The matrix metalloproteinase 14 (MMP14) is identified as a new target gene of miR-150-5p. miR-150-5p markedly inhibits MMP14 expression in hepatoma cells, and miR-150-5p expression is negative correlation with MMP14 expression invivo. More important, re-expression of MMP14 in hepatoma cells partially reverses the effect of miR-150-5p in inhibiting cell invasion.Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide. Despite progress in diagnostics and treatment of HCC, its prognosis remains poor because the molecular mechanisms underlying hepatocarcinogenesis are not well understood. In the study, we focused on identifying the role of miRNAs in HCC progression. miRNA microarray was used to analyze the differentially expressed miRNAs, and the results were validated by qPCR. We found that the miR-150-5p expression is down-regulated in HCC tissues compared with pair non-tumor tissues. miR-150-5p expression is also decreased in metastatic cancer tissues compared with pair primary tissues, indicating that miR-150-5p may be involved in HCC metastasis. Functionally, miR-150-5p inhibition significantly promotes hepatoma cell migration and invasion, whereas miR-150-5p overexpression suppresses cancer cell migration and invasion  Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and the incidence is increasing in the East Asia and in developed countries MicroRNAs (miRNAs) are a recently discovered class of small noncoding RNAs that regulate gene expression at the post-transcription level. miRNAs have been highly conserved among phylogenetically close species during evolution and have emerged recently as potent regulators of cell growth, cell differentiation and carcinogenesis Recent studies showed that miR-150 is abnormally expressed in different type of cancer. Ma et al. found that miR-150 expression is down-regulated in colorectal cancer compared with paired non-cancerous tissue Based on these findings, we investigated whether miR-150-5p involves in HCC progression. We found that decreased expression of miR-150-5p relieves repression of MMP14, which results in an increase of cell migration and invasion.Tumor tissues were obtained with written informed consent from the Ruijin Hospital affiliated to Shanghai Jiaotong University. The study was approved by the Ethics Committee of Shanghai Jiaotong University. Patients with HCC (n = 53) were enrolled in our study , and canTotal RNA was extracted from tissues or cell lines using Trizol reagent , and reverse transcription (RT) reactions were performed using miR-150-5p-special prime. The specific stem–loop RT primers for miR-150-5p were designed as previously described Human miR-150-5p (UCUCCCAACCCUUGUACCAGUG) or 2′-O-methyl modified miR-150 inhibitor (CACUGGUACAAGGGUUGGGAGA) were transfected to hepatoma cells by using Lipofectamine 2000 (Invitrogen) according to the manufacturer.Western blot analysis to assess MMP14 and β-actin expression was performed as previously described 6 cells/well) were treated with indicated reagents, and wounds were made using a 1000 µl plastic pipette tip. The size of wound was measured after 48 h of wound formation and photographed.HCC cells (1–2×10Cell invasion assay was carried out using Transwell chambers with inserts of 8-µm pore size (Corning Costar) as described previously 5 cells/well) were plated in a 24-well plate and cotransfected with 40nM of either miR-150 or miRNA control, 20 ng of either pGL3-MMP14-3′-UTR or pGL3-MMP14-3′-UTR-Mutation, and 2 ng of pRL-TK  by using Lipofectamine 2000. The pRL-TK vector was used as an internal control to correct the differences in both transfection and harvest efficiencies. HepG2 cells were collected 48 h after transfection and analyzed using the Dual-Luciferase Reporter Assay System (Promega).pGL3 plasmid encoding a luciferase reporter gene was purchased from Promega . Recombinant plasmid of pGL3-MMP14-3′-UTR (wildtype) or pGL3-MMP14-3′-UTR-Mutation was constructed in our laboratory. HepG2 cells (1–2×10The athymic BALB/C mice (4–6 weeks old) were purchased from the Chinese Academy of Sciences , and were bred in a specific pathogen-free facility. The protocol was approved by the Animal Ethics Committee of Shanghai Jiaotong University. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Huh7 cells overexpressed with miR-150-5p were subcutaneously injected into the flanks of nude mice, and Huh7 cells were used as mock control. After 6 weeks, the mice were killed and lung metastases were monitored after inducing deep anesthesia.t test. Differences were deemed statistically significant at p<0.05.Data are expressed as mean ± standard deviation (SD) from at least three separate experiments. The differences between groups were analyzed using Student’s To study the roles of miRNAs in disease progression of HCC, we searched GEO database and a miRNA microarray datasets (GSE54751) was obtained to analyze differentially expressed miRNAs between HCC tissues and pair non-tumor tissues. miRNA microarray data revealed that miR-150-5p expression is lower in HCC tissues compared with adjacent normal tissues .. To conThe frequent down-regulation of miR-150-5p in HCC tissues implies that miR-150-5p may play important roles in HCC progression. The biological consequences of miR-150-5p knockdown in regulating cancer cell migration and invasion were then examined using cell biology assays. miR-150-5p expression is significantly decreased in Huh7 or HepG2 cells after treatment with miR-150-5p inhibitor , and miRMMP14 gene, an important regulator involving in tumor progression  are zinc-dependent endopeptidases that degrade extracellular macromolecules  and are known to play important roles in angiogenesis, cancer cell invasion, and cancer metastasis In vivo, a significant negative correlation is also observed between miR-150-5p and MMP14 expression. Functionally, miR-150-5p inhibits hepatoma cell invasion, whereas re-expression of MMP14 partially reverses the effect of miR-150-5p in inhibiting cell invasion.Here we identified that MMP14 is a novel target gene of miR-150-5p. Bioinformatics analysis showed that miR-150-5p targets 3′UTR of MMP14. The reporter assay showed that miR-150-5p markedly suppresses luciferase expression, whereas mutation of 4 nucleotides in 3′-UTR of MMP14 results in complete abrogation the suppressive effect. Overexpression of miR-150-5p decreases the MMP14 protein level in Huh7 and HepG2 cells. miR-150-5p is an important mediator of HCC metastasis, thus offering a new target for the development of therapeutic agents against HCC.S1 FigmiR-150-5p expression is down-regulated in HCC tissues. miR-150-5p expression analysis showed in miRNA microarray datasets (GSE54751).(TIF)Click here for additional data file.


Caenorhabditis elegans to modify their chemosensory behavior, but the mechanisms essential for these expression changes remain poorly characterized. We had previously shown that expression of a feeding state-dependent chemoreceptor gene, srh-234, in the ADL sensory neuron of C. elegans is regulated via the MEF-2 transcription factor. Here, we show that MEF-2 acts together with basic helix-loop-helix (bHLH) transcription factors to regulate srh-234 expression as a function of feeding state. We identify a cis-regulatory MEF2 binding site that is necessary and sufficient for the starvation-induced down regulation of srh-234 expression, while an E-box site known to bind bHLH factors is required to drive srh-234 expression in ADL. We show that HLH-2 (E/Daughterless), HLH-3 and HLH-4 (Achaete-scute homologs) act in ADL neurons to regulate srh-234 expression. We further demonstrate that the expression levels of srh-234 in ADL neurons are regulated remotely by MXL-3 (Max-like 3 homolog) and HLH-30 (TFEB ortholog) acting in the intestine, which is dependent on insulin signaling functioning specifically in ADL neurons. We also show that this intestine-to-neuron feeding-state regulation of srh-234 involves a subset of insulin-like peptides. These results combined suggest that chemoreceptor gene expression is regulated by both cell-autonomous and non-cell-autonomous transcriptional mechanisms mediated by MEF2 and bHLH factors, which may allow animals to fine-tune their chemosensory responses in response to changes in their feeding state.Food and feeding-state dependent changes in chemoreceptor gene expression may allow  Caenorhabditis elegans that act together in ADL sensory neurons to properly regulate expression of a feeding-state dependent chemoreceptor gene. We also showed that chemoreceptor gene expression in ADL neurons are regulated remotely by bHLH factors acting in the intestine through an insulin-mediated signaling pathway, implying a sensory neuron-gut interaction for modulating chemoreceptor gene expression as a function of feeding state. This work describes transcriptional mechanisms mediated by MEF-2 and bHLH factors by which the expression of individual chemoreceptor genes in C. elegans are changed in response to changes in feeding state conditions.Plasticity in chemoreceptor gene expression may be a simple strategy by which an animal can modulate its chemosensory responses in changing external and internal state conditions. However, the transcriptional mechanisms required for these chemoreceptor gene expression changes are poorly understood. Here, we describe the identification of a transcriptional module(s) consisting of MEF-2 and basic helix-loop-helix (bHLH) transcription factors and their cognate binding sites in  Animals modify their chemosensory behavior depending on their feeding-state, which allows them, for instance, to optimize their food-search strategy . A simplCaenorhabditis elegans provides an ideal system to identify transcriptional mechanisms underlying chemoreceptor gene regulation in specific chemosensory neurons, and has provided insight into the cis- and trans-regulatory logic for chemoreceptor gene expression. For example, the transcription factor HMBX-1, a C. elegans homolog of HMBOX1, regulates the srsx-3 chemoreceptor gene , HT1690 unc-119(ed3); wwIs26, HT1693 unc-119(ed3); wwExEx63, HT1695 unc-119(ed3); wwEx64, PY4108 mef-2(gv1); Ex, TU3596 sid-1(pk3321)him-5(e1490); lin-15B(n744) and NL2099 rrf-3(pk1426). Transgenic strains used in this study were: VDL3 oyIs56, and daf-2(e1307); oyIs56; Ex[sre-1p::daf-2 cDNA]  and VL507 unc-119(ed3); wwIs20 [Worms were grown at 20°C under standard conditions on nematode growth medium (NGM) with escribed , 57. Thegfp reporters were grown at 20°C on NGM plates seeded with E. coli OP50 as the bacterial food source. Young adult animals were washed with M9 buffer (to remove any bacteria in the gut) and transferred onto plates with E. coli OP50 bacterial food (fed) or without food for 12–24 hours (starved) unless indicated otherwise. Levels of promoter::gfp expression  were imaged and measured under a microscope equipped with epifluorescence as previously described [gfp construct, expression from the same extra chromosomal array was examined in wild-type and mutants. Briefly, we mounted animals on agarose pads containing 3 mM sodium azide unless indicated otherwise, and visualized these on a Leica DM5500 compound microscope or a Leica TCS SP8 confocal equipped with epifluorescence. Microscope and camera settings were kept constant between images of different genotypes and conditions, unless indicated otherwise. The mean pixel intensity of gfp fluorescence in the entire cell-body of ADL was quantified using Volocity software. Prior to measurement, images of ADL were cropped for srh-234 expression analysis. Statistical analyses of srh-234 expression were performed using the non-parametric two-tail Mann-Whitney test.Transgenic animals carrying promoter::escribed . For eachlh-2, hlh-3, mxl-3, or cDNA of hlh-30 (this study), and cloned in a pSM SL2::mCherry (or gfp)::unc-54 3’UTR vector (a kind gift from Cori Bargmann) using cell- and tissue-specific promoters. This resulted in the generation of the expression constructs: pMG25 sre-1p(1 kb)::mxl-3 genomic::SL2::mCherry (ADL::mxl-3), pMG28 ges-1p(1.9 kb)::mxl-3 genomic::SL2::mCherry (intestine::mxl-3), pMG47 mxl-3p(2.1 kb)::mxl-3 genomic::SL2::mCherry (mxl-3p::mxl-3), hlh-30p(2.2 kb)::hlh-30::SL2::mCherry (hlh-30p::hlh-30), pMG65 ges-1p(1.9 kb)::hlh-30 cDNA (intestine::hlh-30), pMG72 sre-1p(1 kb)::hlh-30 cDNA (ADL::hlh-30), pMG30 ges-1p(1.9 kb)::hlh-2 genomic::SL2::mCherry (intestine::hlh-2), pMG73 svh-1p(1.8 kb)::hlh-2 genomic::SL2::mCherry (ADL::hlh-2), pMG71 hlh-3p(1.5 kb)::hlh-3 genomic::SL2::gfp (hlh-3p::hlh-3::SL2::gfp), pJG17 sre-1p(1 kb)::daf-16a::gfp ::mxl-3 genomic::SL2::mCherry (AWC::mxl-3). The construct ges-1p::mxl-3IR (plasmid 249/250) used for knocking down the mxl-3 gene specifically in the intestine via RNAi (intestine::mxl-3 RNAi) is a kind gift from Eyleen O’Rourke [sri-51p(3 kb)::mCherry was a kind gift from Cori Bargmann [Expression vectors were generated by amplifying either the wild-type genomic sequences of O’Rourke , and theBargmann .hlh-10p::gfp and svh-1p::gfp reporters, we fused about 1.5 kb and 1.6 kb promoter sequence of hlh-10 and svh-1, respectively, to gfp as previously described [sre-1p::gfp construct was generated by inserting about 1 kb promoter sequence of sre-1 into the pPD95.77 vector (AddGene). Transgenic animals were generated using either the unc-122p::dsRed (50–100 ng/μl) or the pRF4 rol-6(su1006) co-injection markers injected at 150 ng/μl using a standard microinjection protocol [For generating the escribed . The sreprotocol . ExpressbHLH genes, we crossed either transgenic lines of hlh-2::mCherry::his-11 (strain VL507) [hlh-4p::gfp (strain UL1583) [hlh-3p(1.5 kb)::hlh-3 genomic::SL2::gfp (this study) with transgenic lines of the relevant ADL-specific transgenic reporter lines, either sri-51p(3 kb)::gfp or srh-234p(165 bp)::gfp,For co-expression analysis of n VL507) , hlh-4p: UL1583) , and hlhsrh-234 for transcription factor binding sites using Lasagna 2.0 (http://biogrid-lasagna.engr.uconn.edu/lasagna_search/) [srh-234p::gfp reporter was generated to create site-directed mutations in the candidate binding sites. For this, we cloned 165 bp upstream sequence of the predicted srh-234 translational start codon into the HindIII and BamHI sites of the pPD95.77 vector (Addgene). The resulting srh-234p(WT)::gfp construct was used to perform site-directed mutations in the E-box and MEF2 binding sites with help of the QuickChange II site-directed mutagenesis kit (Agilent Technologies) to generate the following promoter::gfp constructs: srh-234p(-E-box)::gfp, srh-234p(-E-box L)::gfp, srh-234p(-E-box R)::gfp, srh-234p(-MEF2)::gfp, and srh-234p(-E-box/MEF2)::gfp. For generating the sre-1p(+MEF2)::gfp construct, we inserted the extended srh-234 MEF2 site (AATAGTTATATTTAAAAA) at position -100 bp into 1.1 kb promoter sequence of sre-1 using the Q5 site-directed mutagenesis kit (New England Biolabs). The same mutagenesis kit was used to generate the odr-10p(+E-box)::gfp construct by inserting the extended srh-234 E-box motif (AATCACCTGTCC) into the odr-10 promoter sequence at position -70 bp. All created site-directed mutations were confirmed by sequencing. Transgenic animals of promoter::gfp constructs were generated in wild-type animals using a standard microinjection protocol [gfp constructs were injected at 30 ng/μl together with either the pRF4 rol-6(su1006) or unc-122p::dsRed as co-injection markers at 150 ng/μl.Searching the 165 bp upstream sequence of search/)  identifisearch/) . A srh-2protocol . Promote2, glycerol and SDS, we used the drop-test assay as previously described [hlh-2(tm1768) or hlh-3(ot354) mutants about 20 hours before the assay to NGM plates with (on-food) or without food (off-food). For on-food plates, 25 μl of overnight culture of E. coli OP50 in LB was spread on each plate. For off-food plates, 25 μl of LB was spread on each plate. All plates were allowed to dry for 1 hour without lids. Animals were picked using an eyelash pick to a plate without food for less than a min (to prevent food being transferred), before animals were transferred to on-food and off-food assay plates. Animals were allowed to settle for 10 min, and then assayed using a capillary to deliver the repellent drop. About 5 animals were assayed on each plate at room temperature, all animals were stimulated every 60 seconds and the percentage of responding animals were recorded. The avoidance index is the number of positive responses divided by the total number of trials. The tested repellents were 2 mM CuCl2 (Acros Organics), 0.5 M glycerol (Sigma) and 0.1% SDS (Thermo Scientific). All repellents were dissolved and diluted in M13 buffer .For examining avoidance responses to CuClescribed ,61. Briehlh-2(tm1768) or hlh-3(ot354) mutant animal. The time to respond (seconds) is the amount of time it took for the animal to initiate backward movement at room temperature. Octanol (Sigma) was diluted and freshly made in ethanol.For examining the avoidance response to octanol, we used the smell-on-a-stick assay as previously described ,63. BriebHLH genes was performed in the rrf-3(ok1426) background to enhance neuronal RNAi [rrf-3(pk1426) mutant animals carrying a stably integrated srh-234p::gfp transgene (oyIs56) were placed on an NGM agar plate containing 1 mM IPTG and seeded with E. coli HT115 bacterial food producing dsRNA directed against the selected bHLH genes. Bacteria expressing dsRNAs directed against selected bHLH genes were taken from the C. elegans feeding RNAi library (Source BioScience). For hlh-2(RNAi) and hlh-4(RNAi), we cloned the wild-type genomic sequence of hlh-2 and hlh-4 in the L4440 vector (Addgene) because these bacterial RNAi clones in the feeding RNAi library were incorrect. For fed conditions, animals fed with bHLH(RNAi) were scored as adults in the next generation (F1), and expression of srh-234p::gfp was measured and quantified as described above. For starved conditions, animals in the F1 generation were transferred as L4-staged larvae to RNAi plates without food and srh-234p::gfp expression was determined about 12 hours later. The L4440 empty vector was used as the RNAi control. All RNAi bacterial clones used were cultured as previously described [Feeding RNAi of selected nal RNAi . Brieflyescribed , and conbHLH and ILP genes, we employed a previously described feeding RNAi strategy [sid-1 in the pSM SL2::mCherry::unc-54 3’UTR vector (a kind gift from Cori Bargmann) using the ADL-specific promoter, sre-1, and the intestinal-specific promoter, ges-1, resulting in constructs: pMG57 sre-1p::sid-1 genomic::SL2::mCherry (ADL::sid-1), and pMG66 ges-1p::sid-1 genomic::SL2::mCherry (intestine::sid-1). Transgenic animals were generated in sid-1(pk3321) mutant animals carrying the stably integrated srh-234p::gfp transgene (oyIs56) using a standard microinjection protocol [rol-6(su1006) as a co-injection marker at 150 ng/μl. To selectively enhance RNAi in either ADL neurons or in the intestine (but not in other tissues), L4-staged larvae of transgenic animals carrying either ADL::sid-1 or intestine::sid-1 were placed on an NGM agar plate containing 1 mM IPTG and freshly seeded with E. coli HT115 bacterial food producing dsRNA directed against individual bHLH genes or ILP genes as previously described [srh-234p::gfp was measured and quantified in transgenic animals fed with either bHLH(RNAi) or ILP(RNAi) in the next generation (F1) at the adult stage unless indicated otherwise. The L4440 empty vector was used as the RNAi control, and all RNAi bacterial clones used were confirmed by sequencing.For tissue-selective knockdown of strategy . Brieflyprotocol . Expressescribed . Expresssre-1p::daf-16a::gfp reporter as an extra chromosomal array  or without food for 12 hours (starved). Next, animals were mounted on glass slides containing 1 mM levamisol to paralyze the animals, and visualized as described above. Fluorescent images were taken at the same exposure time using Volocity software. The fraction of DAF-16::GFP in the subcellular compartment of ADL neurons was measured by counting the fraction of animals that show nuclear fluorescence in either the red (cytoplasm > nuclear), orange (nucleus = cytoplasm), or yellow color (nucleus > cytoplasm). Statistical analyses of the distribution between genotypes and conditions was performed using the χ2 test.For measuring the fraction of DAF-16::GFP in the compartment (cytoplasmic:nuclear) of ADL, we generated transgenic animals that carry both a srh-234 and odr-10 coding sequences. Primer sequences for srh-234 are 5′-GGACAATTGAAATGCAACACA-3′ and 5′-GACGGGGACAATAAAGAGCA-3′. Primer sequences for odr-10 are 5′-GAGAATTGTGGATTACCCTAG-3′ and 5′-CTCAATATGCATTATAGGTCGTAATATG-3′.As described previously , total RE. coli OP50 bacterial food before examination of ADL sensory neurons.Dye-filling experiments were performed similarly as previously described . A stockS1 Fig(A) The indicated length and position of the inserted E-box motif of srh-234 relative to the translational start site of odr-10 fused to gfp in expression vectors (see (B) Representative expression of AWA and ADL sensory neurons driven by the odr-10p(+E-box)::gfp transgene in well-fed adults. Anterior is left. Ventral view of images were acquired at the same exposure time at room temperature. (C) Relative expression of odr-10p(+E-box)::gfp in adult hlh-2 mutants. Data shown is for one transgenic line with n>20 adult animals. ** indicates values that are different from that of wild-type adult animals at P<0.05. Error bars denote the SEM.tors see . (B) Rep(EPS)Click here for additional data file.S2 Fig(A) Relative expression of srh-234p::gfp in the ADL cell body of adults of the indicated genotypes during well-fed and starved conditions. >20 adult animals for each genotype and condition. (B) Relative expression of srh-234p::gfp in adult animals fed with RNAi directed against bHLH genes. Feeding RNAi was performed in rrf-3(pk1426) RNAi hypersensitive mutant animals (see (C) Data shown is the ratio of endogenous srh-234 message to endogenous odr-10 message as quantified by qRT–PCR in adult wild-type and mxl-3 mutants when fed or starved. Expression of odr-10 is unaffected by starvation in hermaphrodites. The mean of the ratios from two independent experiments is shown. * indicates values that are different from fed wild-type animals at P<0.01 using a two-sample t-test.  Percentage of adult mutant animals (D) and RNAi-treated animals (E) of the indicated genotypes showing normal ADL dye-filling compared to wild-type  Summary of the main bHLH factors that regulate srh-234 expression without altering ADL neuron identity. Proteins in parentheses denote minor bHLH factors that negatively or positively regulate srh-234 expression.mals see . (C) Dattype see . n>20 ad(EPS)Click here for additional data file.S3 Fig(A) Avoidance responses of adult wild-type, hlh-2 and hlh-3 mutants to CuCl2, glycerol and SDS at the indicated concentrations. Drop-test assays were performed at room temperature (see P<0.05. (B) Response  time of adult wild-type, hlh-2 and hlh-3 mutants to either 100% or 30% 1-octanol. Smell-on-stick assays were performed at room temperature (see ture see . n>70 trture see . n>20 tr(EPS)Click here for additional data file.S4 Fig(A) The indicated lengths and positions of predicted regulatory elements relative to the translational start site of sre-1 fused to gfp in expression vectors (see sre-1. (B) Representative expression of ADL sensory neurons driven by wild-type sre-1 regulatory sequences in adult wild-type and mutant animals of the indicated bHLH genes in well-fed conditions. Images are lateral views of ADL sensory neurons with the arrow pointing to the cell body; anterior is left. Images were acquired at the same exposure time at room temperature. The same sre-1p::gfp transgene was examined in adult wild-type and mutant animals.  Relative expression of wild-type sre-1p::gfp(C) and sre-1p(+MEF2)::gfp(D) in mutants of bHLH genes compared to wild-type adults when fed or starved. n>25 adult animals for each genotype. Error bars denote the SEM.tors see . The seq(EPS)Click here for additional data file.S5 Fig(A) The indicated length and position of a predicted endogenous E-box motif of svh-1 relative to the translational start site of svh-1 fused to gfp in expression vectors (see (B) Representative expression of ADL sensory neurons (arrow) driven by the svh-1p::gfp transgene in well-fed adult animals. The same svh-1p::gfp transgene was examined in adult wild-type and mutant animals. Anterior is left. Ventral view of images was acquired at the same exposure time and at room temperature.tors see . (B) Rep(EPS)Click here for additional data file.S6 Fig(A-C) Expression of hlh-2(A)hlh-3(B) or hlh-10(C) with either sri-51p::mCherry or srh-234p::gfp as ADL-specific reporters in larvae and embryos. ADL neurons are indicated with arrows. When co-expressed, ADL neurons are shown as a yellow cell in a merged fluorescent image. For animals carrying the hlh-2p::mCherry::his-11 transgene expressing hlh-2 in the nucleus ADL appears with a green cytoplasm and yellow nucleus in a merged fluorescent image. int, intestine; ph, pharynx.(EPS)Click here for additional data file.S7 Fig(A) Overview of tissue-specific knockdown of bHLH genes by RNAi. (B-C) Representative expression of srh-234p::gfp in adult animals by feeding either hlh-2(RNAi), hlh-3(RNAi), hlh-4(RNAi), mxl-3(RNAi) or control RNAi. Feeding RNAi was performed in sid-1(pk3321)him-5(e1409) mutants with the stably integrated oyIs56 transgene carrying either a ADL::sid-1(B) or intestine::sid-1(C) extra chromosomal transgene to selectively enhance RNAi in ADL neurons or in the intestine, respectively, but not in other tissues (see (D-E) Relative expression of srh-234p::gfp in the ADL cell body of adult animals when fed either hlh-2(RNAi), hlh-3(RNAi), hlh-4(RNAi), or mxl-3(RNAi) compared to adult animals fed with control RNAi. Data shown is the average of at least two independent transgenic lines with n>20 adult animals for each line. * indicates values that are different from that of wild-type adult animals at P<0.001, and # between the genotypes compared by brackets at P<0.001. Error bars denote the SEM.sues see . Images (EPS)Click here for additional data file.S8 Fig(A) Representative expression of mef-2::mef-2::gfp in adult wild-type animals when fed and starved (upper panels), as well as adult daf-2 and mxl-3 mutants (lower panels). (B) Representative expression of hlh-2::mCherry::his-11 in adult wild-type animals when fed and starved (upper panels) and adult daf-2 and daf-16 mutants (lower panels). In all experiments, images were taken at the same exposure time at room temperature. Anterior is right. The same transgene was examined in each genotype and condition.(EPS)Click here for additional data file.S9 Fig(A) Relative expression of srh-234p::gfp in the ADL cell body of adult animals by feeding individual ILP(RNAi) compared to adult animals fed with control RNAi. Feeding RNAi was performed in sid-1(pk3321)him-5(e1409) mutants with the stably integrated oyIs56 transgene carrying the intestine::sid-1 extra chromosomal transgene to selectively enhance RNAi in the intestine, but not in other tissues  Relative expression of srh-234p::gfp in ADL neurons of adult daf-28, ins-26 and mxl-3 mutants (ILPs) in well-fed conditions. n>25 adult animals for the indicated genotypes. Error bars denote the SEM. (C) Images are lateral views of ADL with the arrow pointing to the cell body. Anterior is left. Images were acquired at the same exposure time at room temperature. (D) Summary of the main insulin-like peptides (ILPs) that regulate srh-234 expression in ADL. Proteins in parentheses denote minor ILPs that negatively or positively regulate srh-234 expression.sues see . Data sh(EPS)Click here for additional data file.S10 Fig(A-C) Representative expression of selected ILP genes ins-3p::gfp(A), ins-4p::gfp and ins-5p::gfp(B) and ins-26p::gfp(C) in adult wild-type (left panels) and mxl-3 mutant animals (right panels) during fed conditions. White box represents intestinal area used for comparative analysis of expression. In all experiments, images were taken at the same exposure time at room temperature. The same transgene was examined in each genotype. Arrow indicates ASE neuron.(EPS)Click here for additional data file.S1 Tablehttp://biogrid-lasagna.engr.uconn.edu/lasagna_search/) with a cut-off value of P<0.01.A search of possible MXL-3 and HLH-3 binding sites present in promoter sequences of insulin-like peptide genes using Lasagna 2.0 ((XLSX)Click here for additional data file.


Escherichia coli sequence type 131 (ST131) strain JJ1887. The strain was isolated in 2007 in the United States from a patient with recurrent cystitis, whose caregiver sister died from urosepsis caused by a nearly identical strain.We report here the complete genome sequence, including five plasmid sequences, of  Escherichia coli sequence type 131 (ST131) is currently the most prevalent extraintestinal E. coli lineage (H30 sublineage accounts for most E. coli isolates that are fluoroquinolone resistant and produce extended-spectrum β-lactamases (ESBLs) , from the ST131-H30Rx sublineage (E. coli strain (JJ1887), which is also from ST131-H30Rx and nearly identical to JJ1886. Whether the sisters’ distinct clinical outcomes are attributable to host response differences, strain-specific features, or other factors, the JJ1887 genome represents a valuable added resource for understanding the pathogenicity of the ST131-H30Rx lineage. (ESBLs) . Carriagrosepsis –7. We setrain JJ186, from ) Portal, which produced a circular chromosome and five closed plasmids, with a mean coverage of 118×. The short-read mapping was further analyzed with Delly (http://TGenNorth.github.io/NASP). The verified sequences were annotated using Prokka 1.10 (JJ1887 was sequenced on the PacBio platform (P5-C3 chemistry), which generated 66,442 raw PacBio reads . The PacBio sequence reads were manually error corrected using Illumina short reads  using CLC Genomics Workbench version 7. The resultant data were assembled using the Hierarchical Genome Assembly Process (HGAP) version 3 in the PacBio single-molecule real-time , 89 tRNAs, 22 rRNA features, and 7 intact prophage regions, one of which (57.4 kb) harbors blaTEM-1B, blaOXA-1 in pJJ1887-4 and blaTEM-1B in pJJ1887-5 confer resistance to β-lactams, and the aac(6′)-Ib-cr gene in pJJ1887-4 codes for fluoroquinolone resistance. pJJ1887-5 also carries genes conferring resistance to aminoglycosides [aac-(3)-IIa], macrolides [mph(A)], tetracyclines [tet(B)], trimethoprim (dfrB4), sulfonamides (sul1), and fluoroquinolones (qepA).JJ1887 contains five plasmids, of which pJJ1887-1, -2, and -3 are identical to plasmids in JJ1886 , pJJ1887E. coli JJ1887 have been deposited in GenBank under accession numbers CP014316 to CP014321.The complete sequences of the chromosome and plasmids of


Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) 6-oxopurine phosphoribosyltransferases (PRTs) propane-1,3-diyl)bis(oxy)]bis (me thy-lene)} diphosphonic acid exhibited a KThe crystal structure of {[(2-[(Guanine-9Hyl)methyl] propane-1,3-diyl)bis(oxy)]bis(methylene)} diphosphonic acid in complex with human HGPRT provides a template for chemical modifications to increase both potency and selectivity for the parasite enzymes.


In severe sepsis (SS) or trauma-related systemic inflammatory response syndrome (SIRS), induction of heat-shock-protein-72 (HSP72) may protect cells from stress.We compared the heat-shock (HS) with the lipopolysaccharide (LPS) induction/repression effect on HSP72 of peripheral blood mononuclear cells (PBMCs) in SS or SIRS patients and healthy-controls (H) and investigated any possible modulating glutamine (Gln) effect.o HS vs. no stimulation for 4h. In each group 3 experiments involved L-Ala-Gln10mM incubation 1h before (Gln-b) or after (Gln-a) induction, or no glutamine (1088 measurements). Intracellular Mean Fluorescence Intensity (MFI) levels of monocytes (mHSP72) or lymphocytes (lHSP72) were determined using Flow Cytometry.PBMCs from 16/H, 11/SS, and 7/SIRS were incubated with 1µg/ml LPS or 43In H-PBMCs, LPS did not affect mHSP72 (79±10 MFI vs. 78±13) or lHSP72 (7±1.7 vs. 7±2). HS induced mHSP72  and lHSP72  with or without Gln (p< 0.0001). Basal mHSP72 was higher in SIRS compared to H . A HS-induction effect on SIRS-mHSP72  and lHSP72  was further enhanced by Gln-b . LPS suppressed SIRS-mHSP72  especially in the Gln-b group . Basal Gln-b mHSP72 in SS was higher compared to H . In SS-PBMCs HS, but not LPS, induced mHSP72 . LPS repressed the SS-lHSP72  an effect attenuated by Gln-b (13±5).Heat shock greatly induces mHSP72 and lHSP72 of ICU patients' PBMCs. LPS may repress lHSP72 in septic or trauma patients. Glutamine pre-treatment may either enhance HS-induction or LPS-repression on mHSP72 or attenuate LPS-repression on lHSP72 in SS and SIRS groups.


L support survival following PI3K pathway inhibition, and that the dual PI3K/mTOR inhibitor BEZ235 strongly synergizes with BCL-2 antagonists in DLBCL. However, we identify an alternative mechanism of synergy between PI3K/mTOR and BCL-2 inhibitors, independent of MCL-1 down-regulation. Instead, we show that suppression of AKT activation by BEZ235 can induce the mitochondrial accumulation of pro-apoptotic BAD and BIM, and that expression of a constitutively active form of AKT prevents sensitization to BCL-2 antagonism. Thus, our work identifies an additional mechanism of synergy between PI3K pathway inhibitors and BCL-2 antagonists that strengthens the rationale for testing this combination in DLBCL.The PI3K/AKT/mTOR axis promotes survival and is a frequently mutated pathway in cancer. Yet, inhibitors targeting this pathway are insufficient to induce cancer cell death as single agents in some contexts, including diffuse large B cell lymphoma (DLBCL). In these situations, combinations with inhibitors targeting BCL-2 survival proteins (ABT-199 and ABT-263) may hold potential. Indeed, studies have demonstrated marked synergy in contexts where PI3K/mTOR inhibitors suppress expression of the pro-survival protein, MCL-1. In this study, we use BH3 profiling to confirm that BCL-2 and BCL-X PI3K inhibitors have recently received increased attention in blood cancers, where an inhibitor of the p110δ catalytic isoform  elicits significant patient responses in both chronic lymphocytic leukemia (CLL) and indolent non-Hodgkin's lymphoma , highligThe potential of simultaneously inhibiting the PI3K pathway and BCL-2 survival proteins has been previously demonstrated in several contexts -12. In DL for survival in the absence of PI3K pathway activity. In accord, our data show that the combination of PI3K pathway and BCL-2 inhibitors synergistically induce apoptosis in a panel of GCB-DLBCL cell lines, with dual PI3K/mTOR inhibition providing the greatest effect. Importantly, we also demonstrate synergy between PI3K/mTOR and BCL-2 inhibition in chemo-resistant DLBCL lines over-expressing BCL-2, but show that this combination lacked toxicity in normal T lymphocytes. Contrary to other tumor cell contexts, MCL-1 expression in GCB-DLBCL cells did not decrease following PI3K/mTOR inhibition. Instead, treatment with dual PI3K/mTOR inhibitors resulted in a significant accumulation of the pro-apoptotic factors BAD and BIM at the mitochondria. These effects were dependent on suppression of AKT activity, as a constitutively active mutant of AKT opposed the synergy between PI3K pathway inhibitors and BCL-2 antagonists. These findings identify a promising combination approach to achieve selective GCB-DLBCL death, and highlight a previously unpredicted mechanism of synergy.To gain insight into survival signaling in GCB-DLBCL, we used BH3 profiling to identify any changes in the relative balance of BCL-2 family proteins following PI3K pathway inhibition . This anTo evaluate the impact of PI3K pathway inhibitors as single agents, we used several classes of chemical inhibitors targeting distinct nodes in the PI3K/AKT/mTOR axis. For each class of inhibitor, we compared the effects of two chemically distinct compounds to limit the contribution of off-target effects Table . Using tDespite lacking single-agent cytotoxicity, PI3K pathway inhibitors can synergize with BCL-2 antagonists by priming cells for undergoing apoptosis -13. ThusL) to maintain survival following PI3K pathway inhibition. Consistent with this interpretation, previous studies have shown that increased sensitivity to the BAD peptide correlates with higher efficacy of the dual BCL-2/BCL-XL antagonist, ABT-737 . Wh. Wh50]. Despite showing promising clinical efficacy in some blood cancers , PI3K/mTIt is surprising that unlike in other contexts -12, suppContrary to previous reports, in this study we describe an alternative mechanism of synergy between PI3K/mTOR inhibitors and BCL-2 antagonists in GCB-DLBCL, which involves the mitochondrial accumulation of BAD and BIM. We identify that this effect stems from suppression of AKT, as inducible expression of constitutively active AKT prevented accumulation of BAD and BIM and abolished the synergy. While these particular survival outputs from AKT have been described previously , 17, theIn conclusion, while the mechanism may differ depending on the context, the combination of PI3K/AKT/mTOR inhibitors and BCL-2 antagonists strongly synergizes to kill DLBCL cells. In fact, the existence of multiple mechanisms of synergy may prove beneficial in combating tumor heterogeneity and preventing acquired resistance in a clinical setting. In addition, our results define alternative markers of response and identify situations in which tracking MCL-1 expression may not be predictive of patient responses. Thus, the results of this, and other, studies provide a strengthened rationale for testing dual PI3K/mTOR inhibitors with BCL-2 inhibitors in GCB-DLBCL patients.We obtained rapamycin, MLN0128, GDC-0941, and NVP-BEZ235 from LC Laboratories ; ABT-263, ABT-199, MK2206 and GDC-0980 from Active Biochem , and AKT inhibitor VIII from Chemdea . InSolution Q-VD-OPh was obtained from EMD Millipore , vincristine was obtained from Sigma-Aldrich , dimethyl sulfoxide (DMSO) from Fisher Scientific  and doxycycline from Sigma-Aldrich .6 cells/mL. Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco's Modified Eagle Medium  supplemented with 10% calf serum, 100 I.U. penicillin, and 100 μg/mL streptomycin. Human peripheral blood mononuclear cells (PBMCs) were isolated from blood samples by centrifugation through Ficoll-Paque™  and were grown in RPMI  with 10% FBS, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 mM L-Glutamine, 100 I.U. penicillin, 100 μg/ml streptomycin and 55 μM BME.OCI-LY1, OCI-LY7, OCI-LY8, and SU-DHL4 cell lines  were cultured in IMDM  supplemented with 10% FBS, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 mM L-Glutamine, 100 I.U. penicillin, and 100 μg/ml streptomycin. Cells were grown in a humidified 37°C incubator with 5% CO2. Cells were routinely tested to ensure absence of mycoplasma, and were maintained at or below 2 × 10® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0, 2 mM EDTA, 50 mM NaF) supplemented with protease inhibitor cocktail  and phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich). Protein concentrations were normalized using a Bradford protein assay (Bio-Rad). Lysates were prepared at 1 μg/μl concentration in 1X XT Sample Buffer (Bio-Rad) and 5% 2-mercaptoethanol (Sigma-Aldrich). Lysates were run on 4-12% Bolt® Bis-Tris Plus gels (Life Technologies), and transferred onto nitrocellulose membranes. The following antibodies were used: phospho-AKT (S473), phospho-PRAS40 (T246), phospho-rS6 (S240/244), phospho-BAD (S136), 4E-BP1, GAPDH, PARP, caspase 9, cleaved caspase 3, cleaved caspase 8, MCL-1, BIM, COX IV, ERK, phospho-FOXO1 (T24)/FOXO3 (T32), HA-Tag, BCL-XL, Bad , BCL-2 , and Bad . The following secondary HRP-conjugated antibodies were used: anti-mouse IgG, anti-rabbit IgG , and Protein A (BD Pharmingen). Blots were developed using Pierce ECL Western Blotting Substrate or SuperSignal West Femto Maximum Sensitivity Substrate (Life Technologies) and detected using a Nikon D700 SLR camera as described previously [Cells were lysed in radio-immunoprecipitation assay (RIPA) buffer  in Hank's Balanced Salt Solution  supplemented with 2.5% bovine serum albumin for 10 min at room temperature. Cell fluorescence was assessed using a FACSCalibur flow cytometer . Analysis of the data was completed using FlowJo Software v10.0.7 .Cell viability assays were performed in 96-well plates, with 6 × 106 cells per 10 ml of media and treated with inhibitors for 16 hours. 4 × 105 cells were incubated in T-EB buffer  with 200 nM JC-1 (Life Technologies), 0.001% digitonin (Sigma-Aldrich), and 10 μg/ml oligomycin (Sigma-Aldrich) with either DMSO or BH3-only peptides for 60 minutes prior to analysis using a FACScalibur (Becton-Dickinson). The sequences and method of synthesis of BH3-only peptides were described previously [OCI-LY1 and SU-DHL4 cell lines were profiled as previously described,  with modeviously . PercentFor all viral productions, 293T HEK cells were transfected using X-tremeGene HP DNA Transfection Reagent . 293T cells were incubated for 24 hours prior to replacing medium with IMDM. These virus-containing media were then harvested after an additional 24 hours and used to transduce DLBCL cell lines. For retroviral production, 293T cells were co-transfected with pCL-ampho viral packaging vector  whereas pCMV-VSVG (Addgene plasmid 8454) and psPAX2 (Addgene plasmid 12260) were co-transfected for lentivirus production. To transduce DLBCL cell lines, we incubated cells in viral supernatants for 72 hours (changing supernatant every 24 hours) with 10 μg/ml 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide . Cells were treated with either blasticidin (8 μg/ml) or puromycin (2 μg/ml) for 5 days after transduction to select for stably transduced cells. Plasmid-positive cells were maintained with blasticidin (4 μg/ml) or puromycin (1 μg/ml).To generate DLBCL cells with doxycycline-inducible expression of a gene of interest, cells were first transduced with pMA2640 (Addgene plasmid #25434) and selected for blasticidin resistance. Expression of the improved tetracycline-controlled transactivator (rtTA-Advanced) allowed for doxycycline-inducible expression of genes downstream of the modified Tet-responsive element provided in the pLVX-tight-puro vector (Clontech). To generate MCL-1 expression plasmid, the human MCL-1 cDNA was cloned from pCMV-Flag-hMCL-1 (Addgene plasmid #25392) into pUC118 using BamHI and EcoRV. MCL-1 was then cloned into plvx-tight-puro using BamHI and NotI. To generate the AKT(S473D)-pLVX-tight-puro plasmid, we cloned AKT (S473D) from a plasmid received from Dr. Bing Su  into pLVX-tight-puro using NotI and EcoRI. To generate BAD expression plasmids, murine Bad (S136A) in pcDNA3 (Addgene plasmid #8798) was cloned into plvx-tight-puro using EcoRI. WT murine Bad was generated using the QuikChange II XL Site-Directed Mutagenesis Kit  to introduce a point mutation to restore expression of a serine rather than alanine at position 136. The following primers were used for this purpose 5′-AGGACGCTCGCGTTCGGCTCCCC-3′ and 5′-GGGAGCCGAACGCGAGCGTCCT-3′. To generate BCL-2 expression plasmid, the human BCL-2 cDNA was cloned from pMIG-BCL-2 (Addgene plasmid #8793) into pLVX-tight-puro using EcoRI. pLKO.1 shRNA expression plasmids containing MCL-1 hairpins  were a gift from Dr. Anand Ganesan (UC Irvine). All pLVX-tight-puro plasmids were sequenced using the following primer, 5′-AGCTCGTTTAGTGAACCGTCAGATC-3′.Subcellular fractionation was performed as described previously . In brieCo-immunoprecipitations were performed as described previously . BrieflyThe number “n” of biological replicates for each experiment is indicated in the Figure Legends. Student t-tests were applied to population means assuming equal variance (standard deviations within two-fold). The use of one- versus two-sample tests, and paired versus unpaired comparisons, was justified by the experimental design as indicated in the Figure Legends.Additional Materials and Methods are provided in


Kim-1 and Ngal, and promoted tubular injury in M-Rac1 FC mice. By contrast, deletion of myeloid Rac1 almost completely prevented the LPS-mediated renal impairment. LPS triggered a marked induction of macrophage-derived inflammatory cytokines, IL-6 and TNFα, in M-Rac1 FC mice, which was accompanied by Rac1 activation, stimulation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, and reactive oxygen species overproduction. These changes were inhibited in M-Rac1 KO mice. LPS evoked F4/80-positive macrophages accumulation in the kidney, which was not affected by myeloid Rac1 deficiency. We further tested the role of Rac1 signaling in cytokine production using macrophage cell line, RAW264.7. Exposure to LPS increased IL-6 and TNFα mRNA expression. The LPS-driven cytokine induction was dose-dependently blocked by the Rac1 inhibitor EHT1864, NADPH oxidase inhibitor diphenyleneiodonium, and NF-κB inhibitor BAY11-7082. In conclusion, genetic ablation of Rac1 in the myeloid lineage protected against LPS-induced renal inflammation and injury, by suppressing macrophage-derived cytokines, IL-6 and TNFα, without blocking recruitment. Our data suggest that Rac1 in macrophage is a novel target for the treatment of kidney disease through inhibition of cytokine production.Macrophage-mediated inflammation has been implicated in various kidney diseases. We previously reported that Rac1, a Rho family small GTP-binding protein, was overactivated in several chronic kidney disease models, and that Rac1 inhibitors ameliorated renal injury, in part via inhibition of inflammation, but the detailed mechanisms have not been clarified. In the present study, we examined whether Rac1 in macrophages effects cytokine production and the inflammatory mechanisms contributing to kidney derangement. Myeloid-selective Rac1 flox control (M-Rac1 FC) and knockout (M-Rac1 KO) mice were generated using the cre-loxP system. Renal function under basal conditions did not differ between M-Rac1 FC and KO mice. Accordingly, lipopolysaccharide (LPS)-evoked kidney injury model was created. LPS elevated blood urea nitrogen and serum creatinine, enhanced expressions of kidney injury biomarkers,  A numbergenitors. Strategigenitors., 9 or magenitors., 11 or fgenitors., 13 are in vivo significance of Rac1 was previously substantiated using gene targeted mice. Systemic Rac1 knockout (KO) mice were embryonic lethal due to germ-layer formation defects.[Rac1, a member of the Rho-family of small GTPases, is a multi-functional molecule that regulates actin cytoskeletal dynamics, generate defects. Subseque defects. Dorsal t defects. Vascular defects. while he defects. Cardiomy defects. These fiIn the kidney, Rac1 is shown to act as a regulatory subunit of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase, and the resultant ROS exert proinflammatory responses via NF-κB activation., 24 We pThis study was performed in strict accordance with the guidelines of the Animal Research Committee of Juntendo University, and protocols were approved by the Animal Research Committee of Juntendo University . Animals were sacrificed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.2 Fragment ; sheep anti-Mouse IgG, HRP-Linked F(ab’)2 Fragment , and donkey anti-goat IgG-HRP .We used the following antibodies; rat anti-mouse F4/80 monoclonal antibody ; rat anti-mouse Ly-6B.2 monoclonal antibody ; mouse anti-human Rac1 monoclonal antibody ; mouse anti-β actin monoclonal antibody ; goat anti-p47phox polyclonal antibody ; rabbit anti-human NF-κB p65 monoclonal antibody ; rabbit anti-human AIF monoclonal antibody ; rabbit anti-human histone H3 monoclonal antibody ; donkey anti-Rabbit IgG, HRP-Linked F(ab’)flox/flox) mice containing loxP sites flanking the entire exon 1 of the Rac1 gene  [cre/+) (C57BL/6J background),[cre/+ Rac1flox/flox: M-Rac1 KO) mice. LysM+/+ Rac1flox/flox littermates were used as flox control (M-Rac1 FC). Genotype was determined by PCR using tail genomic DNA and the following primers: cre  and Rac1 . Animals were housed in a room maintained at constant temperature, humidity, and light cycle (12-h light/dark).Rac1-floxed (Rac1C57BL/6)  were crokground), generatiLPS from Escherichia coli 0111:B4 (Sigma) was dissolved in phosphate buffered saline (PBS) at a concentration of 0.25 mg/ml. Male M-Rac1 FC and KO littermates (16 to 24 weeks old) were given a single intraperitoneal injection of LPS (5 mg/kg) or PBS (Vehicle). To avoid dehydration, PBS was injected at 24 h. At 48 h after LPS injection, blood was collected. Kidneys were harvested after perfusion with 50 ml of saline. Blood urea nitrogen (BUN) and serum creatinine were measured at SRL laboratory .Mouse monocyte/macrophage cell line, RAW264.7 , were cultured in RPMI1640 medium (Life Technologies) supplemented with 10% fetal bovine serum (FBS: HyClone) and antibiotics. Cells were stimulated with LPS (1 to 1000 ng/ml) for the indicated time. In some experiments, cells were pretreated with the Rac1 inhibitor EHT1864 (Tocris), the NF-κB inhibitor BAY11-7082 , or the NADPH oxidase inhibitor diphenyleneiodonium (DPI: Sigma) for 2 h, then subjected to LPS. EHT1864 associates with Rac1 tightly and causes the release of bound GTP, thus prevents effector interaction. BAY11-70L929 cells (CCL-1) were purchased from ATCC. L929-cell conditioned medium (LCCM) was obtained as a source of granulocyte/macrophage colony stimulating factor, by cultivating L929 cells in RPMI supplemented with 10% FBS and antibiotics for 4 days and additional 3 days. Bone marrow cells were collected in a sterile condition from the femur and tibia of M-Rac1 FC and KO mice, and cultured in RPMI containing 30% LCCM, 20% FBS, and antibiotics for 7 days.[cre expression, we used SYBR Green chemistry and the following primers .Total RNA was extracted using an RNeasy mini kit (Qiagen), and reverse-transcribed to cDNA with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Gene expression was determined by real-time quantitative RT-PCR using 7500 Fast Real-Time PCR System (Applied Biosystems). We basicKidneys were fixed in 4% paraformaldehyde solution. For morphologic evaluations, we stained paraffin sections (4 μm) with hematoxylin-eosin. The degree of tubular injury was semiquantitatively assessed using at least 10 randomly selected fields in each specimen in a blinded manner. Tubular injury was graded (0 to 4) on the basis of the percentage of tubular dilation, atrophy, cellularity, basement membrane thickening, or sloughing, as follows: 0, intact; 1, <10%; 2, 10–25%; 3, 25–50%; 4, 50–75%; 5, >75% of the tubules were affected.Paraformaldehyde-fixed cryosections (5 μm thick) were treated with 3% hydrogen peroxide, avidin-biotin blocking solution, 0.5% blocking reagent (Sigma), then incubated with rat anti-mouse F4/80 (1:200) or rat anti-mouse Ly-6B.2 , and subsequently with biotin-labeled secondary antibodies., 32 Immu2, and Protease Inhibitor cocktail. Nuclear protein was obtained using Nuclear/Cytosol Fractionation Kit (BioVision). For membrane fractionation, samples were homogenized in M1 buffer that contained 20 mM Tris (pH 7.4), 2 mM MgCl2, 250 mM sucrose, and Protease Inhibitor cocktail. Nuclei and unlysed cells were removed by low-speed centrifugation (500 x g), and the supernatant was centrifuged at 100,000 x g. The pellet was resuspended in M2 buffer that contained 40 mM Tris (pH 7.9), 260 mM sucrose, 1% Triton X-100, and Protease Inhibitor cocktail.[http://www.sigmaaldrich.com/catalog/product/sigma/sab2500674?lang=en&region=US), rabbit anti-NF-kB p65 , rabbit anti-AIF , or rabbit anti-human histone H3 . After incubation with HRP-linked anti-rabbit IgG (1:4000), anti-mouse IgG (1:4000), or anti-goat IgG (1:5000),[Western blotting was performed as described previously. Whole cecocktail. The nitrcocktail. mouse an(1:5000), signals Rac1 activity was evaluated by GST pull-down assay. Briefly,NADPH oxidase activity was measured using bis-N-methylacridinium nitrate (lucigenin) chemiluminescence. Briefly,ROS in the kidney were evaluated as malonyldialdehyde (MDA) using TBARS Assay Kit (Zeptometrix). Briefly, the kidney homogenate (50 mg/ml PBS) was mixed with sodium dodecyl sulfate and thiobarbituric acid-buffer reagent, and incubated at 95°C for 60 min. The sample was cooled to room temperature, and centrifuged at 3,000 rpm for 15 min. The absorbance of the supernatant was measured at 532 nm in a spectrophotometer . The concentration of TBARS (μM) was calculated from a standard curve of MDA.post hoc test. Student’s unpaired t-test was used for comparison between the two groups. Histological data were analyzed using nonparametric analysis with Kruskal-Wallis test, followed by Mann-Whitney U test. P Values < 0.05 were considered to be significant.Values are expressed as means ± s.e.m. For multiple comparisons, statistical analysis was performed by two-way ANOVA or one-way ANOVA followed by Bonferroni's cre in BMDM, validating cell type-specific Rac1 depletion in the myeloid cells , reflecting the presence of a small number of cre-positive tissue macrophages in the kidney.M-Rac1 FC and KO mice were born in the Mendelian ratio. Immunoblotting and qPCR analyses revealed reduced expressions of Rac1 and abundant and selective induction of id cells . Cre traUnder basal conditions, renal function  and histology did not significantly differ between M-Rac1 FC and KO mice . AccordiKim-1) and neutrophil gelatinase-associated lipocalin  in M-Rac1 FC mice, while myeloid Rac1 ablation prevented the LPS-invoked Kim-1 and Ngal increment , while LPS-evoked IL-6 induction was prominently suppressed in M-Rac1 KO mice (only 8-fold vs. Vehicle group)  . Gene exRac1 is a component of NADPH oxidase complex (NOX2 isoform) in macrophages, and upon activation, Rac1 stimulates translocation of NADPH oxidase regulatory subunits p47phox and p67phox from cytosol to membrane, resulting in NADPH oxidase activation and ROS production., 24 We fThese data suggest the involvement of Rac1-NADPH oxidase-ROS-cytokine cascade in LPS-evoked renal tubular injury.cre mRNA expression in the kidney of M-Rac1 KO mice -mediated IL-6 and TNFα mRNA induction was significantly attenuated by pretreatment with the Rac1 inhibitor EHT1864  in a dose-dependent manner , Cxcl1, and Cxcl2. MCP-1 is a major monocyte/macrophage chemoattractant, while Cxcl1 and Cxcl2 enhance neutrophil recruitment. These chemokines are produced by tubular epithelial cells, vascular endothelial and smooth muscle cells, fibroblasts, neutrophils, monocytes, and macrophages. We quantitatively analyzed the mRNA expression profiles of these chemokines in the kidneys of Vehicle- and LPS-injected M-Rac1 FC and KO mice . The MCPIn contrast to the myeloid-selective Rac1 KO mice, we previously reported that systemic inhibition of Rac1 by drug treatment blocked not only cytokine overexpression but also macrophage infiltration and MCP-1 mRNA induction in the kidney of obese diabetic mice, and salt-loaded angiotensin II-overproducing mice., 28 SystIn the systemic Rac1 inhibition models, it is difficult to evaluate the influence of Rac1 inhibition on cytokine production within macrophages, because Rac1 inhibitor interrupts macrophage infiltration itself. On the other hand, macrophage accumulation occurred in myeloid cell-selective Rac1 KO mice subjected to LPS. In this sense, our model has an advantage in enabling analysis of the intracellular events within macrophages. We successfully identified the intracellular crosstalk between Rac1-NADPH oxidase-ROS-NF-κB pathway and LPS-triggered cytokine production cascade in macrophages.IL-6 and TNFα mRNA upregulation via NADPH oxidase and NF-κB activation using cultured macrophage cell line. Taken these findings together, our data suggest that Rac1 in myeloid cells, especially macrophages, plays an important role in LPS-mediated cytokine overproduction, inflammation, and kidney injury. Rac1 in myeloid cells has potential as a novel target for the treatment of kidney disease.In conclusion, M-Rac1 KO mice were resistant to LPS-evoked kidney dysfunction and tubular damage. Renoprotection correlated with suppression of Rac1 activation, NADPH oxidase activity, oxidative stress, and macrophage proinflammatory cytokines, IL-6 and TNFα, while M-Rac1 deletion did not block macrophage recruitment to the kidney. Indeed, we demonstrated that Rac1 signaling has crucial role in mediating LPS-evoked S1 FigSurvival rate was analyzed by Kaplan Meier method after LPS injection up to 14 days in M-Rac1 FC and KO mice (n = 10 for each group). Statistical analysis was performed by log rank test. n.s., not significant.(TIF)Click here for additional data file.S2 FigP < 0.01 genotype effect, P < 0.01 treatment effect, P < 0.01 interaction effect. **P < 0.01 by Bonferroni's post hoc test. n.s., not significant. n = 8 per each group.The mRNA levels were compared using real-time quantitative RT-PCR and expressed relative to M-Rac1 FC Vehicle group. Statistical analysis was performed by two-way ANOVA, (TIF)Click here for additional data file.S3 Fig(A) Original blots of (TIF)Click here for additional data file.S1 TableRenal function was measured by collecting serum and urine from M-Rac1 FC and KO mice at 4 months of age. Data are means was.e.m (n = 6 for each group). Statistical analysis was performed by Student’s unpaired t-test. n.s., not significant.(TIF)Click here for additional data file.


ETV6/RUNX1 fusion gene represents the most frequent chromosomal rearrangement in childhood leukemia. Presence of ETV6/RUNX1 alone is usually not sufficient for leukemia onset, and additional genetic alterations have to occur in ETV6/RUNX1-positive cells to cause transformation. We have previously generated an ETV6/RUNX1 transgenic mouse model where the expression of the fusion gene is restricted to CD19-positive B cells. Since BCL2 family members have been proposed to play a role in leukemogenesis, we investigated combined effects of ETV6/RUNX1 with exogenous expression of the antiapoptotic protein BCL2 by crossing ETV6/RUNX1 transgenic animals with Vav-BCL2 transgenic mice. Strikingly, co-expression of ETV6/RUNX1 and BCL2 resulted in significantly shorter disease latency in mice, indicating oncogene cooperativity. This was associated with faster development of follicular B cell lymphoma and exacerbated immune complex glomerulonephritis. ETV6/RUNX1-BCL2 double transgenic animals displayed increased B cell numbers and immunoglobulin titers compared to Vav-BCL2 transgenic mice. This led to pronounced deposition of immune complexes in glomeruli followed by accelerated development of immune complex glomerulonephritis. Thus, our study reveals a previously unrecognized synergism between ETV6/RUNX1 and BCL2 impacting on malignant disease and autoimmunity.The t translocation generating the  ETV6/RUNX1 (TEL/AML1) fusion gene. ETV6/RUNX1 represents the most abundant translocation product in pediatric cancers with an incidence of up to 25% in children with B lymphoblastic leukemia (B-ALL)   was used as a marker for germinal center cells. Characterization of infiltrates was performed using polyclonal antibodies against Kappa light chain (Ref. NB7549), Lambda light chain (Ref. NB7552), IgM (Ref. NB7497) and IgA (Ref. NB7504), all Novus Biologicals  as well as an anti-polyvalent biotinylated antibody against IgG . To detect renal IgG deposits, an F(ab’)2 anti-mouse IgG-fluorescein isothiocyanate (FITC) antibody (eBioscience) was used as previously described .TM software . Analysis was performed using HistoQuestTM software  as infiFor electron microscopy, 1-2 mm thin pieces of kidney were fixed in 4% paraformaldehyde resolved in 0.1 M cacodylate buffer and embedded in epoxy resin. Ultrathin sections were cut with an Ultracut EM UC7 , stained with uranylacetate and examined using a Jeol JEM 1010  electron microscope.After incubation of single-cell suspensions with an anti-CD16/CD32 (93) antibody to prevent nonspecific Fc receptor-mediated binding, cells were stained with antibodies directed against murine Ly6G/Gr-1 (1A8-Ly6G), CD11b (M1/70), CD3e (145-2C11), B220 (RA3-6B2), CD19 (1D3), CD138 (281-2), CD4 (GK1.5) or CD8 (53-67). Fluorescein isothiocyanate (FITC)- phycoerythrin (PE)- or allophycocyanin (APC)-labeled Streptavidin was used for the detection of biotinylated antibodies. All antibodies were purchased from eBioscience or BD Biosciences , respectively. Subsequently, samples were acquired on a BD FACSCanto II flow cytometry device (BD Biosciences), and data were analyzed with FACSDiva (BD Biosciences) or FlowJo software .Serum levels of urea were measured using Reflotron-based test strips . Blood urea nitrogen was calculated using the following formula: Urea (mg/dl) x 0.467 = BUN (mg/dl).An ELISA-based immunoglobulin (Ig) clonotyping system  was used to determine Ig titers in the serum of mice. For quantification of IgA and IgG levels in serum the Mouse IgA and IgG ELISA Ready-SET-Go! kits (eBioscience) were utilized according to the manufacturer's protocol.® ANA/HEp-2 IgG cells  as a substrate coated on microscope slides. Mouse serum was added to the cells and ANA were visualized by addition of a FITC-labeled polyclonal rabbit anti-mouse immunoglobulin antibody  as described [Anti-nuclear antibodies (ANA) were detected using VIRGOescribed .Anti-histone and anti-chromatin (nucleosome) antibodies were measured by ELISA  using horseradish peroxidase-conjugated anti-mouse antibodies  as secondary antibody as previously described .® ANA Update immunoassay  was used for the detection and identification of autoantibodies against nuclear and cytoplasmic antigens in serum according to the manufacturer's protocols.INNO-LIAp values are considered as follows: p < 0.05 *, p < 0.01 **, and p < 0.001 ***.Statistical analyses were performed using GraphPad Prism 5 . Differences were investigated for statistical significance by one-way analysis of variance with Tukey's Multiple Comparison Test if not otherwise mentioned. For Kaplan-Meier plots significant differences were calculated by Log-rank test. Error bars represent means ± SEM if not stated otherwise.


Caenorhabditis elegans. Both genetically silencing and ChR2-mediated activation of ADL sensory neurons significantly affected preference choice. ADL regulated preference choice by inhibiting function of G protein-coupled receptor (GPCR)/SRH-220. ADL sensory neurons might regulate preference choice through peptidergic signals of FLP-4 and NLP-10, and function of FLP-4 or NLP-10 in regulating preference choice was regulated by SRH-220. FLP-4 released from ADL sensory neurons further regulated preference choice through its receptor of NPR-4 in AIB interneurons. In AIB interneurons, NPR-4 was involved in the control of preference choice by activating the functions of ASH-2 trithorax complex consisting of SET-2, ASH-2, and WDR-5, implying the crucial role of molecular machinery of trimethylation of histone H3K4 in the preference choice control. The identified novel neuronal circuit and the underlying molecular mechanisms will strengthen our understanding neuronal basis of preference choice in animals.Preference choice on food is an important response strategy for animals living in the environment. Using assay system of preference choice on bacterial foods, OP50 and PA14, we identified the involvement of ADL sensory neurons in the control of preference choice in  Caenorhabditis elegans is an attractive model animal for behavioral study due to its simple nervous system and ability to sense diverse environmental stimuli, such as touch, smell, taste and temperature3C. elegans nervous system makes it a useful assay system for seeking the biological mechanisms of behavioral choice5C. elegans can exhibit several types of behavioral choice based on its response to paired or multiple stimuli, such as attractants versus aversive stimuli or different attractants7891011Animals’ decision making includes two main levels of complexity, behavioral choice and value-based decision making1C. elegans, olfactory chemotaxis towards food-associated odors is an innate behavior, and highly reproducible among animalsC. elegans121212C. elegans, AWB and AWC sensory neurons are necessary for innate preference for Pseudomonas aeruginosaC. elegans.In C. elegans, ASH and ADL are important sensory neurons functioning to sense and mediate avoidance of noxious environmental stimuli, such as Cu2+ ion and garlic1819C. elegans. The new identified neuronal circuit and the related underlying mechanism for preference choice on bacterial food will strengthen our understanding neuronal basis of behavioral choice in animals.Among the well-known 12 kinds of amphid neurons  in egl-1 gene encodes a cell-death activator , and can be used to genetically ablating specific neuronsegl-1 under the control of srh-220 promoter. Interestingly, genetically ablating ASH sensory neurons did not significantly affect preference choice compared with wild-type N2  mutant had the similar preference choice to wild-type (data not shown). We observed that nematodes with channelrhodopsin-2 (ChR2)-mediated activation of ADL sensory neurons on assay plates with all-trans retinal (ATR) exhibited significantly enhanced choice index compared with wild-type N2  mutant had the similar leaving behavior from OP50 or PA14 lawns and chemotaxis to OP50 to wild-type (data not shown). Meanwhile, nematodes with optogenetically activating ADL sensory neurons showed the similar leaving behavior from OP50 or PA14 lawns and chemotaxis to OP50 compared with wild-type N2  adaptor protein, and plays important roles in both longevity and immunityarr-1 gene caused the deficit in preference choice behavior compared with wild-type N2 (arr-1(ok401) mutant nematodes had similar leaving behavior from OP50 or PA14 lawns and chemotaxis to OP50 to those in wild-type N2 (arr-1 gene under the control of srh-220 promoter rescued the deficit in preference choice in arr-1(ok401) mutant (In -type N2 . arr-1(o-type N2 . Moreove) mutant , suggestC. elegans, some genes encoding GPCRs such as SRE-1, SRI-51, SRH-132, and SRH-220 are expressed in ADL sensory neurons2425srh-220 gene resulted in enhanced choice index compared with wild-type N2 (srh-220(tm3939) mutants had normal leaving behavior from OP50 or PA14 lawns and chemotaxis to OP50 (srh-220(tm3939) could be rescued by the expression of srh-220 under the control of its own promoter  releaseunc-31 gene significantly decreased choice index compared with wild-type N2  is a gain-of-function mutant, and pde-4(ok1290) is a loss-of-function mutant. We further found that the gsa-1(ce94) mutant had the increased choice index, whereas the pde-4(ok1290) mutant has the decreased choice index compared with wild-type N2  mutants had normal leaving behavior from OP50 or PA14 lawns and chemotaxis to OP50 (In -type N2 . Nematod to OP50 . Therefosrh-220 and flp-4 or nlp-10 in regulating preference choice in nematodes. We found that the preference choice phenotype in double mutant of flp-4(RNAi);srh-220(tm3783) was similar to that in flp-4(RNAi) nematodes (nlp-10(tm6232);srh-220(tm3783) was similar to that in nlp-10(tm6232) mutant nematodes  mutants had normal leaving behavior from OP50 or PA14 lawns and chemotaxis to OP50  mutants (6npr-4(tm1782) mutants . Among 24 genes expressed in AIB interneurons, we found that mutation of npr-4 gene caused the significant increase in expression levels of ptp-3, and ced-10 genes, and the significant decrease in expression levels of glr-2, tax-6, cdc-42, and set-2 genes  methyltransferase.To identify candidate targeted genes for -2 genes . In C. enpr-4 to investigate their possible function in regulating preference choice in nematodes. Among 7 candidate genes examined, mutation of set-2 gene caused the significant decrease in choice index compared with wild-type N2 (set-2(ok952);npr-4(tm1782) was similar to that of single mutant of set-2(ok952) or npr-4(tm1782) (set-2(ok952) mutants had similar phenotypes of leaving behavior from OP50 or PA14 lawns and chemotaxis to OP50 to those in wild-type N2 (We further used the available mutants for candidate targeted genes of -type N2 . Moreove(tm1782) , suggest-type N2 .C. elegans, it was reported that ASH, WDR-5, and SET-2 constitute an ASH-2 trithorax complex, which trimethylates histone H3K4ash-2 or wdr-5 gene caused the significant decrease in choice index compared with wild-type N2; however, mutation of rbr-2 did not significantly affect preference choice behavior (In behavior . These rash-2(tm1726) or wdr-5(ok1417) mutants had similar phenotypes of leaving behavior from OP50 or PA14 lawns and chemotaxis to OP50 to those in wild-type N2  mutants  X, arr-1(ok401) X, srh-220(tm3939) IV, flp-21(ok889) V, nlp-7(tm1970) X, nlp-8(ok1799) I, nlp-10(tm6232) III, npr-4(tm1782) X, tax-6(ok2065) IV, glr-2(ok2342) III, ced-10(n1993) IV, ptp-3(ok244) II, cdc-42(ok825) II, gsa-1(ce94) I, pde-4(ok1290) II, set-2(ok952) III, ash-2(tm1726) II, wdr-5.1(ok1417) III, and rbr-2(ok2544) IV, and transgenic strains of quEx128[npr-9::GFP + pRF4 rol-6(su1006)] expressing npr-9 in AIB interneurons and ASH::TeTxNematodes were grown on nematode growth medium (NGM) plates seeded with E. coli OP50 and P. aeruginosa PA14. E. coli and P. aeruginosa cultures were grown in Luria-Bertani (LB) broth overnight at 37 °C.Bacterial strains used were E. coli OP50. All the behavioral experiments were performed with young adults maintained at 20 °C. Behavioral choice assay was performed on 9-cm NGM plates. Bacterial food with a diameter of 0.5-cm was seeded at the distance of 1.5 cm from periphery /total number. Ten replicates were performed.C. elegans genomic DNA  was inserted into 95_77-mcherry vector and then transformed into the strain of quEx128. Germline transformation was performed as describeddop-1::rfp or Plin-44::gfp at a concentration of 60 μg/mL into the gonad of nematodes.To generate entry vectors carrying promoter sequences, promoter regions were amplified by polymerase chain reaction (PCR) from wild-type Chlamydomonas reinhardtii, can utilize chromophore ATR to enable the activity of specific neurons in C. eleganssrh-220 promoter was used for the ADL-specific expression, and the npr-9 promoter was used for the AIB-specific expression. Nematodes expressing ChR2 in ADL sensory neurons or AIB interneurons were grown on OP50-seeded NGM agar plates containing 50 μM of ATR. Plates were seeded on day 0. Nematodes were transferred to OP50-seeded plates containing 50 μM of ATR on day 1 in the dark. After starvation for 5 h, nematodes were transferred to assay plates containing 50 μM of ATR for behavioral analysis on day 2. During the assay, whole field illumination was performed, and ChR2 was excited by a round blue light in diameter of 9.5 cm sourced from LED array  constructed in a LED light source . Light intensity measured at the sample was 5 mW/mm2 of 465 nm light. For optogenetic experiments, light intensity was monitored using an optical power meter . All work was carried out under low-illumination conditions to prevent the preactivation of ChR2-expressing neurons. Nematodes without ATR treatment were used as the control. Optogenetical activation was performed under the lite-1 mutation background. Ten replicates were performed.Light-activated ChR2, a directly light-gated cation channel from the green alga E. coli strain HT115 (DE3) expressing double-stranded RNA that is homologous to a target gene as describedE. coli HT115 (DE3) grown in LB broth containing ampicillin (100 μg/ml) at 37 °C overnight was plated onto NGM containing 100 μg/mL ampicillin and 5 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG). L2 larvae were placed on RNAi or vector control plates for 2 days at 20 °C until nematodes became gravid. Gravid adults were transferred to fresh RNAi-expressing bacterial lawns and allowed to lay eggs for 2 h to obtain the second generation of RNAi population. Eggs were allowed to develop at 20 °C to young adults for the subsequent assays. unc-22 RNAi was included as a positive control.RNAi was performed by feeding nematodes with flp-4, pde-4, unc-31, or gsa-1 gene was amplified by PCR to yield product A. The srh-220 promoter was used for ADL-specific expression, and amplified in two different reactions with promoter reverse sense primer or promoter reverse antisense primer together with promoter forward primer to yield product B and C. Product A was further fused by amplification to B or C using the nested primers to yield fragments D and E. In fragment D, the flp-4, pde-4, unc-31, or gsa-1 gene was transcribed by ADL-specific promoter in the sense orientation. In fragment E, the flp-4, pde-4, unc-31, or gsa-1 gene was transcribed by the ADL-specific promoter in the antisense orientation. The fragments D and E were mixed in equimolar amounts and injected at 25–100 ng/μL into the wild-type nematodes. The Plin-44::gfp was used as a transgenic marker.The method was performed basically as describedact-1 gene was chosen as a reference gene. Relative quantification of targeted genes in comparison to reference act-1 gene was determined, and the final results were expressed as relative expression ratio between targeted gene and reference gene. The primer information was shown in Total RNA of nematodes was extracted using RNeasy Mini Kit . Purity and concentration of RNA were evaluated by OD260/280 in a spectrophotometer. Total RNA was reverse-transcribed using cDNA synthesis kit . After cDNA synthesis, real-time PCR was performed using SYBR Premix Ex Taq™ (Takara) for amplification of the PCR products. The All data in this article were expressed as means ± standard error of the mean (S.E.M.). Graphs were generated using Microsoft Excel . Statistical analysis was performed using SPSS 12.0 . Differences between groups were determined using analysis of variance (ANOVA). The probability levels of 0.05 and 0.01 were considered to be statistically significant.How to cite this article: Yu, Y. et al. FLP-4 neuropeptide and its receptor in a neuronal circuit regulate preference choice through functions of ASH-2 trithorax complex in Caenorhabditis elegans. Sci. Rep.6, 21485; doi: 10.1038/srep21485 (2016).


HTLV-1 associated myelopathy/tropic spastic paraparesis (HAM/TSP) is induced by chronic inflammation in spinal cord due to HTLV-1 infection. Cerebrospinal fluid (CSF) neopterin or proviral load are clinically measured as disease grading biomarkers, however, they are not exactly specific to HAM/TSP. Therefore, we aimed to identify HAM/TSP-specific biomarker molecules and establish a novel less-invasive plasma diagnostic model for HAM/TSP. Proteome-wide quantitative profiling of CSFs from 6 asymptomatic HTLV-1 carriers (AC) and 51 HAM/TSP patients was performed. Quantitative analysis for 1,871 nonredundant CSF proteins identified from 57 individuals defined 14 CSF proteins showing significant correlation with Osame's motor disability score (OMDS). The 14 severity grade biomarker proteins were further examined plasma ELISA assays (n = 71). This results confirmed secreted protein acidic and rich in cysteine (SPARC) and vascular cell adhesion molecule-1 (VCAM-1) demonstrated the same correlations in plasma . Finally, we constructed three-factor logistic regression model and evaluated the diagnostic power using 105 plasma samples. In this training set, we constructed a HAM/TSP diagnostic model using SPARC, VCAM1, and viral load. Sensitivity and specificity to diagnose HAM/TSP patients from AC (AC vs. OMDS 1–11) were 85.3% and 81.1%, respectively. Importantly, this model could be also useful for determination of therapeutic intervention point (OMDS 1–3 + AC vs. OMDS 4–11), exhibiting 80.0% sensitivity and 82.9% specificity. We propose a novel less-invasive diagnostic model for early detection and clinical stratification of HAM/TSP.


Fbxo45) through the ubiquitin proteasome system (UPS). The key step is recognition of EMT-TFs by Fbxo45 through its SPRY domain for Zeb2, or F-box domain for the other three EMT-TFs Snai1, Snai2 and Twist1, respectively. The K48-linkaged ubiquitination capability on Zeb2 relies on its functional SBD domain. In addition, miR-27a* can directly down-regulate the expression of Fbxo45, preventing degradation of EMT-TFs and thus ensuring EMT phenotype. We suggest that Fbxo45 is a key node of the miR-27a*/Fbxo45/EMT-TFs signaling axis.Epithelial-mesenchymal transition (EMT) plays a critical role in the development of tumor metastases by enhancing migration/invasion. One of the hallmarks of EMT is loss of E-cadherin and gain of N-cadherin expression, which are regulated by the core EMT-inducing transcription factors (EMT-TFs), such as Zeb1/2, Snai1/2 and Twist1. Here, we find that EMT-TFs can be dynamically degraded by an atypical ubiquitin E3 ligase complex Skp1-Pam-Fbxo45 (SPF The trapromoter , and alspromoter  and Snaipromoter . The zingression -23.i.e., SCF complex) is a multisubunit E3 ubiquitin ligase catalyzing the ubiquitination of proteins destined for proteasomal degradation [Fbxo45, plays a part in the embryogenesis, synapse formation or apoptosis in tumor [At the transcriptional regulatory level, EMT-TFs can be either regulated by transcription factors of themselves or others through binding to their promoter regions , or suppradation . Canonicradation , 34. Appradation . Exceptiradation . Insteadin tumor -37.Fbxo45 mediates the degradation of EMT-TFs through the ubiquitin-proteasome pathway, which directly affects the EMT processes in mammalian cancer cells. We also find that miR-27a* transcriptionally suppresses the expression of Fbxo45 to influence the formation of SPFFbxo45 complex, resulting in stabilization of EMT-TFs which contribute the EMT initiation and cancer progression.Here, we have identified that the atypical E3 ligase SPF2C2 or C2H2 zinc finger domains whereas Twist1 contains only the helix-loop-helix (HLH) domain, which are all responsible for binding to the specific DNA motif to regulate transcription activities for further downstream signaling or cellular actions , Okayama Lung (246 cases), Taylor Prostate (160 cases) and Roessler Liver (445 cases) were collected for analysis, showing that the mRNA levels in carcinoma samples were not obviously increased when compared to those of normal tissue or benign neoplasm; on the contrary, they were decreased to some extent Fig . HoweverXenopus laevis, the protein stabilities of Twist1, Snai1, Snai2 and even Zeb2 are associated with the F-box protein partner of paired (Ppa), a component of E3 ubiquitin enzyme, which functions on the embryonic or neural crest development [Fbxo45 complex might also make contributions to EMT processes by mediating the ubiquitin-dependent degradation of EMT-TFs, particularly Zeb1 and Zeb2 proteins in cancer cells.The stability of Snai1 has been confirmed to be mediated through ubiquitin-proteasome system . In Xenoelopment , 41. ThuFbxo45 complex executing the function of ubiquitin E3  was subcloned downstream of the reporter Renilla luciferase gene of psiCheck2 vector  on matrix-gel can illustrate the interrelationships between the genetically dysregulated invasive tumor cell, the microenvironment and the malignant switch , and VM via directly targeting Fbxo45 to disrupt the SPFFbxo45 complex, thereby maintaining the protein stabilities of EMT-TFs, along with characteristic phenotypes of malignant cells  contains a typical RING-finger structure at its COOH-terminal, which is responsible for the E2-Ubiquitin recognition and transferring ubiquitin molecular to specific substrates [via its NH2-terminal F-box domain, respectively [Fbxo45 for ubiquitin-dependent degradation of those EMT-TFs. Consistently, ablation of either Pam or Fbxo45 stabilized EMT-TFs through reducing ubiquitination on substrates, such as Zeb2, which strongly implied that the SPFFbxo45 complex was the specific ubiquitin E3 ligase of EMT-TFs. We also demonstrate that Fbxo45 could recognize diverse substrates  mainly depends on its SPRY domain, which is in line with the previous reports [Fbxo45 on specific substrates Fbxo45 co reports , 56. Lik reports , 58, canFbxo45. Our data demonstrate that the protein level of Zeb2 is strictly controlled by the SPFFbxo45complex and its degradation relies on the formation of K48-linkage poly-ubiquitin chains. Deletions of either F-box or SPRY domain of Fbxo45 significantly weakened the ubiquitination on Zeb2, suggesting that Zeb2 protein degradation is connected with the correct formation of SPFFbxo45-Zeb2 complex. On the other hand, Zeb2 protein comprises multiple functional domains, including the DNA binding domain , CtBP protein interaction domain (CID), homeodomain (HD) and Smad protein interaction domain (SBD). Our results suggest that Zeb2 interacts with Fbxo45 by the N-terminal region Zeb2-ZSH other than the C-terminal region Zeb2-CZ. Moreover, SBD is essential for K48-linkage poly-ubiquitination on Zeb2, probably due to its containing precise lysines for the ubiquitination , HA-probe (SC-57592), goat anti-Snai2 (SC-10437) and mouse anti-N-Cadherin (SC-8424) were from Santa Cruz Biotechnology. Rabbit antibodies against Pam (ab86078), Fbxo45 (ab126521), GAPDH (ab37168), and mouse anti-Twist1 (ab50581) were from Abcam. Rabbit anti-E-cadherin (#3195), -Zeb1 (#3396), and mouse anti-Myc (#2276) were from Cell signaling technology. Rabbit anti-Zeb2 (NBP1-77179) was from Novus. α-tubulin (#66031-1-1g) antibody was from Proteintechnology. Anti-Flag M2 (F1804), -HA (MMS-101P) and -Vimentin V9 (V6630) mouse antibodies were from Sigma-Aldrich. Snai1 (A52437) rabbit antibodies were from ABclonal. Rabbit anti-fibronectin (GTX112794) was from GeneTex. Protein G Plus/A agarose suspension (#IP05) was purchased from Calbiochem.mut forward and reverse. All plasmids were checked carefully by sequencing the entire insert. Oligo nucleotide sequences are provided in the The human full-length Zeb1 and Zeb2 tagged with 3xFlag were cloned into pCDH-CMV-MCS-EF1-GreenPuro vector (System Bioscences) for exogenous expression. Flag-tagged Snai1, Snai2 and Twist1 were stored in our lab. Full-length Fbxo45 and Fbxo9 cDNA were inserted into pCMV-HA, pCMV-Myc or pGEX-4T-1 vectors. HA-Fbxo45 and 3xFlag-Zeb2 mutants were generated by mutagenesis PCR using KOD Hot Start DNA polymerase (TOYOBO). The synthetic DNA oligos (Sangon), miR-27a* (forward and reverse) or zipmiR-27a* (forward and reverse) were used to generate an mRNA transcripts from pGreen-puro vector (System Bioscences) for further lentivirus packaging in HEK293FT cells with pCMV-dR8 and pMD.2G (System Bioscences). To generate psiCheck2-Fbxo45 3′UTR, 810-bp fragment containing the complementary DNA sequence with miR-27a* seed region (4389-4395bp) were cloned between sites XhoI and NotI of the psiCheck2 vector (Promega). Further we mutated the recognition site of miR-27a* by mutagenesis PCR using the following primers Fbxo45 3′UTR2 in DMEM supplemented with 10% fetal bovine serum (FBS), and penicillin/streptomycin at 100U/ml. P69 and M12 were cultured in RPMI-1640 (Hyclone) supplemented with 5% fetal bovine serum (FBS) (Hyclone), 10ng/ml EGF, 0.2μM Dexamethasone, 5μg/ml Insulin, 5μg/ml Transferrin, 5μg/ml Sodium Selenite, 50μg/ml Gentamicin, 2.5μg/ml Fungizone. A549, H1299 and SMMC7721 cells were cultured in RPMI-1640 with 10% FBS, and penicillin/streptomycin at 100U/ml. MCF7 cells were also cultured in DMEM with 10% FBS, but without phenol for the 17β-estrogen inducing experiments. P69 or M12 cells stably expressing miR-27a* or zipmiR-27a*, respectively, were in addition supplemented with 20μg/ml puromycin. Plasmid transfection was performed using Lipofectamine 2000 (Invitrogen) according to manufacturer's instructions.HEK293T, HeLa, U2OS, HepG2, PC3, MCF7 and MDA-MB-231 cells were maintained at 37°C and 5% COE.coli bacteria (Sangon) with 0.5 mM IPTG at 16°C for 10 hrs. Pellets were dissociated on ice to clear in bacteria protein extraction regent (Thermo Fisher) containing 100 μg/ml lysozyme (Sigma), 12.5 μl/ml protease inhibitor cocktails with EDTA (Sangon), 2 U/ml DNase I (Thermo Fisher). Glutathione sepharose 4B  were used to purify the GST-tagged protein and eluted in sequential buffer 1 (50mM Tris-HCl (pH8.0), 20mM Glutathion), buffer 2 (50mM Tris-HCl (pH8.0), 10mM Glutathion) and concentrated for further pull down experiments. Endogenous Zeb1/2, Snai1/2, Twist1 from HeLa cell extraction in RIPA buffer (50 mM Tris-HCl (pH7.4), 150 mM NaCl, 0.5% NP-40, and complete protease inhibitor cocktail tablet (Roche) were incubated and rotated with purified GST-tagged proteins and Glutathione sepharose 4B beads at 4°C overnight. RIPA buffer washed precipitations were resolved by SDS-PAGE for western-blot detection using antibodies.The pGEX-4T-1 plasmid  was used to clone pGEX-Fbxo45 and its truncated forms. All plasmids were induced to express insert genes in DE3 Immunoprecipitation (IP) and Co-Immunoprecipitation (Co-IP) were performed with 300 to 500 μg of HEK293T lysates prepared using modified RIPA buffer (50 mM Tris-HCl (pH7.4), 150 mM NaCl, 0.5% NP-40 for Co-IP or 1% NP-40 for IP, and complete protease inhibitor cocktail tablet (Roche). Exogenous Flag-tagged proteins  were precleared with protein A/G-agarose beads (Santa Cruz Biotechnology) prior to immunoprecipitation with mouse anti-Flag M2 antibody. Immunocomplexes bound to agarose beads were washed four times in the same RIPA buffer. Western blotting was performed by resolving whole-cell lysates or immune-complex by SDS-PAGE and blotting onto Immobilon-P membranes (Millipore Corporation). The supersensitive ECL kit (Millipore Corporation) was used and the blotting was developed with LASmini4000  as described before.U2OS cells were co-transfected with 3xFlag-Zeb2 (GFP fragment in the plasmid was truncated by mutation methods) and HA-tagged wide-type or truncated forms of Fbxo45 on 12mm coverslips prior treatments. After 24 hours, cells were fixed in 4% paraformaldehyde/PBS at room temperature for 20 minutes, permeabilized in 0.2% Triton X-100/PBS for 5 minutes, blocked in 10% goat serum in PBS for 1 hr. First antibodies diluted in 5% goat serum in PBS (rabbit anti-HA 1:250 and mouse anti-Flag 1:500) were incubated at 4°C overnight. Further, cells were washed 3 times with PBS and stained with secondary antibodies diluted in 2% goat serum in PBS (Fluor 488 anti-Rabbit 1:500 and Fluor 568 anti-mouse 1:500) for 1hr at room temperature in the dark. Cells were washed one time and then incubated with 0.1μg/ml DAPI for 5 minutes. Covered slides were observed and taken photography under Nikon microscope.The dual luciferase reporter assays were performed as described before, 64. TheThe soft agar assay for colony formation was performed as described before, 64. Bri4 cells/ml) were mixed and transferred into the 8-well slides (IBIDI) and covered with complete cell culture medium after the gel being solidified. The slides were incubated at 37°C and 5% CO2 cell incubator for 6 to 10 days depending on the cell types, then for observation under microscope.The 3D cell culture assay was performed as described before. BrieflyThe vasculogenic mimicry experiment was performed as described before .This method for analysis of migration was conducted as described previously.Cell migration experiments were performed using the xCELLigence RTCA-DP system  as described previously , 63-64.t-test with triplicate or quadruplicate sets. p<0.05 was considered statistically significant.Experiments were performed at least three times, and representative results were shown. Statistical analysis of qRT-PCR or luciferase assay was performed with a two-tailed unpaired


IL-6) expressing IL-6, and generated IL-6 transgene-engineered DC vaccine (DCOVA/IL-6) by transfection of murine bone marrow-derived ovalbumin (OVA)-pulsed DCs (DCOVA) with AdVIL-6. We then assessed DCOVA/IL-6-stimulated cytotoxic T-lymphocyte (CTL) responses and antitumor immunity in OVA-specific animal tumor model. We demonstrate that DCOVA/IL-6 vaccine up-regulates expression of DC maturation markers, secretes transgene-encoded IL-6, and more efficiently stimulates OVA-specific CTL responses and therapeutic immunity against OVA-expressing B16 melanoma BL6-10OVAin vivo than the control DCOVA/Null vaccine. Moreover, DCOVA/IL-6-stimulated CTL responses were relatively maintained in mice with transfer of CD4+25++Foxp3 Tr-cells, but significantly reduced when treated with anti-IL-6 antibody. In addition, we demonstrate that IL-6 down-regulates Foxp3-expression of CD4+25++Foxp3 Tr-cells in vitro. Taken together, our results demonstrate that AdV-mediated IL-6 transgene-engineered DC vaccine stimulates potent CTL responses and antitumor immunity by counteracting CD4+25+ Tr immunosuppression via IL-6-induced Foxp3 down-regulation. Thus, IL-6 may be a good candidate for engineering DCs for cancer immunotherapy.Dendritic cells (DCs), the most potent antigen-presenting cells have been extensively applied in clinical trials for evaluation of antitumor immunity. However, the efficacy of DC-mediated cancer vaccines is still limited as they are unable to sufficiently break the immune tolerance. In this study, we constructed a recombinant adenoviral vector (AdV CTLs are also essential for effective immunity against tumors .,53.IL-6 boratory  .3.3.OVA.C57BL/6 mouse bone marrow (BM)-derived dendritic cells (DCs) were prepared as described previously . Briefly3.4.+ T cells were purified by using Dynal CD8 microbeads , and CD4+25++Foxp3 Tr cells were then further purified from naïve CD4+ T cell population using biotin-anti-CD25 antibody and anti-biotin MACS beads , as previously described  were stained with PE-labeled H-2Kb/OVA257–264 tetramer and FITC-labeled anti-CD8 antibody on day six after immunization followed by flow cytometric analysis. In another set of experiments, mice were first i.v. transferred with CD4+25++Foxp3 Tr cells (1 × 106 cells/mouse). One day after Tr cell transfer, mice were i.v. immunized with DCOVA/IL-6, DCOVA/Null with or without i.v. treatment of anti-IL-6 antibody (0.5 mg/mL), and the CTL responses were analyzed six days after immunization by flow cytometry. To assess Foxp3 expression, CD4+25++Foxp3 Tr cells were incubated in culture medium with IL-2 (40 units/mL) in the presence or absence of IL-6 (40 ng/mL) for overnight. The cells were permeabilized with cytofix/cytoperm solution  and then stained with PE-Cy5-conjugated anti-Foxp3 antibody followed by flow cytometric analysis.For phenotypic analysis, DC3.7.in vivo cytotoxicity assay was performed as described previously [high) or low  concentrations of CFSE. CFSEhigh cells were further pulsed with OVAI (OVA257–264) peptide (SIINFEKL), and washed extensively to remove free peptide. However, the CFSElow cells were pulsed with the control Mut peptide (FEQNTAQP) to become the internal controls. CFSEhigh and CFSElow target cells were co-injected i.v. at a ratio of 1:1 into the above immunized mice six days after immunization. Sixteen hours after injection, spleens were removed from the immunized mice to analyze the residual OVA-specific CFSEhigh and irrelevant control CFSElow target cells remaining in recipients’ spleens by flow cytometry.The eviously . Briefly3.8.6 OVA-expressing BL6-10OVA tumor cells. Six days after tumor cell injection, mice were vaccinated i.v. with 1 × 106 engineered DCOVA/IL-6, DCOVA/Null, and PBS, respectively. Three weeks after tumor cell challenge, mice were sacrificed, and numbers of lung metastatic tumor colonies were counted. The metastasis on freshly isolated lungs appeared as discrete black pigmented foci that can be easily distinguishable from normal lung tissues and further confirmed by histopathological examination. Tumor metastatic foci too numerous to count were assigned an arbitrary value of >300.For evaluation of therapeutic antitumor immunity, C57BL/6 mice were first challenged by i.v. injection with 1 × 103.9.t-test or Mann-Whitney U test to compare variables from different groups [p < 0.05 is considered significant.Statistical analyses were performed using Student’s t groups . A value4.IL-6 transgene-engineered DC vaccine stimulates potent CTL responses and antitumor immunity by counteracting CD4+25+ Tr immunosuppression via IL-6-induced Foxp3 down-regulation. Thus, IL-6 may be a good candidate for engineering DCs for effective cancer immunotherapy.The results of our study demonstrate that AdV-mediated