 24 h prior to infection. Approximately 100 plaque-forming units (PFU) of DENV per well were incubated with or without serial dilutions of aptamer in Hank’s Balanced Salt Solution (HBSS)  which contained 0.4% (w/v) bovine albumin fraction V (BSA fraction V) (GIBCO) in a 5% CO2 incubator at 37°C for 1 h. Then Virus-aptamer or virus-control mixtures were allowed to infect the confluent monolayer of Vero E6 cells in a 5% CO2 incubator at 37°C for an additional 1 h, inoculated mixtures were removed and the cells were washed once with phosphate buffered saline (PBS) and overlaid with overlay medium . After 5 days incubation, the plaques were stained with crystal violet dye   vs. aptamer concentration [log(aptamer)] of aptamers were fitted with Stern-volmer equation, as described in Materials and Methods. The best fit of fluorescence data was found by setting Kd = 200 nM and n = 1.14 for aptamer S15. Aptamer S22 had same binding stoichiometry (n = 1.09), but its binding constant was higher than that of aptamer S15 (Kd = 500 nM).The interactions between the aptamers (S15 and S22) and DENV-2 ED3 were determined by fluorescence quenching experiment. Tryptophan residue in ED3 gave rise to an emission peak at 350 nm when it was exited at 280 nm. Upon the addition of either aptamer S15 or S22, a significant decrease in fluorescence emission was observed . To calcA and βB strand . G318A mutation showed less effect on S15 binding. However, when G318 was substituted with proline [d = 1008 nM). Proline has a cyclic structure. The G318P mutation may reduce the flexibility of the epitope loop, hence affects the binding. Similar decrease of binding was found for the H317A mutation (Kd = 1027 nM). In contrast, T319A mutation showed tiny effect on the binding (Kd = 378 nM) demonstrating that residue T319 may be less important for aptamer S15 binding. Mutations on residue I335 and R345 that were distant from the epitope showed no significant effect on S15 binding (Kd = 279 and 190 nM).Site-directed mutagenesis was used to alter these residues to confirm their essential role for aptamer S15 binding. As shown in  proline , it causELISA binding assays were conducted to determine the specificity and cross-reaction of the aptamer S15. The results showed that S15 can bind to ED3 in a dose-dependent manner. In contrast, when using fetal bovine serum (FBS) as the target, no cross-reaction was observed . In addi50 was 4.2 μM. Moreover, using structure-based sequence alignment we found that residues  on the binding epitope for aptamer S15 are identical . However, only DENV-2 NGC can be completely neutralized by S15 at 100 μM.Aptamer S15 was able to recognize DENV-2 virion as well as ED3 (data not shown), although the epitope appeared to be poorly accessible in the native virus structure . We perfdentical . We ther2L1-10 profile between G6 and G33, and was able to fold into a G-quadruplex structure composed of two G·G·G·G tetrads and external loops  derived from aptamer S15 were designed. Aptamer S15G2 contained eight guanines separated into four clusters. However, CD spectrum of aptamer S15G2 showed no similarity to aptamer S15 . In contA strand, of which residues are not identical among all four DENVs. They, therefore, failed to treat infections by DENVs due to ADE. In contrast, antibodies  that targeted a highly conserved loop between βA and βB strands exhibited cross-reactive neutralization activity. However, 3D structure of E protein revealed that the loop appeared to have limited accessibility on the mature virion, leading to the poor neutralizing activity of cross-reactive antibodies (IC50 > 100 μg/ml). Since the first aptamer based drug, mercugen, was approved by FDA in 1999 [A and βB strands of ED3. In comparison to antibodies, aptamer S15 has smaller size (~10KDa), which may be helpful for improving the interaction between S15 and the poorly accessible loop. Significantly, it exhibited a neutralization activity (IC50 < 60 μg/ml) stronger than cross-reactive antibodies Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.


Human lung cancer is highly invasive and the most malignant among human tumors. Adenocarcinoma as a specific type of non-small cell lung cancer occurs with high frequency and is also highly resistant to radiation therapy. Thus, how to avoid radiation resistance and improve radiotherapy effectiveness is a crucial question. In the present study, human lung cancer A549 and H1299 cells were irradiated using γ-rays from a Co60 irradiator. Protein expression was detected by Western blotting. Cell cycle and apoptosis were measured by flow cytometry. Surviving fraction was determined by colony formation assay. γH2AX and 53BP1 foci formation were examined by fluorescence microscopy. In the results, we show that CHMP4C, a subunit of Endosomal sorting complex-III (ESCRT-III), is involved in radiation-induced cellular response. Radiation-induced Aurora B expression enhances CHMP4C phosphorylation in non-small cell lung cancer (NSCLC) cells, maintaining cell cycle check-point and cellular viability as well as resisting apoptosis. CHMP4C depletion enhances cellular sensitivity to radiation, delays S-phase of cell cycle and reduces ionizing radiation (IR)-induced γH2AX foci formation. We found that Aurora B targets CHMP4C and inhibition of Aurora B exhibits similar effects with silencing of CHMP4C in radioresistance. We also confirm that CHMP4C phosphorylation is elevated after IR both in p53-positive and-negative cells, indicating that the close correlation between CHMP4C and Aurora B signaling pathway in mediating radiation resistance is not p53 dependent. Together, our work establishes a new function of CHMP4C in radiation resistance, which will offer a potential strategy for non-small cell lung cancer by disrupting CHMP4C. Human lung cancer is highly invasive and most malignant among human tumors, which is classified into non-small cell lung cancer and small cell lung cancer. Non-small cell lung cancer accounts for about 85% of lung cancers and the current therapeutic strategy includes surgical resection together with radiation and/or chemo-treatment. Radiation resistance is the severe outcome of lung cancer radiotherapy and therefore poses a critical barrier for the radio-therapeutic effect. Hence, how to avoid the radiation resistance and to improve its treatment efficiency is a major challenge ,2.Ionizing radiation initiates DNA damage and arrests cell cycle progression, leading to cellular genomic instability and loss of genetic information. In response to the damage stress, the ataxia telangiectasia mutated kinase (ATM) and ataxia telangiectasia and Rad3-related protein (ATR) are activated. ATM and ATR can also engage in cell division control via activating cyclins and cyclin-dependent kinases (Cdks) . A recenAurora B kinase is the key kinase of CPC  essential for the mitotic processes. Aurora B expression and activity are altered during the cell cycle and peaks at the G2-M transition. Aurora B is elevated in a variety of human tumors and its over-expression is susceptible to tumor formation and poor outcomes of non-small cell lung cancer radiotherapy, therefore indicating that Aurora B might be a drug target for lung cancer ,7,8. HowThat ESCRT  is required for the CPC -mediated cytokinetic abscission, during which CPC monitors the right abscission time of two daughter cells. ESCRT has six distinct complexes  engaging in multi-vesicular body formation, cytokinesis and HBV (Hepatitis B virus) budding ,10,11. EIn the study, we first demonstrate that the subunit of ESCRT-III CHMP4C is involved in cellular radiation reactions. Radiation enhances Aurora B expression and CHMP4C phosphorylation in NSCLC cells, collectively directing cell cycle check-point and promoting cell survival. CHMP4C silencing increases cellular sensitivity to radiation, hinders S-phase progression in cell cycle and diminishes ionizing radiation (IR)-triggered γH2AX foci formation. We show that CHMP4C is the major target of Aurora B and has a similar effect with Aurora B upon radiation. We further reveal that the phosphorylation level of CHMP4C is rising both in p53-positive and-negative cells, showing that the close correlation between CHMP4C and Aurora B signaling pathway in mediating radiation resistance is not p53 dependent. Overall, our work discovers a novel action of CHMP4C in radiation resistance, suggesting CHMP4C as a new drug target for non-small cell lung cancer treatments.To test if CHMP4C is involved in DNA damage response, cells were γ-irradiated with 2, 4, and 6 Gy and CHMP4C expression as well as phosphorylation was checked. We found that CHMP4C protein increases after irradiation (IR) and peaks at 24 h in A549 but not in H1299 cells A,B. HoweNext, we wondered if CHMP4C exerts its regulatory role in cell cycle progression. A549 and H1299 cells were depleted of CHMP4C for 24 h and the S phase delay was checked, indicating that CHMP4C could impact S phase exit A–D,G–J. In addition, CHMP4C knockdown has no influence on p21 expression E, and p2The above data indicate that CHMP4C inhibition can arrest S-phase of cell cycle. Colony formation assays were next employed to detect if CHMP4C acts in cell radioresistance. These assays showed that CHMP4C knockdown decreases cell survival compared to control with IR. Similarly, the result was also found in Aurora B clearing. Inversely, CHMP4C or Aurora B expression can increase cell growth upon irradiation A,B.To verify whether Aurora B regulates CHMP4C, we silenced or overexpressed the Aurora B with siRNA or Aurora B expression plasmids in A549 or H1299. CHMP4C knockdown has no effect on Aurora B protein level, whereas Aurora B silencing inhibits CHMP4C protein level indicating that Aurora B acts upstream of CHMP4C A. MoreovThe results of colony formation assay indicate that the surviving fraction of NSCLC cells was significantly attenuated after IR treatment of 4, 6 Gy. We next want to ascertain whether CHMP4C knockdown can enhance IR-induced apoptosis under three conditions in A549 cells: radiation only (6 Gy), CHMP4C knockdown, combined CHMP4C knockdown and IR (6 Gy). We found that combined CHMP4C silencing and irradiation produce more evident apoptosis than single treatment . Hence, Next, to analyze if CHMP4C is immediately involved in DNA damage signaling, we examined the γH2AX and 53BP1 foci assembly in A549 cells with or without CHMP4C silencing. The IR dose of 4 Gy was selected in this analysis according to the results of colony formation and apoptosis assay. As shown in 1 to S transition [Current investigations on CHMP4C mainly focus its function on endosome generation. However, whether and how it serves its part in cell cycle, viability and apoptosis under irradiation is not clear. It has been reported that p53 regulates transcription of CHMP4C in the autophagy and endosome production . p53 plaansition ,20,21. WFurther, our results also showed that CHMP4C can promote cell survival and cell viability upon IR, indicating that CHMP4C offer the radioresistance to the irradiated cells. We next studied if CHMP4C was connected with DNA repair, and found that CHMP4C inhibition repressed DNA repair by decreasing γH2AX and 53BP1 foci reacting to radiation stress. Hence, the novel functions of CHMP4C in cell cycle control and DNA repair not only enriches the content of the DNA damage pathway but also extends its specific role in cell regulation.p53 is often mutated in NSCLC and its mutation increases sensitivity to ionizing radiation in tumor cells . CHMP4C Several Aurora kinase inhibitors are tested in clinical trials . StudiesCHMP4C can check the cytokinetic abscission to prevent premature and accumulated DNA damage as an abscission timer. CHMP4C deficiency generates the defective abscission check, which makes injured cells quickly pass through M to S phase, leading to DNA repair disability and genomic instability. These data suggests that CHMP4C deficiency disorganizes the cell cycle and enhances cell sensitivity to radiation, thus providing a new combined strategy to diagnosis and treatment of the lung cancer.We propose that ATM is activated by Aurora B, followed by p53-mediated activation of CHMP4C. In turn, CHMP4C promotes cell viability and proliferation. However, there is another major tumor suppressor factor, the alternative reading frame (ARF), which is negatively regulated by ATM in a transcription-independent manner. Contrary to the function of CHMP4C on cell fate, inhibition of ATM enhances ARF levels and stimulates the tumor-suppressive effects of ARF in human oncogene-transformed and cancer cells . These f2 and treated with different radiation of 2, 4, and 6 Gy using Co60 γ-rays with a dose rate of 1 Gy/min in the Irradiation (IR) Center .The human NSCLC cell lines A549 (p53 wild type) and H1299 (p53 deficient)  were cultured in RPMI-1640 containing 10% fetal bovine serum , incubated in 37 °C humidified incubator with 5% COA549 and H1299 cells were grown at 80% confluence and then introduced with siRNAs against CHMP4C , Aurora B (Ambion), p53 and p21  as well as control siRNA (Ambion). CHMP4C, Aurora B, p53 and p21 siRNA sequence is respectively: 5′-CCUGCGUCUCUACAACUAU-3′, 5′-CAUGGAUCUGAACAAAAUATT-3′, 5′-CUACUUCCUGAAAACAACG-3′ and 5′-CCUCUGGCAUUAGAAUUAUTT-3′. siRNA tansfections were performed using Lipofectamine RNAi MAX reagent (Invitrogen). Twenty-four hours after transfection, the cells were irradiated and harvested.pcDNA3.1-CHMP4C or -Aurora B was contrasted and transfected into A549 or H1299 cells using Lipofectamine 3000 reagent (Invitrogen). Twenty-four hours later, cells were collected and performed for subsequent experiments as siRNAs treatment.Aurora B kinase inhibitor AZD1152-HQPA was dissolved in dimethyl sulphoxide (DMSO). The A549 and H1299 cells were treated with AZD1152-HQPA at the concentration of 10, 100 and 1000 nM. After 24 h of treatment, CHMP4C phosphorylation was examined by Western blot.g for 15 min at 4 °C and measured by BCA protein assay kit (Thermo Scientific Pierce). Equal amounts of protein were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and blotted on nitrocellulose membranes for Western blot analysis. The membranes were blocked in 5% nonfat milk and then incubated with the following primary antibodies: CHMP4C , phosphorylated (p)—CHMP4C , Aurora B (Abcam), p53 , p21  and β-actin . The CHMP4C antibody is diluted in 1:500, and the rest were used in 1:1000 dilutions. Membranes were washed in tris-buffered saline containing 0.5% tween-20 and then incubated with goat anti-rabbit lgG  or goat anti-mouse lgG  conjugated to horseradish peroxidase for 1 h at room temperature. The membranes were detected using Chemiluminescence liquid (Thermo Scientific Pierce) according to the manufacturer’s protocol and analyzed by the Image J software .The cells were harvested and lysed in RIPA lysis buffer . The protein was collected at 12,000× Ct−∆∆ method.Total RNA was extracted using SV total RNA isolation system kit  followed by reverse transcription using the GoScript reverse transcription system kit (Promega). The subsequent cDNA products were used as templates to perform the real-time PCR assays. The primers for the amplification of Aurora B or CHMP4C are as follows; Aurora B forward: TTTGAGATTGGGCGTCCTCT and reverse: CGCCCTCCTTCTCTATCTGG; CHMP4C forward: AGAAGCCCTGGAGAACTCAC and reverse: CTTGGGCAGTATCCTGTTGC. The β-actin was used as the internal control using primers forward: TGCCAGAAAACAAGATGAG and reverse: CACCTTCACCGTTCCAGTTT. PCR amplifications were performed in triplicate wells and each experiment was repeated for three times. The relative expression levels of genes were analyzed through the use of the 2Cells were harvested and treated with Vibrant Dyecycle green stain (Invitrogen) at 37 °C for 30 min in a dark place. Then, cell cycle was analyzed on a flow cytometer using 488-nm excitation and green emission.To analyze colony formation, Single cell was plated in the 6 cm dish at a density of 1000 cells/mL for 10 to 14 days to form spheres. Colonies were fixed with 4% paraformaldehyde for 20 min and then stained with 1% crystal violet for 15 min. Colonies were counted and surviving fraction was calculated as the mean number of colonies/(cells seeded × plating efficiency).6 cells/mL and stained with PE AnnexinV and 7-AAD (BD) for 15 min at room temperature in the dark. Samples were analyzed by flow cytometry.Cells were harvested and washed twice with cold PBS. Cells were resuspended in 1× binding buffer (BD) at a concentration of 1 × 10Cells were seeded on the glass sheets into 6-well plates. Cells were transfected with siRNAs and then exposed to 4 Gy of irradiation. 30 min later, cells were washed twice in PBS, and then incubated with anti-γH2AX , anti-53BP1 (Abcam) or anti-CHMP4C antibody at a dilution of 1:200 overnight at 4 °C. After washing twice in washing buffer (0.05% Tween-20 in PBS), the cells were incubated with goat anti-rabbit lgG  at 37 °C for 1 h. Cells were washed twice with washing buffer and once with PBS. The coverslips were then mounted with mounting medium for fluorescence with DAPI (Vectashield). The image of γH2AX and 53BP1 foci was detected using a Leica DMRXA fluorescent microscope . γH2AX and 53BP1 foci were counted in cells with more than five foci.t test using Microsoft Excel . p-value <0.05 indicates statistical significance.Data are presented as the mean ± S.E. of three independent experiments, and the statistics were analyzed by students’ in vivo, for example the function of CHMP4C in xenografts [in vivo evidence for the new action of CHMP4C.In conclusion, we identify that CHMP4C is a new radiation-inducible protein engaged in cell cycle and cell survival. We first confirm CHMP4C offers lung cancer cells radioresistance, which will provide a potential radio-therapeutic strategy for non-small cell lung cancer by disrupting CHMP4C expression. In future research, the role of CHMP4C combined with radiation nografts , will be


Rlf.We recently identified a novel protein, Rearranged L-myc fusion (Rlf), that is required for DNA hypomethylation and transcriptional activity at two specific regions of the genome known to be sensitive to epigenetic gene silencing. To identify other loci affected by the absence of Rlf, we have now analysed 12 whole genome bisulphite sequencing datasets across three different embryonic tissues/stages from mice wild-type or null for Rlf mutants. RNA-seq showed that the numerous effects of the absence of Rlf on the epigenome are associated with relatively subtle effects on the mRNA population. In vitro studies suggest that Rlf’s zinc fingers have the capacity to bind DNA and that the protein interacts with other known epigenetic modifiers.Here we show that the absence of Rlf results in an increase in DNA methylation at thousands of elements involved in transcriptional regulation and many of the changes occur at enhancers and CpG island shores. ChIP-seq for H3K4me1, a mark generally found at regulatory elements, revealed associated changes at many of the regions that are differentially methylated in the This study provides the first evidence that the epigenetic modifier Rlf is involved in the maintenance of DNA methylation at enhancers and CGI shores across the genome.The online version of this article (doi:10.1186/s12915-015-0128-2) contains supplementary material, which is available to authorized users. Rearranged L-myc fusion (Rlf) acts as an epigenetic modifier [Modifiers of murine metastable epiallele Dominant (MommeD) [. We found that three of the lines, MommeD8, MommeD28 and MommeD34, carry mutations in Rlf and have designated the alleles RlfMommeD8, RlfMommeD28 and RlfMommeD34. Of these, RlfMommeD28 and RlfMommeD34 are null alleles and RlfMommeD8 is hypomorphic [agouti viable yellow [Rlf mutations are viable, with no overt abnormalities. Mice homozygous for the null alleles die around birth. Little is known about the function of Rlf, although the predicted presence of 16 widely-spaced zinc fingers suggests a role in transcription [RlfMommeD28/MommeD28 embryos, consistent with its reduced expression [We recently reported that modifier . The genmodifier . This is(MommeD) . We founomorphic . Mice hee yellow . Mice heTo discover whether or not Rlf has functions at other loci, we have carried out whole genome bisulphite sequencing at three different stages of development and provide evidence that Rlf has a role in the maintenance of DNA hypomethylation at thousands of elements across the genome. These regions overlap with those previously found to be differentially methylated across different tissue types, called tissue-specific DMRs (tsDMRs) -8. TissuHere we show that Rlf has a role in maintaining DNA hypomethylation at enhancers across the genome. Increases in DNA methylation that occur in the absence of Rlf, are accompanied by reductions in H3K4me1 occupancy.Rlf+/+ and RlfMommeD28/MommeD28). We chose E14.5 liver because the original screen was carried out using erythroid cells and the liver is the major erythropoietic tissue at this stage of development. Also, since E14.5 embryonic liver is one of ENCODE’s chosen tissues, associations can be made between sites of differential DNA methylation and histone marks. Mice homozygous for the null allele appear grossly phenotypically normal at this stage, reducing the risk of possible changes to DNA methylation that are the consequence of their demise at later developmental stages. Bisulphite sequencing was carried out on E14.5 livers from two wild-type (Rlf+/+) and two homozygous (RlfMommeD28/MommeD28) littermates to an average depth of 30-fold per sample. Approximately 85% of the CpGs in the mouse genome were covered by >5 reads and approximately 75% were covered by >20 reads , occurred at regions of the genome that are less methylated than the surrounding DNA  and half were intergenic (n = 677)  Figure B. This iUsing the ENCODE E14.5 liver dataset, we found that 287 of the 1,329 E14.5 liver Rlf-DMRs were enriched for H3K4me1 ChIP-seq reads . In tot5 Figure F. The dig Figure G.RlfMommeD28/MommeD28 E14.5 fetal livers. For each sample, 28–40 million reads were sequenced. We saw a good correlation between our wild-type datasets and those in ENCODE (data not shown). To enable characterisation of H3K4me1 occupancy at the GFP transgene, a subset of 1x107 reads were selected from each dataset and mapped to a mouse genome containing the sequence of the GFP transgene as a separate chromosome. The GFP transgene showed reduced H3K4me1 read coverage in homozygous mutants, consistent with the observed increase in DNA methylation and decrease in expression  were not enriched for H3K4me1, H3K27ac or H3K4me3, raising the possibility that many Rlf-DMRs might be active enhancers in another tissue or at a different stage of development. Indeed, others have noted differentially methylated regions of the adult genome that are not active enhancers but are likely to have been at an earlier stage in development, termed “vestigial” [+ (erythroblasts) and 23% being Krt8+ Acta2− epithelial cells (hepatoblasts). By E18.5, erythroblasts represent only 7% of the total cells and hepatoblasts become the dominant cell type, making up 82% of the total cell population [Western blot analysis showed that Rlf is expressed in many adult, fetal and embryonic tissues . Because Rlf-DMRs were found at regulatory elements, we searched for enriched consensus DNA binding motifs at Rlf-DMRs. No motifs were found to be significantly enriched over background (data not shown).Given that Rlf was found to be required for normal DNA methylation status at regulatory elements throughout the genome, we were interested to find protein partners. Co-immunoprecipitation (co-IP) experiments were performed. Human HEK293T cells expressing either a Flag-tagged RLF cDNA (RLF-Flag) or an empty vector control (EV) were generated via transient transfection. Anti-Flag conjugated magnetic beads were used to co-IP tagged RLF from nuclear lysates. Eluted proteins were identified by mass spectrometry. Three biological replicates were used in each case and the data were analysed using Scaffold see . Interac431–513 and Rlf1001–1362, that contain two and six zinc fingers, respectively -labelled oligonucleotide probes, called pentaprobes, which have been used previously to test DNA binding [1001–1362 or Gata1CF resulted in a shift in the migration pattern of both probes. No shift was detected with Rlf431–513 fragment  using two non-overlapping sub-fragments of Rlf, Rlf binding . A proteRlf homozygous mutants. A relatively small number of significant changes were detected, using an adjusted p-value <0.05  showed an over-representation of genes involved in the following networks: Cardiovascular System Development and Function, Organismal Development, Tissue Development (p score = 52); Cancer, Cellular Development, Cellular Growth and Proliferation (p score = 30); and Lipid Metabolism, Small Molecule Biochemistry, Nucleic Acid Metabolism (p score = 19). Real-time RT-PCR using RNA from an independent Rlf mutant mouse line, RlfMommeD34/MommeD34, was used to validate RNA-seq findings for four up-regulated and four down-regulated genes  are relatively common in the genome and are well known as DNA-binding factors, proteins with widely-spaced zinc fingers, like Rlf, are less common and are poorly characterised. The significance of the wide spacing is unknown . Interesiegation , and Wiziegation . Only a iegation  and Peg3iegation , both ofiegation ,23. Despcis-acting regulatory regions across the genome. Future studies of this protein will reveal the mechanisms that underlie this process.Overall our data show that Rlf has a prominent role in the modulation of epigenetic state at MommeD28 and MommeD34 mouse lines were produced in an ENU mutagenesis screen for epigenetic modifiers and have been described previously [Line3 FVB/NJ mice carrying a multi-copy GFP transgene under the control of the human alpha-globin promoter and the HS-40 enhancer. All mice used in this study were homozygous for the GFP transgene array. ENU mutant lines have been maintained as inbred colonies by backcrossing to unmutagenised Line3 mice for at least ten generations. All procedures were carried out with approval from the Animal Ethics Committee of the QIMR Berghofer Medical Research Institute.eviously . The ENUMommeD28 Geno F: TCT GCC AGT CTC TGA AGA AAA GGC A, MommeD28 Geno R: CTG TGG CTT TCA TTC TGG AAG GGA A; MommeD34 Geno F: GTTGCCCATTTTAGGGGAG, MommeD34 Geno R: TGGATTGCACCCTGGACTAC.Embryonic tissues were obtained from natural timed-matings of heterozygous individuals, with the presence of a post-coital plug defined as E0.5. Pregnant dams were euthanised via cervical dislocation at the required time point. Whole embryos (E10.5) or fetal livers (E14.5 & E18.5) were removed and homogenised in RLT Plus Buffer  using a syringe and needle. Genomic DNA and total RNA were prepared using an AllPrep DNA/RNA Mini Kit according to manufacturers’ instructions . Genotyping of DNA was performed by Sanger sequencing. Primers: Whole Genome Bisulphite Sequencing was performed by the Centro Nacional de Análisis Genomico . Genomic DNA samples were spiked with λ DNA and sheared by sonication. Libraries were prepared using the TruSeq Sample Prep Kit  and underwent two rounds of sodium bisulphite conversion using the EpiTect Bisulphite Kit . 100 bp paired end sequencing was performed on the Illumina HiSeq 2000. Sequencing reads were trimmed for poor quality at their 3′ ends using the program trim_seq  with theFor the full E14.5 liver datasets, differentially methylated regions were identified using the R package bsseq . The smoThe 10.5 embryo dataset, the E18.5 liver dataset and the sub-sampled E14.5 liver dataset  were mapped to the genome and methylation values obtained, as described above. To enable comparison between the tissues, methylation values for CpGs covered by at least 6 CpGs in all samples were extracted and Rlf-DMRs called across mutants and wild-types for each tissue using bsseq, smoothing with the options ns = 50, h = 500, maxGap = 10,000,000, requiring a coverage of at least 6 in both replicates and a maximum gap of 1,500 bp between CpGs in a Rlf-DMR. Rlf-DMRs with ≥8 CpGs and mean methylation change >15% were used for downstream analyses.Methylation values used for hierarchical clustering were the weighted averages of CpG methylation within each genomic coordinate. Only coordinates containing at least 8 CpGs with coverage of at least 6 reads across all samples, were used.To calculate enrichment of histones at Rlf-DMRs the ENCODE sequencing reads  for bothGene ontology analyses of genes proximal to Rlf-DMRs were carried out using the GREAT tool . The Hom. The PCR product was ligated into pGEM T Easy  and transformed. DNA from individual colonies was sequenced using Sanger sequencing. The bisulphite conversion rate was ≥98% and sequences were analysed using BiQ Analyser software [Bisulphite conversion of genomic DNA was carried out using the EpiTect Bisulphite Kit . Nested PCRs were performed to interrogate methylation at the Smad3-6 intergenic region with the following primers: Smad3-6 bis F1: AAGTGGAATTTTTTAGTGGTAGATG; Smad3-6 bis R1: AACTACTTTAATAAAAAATAACATAACC, Smad3-6 bis F2: TTGGTATGTGTTGTTTTTAGTTTTG and Smad3-6 bis R2: ACAATTTAACTATTCATTATATCTCTAACA. Cycling conditions were as follows: Primary PCR, 94°C for 2 minutes for 1 cycle; 94°C for 30 secs, 53°C for 30 secs, 72°C for 45 secs for 35 cycles and 72°C for 6 mins for 1 cycle. The secondary PCR was performed using an annealing temperature of 51°Csoftware .H3K4me1 ChIP-seq library preparation, sequencing and initial bioinformatical analyses were carried out by ActiveMotif. Briefly, the prepared libraries were sequenced on the Illumina NextSeq 500 platform using a read length of 75 nt. Each resulting dataset was sub-sampled to the read-count of the library with the smallest number of reads and aligned to the mouse genome (mm9) using BWA  with defMapped reads were then extended from their 3′ ends to a length of 200 bp to account for the length of the ChIP fragments used to prepare the libraries. The density of fragments along the genome was obtained by dividing the genome into 32-nt bins and the number of fragments overlapping each bin was determined.Regions enriched for H3K4me1 (i.e. peaks) were determined using the program MACS  using deThe density of H3K4me1 reads across the GFP transgene was determined by mapping 10,000,000 reads from each library to a mouse genome (mm9) with the sequence of the transgene added as an additional chromosome. The reads were mapped using the program Bowtie2  using dep-value of <0.05, with at least five peptides detected.HEK293T cells  were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) . Cells were transfected with a pCMV6-Entry vector containing Myc-DDK-tagged-Human rearranged L-myc fusion, RLF-Flag or vector alone, EV  using Lipofectamine 2000 . After 48 hrs, the cells were lysed with a hypotonic lysis buffer, nuclei were isolated and resuspended in digestion buffer with PMSF and protease inhibitors . For each replicate, 4 mg of nuclear lysate was immunoprecipitated using anti-FLAG M2 magnetic beads . IP reactions were carried out in High stringency IP buffer supplemented with protease inhibitor cocktail, for 1 hr at 4°C. Samples were eluted with 8 M Urea. Digestion and IP buffers were from the Nuclear Complex Co-IP kit . Eluted proteins were analysed via mass spectrometry at the La Trobe University Mass Spectrometry Facility. Samples were digested with 1 μg of trypsin overnight at 37°C and peptide fragments analysed via tandem mass spectrometry on an Orbitrap Elite mass spectrometer (Thermo Scientific). Three biological replicates were analysed per condition, with three replicate injections performed for each. MS/MS spectra were queried using Thermo Scientific Proteome Discoverer software v. 1.4 with Mascot (v. 2.4.0 Matrix Science) search engine. Result files were imported into Scaffold (Proteome Software Inc.) for visualisation and validation. Putative interacting proteins were considered significant if they showed at least a five-fold enrichment in RLF-Flag samples compared to EV, with a Protein lysates from whole tissues were prepared by homogenising in ten volumes of urea lysis buffer, as described . Samples431 F: GCGGATCCGGTTCCTCTGAGAGATACCAGAG, Rlf513 R: GCGAATTCCTATTAAGCGCTTTTCAATAGTAATTTCTT, Rlf1001 F: GCGGATCCAGTCAGTACCTTGCACAGTTGGC, Rlf1362 R: GCGAATTCCTATTA GCTGCTGAACAGGTTGTCATAATA and cloned into pGEX-6P, for expression with an N-terminal GST tag, using BamHI and EcoRI restriction sites. GST-Rlf protein sub-fragments were overexpressed in Escherichia coli Rosetta II cells with induction by IPTG. Cells were lysed by sonication in lysis buffer , incubated with 1 mg/mL DNase for 30 mins at 4°C, and further sonicated. The soluble fraction was loaded onto glutathione-sepharose resin and washed  glycerol, 1.4 mM β-mercaptoethanol, 1 mM PMSF). GST-Rlf recombinant protein sub-fragments were eluted  and further purified by size exclusion chromatography on a HiLoadTM Superdex 75 column .Rlf sub-fragments were amplified from mouse cDNA using the following primers, Rlf2, 20 μM EDTA) containing 10 mM DTT, 10 μM ZnSO4, 67 μg/mL acetylated bovine serum albumin and 4% Ficoll. All reactions contained 5 nM oligonucleotide and either 0.5 μM, 5 μM or no protein. Samples were incubated on ice for 30 min, separated on non-denaturing polyacrylamide TBE gels and visualised using a Typhoon FLA 9500 scanner .EMSAs were performed using FAM labeled pentaprobe oligonucleotides . PP2: FAM-TATCTTACTTTAGTTTCATTTAATTGT GTTGTACTCTCCTCTGCGTTCACTTAGCTTAACTTGGTTTGGCTTGATTTGACTTCAGTTGCGCTCTATTCTA; PP4: FAM-TATCCTACCCATTGGGCTCATCTGATCCATCCGGTCCCGTCCACTCGGCTATGTTATGCTGTATTGCAGTCGTGTCGCGTCGAGCTGCCCTAATCCCACC. Reactions were performed in MSB buffer  and Illumina HiSeq 2000 Sequencing with 50 bp single-end reads was performed by the Australian Genome Research Facility . Reads were aligned to the mouse genome (NCBI37/mm9) using Tophat  with thet test. Primer sequences used are as follows: Aldh1a7 F: GCAGGGAAAAGCAATCTGAA, R: TCTGACCCTGGTGGAAGAAC; Vldlr F: TCGGGCTTTGTTTACTGGTC, R: AGTAGAGGCGGCTTTTGACA; Mgam F: TTGTTCTGCTGCTTGTCCTG R: ACTGGGCAATTGGGAGAGTT; Myo5c F: GGCTGAAATCGCAAAGGACT R: CTCATGGAGGTAGCTGAGGG; Hpd: F: AGGTAGTCAGCCACGTCATC, R: CAATGTGGTCGCAGTCCAGC; Prss50: F: GGTTCATTCCAGCAACCTCC, R: GAAGCGATAAGGATGCCAGC; Atp2b2 F: CCTCAAAACCTCGCCTGTTG, R: GTGGGTGGTAGAAGGACAGT; Upb1 F: AGGAATCTCGATCTGCCCAG, R: ATTGACTCCACACATTGCGG.RNA was reverse transcribed using iScript Reverse Transcription Supermix . Quantitative real-time reverse transcription (RT)-PCR analysis was performed on a Corbett Research Rotor-Gene  using Platinum SYBR Green qPCR Supermix-UDG . Expression of β-actin was used for normalisation. Statistical analysis was performed using Student’s http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE58108.All methylome and RNA-seq datasets used in this study have been submitted to the NCBI Gene Omnibus (GEO;


P < 0.0001) in K8 PS74 and PS432 levels by 59% and 66%, respectively, resulting in increased apoptosis.Hyperphosphorylated keratin (K) 8 acts as a phosphate “sponge” for stress-activated protein kinases thereby inhibiting pro-apoptotic molecules and thus apoptosis. MAP kinase/ERK1 has increased activity in colorectal cancer (CRC) and is known to phosphorylate K8. The aims were to identify the K8 isoforms abundantly present in colon tumors, using 2D difference gel electrophoresis (DIGE), to identify the modifications using mass spectrometry, and to validate the differential abundance of these isoforms in tumors relative to matched normal mucosae. 2D DIGE showed 3 isoforms of K8 significantly increased in tumor ≥2-fold in 6/8 pairs. Metal oxide affinity chromatography mass spectrometry and bioinformatics were used to identify phosphorylated serine residues. Levels of PS24, PS432, and PS74 by western blotting were found to be significantly increased in tumor versus matched normal. Blocking of EGFR signaling in Caco2 cells showed a significant decrease ( Partial sequence and the mass loss of a phosphate were obtained from the [M+H]+-ion 3805.269 ([415]–[454] TTSGYAGGLS(s)A(y)GGL(t)(s)PGLSYSLGSSFGSGAGSSSFSR) . The residue that had lost the phosphate group was assigned by using BioTools, where all possible phosphorylation sites within the peptide were analysed and ranked according to probability. The peptide contains 4 potential phosphorylation sites according to the Uniprot data base (http://www.uniprot.org/uniprot/P05787) of which 2 have been assigned by similarity and 2 by annotation. The serine residue 432 was scored as having the highest probability of phosphorylation using BioTools. Using phospho-specific antibodies, 2D western blots confirmed the presence of the serine phosphorylated residues 24, 432, and 74 in the K8 spots of interest .The K8 isoforms detected as increased in tumor were excised from a preparative 2D gel and digested with trypsin. Phosphorylated peptides were enriched using Phos-Trap TiOP = 0.053 Wilcoxon signed rank test). For the phospho-K8 isoforms the median level between the tumor and matched normal was significantly different: PS24 0.667 and 0.29, respectively, P = 0.004; PS432 1.02 and 0.30, respectively, P = 0.03; PS74 0.18 and 0.06, respectively, P = 0.0005 (Wilcoxon signed rank test). Among the 30 patients, 16/30 (53%) showed overabundance  for PS24, 17/30 (57%) for PS432, and 19/30 (63%) for PS74 (The results of western blot quantification are shown in for PS74 .P < 0.0001) and PS432 (P < 0.0001) (ANOVA) at 6 and 8 hours after treatment  that blocks ligand binding and hence activation of EGFR. Treated and untreated cell lysates were then analysed by western blotting for PS24, PS74, and PS432. Treatment of Caco2 cells with the sc-120 antibody led to a significant decrease in the levels of PS74 (reatment . At the P = 0.0005 (ANOVA)  compared to the DOX alone (0.11 ± 0.002), sc-120-treated (0.081 ± 0.008) or -untreated (0.071 ± 0.006),  (ANOVA) . The resThis study found K8 isoforms, CK18, and CK19 to be significantly upregulated in laser microdissected tissue from 8 stage II tumor samples compared to matched normal by 2D DIGE. The advantage of 2D DIGE over standard 2D gels is the greatly improved statistical validity obtained due to the presence of the tumor, normal and pooled reference sample, to which abundance of each spot is normalised, on every gel. Spot matching within a gel is therefore exact. The software uses a novel algorithm used to match spots gel to gel and, through spot normalisation, greatly reducing gel-gel variation effects and increasing the statistical value of results. We hypothesised that the K8 isoforms present in abundance in the tumor samples compared to matched normal were a result of increased phosphorylation. Indeed, this was confirmed using mass spectrometry, bioinformatics, and 2D western blotting. In a larger cohort of human colon cancer tissues, levels of all 3 K8 isoforms PS24, PS432, and PS74 were found to be significantly increased in tumor compared to matched normal mucosa across all stages.P < 0.0001) in support of the earlier work. The MAP kinase p38 and JNK are also known to phosphorylate serines 431 and 73 [Mutation of genes in the EGFR signaling cascade results in increased RAS/RAF/MEK/ERK pathway activation and increased MAPK (ERK1/ERK2) activity in particular . The kin1 and 73 , 13, butP = 0.004). Treatment of Caco2 cells with the sc-120 antibody did not significantly affect K8 PS24 levels, suggesting that kinases involved in the EGFR signaling pathway are not involved in the phosphorylation of this residue. One study has shown that protein kinase C ε phosphorylates K8 S24 in rat pituitary cells stimulated by thyrotropin-releasing hormone [In our study the level of K8 PS24 was significantly increased in the tumor samples compared to matched normal  laser microdissection of tumour section, (2) flow cytometry of Caco2 cells to determine saturating amount of anti-EGFR SC-120 antibody, (3) MALDI-TOF MS spectra showing detection of PS432 after Phos-Trap™ enrichment, (4) Tandem MS analysis showing a mass loss of 98 (a phosphate group) conferring to K8 PS74, (5) Tandem MS analysis identifying K8 PS432 as the most likely residue, (6) 2D western blots confirming the phosphorylated serine residues of K8 isoforms as PS24, PS432 and PS74. Finally, the 2D DIGE method is described in further detail.


Shewanella algae strain C6G3, isolated from the 2 uppermost centimeters of muddy sediment of Arcachon Bay  has the capability to use a large panel of terminal electron acceptors under anaerobic condition, such as nitrate, nitrite and metal-oxide, and presents a great metabolic versatility. Here, we present the non-contiguous draft-genome sequence of Shewanella algae C6G3, which consists of a 4,879,425 bp. The chromosome contains 5792 predicted genes. In total, the genome consists of 24 rRNA genes, 86 tRNA genes and 5660 genes assigned as protein-coding genes.The online version of this article (doi:10.1186/s40793-015-0022-0) contains supplementary material, which is available to authorized users. Shewanella comprises several Gram-negative species which are widely distributed in marine and freshwater environments. Shewanella algae (formerly classified as S. putrefaciens) has been frequently isolated from marine water samples and spoiling fish [S. putrefaciens showed beta-hemolysis on sheep blood agar whereas environmental strains were nonhemolytic [Shewanella genus and were able to reduce Mn(III/IV) and/or nitrate. The genus seems to play an important role in the turnover of organic matter coupled to anaerobic respiration electron acceptors, such as Fe(III), Mn(III/IV) and NO2/3. Here we report on further taxonomic and physiological studies on strain Shewanella algae strain C6G3 and present its main genomic features.The genus ing fish –3. They ing fish , 5. The emolytic , 7. Duriemolytic , a largeShewanella currently contains 62 species [Shewanella algae ATCC 51192T, the first described [Shewanella algae . Among the two strains of S. algae, slightly different patterns of carbon source were noticed; however, profil of S oneidensis MR-1T was different (16/95). Some electron acceptors were also tested for strain C6G3 according to genome annotation and Shewanella algae literature [The genus  species  includin2T, ACDC , BrY [10is MR-1T , the useS. algae  and on S. oneidensis MR-1T , C16:0 (34.6 %) . S. algae C6G3 presents also a relatively higher percentage of short-chain fatty acids  than both S. oneidensis MR-1T and S. algae ATCC 51192T (8.5 %).The fatty acid analysis was performed on two strains of ectively ) and 3OHS. algae C6G3 was isolated from intertidal marine sediment on the basis of its ability to use large range of electron acceptors particularly nitrate, nitrite and metal-oxides [S. algae C6G3 is the second to be reported from that species, the other one being S. algae ACDC [S. algae C6G3 is deposited in the Genome On Line Database [l-oxides . The gengae ACDC . The genDatabase . A summaDatabase  are showS. algae C6G3 was grown aerobically at 30 °C under stirring condition on artificial sea water [ea water  amended ea water . DNA conS. algae C6G3 was generated at the Molecular Research LP MR DNA Laboratory (USA). De novo whole-genome shotgun sequencing was performed using the Ion Torrent PGM . The final assembly identified 43 contigs generating a genome size of 4,9 Mb.The genome sequencing of nologies ) sequencGenome annotation was performed on two platforms: on RAST  and on IThe assembly of non-contiguous finished draft genome consists of 43 contigs representing overall 4,879,425 pb. The DNA G + C content was 53.08 %. Using RAST and IMG/ER, 5770 and 5795 genes were respectively predicted. Among them 4149 and 5660 protein-coding genes and 108 and 135 RNAs were identified by RAST and IMG/ER, respectively. The properties and the statistics of the genome (IMG/ER data) are summarized in Table S. algae C6G3 encodes genes for complete glycolysis and tricarboxylic acid (TCA) cycle. A focus has been made on the enzymes involved in the reductive respiratory reactions of the N-cycle  and in extracellular electron transfer through the outer-membrane  and Mn(III/IV)) on the basis of protein system described in S. oneidensis MR-1T.Genome of S. algae C6G3 is NapAB  by a periplasmic nitrite reduction system (NRF) [Shewanella strains are known for encoding NrfABCD system only. Genome annotations of S. algae C6G3 and S. oneidensis MR-1T identify nrfA in both cases but nrfBCD were found in S. algae C6G3 only . Indeed, nrfB is lacking and nrfCD are present but proposed to be pseudogene because of truncation [, [c-Cyt CymA, a cytoplasmic membrane electron transport protein, is likely to be the functional replacement of both NapC and NrfBCD/NrfH allowing to NrfAH-like system to be efficient in S. oneidensis MR-1T.Nitrite can be reduced to ammonium (NHem (NRF) . As NAP nly Fig. . As prevuncation . Gao et ation [,  suggesteS. algae C6G3 can utilize extracellular mineral metal oxides of Fe(III) and Mn(III/IV) as respiratory electron acceptors (unpublished data). Inspection of its genome confirmed the presence of genes involved in pathway  for transferring electrons from the inner membrane through the periplasm and across the outer membrane where metal oxides are reduced [S. algae C6G3 genome, genes that encode MtrCBA and OmcA are located in the same region, which also includes mtrD (an mtrA homologue), mtrE (an mtrB homologue) and mtrF (an mtrC homologue)  and Mn(III/IV) as terminal electron acceptors. Reduction of these particulates occurs at the cell surface and is catalyzed by multihaem cytochromes whose properties are beginning to emerge.The ronments , 42, 43.


Our data suggest that RAD18 can function as a mediator for DNA damage response signals to activate the G2/M checkpoint in order to maintain genome integrity and cell survival after IR exposure.The ubiquitin ligase RAD18 is involved in post replication repair pathways via its recruitment to stalled replication forks, and its role in the ubiquitylation of proliferating cell nuclear antigen (PCNA). Recently, it has been reported that RAD18 is also recruited to DNA double strand break (DSB) sites, where it plays novel functions in the DNA damage response induced by ionizing radiation (IR). This new role is independent of PCNA ubiquitylation, but little is known about how RAD18 functions after IR exposure. Here, we describe a role for RAD18 in the IR-induced DNA damage signaling pathway at G2/M phase in the cell cycle. Depleting cells of RAD18 reduced the recruitment of the DNA damage signaling factors ATM, γH2AX, and 53BP1 to foci in cells at the G2/M phase after IR exposure, and attenuated activation of the G2/M checkpoint. Furthermore, depletion of RAD18 increased micronuclei formation and cell death following IR exposure, both  The RING-type (E3) ubiquitin ligase RAD18 is a key player involved in post-replication repair (PRR) that regulates ubiquitylation of proliferating cell nuclear antigen (PCNA) in response to replication stresses. RAD18 isMammalian cells use two major DNA repair systems, non-homologous end joining (NHEJ) and homologous recombination (HR), which are employed to repair DNA damage depending on phases of the cell cycle. During tin vivo. These data suggest a crucial role for RAD18 in the DNA damage response induced by IR.In this study, we describe a new role for RAD18 in radioresponse of cells in the G2/M phase after IR exposure. RAD18 facilitates the DNA damage signaling pathway to activate the G2/M checkpoint. Rad18-depleted cells showed increased sensitivity to IR, in term of micronuclei formation and apoptosis after irradiation; similar IR sensitivity was observed HT1080, HeLa, HEK293 and H1299 human cancer cell lines were cultured in alpha Eagle’s medium (Sigma) supplemented with 10% fetal bovine serum (FBS) and streptomycin sulfate and penicillin. Transfections with siRNA at a final concentration of 100nM were performed using 0.5% lipofectamine RNAiMAX reagent (Life Technologies). Cells were transfected with a non-targeted siRNA negative control (si-ctrl) or RAD18 siRNA (si-RAD18), both purchased from Life Technologies. Transfected cells were cultured for 48 hrs before being used in individual experiments.Cells were separated into soluble and insoluble fractions using the complete Lysis-M, EDTA-free reagent (Roche). Proteins prepared from the insoluble fractions were electrophoresed on 5–20% sodium dodecyl sulfate-polyacrylamide gels (Atto), transferred to nitrocellulose membranes (Bio-Rad) and analyzed by immunoblotting with the indicated antibodies (Abs). Primary Abs used recognized ATM phosphorylated at Ser1981 (Rockland); p53 phosphorylated at Ser15 ; H2AX phosphorylated at Ser139 (Merck Millipore); histone H3 phosphorylated at Ser10 (Merck Millipore); histone H1 (Santa Cruz Biotechnology). The rabbit polyclonal Abs against human and mouse RAD18 are described elsewhere. The inteII (BD Biosciences). The percentage of each cell cycle phase was measured using the FLOW JO software.For cell cycle distribution analyses, HT1080 cells were transfected with si-ctrl or si-RAD18. After 48 hrs, the cells were exposed to IR or UV and cultured for 0, 6, 12 and 18 hrs. The cells were then fixed with 70% ethanol at-20°C. The fixed cells were washed twice with PBS and then treated with propidium iodide (PI) (BD Biosciences) for 30min at room temperature and analyzed using a FACSCanto 2, 1 mM phenylmethylsulfonyl fluoride, and 0.5% NP40) for eight min on ice. Cells were fixed with 4% paraformaldehyde in PBS for 15 min, and then treated with 0.5% Triton X-100 in PBS for 30min. Cells were stained with a specific primary Ab, then stained with species-specific Cy3 (Jackson ImmunoResearch Laboratories), Alexa Flour 488 (Life Technologies) or Alexa 647 (Life Technologies) secondary Abs. Nuclei were labeled with Hoechst 33258. Fluorescence images were obtained using an automated fluorescence microscope and IN Cell Analyzer 2000 . Cell cycle distribution was determined by the intensity of Hoechst 33258 and EdU labeling. The number of foci per cell at each cell cycle phase were determined using the image-analysis software provided with the IN Cell Developer .Cells were plated into 96-well plates (Ibidi). Cells in the S phase were labeled using the Clik-It EdU Alexa Fluor 488 imaging kit (Life Technologies). Cells were washed twice with PBS then treated with hypotonic buffer . Following incubation with an anti-γH2AX antibody for 20 min at room temperature, a mouse-specific Alexa Flour 488 secondary Ab (Life Technologies) was added to the cell pellet. The fluorescence level of γH2AX was analyzed by using the FACS Canto II and γH2AX levels induced by IR were determined by comparing the relative mean γH2AX fluorescence intensities between non-irradiated and irradiated cells.Irradiated cells were fixed in 70% ethanol at-20°C. Fixed cells were washed with PBS and then treated with permeabilization buffer  respectively. The numbers of micronuclei were determined using the IN Cell Analyzer 2000. Quantitative analyses of the frequency of micronuclei were performed using the IN Cell Developer.Rad18-/- mice were generated as previously described and the crosses and genotyping of experimental mice were carried out as previously described.[escribed.,15 This PLUS kit (mouse) (BD biosciences), according to the manufacturer’s instructions. More than 20,000 reticulocytes were analyzed to determine MN frequencies using the FACS Canto II.Peripheral blood was withdrawn from the tail vein in each experimental group at 0, 24 and 48 hrs after IR exposure. Blood samples (20 μl) were analyzed using the MicroFlowThymocytes were isolated from each experimental group at 0, 3, 6, 9 and 12 hrs after IR exposure. The distributions of apoptotic thymocytes were then identified using a PE Annexin V Apoptosis Detection kit I (BD Biosciences). More than 10,000 thymocytes per mouse were analyzed to determine the frequency of apoptosis using the FACS Canto II. All results are presented as the percentage of apoptotic cells per sample.t test was used for statistical analysis. Chi-squared test was used for cell cycle distribution. Single and double asterisks indicate significant differences as p<0.05 and p<0.01.Error bar represent standard deviations. Student To investigate roles of RAD18 in cell cycle checkpoint activation after IR exposure, we analyzed the distribution of the cell cycle using flow cytometry in HT1080 human cancer cell line treated with a non-targeted siRNA (si-ctrl.) or siRNA that targeted RAD18 (si-RAD18). Treatment with si-RAD18 depleted the expression of RAD18 and ubiquitylated RAD18 in these cells to less than 10%  but did We further investigated roles of RAD18 on G2/M checkpoint activation by measuring the mitosis marker histone H3 phosphorylated at Ser10 (phospho-histone H3) to identify the M phase cells in HT1080 cell line. The percentage of phospho-histone H3-positive M phase cells was similar between non-irradiated control and RAD18-depleted cells . In contM-phase cells were quantified with 4N DNA content and positive-staining for phospho-histone H3 using flow cytometry. Before IR exposure, the percentages of M phase cells were about 1.6% and 2.3% in the control and RAD18-depleted cell populations respectively . HoweverTo investigate effects of depleting RAD18 on the activation of the DNA damage signaling pathway in response to IR, we examined levels of ATM phosphorylated at Ser1981, H2AX phosphorylated at Ser139 (γH2AX) and p53 phosphorylated at Ser15, in RAD18-depleted cells compared to control cells in HEK293 cell line. Phosphorylated ATM, H2AX and p53 were evident in control cells 30 min after 2 Gy or 4 Gy irradiation, whereas phosphorylated ATM, H2AX and p53 levels were about 2~7 times lower than that in RAD18-depleted cells . In addiNext, we aimed to confirm the role of RAD18 for DNA damage response at the G2/M phase. We measured levels of γH2AX as a marker for DNA damage signaling at the G2/M phase in the cells irradiated with 2 Gy of IR. Cell cycle stage was determined by DNA content . As showWe next examined effects of depleting RAD18 on recruitment of DNA repair proteins and formation of foci in G2/M phase cells using an IN Cell analyzer. Cell cycle stage was determined by DNA content and levels of 5-ethynyl-2’-deoxyuridine (EdU), with which S phase cells were specifically labeled. Under non-irradiated conditions, phosphorylated ATM (phospho-ATM), γH2AX and 53BP1 foci at the G2/M phase were more evident in RAD18-depleted cells than control cells . In the in vitro, we compared the frequency of micronucleus formation in control cells and RAD18-depleted cells exposed to IR. Depleting RAD18 increased micronucleus formation in HT1080 cells even without irradiation  and the cell cycle distribution was analyzed using flow cytometry. (B) HT1080 Cells were exposed to 1, 2 or 4 J/m2 UV, and then fixed at 12 hrs after UV treatment. Fixed cells were stained with PI and analyzed using flow cytometry. (C) Cells were exposed to 1 or 2 J/m2 UV and lysed at the time points indicated after UV treatment. Samples prepared from the insoluble fractions were analyzed by western blotting with the indicated antibodies.(A) HT1080 cells transfected with si-ctrl or si-RAD18 were exposed to 2J/m(DOCX)Click here for additional data file.S2 FigH1299, HEK293 and HeLa human cancer cells transfected with si-ctrl or si-RAD18 were exposed to 2 Gy of IR and lysed at the time points indicated after irradiation. Samples prepared from the insoluble fractions were analyzed by western blotting with the indicated antibodies.(DOCX)Click here for additional data file.S3 FigHT1080 cells transfected with si-ctrl or si-RAD18 were exposed to 2 Gy IR, labeled with EdU, and then fixed 90 min after irradiation. The cells were co-immunostained with anti-BrdU and anti-γH2AX, anti-phospho-ATM or anti-53BP1 antibodies. The G1, S, G2/M phase cells were distinguished using the IN Cell Analyzer. The number of foci per cell was determined using the image-analysis software of the IN Cell Developer. Each value represents the mean (+standard deviation) of the results from three independent experiments.(DOCX)Click here for additional data file.S4 FigThe sensitivity to IR (A) or UV (B) was analyzed using colony formation assays. HT1080 cells transfected with si-ctrl or si-RAD18 were exposed to increasing doses of IR or UV. Each value represents the mean (+standard deviation) of the results from three independent experiments.(DOCX)Click here for additional data file.S5 FigHT1080 cells were exposed to 4Gy IR, labeled with EdU, and then fixed at 60 min after irradiation. The cells were co-immunostained with anti-EdU and the indicated antibodies, then the G1, S, G2/M phase cells were distinguished using an IN Cell Analyzer.(DOCX)Click here for additional data file.S6 FigHT1080 cells transfected with si-ctrl or si-RAD18 were exposed to 2 Gy IR, labeled with EdU, and then fixed at 90 min after irradiation. The cells were co-immunostained with anti-BrdU and anti-NBS1 or anti- MDC1 antibodies. The G1, S, G2/M phase cells were distinguished using the IN Cell Analyzer. The number of foci per cell was determined using the image-analysis software of the IN Cell Developer. Each value represents the mean (+standard deviation) of the results from three independent experiments.(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


New reassortant H5N8 highly pathogenic avian influenza viruses were isolated from waterfowl in Southern China. Blast analysis demonstrated that the PB2 gene in these viruses were most closely related to A/wild duck/Shangdong/628/2011 (H5N1), while their NP genes were both more closely related to A/wild duck/Shandong/1/2011 (H5N1) and A/duck/Jiangsu/k1203/2010 (H5N8). However, the HA, NA, PB1, PA, M, and NS genes had the highest identity with A/duck/Jiangsu/k1203/2010 (H5N8). Phylogenetic analysis revealed that their HA genes belonged to the same GsGd H5 clade 2.3.4.4 detected in China in 2010. Therefore, we supposed that these H5N8 viruses might be novel reassortant viruses that have a H5N8 backbone while acquiring PB2 and NP genes from H5N1 viruses. This study is useful for better understanding the genetic and antigenic evolution of H5 avian influenza viruses in Southern China. Aquatic birds, including wild and domestic waterfowl, are the original reservoir of influenza A viruses, which provide the genetic diversity of influenza viruses and contribute to create new human and bird pandemic influenza viruses . About tIn 1996, the H5N1 highly pathogenic avian influenza virus (HPAIV) (A/goose/Guangdong/1/1996) was first isolated from sick geese during an outbreak in Guangdong of China . In 1997In our study, two new H5N8 viruses were isolated from waterfowl in Southern China between 2013 and 2014. We sequenced the entire viral genome and performed the phylogenetic analysis and determined the molecular characteristics of these viruses.50) and 50% egg lethal doses (ELD50) were calculated using the Reed–Muench method  (GDs13124) and A/duck/Guangdong/s14044/2014 (H5N8) (GDs14044), were isolated from fecal samples of healthy white ducks and black mane geese in live bird market of Guangdong, Southern China between 2013 and 2014. These H5N8 viruses were identified by reverse-transcription polymerase-chain reaction (RT-PCR), hemagglutination test, and hemagglutination inhibition (HI) test as per the standard protocol . Moreoveh method . All expThe eight segments of the two viruses were generated using primers as described above. The PCR procedure was alike as described above. The PCR products were purified with the QIAquick PCR purification kit (QIAGEN) following the manufacturer’s instructions and sequencing was performed by using an ABI Prism 3730 genetic analyser (Applied Biosystems) by Shanghai Invitrogen Biotechnology Co., Ltd. All sequences were assembled, edited, alignment, and residue analysis using Lasergene 7.1 (DNASTAR) . Neighbo10 50% egg infectious dose (EID50)/0.1 mL, respectively. The viruses were highly pathogenic (HP) in eggs with titres of 6.75 and 7.50 log1050% egg lethal dose (ELD50)/0.1 mL, respectively.GDs13124 and GDs14044 grew efficiently in eggs with virus titres of 8.83 and 8.38 logTo understand the genetic properties of these viruses, all eight genes of GDs13124 and GDs14044 were characterized and phylogenetically analyzed, respectively. Our sequence data were compared with the reference sequence from the NCBI Influenza Virus Resource.Figure 1)  in the HA molecule. The HA gene of both encoded 567 amino acids. 182, 222, and 224 amino acid sites were still Asn, Gln, Gly in the receptor binding pocket of the HA1 (H5 numbering used throughout), respectively, suggesting that the two viruses preferentially bind to the avian-like NeuAca2,3-Gal receptor rather than the human-like NeuAca2,6-Gal receptor  . The HA igure 1) . As showreceptor .Figure 3E). As shown in Tables 1 and 2 and Figure 2, the NA genes were closest to A/duck/Jiangsu/k1203/2010 (H5N8), with a nucleotide identity of 97.6 and 97.7%, respectively. No stalk deletion was found in the NA gene. Both viruses had 96-Ala and 258-Gln in the NA gene, indicating that they may reduce susceptibility to oseltamivir or zanarnivir . The PB2 gene of both viruses had 98.6% identity compared with the A/wild duck/Shangdong/628/2011 (H5N1). The NP genes were both more closely related to A/wild duck/Shandong/1/2011 (H5N1) and A/duck/Jiangsu/k1203/2010 (H5N8), with a nucleotide identity of 98.4%, respectively . The PB2 is an important factor related to the host range and virulence of influenza viruses. E627K or D701N substitutions were thought to contribute to the adaptation, replication, and virulence of H5N1 viruses in humans and mice . As shown in Tables 1 and 2 and Figure 2, the PB1, PA, and NS genes had the highest identities with A/duck/Jiangsu/k1203/2010 (H5N8) . The M genes were both closest to the A/duck/Jiangsu/k1203/2010 (H5N8) virus with nucleotide identities of 99.2 and 98.6%, respectively. There was an S31N mutation in the M2 protein indicating that these H5N8 viruses may reduce susceptibility to adamantanes and rimantadine  from waterfowl in Southern China between 2013 and 2014. Our results demonstrated that the PB2 gene of these viruses had the highest identity with A/wild duck/Shangdong/628/2011 (H5N1); their NP genes were both more closed to A/wild duck/Shandong/1/2011 (H5N1) and A/duck/Jiangsu/k1203/2010 (H5N8); the other genes were more close to A/duck/Jiangsu/k1203/2010 (H5N8). The HA gene of these viruses were both clustered into clade 2.3.4.4 . The NA Even though massive slaughter of poultry and vaccination strategies were used to prevent and control the H5 HPAI viruses in China, the H5 HPAI viruses still circulated continuously in waterfowl in Southern China and frequently provided gene segments to generate new strains . BetweenIn summary, our results showed that the new H5N8 HPAI viruses may come from reassortant between the A/duck/Jiangsu/k1203/2010 (H5N8) virus and other H5N1 viruses, and their HA genes belonged to the same GsGd H5 clade 2.3.4.4. Therefore, this study is useful for better understanding the genetic and antigenic evolution of H5 AIVs in Southern China.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.