IL10 polymorphisms: -3575 T>A (rs1800890), -2849 G>A (rs6703630), -2763 C>A (rs6693899), -1082 G>A (rs1800896) and -592 C>A (rs1800872). Results from meta-analysis supported a significant susceptibility association for the -819T allele, with pooled Odds Ratio of 1.22 (CI = 1.11–1.34) and P-value = 3x10–5 confirming previous data. This result remained unaltered after inclusion of the Brazilian family-based groups . Also, meta-analysis confirmed association of -592 A allele and leprosy outcome . In support of this, linkage disequilibrium analysis in 1000 genomes AFR, EUR, ASN and AMR populations pointed to r2 = 1.0 between the -592C>A and -819C>T SNPs. We found no evidence of association for the other IL10 polymorphisms analyzed for leprosy outcome. Our results reinforce the role of the -819C>T as a tag SNP (rs1800871) and its association with leprosy susceptibility.Leprosy is a chronic infectious disease that depends on the interplay of several factors. Single nucleotide polymorphisms (SNPs) in host immune related genes have been consistently suggested as participants in susceptibility towards disease. Interleukin-10 (IL-10) is a crucial immunomodulatory cytokine in mycobacterial pathogenesis and especially the -819C>T SNP (rs1800871) has been tested in several case-control studies indicating association with leprosy risk, although a recent consensus estimate is still missing. In this study, we evaluated the association of the -819C>T SNP and leprosy in two new Brazilian family-based populations. Then, we performed meta-analysis for this polymorphism summarizing published studies including these Brazilian family-based groups. Finally, we also retrieved published studies for other distal and proximal  Interleukin-10 (IL-10) is mainly secreted by monocytes and lymphocytes and exhibits an important immunomodulatory activity regulating mainly antibody secretion or inflammation, which if sustained could provoke tissue injury during chronic diseases  and andIL10 razilian  were subIL10 polymorphisms . Cochran´s Q statistic . After removing one study at a time to assess its individual influence over the pooled result, we found no evidence of alteration in OR values for neither -3575 T>A (rs1800890), -2849 G>A (rs6703630), -2763 C>A (rs6693899), -1082 G>A (rs1800896) polymorphisms (data not shown).When testing for publication bias results did not indicate funnel plot asymmetry for none of the six tatistic  suggesteP-value from 0.01 to 3x10–5. In the case of the -592 polymorphism, after exclusion of Cardona et al., 2012, P-value was altered to 0.02. Pooled OR values for the -592 polymorphism increased from 1.14 (CI = 0.91–1.43) to 1.24 (CI = 1.03–1.50) after removal of this study from meta-analysis. Therefore, after sensitivity analysis we considered this as an outlier study and decided to remove it from the final quantitative synthesis for -592C>A and -819C>T SNPs detailed in Meta-analysis results  showed aP-value = 3x10–5). After inclusion of the two Brazilian family-based studies data for this SNP summarized 10 studies and pooled results remained similar with OR = 1.20, (95% CI = 1.10–1.31) and P-value = 2x10–5 reinforcing the role of the -819 T allele and leprosy susceptibility (We found significant evidence of association with leprosy susceptibility and the -819 C>T (rs1800871) polymorphism first when considering the available articles from literature , -2849 G>A (rs6703630), -2763 C>A (rs6693899) and -1082 G>A (rs1800896) showed no significant association with leprosy outcome  (In contrast the remaining s >0.05) –S6 Figs.2 = 1) between -819 (rs1800871) and -592 (rs1800872) polymorphisms. Specifically in ASN we also observed high LD between the -1082 (rs1800896) and -2763 (rs6693899) polymorphisms. For both EUR and AMR groups the distal polymorphism -3575 (rs1800890) had moderate LD values (r2>0.6) with —2763 (rs6693899) and -1082 (rs1800896) SNPs.Finally to better understand these results, we performed linkage disequilibrium analysis to test for the presence of bins in the region encompassing these SNPs. LD plots for AFR, EUR, ASN and AMR populations from 1000 genomes  presenteOur goal was to update the previous meta-analysis  which suInterestingly, unrelated household contacts (HC) constituted the control group in the Cardona study, differently from control groups of other reviewed articles that consisted of blood bank (BB) donors or healthy individuals (HI). We cannot directly infer that the control group composition could influence the results, but some cryptic consanguineous relations between controls and cases could introduce bias.IL10 –819C>T and -592C>A were associated with tuberculosis. Both studies pointed to the -819T allele as significantly associated in Asians when subgroup analyses were performed [IL10 polymorphisms did not allow us to explore possible sources of heterogeneity such as subgroup analysis, this, for instance, could allow stratifying multibacillary or paucibacilary groups. It would be interesting that the publication of future studies evaluating such polymorphisms in well-defined clinical groups could aid in confirming results for distal polymorphisms and to perform clinical stratification analysis in proximal IL10 polymorphisms.Recently, two meta-analysis also showed that erformed , 29. Theerformed  differenIL10 promoter polymorphisms and its association with leprosy. A better understanding of the genomic arrangements of this region indicated perfect LD between -819C>T and -592C>A, and, consequently, both SNPs were associated with leprosy.In the present study, we provided an updated pooled OR estimate strengthening previous findings for S1 Meta-Analysis Checklist(PDF)Click here for additional data file.S1 Fig(A) Forest plot for IL10 –592 C>A (rs1800872). Bars represent 95% confidence intervals and boxes represent OR values. (B) Forest plot for IL10 –819 C>T (rs1800871). Bars represent 95% and boxes represent OR values(TIF)Click here for additional data file.S2 Fig(A)IL10 –592 C>A (rs1800872). (B)IL10 –819 C>T (rs1800871)(TIF)Click here for additional data file.S3 Fig(A) Forest plot summarizing association. Bars represent 95% confidence intervals and boxes represent OR values. (B) Funnel plot for publication bias(TIF)Click here for additional data file.S4 Fig(A) Forest plot summarizing association. Bars represent 95% confidence intervals and boxes represent OR values. (B) Funnel plot for publication bias(TIF)Click here for additional data file.S5 Fig(A) Forest plot summarizing association. Bars represent 95% confidence intervals and boxes represent OR values. (B) Funnel plot for publication bias(TIF)Click here for additional data file.S6 Fig(A) Forest plot summarizing association. Bars represent 95% confidence intervals and boxes represent OR values. (B) Funnel plot for publication bias(TIF)Click here for additional data file.S7 Fig(A) African-AFR. (B) European-EUR. (C) Asian-ASN and (D) Amerindian-AMR. Values shown in each box and the intensity of shading are proportional to r2. IL10 –592 C>A (rs1800872) and IL10 –819 C>T (rs1800871) promoter polymorphisms are shown with *.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(DOC)Click here for additional data file.


Therapeutic hypothermia (HT) is standard treatment after perinatal hypoxic-ischemic (HI) injury. However, around 50% of asphyxiated newborns still suffer poor outcome, some of which may have been exposed to perinatal infection prior to HI, as infection may increase vulnerability to HI injury.To develop an infection-sensitized newborn animal model of HI injury and to investigate whether HT is neuroprotective in this setting.Seven day old rat pups were injected either with vehicle (NaCl 0.9%) or E.coli lipopolysaccharide (LPS). Subsequently, they were exposed to left carotid ligation followed by global hypoxia inducing a unilateral HI injury. Pups were randomized to the following treatments: 1) Vehicle treated HI-pups receiving normothermia treatment (NT) (Veh-NT); 2) LPS treated HI-pups receiving NT treatment (LPS-NT); 3) vehicle treated HI-pups receiving HT treatment (Veh-HT); or 4) LPS treated HI-pups receiving HT treatment (LPS-HT). Relative area loss of the left/right hemisphere and the areas of hippocampi were measured after one-week survival.Mean brain area loss in the Veh-NT group was 11.2%±14%. The brain area loss in LPS-NT pups was 29.8%±17%, being significantly higher than in the Veh-NT group (p = 0.002). The Veh-HT group had a significantly smaller brain area loss (5.4%±6%) when compared to Veh-NT group (p = 0.043). The LPS-HT group showed a brain area loss of 32.5%±16%, being significantly higher than in the Veh-HT group (p < 0.001). LPS-HT group also had significantly smaller size of the left hippocampus. LPS-sensitization significantly decreased the sizes of the right, unligated-hemispheres, independent of post-HI treatment.Therapeutic hypothermia is not neuroprotective in this infection-sensitized HI brain injury model. Lack of neuroprotection was particularly seen in the hippocampus. The exact mechanisms remain unclear and further investigation is needed to explain why a neuroprotective therapy loses its efficacy and how hypothermia may be optimized.


Caenorhabditis elegans gene tbx-2 to understand how T-box gene expression is regulated, and here we demonstrate TBX-2 itself directly represses its own expression in a negative autoregulatory loop. tbx-2 is essential for normal pharyngeal muscle development, and a tbx-2 promoter gfp fusion (Ptbx-2::gfp) is transiently expressed in the pharynx during embryogenesis and in a small number of head neurons in larvae and adults. Reduced tbx-2 function resulted in ectopic Ptbx-2::gfp expression in the seam cells and gut in larvae and adults. Mutation of potential T-box binding sites within the tbx-2 promoter resulted in a similar pattern of ectopic Ptbx-2::gfp expression, and chromatin immunoprecipitation analyses show TBX-2 binds these sites in vivo. This pattern of ectopic Ptbx-2::gfp expression in tbx-2 mutants was very similar to that observed in mutants affecting the NF-Y complex, and our results comparing tbx-2 and nfyb-1 single- and double mutants suggest TBX-2 and NF-Y function in a single pathway to repress the tbx-2 promoter. The tbx-2 promoter is the first direct target identified for TBX-2, and we used it to ask whether SUMOylation is essential for TBX-2 repression. RNAi knockdown of SUMOylation pathway components led to ectopic Ptbx-2::gfp expression in the seam cells and gut. Ectopic Ptbx-2::gfp also was observed in the syncytial hypodermis, suggesting either the tbx-2 promoter is repressed by other SUMOylation dependent mechanisms, or that decreased SUMOylation leads to stable changes in seam cell nuclei as they fuse with the syncytial hypodermis. We suggest negative autoregulation is an important mechanism that allows precise control of tbx-2 expression levels and may allow rapid changes in gene expression during development.T-box genes often exhibit dynamic expression patterns, and their expression levels can be crucial for normal function. Despite the importance of these genes, there is little known about T-box gene regulation. We have focused on the  Tbx2 and Tbx3 genes are expressed at various developmental stages in tissues as different as the allantois, heart, limbs, eye, mammary gland, and brain , OK0857 proxcuEx686[Ptb-2::gfp], OK0912 distcuEx730[Ptbx-2::gfp], OK0914 distcuEx732[Ptbx-2::gfp], OK0916 prox+distcuEx734[Ptbx-2::gfp], OK0919 prox+distcuEx737[Ptbx-2::gfp], OK0873 tbx-2(ok529); wgIs159, OK0814 nfyb-1(cu13) II (nfyb-1(cu13) II; tbx-2(bx59) III, OK0898 nfyb-1(cu13) II; tbx-2(bx59) III; cuIs25 X, and OK1030 tbx-2(ok529)/dpy-17(e164) unc-32(e189) III; cuIs25 X. OK1029 nfyb-1(cu13) II; cuIs25 X. Newly constructed strains containing tbx-2(ok529), tbx-2(bx59), or nfyb-1(cu13) were molecularly genotyped as previously described (nditions . Germlin0 ng/μL) . The folPtbx-2::gfp plasmid pOK206.33 (AGGTGGCA to ATTGTGC) using the Stratagene QuikChange II kit to produce proxPtbx-2::gfp plasmid pOK255.04. A 42-bp fragment (−1157 to −1116 upstream of the tbx-2 ATG) containing the pair of distal T-box sites was deleted by inverse polymerase chain reaction (PCR) of pOK206.33 or pOK255.04 with primers PO1256 [GACTCTAGAAGTGATAAGAAGCCGCGAG] and PO1257 [GACTCTAGATGCAGCACTGAATTGATGA], digested with XbaI, and recircularized by ligation, to produce distPtbx-2::gfp plasmid pOK278.02 and prox+distPtbx-2::gfp plasmid pOK278.03, respectively. All plasmids were sequenced to verify the presence of the mutations.Standard methods were used to manipulate plasmid DNAs and oligonucleotides , and allTBX-2 binding sites were identified by scanning the Ptbx-2 promoter with the WormBase function Annotate Sequence Motif using the GBrowse plugin MotifFinder  with a T-box half-site position frequency matrix derived from JASPAR MA0009.1 (jaspar.genereg.net) at a threshold of 0.85.Candidate A [40 0 0 0 0 0 1 40 31 ]C [ 0 0 0 0 0 2 7 0 5 ]G [ 0 40 40 0 40 0 28 0 0 ]T [ 0 0 0 40 0 38 4 0 4 ]the_brain.bwh.harvard.edu/uniprobe/) with reduced threshold identified the second distal T-box site at −1135 (ATGTGTGA).This position frequency matrix is nearly identical to those experimentally derived for T-box factors from the mouse and two echinoderm species (Escherichia coli strains expressing dsRNA from transcription factor genes on OK0592cuIs23[Ptbx-2::gfp] III or OK0969 rrf-3(pk1426) II; cuIs23 III at 20° or 16°, respectively, as previously described (in vitro transcription (Ambion) of cDNA from tbx-2 (yk112c4/pOK165.07) was injected into OK0592cuIs23[Ptbx-2::gfp] strain at approximately 200−500 ng/μL (Ptbx-2::gfp expression.The feeding RNAi screen was performed with the use of N2 and tbx-2(bx59) L1 larvae were prepared by bleaching mixed-stage animals and allowing the isolated embryos to grow 12 hr on OP50 seeded NGM plates at 20° (tbx-2  and the control mRNA ama-1  in real-time PCR reactions. Three to four replicates were performed for each RNA sample, and three independently isolated RNA samples were assayed for each genotype. The tbx-2 mRNA level relative to that of ama-1 was compared in N2 and tbx-2(bx59) animals using the 2-ΔΔCt method ; wgIs159 animals as described  at 30% amplitude for 8 cycles of 12-se pulses , with cooling between cycles, and 10% of the lysate was saved for input. Preclearing of the lysate was performed twice, once with Dynabeads protein G (Invitrogen) bound to 2.5 μg of IgG preserum followed by preclearing with Dynabeads alone. Immunoprecipitation reactions containing approximately 2−3 mg of total protein were performed with 3 μg of anti-green fluorescent protein  or IgG preserum (Invitrogen 02-6502). No antibody controls were performed in parallel to experimental assays. Three independent assays were performed for each strain.ChIP assays were performed on mixed-stage sax-2  were used.Crosslinking was reversed and the precipitated DNA was analyzed by semiquantitative PCR for 33 and 35 cycles to verify linear amplification. Primers flanking the proximal T-box site , the distal T-box sites , or rsb.info.nih.gov/ij/). Gel images were inverted and background was subtracted with a rolling ball radius of 20. Enrichment of DNA was measured by averaging anti-GFP/Input or IgG/Input. Fold difference was calculated by difference of averaged GFP over IgG. Statistical significance was determined by two-tailed t-test with unequal variance comparing fold enrichment of the proximal or distal T-box sites to relative to sax-2 in N2 and tbx-2(ok529); wgIs159 using Microsoft Excel.DNA was electrophoresed on 2–2.8% agarose gels and band intensities were measured using ImageJ   . In wilddulthood  (Roy ChoPtbx-2::gfp expression in tbx-2(RNAi) animals and tbx-2 mutants. Although elimination of tbx-2 results in severe pharyngeal defects and L1 arrest, partially affected animals grow to adulthood (tbx-2(RNAi) animals and mutants homozygous for the temperature-sensitive hypomorphic allele tbx-2(bx59) exhibited highly penetrant ectopic Ptbx-2::gfp expression in the seam cells and gut in late larval and adult stages (tbx-2(bx59) mutants exhibited this ectopic expression at both the permissive and nonpermissive temperatures , suggesting that even a relatively small reduction in TBX-2 activity derepresses the tbx-2 promoter. This ectopic expression is specific for Ptbx-2::gfp, as we have examined expression of numerous other gfp reporters in tbx-2(bx59) mutants, including those regulated the D2096.6, T25E4.1, pqn-71, myo-5, nmgp-1, T12A7.6, cpn-4, and F41H10.5 promoters, but have never observed ectopic expression in the seam cells and gut [(We more carefully examined t stages . tbx-2(btbx-2(ok529) null mutants are completely viable as heterozygotes (tbx-2(ok529)/+ heterozygotes exhibited ectopic Ptbx-2::gfp expression in the seam cells (tbx-2(ok529) also exhibits a mild haploinsufficient phenotype.In humans, T-box gene mutations often are haploinsufficient, with the loss of one functional allele underlying congenital diseases such as Holt-Oram and DiGeorge syndromes . Althougam cells . No ectotbx-2(ok529) homozygotes and approximately half of tbx-2(bx59) animals grown at the nonpermissive temperature arrest as L1 larvae, and we examined Ptbx-2::gfp expression in these L1 animals. At this stage, no ectopic Ptbx-2::gfp was detectable in the seam cells, and only occasional expression was observed in the gut. However, these animals exhibited a strong up-regulation of Ptbx-2::gfp expression in the head neurons where Ptbx-2::gfp is normally expressed  L1 animals grown at the nonpermissive temperature. We found tbx-2 mRNA was overexpressed fourfold in tbx-2(bx59) compared with the wild type (tbx-2 mRNA levels are increased in tbx-2(bx59) mutants  or deletion of a pair of closely linked distal sites (Ptbx-2::distgfp) led to ectopic expression in seam cells and gut in a pattern similar to that observed when wild-type Ptbx-2::gfp expression was examined in tbx-2 mutants and tbx-2(RNAi) animals  would exhibit an additive expression pattern, but prox+distPtbx-2 was expressed in a pattern very similar to the Ptbx-2::distgfp single mutant, with very frequent expression in the gut but very little expression in the seam cells  . The modENCODE Consortium has used similar transgenes to map transcription factor binding sites throughout the genome by ChIP using anti-GFP antibodies (The modENCODE Consortium provided a tbx-2(ok529); wgIs159 hermaphrodites and found TBX-2 specifically binds both the proximal and distal sites in the tbx-2 promoter  and smo-1(RNAi)  . We obsePtbx-2::gfp expression in seam cells and the gut in a pattern similar to what we observed in tbx-2(RNAi) and tbx-2 mutants, and we did observe Ptbx-2::gfp expression in both of these tissues in ubc-9(RNAi) and gei-17(RNAi) animals (Ptbx-2::gfp expression was found in the seam cells of smo-1(RNAi) animals but was not observed in the gut. Surprisingly, we also observed frequent ectopic Ptbx-2::gfp expression in the nonseam, syncytial hypodermis in ubc-9(RNAi) and smo-1(RNAi) animals, and at a low frequency in gei-17(RNAi) animals ; tbx-2(bx59) double mutants with that of wild-type animals and tbx-2(bx59) and nfyb-1(cu13) single mutants. nfyb-1(cu13); tbx-2(bx59) double mutants exhibit temperature-sensitive, synthetic lethality at the L1 stage, so we examined expression in L1 animals grown at the nonpermissive temperature (25°).We have previously shown that the NF-Y complex also represses Ptbx-2::gfp was expressed most frequently in head neurons with less frequent expression in the anterior-most seam cell H0 and the syncytial head hypodermal cell hyp6 (tbx-2(bx59) and nfyb-1(cu13) single mutants exhibited a strong increase in the level of Ptbx-2::gfp expression in head neurons  mutants exhibited Ptbx-2::gfp expression in H0 and the gut. The nfyb-1(cu13); tbx-2(bx59) double mutant expressed Ptbx-2::gfp in a pattern that was very similar to the nfyb-1(cu13) single mutant, with very strong expression in the head neurons and frequent expression in the gut ; tbx-2(bx59) double mutant does not result from synergistic increase in tbx-2 promoter activity.In wild-type L1s, ell hyp6 . Both tb neurons . In addionstruct . Notably the gut . There wTBX-2 binds sites within its own promoter and negatively autoregulates its own expression. Knockdown of TBX-2 activity or mutation of T-box binding sites within the promoter leads to ectopic Ptbx-2::gfp expression in the hypodermal seam cells and the gut of larvae and adults and increased expression in head neurons in the L1 stage. TBX-2 protein binds these sites in the tbx-2 promoter in vivo, indicating this negative autoregulation is direct. This regulation also affects endogenous tbx-2 expression as tbx-2 mRNA is increased in tbx-2 mutants, although genome editing experiments could be done to verify this regulation occurs through the sites we have identified using Ptbx-2::gfp . SUMOylation is required for tbx-2 repression, as RNAi knockdown of the UBC-9 E2 SUMO-conjugating enzyme, the GEI-17 E3 SUMO-ligase, or the SMO-1 SUMO peptide also resulted in ectopic Ptbx-2::gfp expression. These studies identify tbx-2 as the first target regulated by TBX-2, and they demonstrate directly that TBX-2 functions as a transcriptional repressor whose function in vivo depends on SUMOylation. We note that using the Ptbx-2::gfp reporter as a molecular readout for TBX-2 activity facilitated our ability to detect defects in negative autoregulation, because it uncouples increased promoter activity from increased TBX-2 repressor activity.Here we show that E. coli  hypomorphic mutant, indicating SUMOylation is crucial for TBX-2 function.Our previous results led us to hypothesize that tbx-2 promoter as a direct readout of TBX-2 repressor activity to provide additional genetic evidence that TBX-2 requires SUMOylation in vivo. We found that knocking down expression of ubc-9, smo-1, or gei-17 derepresses Ptbx-2::gfp expression similarly to what we observed in tbx-2 mutants and tbx-2(RNAi). Although these results are consistent with our hypothesis, we cannot rule out the possibility that other SUMO-dependent mechanisms repress tbx-2. We did observe tissue specific differences in this derepression in these experiments. For example, while tbx-2(RNAi), ubc-9(RNAi) and gei-17(RNAi) resulted in ectopic Ptbx-2::gfp expression in both seam cells and the gut, smo-1(RNAi) resulted in expression in seam cells but not in gut. This difference might result from less efficient RNAi knockdown of smo-1 in gut than in seam cells. More interestingly, we also observed ectopic expression of Ptbx-2::gfp in the syncytial hypodermis when SUMOylation was knocked down, particularly in ubc-9(RNAi) or smo-1(RNAi) animals, but this expression was only very rarely observed in tbx-2 mutants. This pattern of ectopic Ptbx-2::gfp expression may indicate that additional SUMO-dependent mechanisms repress expression in the syncytial hypodermis. Such mechanisms could involve transcription factors that directly repress Ptbx-2::gfp expression in the syncytial hypodermis, or mechanisms that indirectly affect Ptbx-2::gfp by affecting normal seam cell or hypodermal differentiation. These mechanisms could affect factors such as the PcG component SOP-2 or the nuclear hormone receptor NHR-25, which are SUMOylated and play important roles in seam cell and hypodermal differentiation ; tbx-2(bx59) double mutants compared to each of the single mutants, strongly suggesting NF-Y and TBX-2 function together to repress Ptbx-2::gfp expression rather than through independent, parallel pathways. NF-Y has been suggested to function as a “pioneer” factor that binds its sites in chromatin, regardless of its modification state, and recruits histone modifiers to facilitate binding of other transcription factors ; tbx-2(bx59) double mutants exhibit a highly penetrant synthetic lethal phenotype indicating these factors have common functions in vivo, but we do not yet know the cause of this lethality (TBX-2 and NF-Y regulation leads to double mutant lethality. This and other hypotheses can be tested by identifying and characterizing addition genes regulated by these factors.


In vitro anti-inflammatory actions of IL-6 are mediated through suppression of interferon gamma (IFNγ) [The post-operative period is characterised by increased IL-6 production and clinical features of immune suppression. a (IFNγ) . The cliTo evaluate the role of IL-6 pathways in post-operative immune suppression and the reversibility of this phenomenon.+HLA-DR (mHLA-DR) geometric mean florescent intensity (MFI) measured in the presence and absence of interferon gamma (IFNγ) and IL-6 neutralising antibody. Data were analysed with non-parametric statistics.Patients over 45 years old undergoing elective surgery involving the gastrointestinal tract and requiring at least an overnight hospital stay were recruited. The primary outcome was hospital-acquired infection. IL-6 and IFNγ levels were assayed using ELISA preoperatively and at 24 and 48 hours. Pooled healthy control peripheral blood mononuclear cells (PBMCs) were cultured in perioperative serum and CD14P < 0.0001, Figure P = 0.06, Figure P = 0.009). Higher preoperative IL-6 levels were observed in patients with cancer (P = 0.02). IL-6 levels at 24 (P = 0.0002) and 48 hours (P = 0.003) were associated with the later occurrence of infectious complications. This pattern remained similar after adjustment for baseline characteristics. Healthy donor PBMCs incubated with postoperative serum downregulated mHLA-DR MFI when compared with serum from baseline . Culturing in the presence of IFNγ 250IU (n = 4) prevented this decrease whereas culturing in the presence of IL-6 neutralising antibody 15ng/ml (n = 8) did not.119 patients were recruited and 44 (37%) developed a post-operative infection a median of 9 (IQR 5-11) days postoperatively


Adenocarcinoma, the most common non-small cell lung cancer is a leading cause of death worldwide, with a low overall survival (OS) despite increasing attempts to achieve an early diagnosis and accomplish surgical and multimodality treatment strategies. Cellular retinol binding protein-1 (CRBP-1) regulates retinol bioavailability and cell differentiation, but its role in lung cancerogenesis remains uncertain.CRBP-1 expression, clinical outcome and other prognostic factors were investigated in 167 lung adenocarcinoma patients. CRBP-1 expression was evaluated by immunohistochemistry of tissue microarray sections, gene copy number analysis and tumor methylation specific PCR. Effects of CRBP-1 expression on proliferation/apoptosis gene array, protein and transcripts were investigated in transfected A549 lung adenocarcinoma cells.High expression was observed in 62.3% of adenocarcinomas and correlated with increased tumor grade and reduced OS as an independent prognostic factor. CRBP-1 gene copy gain also associated with tumor CRBP-1High status and dedifferentiation. CRBP-1-transfected (CRBP-1+) A549 grew more than CRBP-1− A549 cells. At >1μM concentrations, all trans-retinoic acid and retinol reduced viability more in CRBP-1+ than in CRBP-1− A549 cells. CRBP-1+ A549 cells showed up-regulated RARα/ RXRα and proliferative and transcriptional genes including pAkt, pEGFR, pErk1/2, creb1 and c-jun, whereas RARβ and p53 were strongly down-regulated; pAkt/pErk/ pEGFR inhibitors counteracted proliferative advantage and increased RARα/RXRα, c-jun and CD44 expression in CRBP-1+ A549 cells.CRBP-1High expression in lung adenocarcinoma correlated with increased tumor grade and reduced OS, likely through increased Akt/Erk/EGFR-mediated cell proliferation and differentiation. CRBP-1High expression can be considered an additional marker of poor prognosis in lung adenocarcinoma patients.CRBP-1 Non-small-cell lung cancer (NSCLC) is a leading cause of cancer death worldwide . Lung caHigh expression was detected in 62.3%. EGFR and Ki-67 expression were comparable to the literature [High expression was detected in 51.85% of tumors. Immunohistochemistry also documented that CRBP-1 expression was absent in non-neoplastic lung parenchyma and present in bronchial epithelium . As concerning the tumor grade, CRBP-1High status associated to the loss of differentiation of lung adenocarcinomas (p<0.001).Representative immunostainings of CRBP-1 expression and other lung adenocarcinoma biomarkers are reported in figure terature ,18. CRABithelium . CRBP-1 ithelium . In partHigh expression in lung adenocarcinoma associated with reduced patients' OS (p<0.01). High RARα, RARβ and Ki-67  and low CRABP-2 and EGFR expression  also correlated with lower OS, similarly to that reported in the literature [High and CRBP-1High coexpression associated with reduced OS (p<0.01). CRBP-1High expression strongly also associated with reduced OS in smoker (p<0.001) but not in no-smoker patients. Multivariate analysis .As reported in figure terature . As repo OS p<0.0. High RAHigh correlated positively with EGFRHigh expression , and negatively with p53 expression . CRABP-2High inversely correlated with bcl-2High expression  and patients' age . An inverse correlation between EGFR and bcl2 expression  also existed. As reported in High was increased and almost double compared with CRBP-1Low tumors. Instead the percentage of nox4 expression was similar. Screening for TK domain .CRBP-1High status and dedifferentiation . Since CRBP-1 expression was absent in a subset of tumors, we analyzed the methylation of the promoter region flanking the CRBP-1 gene .As reported in P-1 gene . Promote+ grew more than CRBP-1− A549 cells. Cell count gave similar results (not shown). At concentrations >1μM  slightly increased compared to CRBP-1− cells (58.13% ± 2.92%), although the difference was not significant. Treatments with atRA and ROL reduced clonogenicity in both CRBP-1+ and CRBP-1− A549 compared to control cultures (data not shown).A mammalian CRBP-1-expressing vector was used to generate stable transfectant A549 lung adenocarcinoma cell lines. Wild A549 cells did not display appreciable CRBP-1 mRNA and protein levels . After 6M figure , atRA an+ compared to CRBP-1− A549 cells; keratin 7 level was unchanged. Keratin 5 and 14 up-regulation was confirmed by blots (data not shown). CRBP-1+ A549 cells also showed the strong up-regulation of nox4 and CD44 expression compared to CRBP-1− A549 cells  construction, tissue samples from diagnostic biopsies and operative procedures were obtained from representative paraffin blocks maintaining patients' anonymity. All tumor slides were reviewed by light microscopy examination of Haematoxylin&Eosin (H&E)-stained sections. The most representative tumor areas were carefully selected and TMA constructed using positive and negative controls . Serial For immunohistochemistry, sections were incubated with mouse monoclonal anti-human Ki-67 (clone 30-9), bcl2 (clone 124), p53 (clone DO-07), EGFR (clone 3C6), keratin 5/6 (clone D5/16B4) and keratin 14 (clone SP53) antibodies using an automatic immunostaining device  . Serial CRBP-1 gene copy number was analyzed by using real-time PCR and TaqMan Genotyping Master Mix (n. 4317355), with RNase control reagents n. 4316844) and CRBP-1  as probe. PCR amplification was performed in an ABI PRISM 7500 (Applied Biosystem) according to manufacturer's instructions. Data analysis was performed using the manufacturer's integrated web-based software package. Genomic DNA, methylation specific PCR and copy number assay were performed as reported [44 and CREGFR mutational status was analized by pyrosequencing . The TK +, Promega, Italy), or the G418-resistance gene alone (CRBP-1-). After 20 days, stable transfected clones were collected and tested by PCR and western blot. The correct plasmid sequence was confirmed by Sanger sequencing. Experimental procedures were repeated by using two different transfected clones, which gave similar results (not shown).Human A549 adenocarcinoma cells  maintained in RPMI 1640  were transfected  by usinatRA and ROL (Sigma-Aldrich) in 0.1% FBS up to 6 days. For cell viability, 3--2,5diphenyl-tetrazolimbromide assay  was carried out in triplicate , phosphatidylinositol 3-kinase/ AKT  and the mitogen-activated protein kinase kinase  inhibitors.For proliferation studies, overnight serum-starved cells were treated with different concentrations of plicate  in 0.1% methanol and their percentages as plating efficiency (PE) calculated.For the clonogenic assay , anti-AKT (pan), anti-phosphorylated extracellular-signal-regulated kinases (pErk1/2), anti-phosphorylated epidermal growth factor receptor (anti-EGFR Thr669), anti-EGFR antibody , anti-keratin 5 , mouse anti-vimentin , anti-keratin 14  and anti-total tubulin antibody (Sigma-Aldrich). Revelation and densitometric blot analysis were performed in three independent experiments and Akt and EGFR activity expressed as phospho/total protein ratio [After isolation, content determination and electrophoresis, proteins were elettroblotted  and incut fold-change calculation (Qiagen) in triplicate experiments.Total RNA was extracted , reverseHigh expression [t test. Univariate analysis of relationship among CRBP-1 and clinicopathological variables was performed by using χ2 test, whereas correlations between CRBP-1 and the other biomarkers by Spearman's rank correlation test. Survival curves were analyzed by Kaplan-Meier method and significant differences between subgroups were calculated by the log-rank test. Independent prognostic factors were identified by multivariate analysis using Cox proportional hazards model. Only factors showing prognostic significance in univariate analysis were adopted in multivariate analysis. Differences were considered statistically significant for value of p<0.05. SPSS 16 software program  was used for statistical analysis.CRBP-1, CRABP-2, nox4 and keratin expression was estimated at 400X magnification by two of the Authors by using the following semiquantitative grading system: absent (0), weakly positive <50% (0.5), moderately positive <50% or weakly positive >50% (1), strongly positive <50% or moderately positive >50% (2), strongly positive >50% (3), as reported . Inter-opression . For eac


PCTAIRE1 sensitized prostate cancer PPC1 and Du145 cells, and breast cancer MDA-MB-468 cells to TNF-family cytokines, including TNF-related apoptosis-inducing ligand (TRAIL). Meanwhile, PCTAIRE1-knockdown did not sensitize non-malignant cells, including diploid fibroblasts IMR-90 and the immortalized prostate epithelial cell line 267B1. PCTAIRE1-knockdown did not up-regulate death receptor expression on the cell surface or affect caspase-8, FADD and FLIP expression levels. PCTAIRE1-knockdown did promote caspase-8 cleavage and RIPK1 degradation, while RIPK1 mRNA knockdown sensitized PPC1 cells to TNF-family cytokines. Furthermore, the kinase inhibitor SNS-032, which inhibits PCTAIRE1 kinase activity, sensitized PPC1 cells to TRAIL-induced apoptosis. Together these results suggest that PCTAIRE1 contributes to the resistance of cancer cell lines to apoptosis induced by TNF-family cytokines, which implies that PCTAIRE1 inhibitors could have synergistic effects with TNF-family cytokines for cytodestruction of cancer cells.While PCTAIRE1/PCTK1/Cdk16 is overexpressed in malignant cells and is crucial in tumorigenesis, its function in apoptosis remains unclear. Here we investigated the role of PCTAIRE1 in apoptosis, especially in the extrinsic cell death pathway. Gene-knockdown of  The N-tc domain , while iWe recently discovered that PCTAIRE1 plays an indispensable role in cancer cell proliferation ,10. We aApoptosis induced by TRAIL, Fas-ligand (FasL) and TNF-alpha proceeds through a series of receptor-mediated protein interactions that minimally require the adapter protein FADD and cysteine proteases such as caspase-8 or-10. While these death receptor signaling complex components are retained in most cancers, resistance to apoptosis remains common. FADD and caspase-8 are among the mediators of the extrinsic pathway that are known to be modulated by protein phosphorylation, which suggests a role for kinases in resistance to pro-apoptotic TNF-family cytokines. Protein kinases are also attractive targets for cancer drug discovery. Moreover, considerable evidence has suggested a role for protein phosphorylation in modulating proximal signaling events induced by TNF-family death receptors –19, as wPCTAIRE1 sensitized prostate and breast cancer cells to TNF-family cytokines, including TNF-related apoptosis-inducing ligand (TRAIL) and Fas, but did not sensitize normal or non-transformed cells to TRAIL. PCTAIRE1-knockdown promoted caspase-8 cleavage and degradation of receptor-interacting serine-threonine protein kinase 1 (RIPK1). The siRNA-mediated knockdown of RIPK1 mRNA also sensitized PPC1 cells to TNF-family cytokines. Our findings suggest that PCTAIRE1 could be an important target for cancer therapy, and that PCTAIRE1 inhibitors could have synergistic effects with TNF-family cytokines to kill cancer cells.In this study, we further characterized the role of PCTAIRE1 in cancer cells, and particularly its function in the extrinsic cell death pathway. We provide evidence suggesting that PCTAIRE1 plays a crucial role for resistance to TNF-family cytokines in cancer cells. Gene knockdown of The cell viability assay kit Cell Titer Glo was purchased from Promega. RNAiMAX was obtained from Life Technologies. Pre-designed short interfering-RNA (siRNA) directed against human PCTAIRE1 , PCTAIRE2 (s10160), PCTAIRE3 (s10162), caspase-8 (s2427), RIPK1 (s16653), p27 (s2837) and scramble-control  were purchased from Life Technologies. Recombinant TRAIL was purchased from Enzo Bioscience. SNS-032 was purchased from Selleck Chemicals. Antibodies against PCTAIRE1 , RIPK1 , caspase-8 , cleaved caspase-8 , phospho-caspase-8 , FADD , phospho-FADD , Fas/CD95 , DR4 , DR5 , FLIP , phospho-Serine , p27 , HA , HA , Myc , beta-actin , alpha-tubulin  and horseradish-peroxidase-conjugated secondary antibodies  were purchased from the indicated sources.Z-score was then assigned to each siRNA. P values were also calculated for each siRNA using a 2-tail T-test assuming a normal distribution of the data. To produce a final gene score, we averaged the Z values of the 2 or 3 siRNAs targeting each gene as well as a P value using viability ratios. Values for siRNAs showing high standard deviation (> 0.25) among duplicates of either DMSO or CH-11 treated cells were removed from the analysis.The Ambion Silencer Human Druggable Genome siRNA Library V2 was delivered into PPC1 cells by reverse transfection in 384 well format at 10 nM siRNA using RNAiMAX (Life Technologies). After 48 hours, cells were treated with 3 ng/ml anti-FAS antibody (CH-11) or DMSO for an additional 24 hours, time after which viability was read using ATP-lite (Perkin Elmer) in an Envision Plate reader (Perkin Elmer Inc.). Raw data readings were normalized to the average of negative controls included in each plate and duplicates averaged. The Effect of FAS activation was measured by calculating viability ratios CH-11/DMSO for each siRNA. A robust Pfu polymerase (Agilent). Expression plasmids for various proteins were constructed in the pcDNA3 vector for transfection or pRDI292-puro vector for lentivirus infection. Appropriate plasmid construction was confirmed by restriction enzyme digestion and DNA sequencing. The details of the primer sequences used to generate the point mutations are available upon request.Point mutations of PCTAIRE1 (K194M) was made with a PCR-based site-directed mutagenesis method using PPC1, Du145, MDA-MB-468, T47D, MCF7, IMR-90, HeLa and HEK293T cells were purchased from ATCC. 267B1 and 267B1/K-ras cells were kind gifts from Dr. Dritschilo [Cell Titer Glo was used for cell viability estimation. Cells were plated in 96-well solid white plates at a density of 5,000 ~ 10,000 cells per well in 100 μl complete medium with or without siRNAs and cultured for 48 hours. The cells were then treated with various concentrations of cytokines or compounds for 24 hours. Cell Titer Glo solution was added at 100 μl per well and the plates were kept in the dark for 15 minutes before measuring luminescence with a luminometer .Cells were processed using an AnnexinV-PI apoptosis detection kit according to the manufacturer’s protocol (Life Technologies) and analyzed by flow cytometry using a FACS Canto device (Becton Dickinson).For transient knockdown, cells were transfected with siRNA duplexes by a reverse transfection method using Lipofectoamine RNAiMAX according to the manufacturer’s instructions (Life Technologies).PCTAIRE1 shRNA#1 (GCTCTCATCACTCCTTCACTT), PCTAIRE1 shRNA#2 (GACCTACATTAAGCTGGACAA), and scramble-control (CAACAAGATGAAGAGCACCAA) were cloned into the inducible pLKO-Tet-On puromycin vector . LentiviTotal RNA was isolated from cultured cells using the RNeasy plus mini kit (Qiagen). The RNA concentration was measured spectrophotometrically and samples were stored at-80°C until use with RT-PCR. RNA was reverse-transcribed using Superscript III (Life Technologies) and complementary DNA samples were analyzed with the SYBR green system (Promega). Primers specific for human PCTAIRE1, PCTAIRE2, PCTAIRE3, RIPK1 and control housekeeping human GAPDH are listed below.Human PCTAIRE1Forward: 5′- GCAGTGACCCTGGAGAGG-3′Reverse: 5′- TCAAGTCCTCGTGCACAATC-3′Human PCTAIRE2Forward: 5′- TGTTATTGGAGGGAGCCTTG-3′Reverse: 5′- TCTCACCATCTGATGCCATTT-3′Human PCTAIRE3Forward: 5′- ATGGCATCCACCTCCTGA-3′Reverse: 5′- TCTGCTGACATGCGACTCTT-3′Human RIPK1Forward: 5′- GTGTACAAGGGGCCCAACT-3′Reverse: 5′- CGGCTGTGTCTCAGTCTGTT-3′Human GAPDHForward: 5′- GAAGGTGAAGGTCGGAGTC-3′Reverse: 5′- ATGGGATTTCCATTGATGAC-3′All experiments were performed in duplicate and normalized relative to GAPDH levels.g for 10 minutes. Protein concentrations were measured using a Bio-Rad protein assay kit (Bio-Rad). For Laemmli SDS-PAGE, proteins were separated on SDS-PAGE 4–15% gradient gels (Life Technologies) and transferred onto nitrocellulose membranes (Bio-Rad). Phos-tag SDS-PAGE was performed with precast phostag gels . After electrophoresis, phos-tag acrylamide gels were washed with running buffer  containing 5 mM EDTA for 10 min with gentle shaking, and then washed again with running buffer without EDTA for 10 min according to the manufacturer’s protocol . Proteins were transferred to nitrocellulose membranes. After blocking for 1 hour in Tris-buffered saline (TBS) with 0.05% Tween-20 and 5% non-fat dry milk, membranes were incubated overnight at 4°C with primary antibodies diluted in blocking buffer. Membranes were rinsed three times in TBS with 0.05% Tween-20 and incubated with secondary HRP-conjugated antibodies for 1 hour at room temperature. An enhanced chemiluminescence (ECL) method  was used for detection.Cells were washed twice with PBS and harvested with radioimmunoprecipitation assay (RIPA) buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 1% Nonidet P-40, 0.1% SDS, 5 mM NaF and EDTA-free cOmplete protease cocktail tablet (Roche). Cells were left on ice for 20 minutes and centrifuged at 14,000 x For immunoprecipitation (IP), cells were lysed in 1% NP40 buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 1% Nonidet P-40, 5 mM NaF and EDTA-free cOmplete protease cocktail tablet. Three milligrams of protein lysate were used for immunoprecipitation with overnight incubation at 4°C and 2 μg of the given antibody. The following day, 30 μL of protein G resin (Life Technologies) was added and incubated for 1 hour at 4°C. The IPs were then washed four times with lysis buffer whereupon sample buffer was added, and the beads were boiled for 10 minutes at 100°C.5 cells per well and then transfected with control or PCTAIRE1-targeting siRNAs. After 2 days, agonistic anti-Fas ab (CH-11) or TRAIL was added and the cells were cultured for 3 days before fixing and staining with 0.5% crystal violet dye. For fixation, cells were incubated with methanol at-20°C for 20 minutes, washed with PBS, and incubated with 0.5% crystal violet dye in 25% methanol for 15 minutes.Cells were seeded in 35 mm dishes at 1.0 x 105) that were reverse-transfected with siRNAs targeting PCTAIRE1 and scramble-control were seeded in 6 cm dishes. Forty-eight hours after reverse-transfection, adherent cells were washed once with PBS, detached using TripleExpress (Gibco), and resuspended in ice-cold FACS buffer (1% FBS in PBS). Following centrifugation, cells were resuspended in FACS buffer at 3.0 x 105 cells/100 μl. The cells were then incubated in the dark on ice with saturating concentrations of phycoerythrin-labeled anti-DR4, anti-DR5 (eBioscience), FITC-labeled anti-Fas (BD Pharmagen), or immunoglobulin G1 isotype control antibodies for 1 hour as per the manufacturer’s instructions. A total of 10,000 events were analyzed by flow cytometry for each treatment.PPC1 and MDA-MB-468 cells  were calculated statistically from three determinations. The data are expressed as mean ± SD. Statistical significance of differences between various samples was determined by Student’s t-test. P < 0.05 was considered significant.To identify candidate suppressors or enhancers of Fas-induced apoptosis in tumor cells, we optimized two types of high throughput screening (HTS) assays for siRNA library screening. Using PPC1 prostate cancer cells, we determined that stimulation with low dose anti-Fas antibody CH11 (3 ng/ml) did not result in cytotoxicity, while stimulation with high dose anti-Fas antibody (25 ng/ml) killed these tumor cells. Using control siRNAs to validate the assay, we determined that 3 of 3 siRNAs targeting FLIP sensitized PPC1 cells to low-dose (3 ng/ml) anti-Fas antibody whereas 3 of 3 siRNAs targeting FADD inhibited PPC1 killing induced by high-dose (25 ng/ml) anti-Fas antibody. After using these control siRNAs to optimize the HTS assays to a Z’ factor > 0.5, we then screened a library of 16,800 siRNAs targeting 5,600 genes (3-fold coverage), which constitutes the so-called “druggable genome”. PPC1 cells in 384 well plates were “reverse” transfected with siRNAs, then 2 days later CH11 antibody was added and cell viability assessed using a bioluminescence assay to determine ATP levels one day later . The effTo further assess the role of PCTAIRE1 on TNF-family cytokines in prostate cancer PPC1 cells, we used siRNAs targeting PCTAIRE1. Immunoblotting was first performed to confirm knockdown at the protein level . Using 3The activation of caspase-8 induced by TNF-family death receptors is accompanied by proteolytic processing of the ~50 kDa pro-caspase-8 molecule initially to yield ~43/41 kDa partially processed molecules and finally to produce the ~18 and ~10 kDa subunits of the catalytically active protease. We used immunoblotting to monitor the effects of siRNA-mediated knockdown of PCTAIRE1 on the proteolytic processing of pro-caspase-8. The siRNA-mediated silencing of PCTAIRE1 in PPC1 cells indeed increased Fas- and TRAIL-induced processing of caspase-8 compared to control RNA . We alsoThe siRNA-mediated PCTAIRE1-knockdown effects on the extrinsic cell death pathway were also reproduced using tetracycline-inducible shRNA vectors introduced by lentivirus infection . In PPC1Among the TNF-family cytokines, TRAIL is considered the most promising weapon for combating cancer cells because TRAIL sensitivity is observed predominantly in tumor but not normal cells ,29. FurtTo explore the mechanism by which PCTAIRE1 suppresses apoptosis induced by TRAIL/Fas, we examined the impact of PCTAIRE1 knockdown on expression of various proteins that have been implicated in the TNF-family cytokine receptor signaling pathway. Because PCTAIRE1-knockdown sensitized PPC1 cells to death receptor stimuli  but not agents that trigger other cell death pathways, we suspected that PCTAIRE1 may block a proximal step in Fas and TRAIL receptor signaling. The critical event for initiating the apoptosis pathway induced by TNF-family cytokines is activation of caspase-8, which requires the adapter protein FADD, and is opposed by FLIP. As shown above, PCTAIRE1-knockdown activated caspase-8 processing , but PCTNext, we used immunoblotting to assess RIPK1 expression in PCTAIRE1-knockdown cells and found that RIPK1 levels in PCTAIRE1-knockdown PPC1 cells were lower than control cells . HoweverBecause PCTAIRE1 knockdown caused a decrease in RIPK1 protein levels without impacting mRNA levels, we hypothesized an effect on RIPK1 protein stability. To this end, the effect of PCTAIRE1 on RIPK1 protein stability was examined by immunoblotting of cell lysates treated with the proteasome inhibitor MG132, which reduced PCTAIRE1-knockdown-mediated RIPK1 degradation . To examSince RIPK1 degradation or deficiency has been reported to sensitize cells to TNF-induced apoptosis –33, we aOur previous study revealed that PCTAIRE1 phosphorylates p27 and promotes its degradation . To inveet al., which reported that SNS-032 sensitizes cancer cells to TRAIL [In our previous study, we showed that the kinase inhibitor SNS-032 suppresses PCTAIRE1 kinase activity . Based oto TRAIL . Our stuPCTAIRE1 sensitized several prostate and breast cancer cells to TNF-family cytokines, whereas PCTAIRE1-knockdown did not sensitize normal or non-transformed cells to TRAIL.In this study we report a role for the PCTAIRE family member PCTAIRE1 in modulating the extrinsic cell death pathway . Gene knet al. reported that inhibition of Cdk9 overcomes TRAIL resistance via suppression of Mcl-1 and FLIP protein expression [Our study showed that the protein kinase PCTAIRE1 is involved in the extrinsic cell death pathway. To date, the relationship between Cdk-family proteins and the extrinsic cell death pathway has not been characterized in detail. Cdk1 has been reported to phosphorylate caspase-8 at Ser 387, which confers TNF-family resistance to HeLa cells . Since Ppression . They alOur results suggested that RIPK1 is associated with activation of the extrinsic cell death pathway in PCTAIRE1 knockdown cancer cells, which supports findings in several reports indicating that modulation of RIPK1 expression is associated with the extrinsic apoptotic pathway. Furthermore, disruption of heat shock protein 90 (Hsp90) function by geldanamycin causes RIPK1 degradation, which sensitizes tumor cells to TNF-induced apoptosis . AnotherIn our previous study, tumor cells with PCTAIRE1 knockdown show late G2-M phase arrest . To asseIn conclusion, we identified PCTAIRE1 as a contributor to resistance of cancer cell lines to killing by TNF-family cytokines. Although the mechanism of PCTAIRE1 awaits additional characterization, the results presented here suggest the potential of PCTAIRE1 as a possible target for cancer therapy. In this regard, for patients with metastatic cancers, a need exists for more effective and less toxic therapies that improve survival. Recently, immune-based therapies for cancers have attracted great interest, showing promising potential for producing long-term remissions for some patients . In as mS1 Dataset(XLSX)Click here for additional data file.S1 FigTwo parallel HTS campaigns were conducted using low-dose (3 ng/ml) and high-dose (25 ng/ml) anti-Fas CH11 antibody. A library of 16,800 siRNAs constituting the “druggable” genome of 5,600 targets at 3-fold coverage was screened.(TIF)Click here for additional data file.S2 Fig PPC1 cells were transfected with control RNA (purple “x”) or various siRNAs targeting PCTAIRE1 . After 48 hours, cells were stimulated with either TNF (A) or thapsigargin (B) at the indicated concentrations. After 24 hours, cellular ATP levels were measured and the data expressed as the ratio between values for cells cultured with and without TNF (A) and thapsigargin (B) . (C-H) PPC1 cells were transfected with scrambled RNA or siRNAs targeting PCTAIRE1 (si-1472), PCTAIRE2 or PCTAIRE3. At 48 hours after transfection, mRNAs levels of PCTAIRE1 (C), PCTAIRE2 (D) and PCTAIRE3 (E) were measured by qRT-PCR, with normalization relative to GADPH . Forty-eight hours after transfection, cells were stimulated with Fas (CH-11) (F), TRAIL (G), or TNF (H) at various concentrations as indicated. After 24 hours, cellular ATP levels were measured, and the data expressed as the ratio between values for cells cultured with and without treatments . (I-N) PPC1 cells stably containing inducible shRNAs targeting different sites on PCTAIRE1 mRNA  or scramble-control were cultured for 48 hours with doxycycline . PPC1 cells were cultured with (ON) or without (OFF) 100 ng/ml Dox for 48 hours, then stimulated with various concentrations of either cisplatin (I-K) or paclitaxel (L-N). After 24 hours, cellular ATP levels were measured and expressed as a ratio relative to cells cultured without treatments .(TIF)Click here for additional data file.S3 Fig5 cells per well in 6 well (35 mm) dishes, then reverse-transfected with control or PCTAIRE1-targeting siRNAs as indicated. After 48 hours, anti-Fas antibody (250 ng/ml) (E) or TRAIL (100 ng/ml) (F) was added and cells were cultured for 72 hours before fixing and staining with 0.5% crystal violet dye. MDA-MB-468 (A) and MCF7 (G) cells were transfected with scrambled RNA or three different siRNA targeting PCTAIRE1 . After 48 hours, relative levels of PCTAIRE1 mRNA were measured by q-RT-PCR analysis. (B) Cell lysates 48 hours after transfection of siRNAs were prepared, normalized for total protein content, and aliquots were analyzed by immunoblotting using mouse anti-PCTAIRE1 (top) or anti-beta-actin (bottom) antibodies.  MDA-MB-468  and MCF7  cells were transfected with control RNA (purple “x”) or various siRNAs targeting PCTAIRE1 . After 48 hours, cells were stimulated with either anti-Fas antibody or TRAIL at various concentrations as indicated. After 24 hours, cellular ATP levels were measured and the data expressed as the ratio between values for cells cultured with and without anti-Fas  or TRAIL . All data represent mean ± SD (n = 3).  Clonogenic survival assays show that PCTAIRE1-knockdown sensitizes MDA-MB-468 cells to Fas and TRAIL. MDA-MB-468 cells were seeded at 2.0 x 10(TIF)Click here for additional data file.S4 Fig(A) MDA-MB-468 cells stably expressing inducible shRNAs targeting different sites on PCTAIRE1 mRNA  were cultured for 48 hours with various concentrations of doxycycline (Dox) ranging from 10 to 1000 ng/ml. PCTAIRE1 mRNA levels were measured by qRT-PCR, with normalization relative to GADPH . (B) Protein lysates were generated from MDA-MB-468 cells cultured for 48 hours with (ON) or without (OFF) 100 ng/ml Dox, normalized for total protein concentration, and analyzed by SDS-PAGE/immunoblotting using antibodies for PCTAIRE1 (top) and beta-actin (bottom).  MDA-MB-468 cells were cultured with (ON) or without (OFF) 100 ng/ml Dox for 48 hours, then stimulated with various concentrations of either anti-Fas antibody (CH-11) (C) or TRAIL (D). After 24 hours, cellular ATP levels were measured, and the data expressed as the ratio relative to cells cultured without anti-Fas or TRAIL .  Clonogenic survival assays. MDA-MB-468 cells stably containing inducible shRNAs  were cultured with (ON) or without (OFF) 100 ng/ml Dox for 48 hours, then stimulated with anti-Fas antibody  or TRAIL (100 ng/ml). Cells were cultured for 3 days before fixing and staining with 0.5% crystal violet dye. (G) MDA-MD-468 cells containing inducible PCTAIRE1 targeting shRNA vectors were cultured with or without Dox (100 ng/ml) for 48 hours, then stimulated with or without TRAIL (100 ng/ml) for 4 hours. Cell lysates were prepared, normalized for total protein content, and analyzed by immunoblotting using antibodies specific for either proteolytically cleaved caspase-8  or beta-actin (bottom).(TIF)Click here for additional data file.S5 Fig(A) Du145 cells were stably infected with two different PCTAIRE1-targeting (tet-inducible) shRNA lentiviruses. Protein lysates were generated from Du145 cells cultured for 48 hours with (ON) or without (OFF) 100 ng/ml doxycycline (Dox), normalized for total protein concentration, and analyzed by SDS-PAGE/immunoblotting using antibodies for PCTAIRE1 (top) and beta-actin (bottom). (B) Du145 cells were cultured with (ON) or without (OFF) 100 ng/ml Dox for 48 hours, then stimulated with various concentrations of TRAIL. After 24 hours, cellular ATP levels were measured, and the data expressed as a ratio relative to cells cultured without TRAIL . (C) Du145 cells containing inducible PCTAIRE1 targeting shRNA vectors were cultured with or without Dox (100 ng/ml) for 48 hours, then stimulated with or without TRAIL (100 ng/ml) for 4 hours. Cell lysates were prepared, normalized for total protein content, and analyzed by immunoblotting using antibodies specific for either proteolytically cleaved caspase-8  or actin (bottom). The cleaved p43/41 and p18 bands of caspase-8 are indicated by arrows.(TIF)Click here for additional data file.S6 Fig(A) PPC-1 cells were transfected with control RNA or three different siRNAs targeting PCTAIRE1. After 48 hours, cell lysates were prepared, normalized for total protein content, and analyzed by immunoblotting using antibodies against various proteins related to the extrinsic pathway for apoptosis. The red arrow indicates non-specific bands. (B) Cell surface Fas, DR4 and DR5 expression was measured by flow cytometry of PPC1 cells transfected with scramble or siRNA targeting PCTAIRE1 (si-1472). Positive rates were assessed by comparison with IgG isotype control (data not shown). The data are representative of two independent experiments.  PPC1 cells were transfected with plasmids producing HA-tagged caspase-8, Myc-tagged PCTAIRE1 (wild type or kinase dead mutant), empty vector, or siRNAs . Lysates were either loaded directly onto gels (“Input”) or subjected to immunoprecipitation (IP) using anti-HA antibody. Immune complexes were analyzed by SDS–PAGE/immunoblotting. (E) PPC1 cells were transfected with plasmids producing HA-tagged FADD, Myc-tagged PCTAIRE1 (wild type or kinase dead mutant). Lysates were either loaded directly onto gels (“Input”) or subjected to immunoprecipitation (IP) using anti-HA antibody. (F) PPC1 cells were transfected with siRNAs . After 48 hours, cell lysates were prepared, normalized for total protein content, and analyzed by immunoblotting.(TIF)Click here for additional data file.S7 Fig PPC1, MDA-MB-468 and MCF7 cells were transfected with scrambled RNA or three different siRNA targeting PCTAIRE1 . After 48 hours, relative levels of RIPK1 mRNA were measured by q-RT-PCR analysis.  MDA-MB-468 and MCF7 cells were transfected with siRNAs as indicated. Cell lysates 48 hours after transfection with siRNAs were prepared and analyzed by immunoblotting using mouse anti-RIPK1 (top) or anti-actin (bottom) antibodies. (F) HeLa cells were synchronized by a double thymidine block. Cell lysates were subjected to immunoblot analysis with indicated antibodies. (G) PPC1 cells were pretreated with the RIPK1 inhibitor necrostatin-1  for 1 hour, and then cells were treated with TRAIL (10 or 100 ng/ml). Cell viability was quantified by Cell Titer Glo after 24 hours. (H) PPC1 cells were transfected with siRNAs as indicated. Cell lysates 48 hours after transfection with siRNAs were prepared and analyzed by phos-tag SDS-PAGE/immunoblotting.(TIF)Click here for additional data file.S8 Fig(A-C) PPC1 cells stably containing shRNA (#2) were reverse-transfected with 5 nM scramble-control RNA or siRNA targeting p27. After 8 hours, the culture media was changed to media with (Tet-ON) or without (Tet-OFF) 100 ng/ml doxycycline (Dox). After 48 hours, cell lysates were prepared, normalized for total protein content, and analyzed by immunoblotting using antibodies for PCTAIRE1 (top), p27 (middle) and actin (bottom) (A).  PPC1 cells were stimulated with either anti-Fas antibody (B) or TRAIL (C). After 24 hours, cellular ATP levels were measured using Cell Titer Glo reagents, with the data expressed as a ratio between cells cultured with and without anti-Fas (B) or TRAIL (C) .  PPC1 cells stably overexpressing p27  were established by lentivirus infection and then stimulated with TRAIL. After 24 hours, cellular ATP levels were measured .(TIF)Click here for additional data file.


Telomeres, the ends of linear eukaryotic chromosomes, have a specialized chromatin structure that provides a stable chromosomal terminus. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomere length. Here we show that binding of multiple Rap1 proteins stimulates DNA double-stranded break (DSB) induction at both telomeric and non-telomeric regions. Consistent with the role of DSB induction, Rap1 stimulates nearby recombination events in a dosage-dependent manner. Rap1 recruits Rif1 and Rif2 to telomeres, but neither Rif1 nor Rif2 is required for DSB induction. Rap1-mediated DSB induction involves replication fork progression but inactivation of checkpoint kinase Mec1 does not affect DSB induction. Rap1 tethering shortens artificially elongated telomeres in parallel with telomerase inhibition, and this telomere shortening does not require homologous recombination. These results suggest that Rap1 contributes to telomere homeostasis by promoting chromosome breakage. Telomere length is maintained primarily through equilibrium between telomerase-mediated lengthening and the loss of telomeric sequence through the end-replication problem. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomerase recruitment in a dosage-dependent manner. In this paper we provide evidence suggesting an alternative Rap1-dependent telomere shortening mechanism in which binding of multiple Rap1 proteins mediates DNA break induction during DNA replication. This process does not involve recombination events; therefore, it is distinct from loop-mediated telomere trimming. Telomeres are specialized nucleoprotein complexes at the ends of linear eukaryotic chromosomes. The DNA component of telomeres typically comprises a double-stranded DNA (dsDNA) region of a tandem repeat and a 3’ protruding single-stranded DNA (ssDNA) region of the G-rich strand ,2. Both Saccharomyces cerevisiae. Telomere shortening results from incomplete DNA end replication and 5’-resection (end-replication problem) . Con. Con81) d nearby . Severalker loss 0-fold buanalysis .URA3Kl marker loss. Since the RAP1 gene is essential for cell proliferation, we examined the effect of partial Rap1 depletion using a copper-inducible rap1 degron, rap1-(Δ) [rap1-(Δ) mutants grew very poorly in the presence of 0.5 mM CuSO4 (4 did not significantly affect cell proliferation (rap1-(Δ) mutation on URA3Kl marker loss in the presence of 0.05 mM CuSO4 (URA3Kl marker loss. These results are consistent with the hypothesis that Rap1 promotes DSB induction in a copy number-dependent manner.We investigated whether Rap1 is required for rap1-(Δ) . ConsistmM CuSO4  as the emM CuSO4 . In contferation  althoughferation . We thusmM CuSO4 . Partialrap1-ΔN or rap1-ΔC mutation on loss of the URA3Kl marker. However, neither the N-terminal nor the C-terminal deletion significantly affected the generation of Ura- cells Fig 1C). 81 and the TG250 repeat sequence can potentially form two and eight G4 structures, respectively [rap1Δ mutants , thereby maintaining the expression level of LacI-Rap1 protein similar to that of endogenous Rap1 protein (4 cassette behaved like ~80 bp of telomeric TG sequence containing four Rap1 binding sites after LacI-Rap1 expression (see below). The average telomere length is ~300 bp and the Rap1 binding motif appears every 20 bp at telomeres [16 repeat in the presence of LacI-Rap1 could correspond to wild-type length telomere (~320 bp of TG repeat sequence) in terms of Rap1 binding. We introduced a 3’-terminal truncation of URA3Kl adjacent to the lacO sequences at the ADH4 locus and a 5’-terminal truncation of URA3Kl on a different chromosome . LacI alone stimulated recombination near the LacO16 cassette but much less efficiently than LacI-Rap1 fusion  structures at TG repeats, which could cause replication fork blocks and induce DNA breaks . The TG8ectively . Moreove mutants  and func protein . To targ protein . No putaromosome . In thisa+ cells . LacI-Raa+ cells . For exa1 fusion . The obs1 fusion .rif1Δ rif2Δ nor sir3Δ sir4Δ double mutation affected recombination (-11M) [Since the N-terminus or C-terminus of Rap1 was dispensable for TG-mediated chromosome truncation see , we exambination . It is pbination . LacI-Gabination . Both Ran (-11M) ,50. One n (-11M) . We foun16 repeat by Southern blot analysis. We used a strain carrying the LacO16 cassette between KanMX and URA3Kl at the ADH4 locus  has been used to end-label DNA ends including DSBs and telomeres . To conf16 repeat during S phase. To address whether DSB induction is coupled with DNA replication, we examined the effect of temperature-sensitive cdc17-1 mutation on break induction during S phase  and the VII-L telomere . Previous studies showed that the GAL4 binding site adjacent to telomeres behaves in a manner similar to a telomere sequence after expression of a Gal4-Rap1 fusion protein [4-telomere were transformed with pGAL-LacI-RAP1 or pGAL-LacI and cultured in sucrose to stationary phase. The cultures were diluted 1,000-fold in galactose and grown for 24 hr (eight generations) for successive serial dilutions. Cells were collected at each dilution point and the length of LacO4-telomere was monitored by Southern blot analysis  became gradually shorter consistent with the above finding that LacI-Rap1-covered lacO sequence is counted as a telomere sequence. This observation indicates that telomerase activity is inhibited because TG33-LacO16 telomeres behave like an extended telomere in the presence of LacI-Rap1. Second, LacI-Rap1 expression generated shorter telomeres (TG-telomeres) corresponding to those extended from the TG33 repeat, consistent with the hypothesis that DSB induction occurs near or within the LacO16 repeat. As Rap1-mediated DSB induction occurs during S phase, continuous culturing increased the population size of cells containing TG-telomeres. 50% of the cells converted from the TG33-LacO16 telomere to the TG-telomere after three successive dilutions (24 generations); it was estimated that 2% of cells received a DNA break within or adjacent to the LacO16 repeat per generation.We then examined the effect of LacI-Rap1 or LacI expression on the length of TGanalysis . As LacIelomeres , it did EST1, which encodes a regulatory protein for telomerase, in cells containing the TG33-LacO16 telomere. Growth of the est1Δ strains was maintained by wild-type EST1 on a URA3-marked plasmid. Wild-type cells and est1Δ mutant cells were transformed with pGAL-LacI-RAP1 and pGAL-LacI, respectively. To select for loss of the complementing EST1 plasmid, cells were grown on sucrose plates containing 5-FOA. The resulting single colonies were inoculated into galactose medium (1st dilution). After 24 hr culture, it was diluted 1000-fold for successive serial dilutions. Southern blotting analysis confirmed that the inhibition of telomerase activity caused slow telomere shortening (~30 bp during each dilution) but did not promote TG-telomere formation , approximately 50% of cells would receive DNA breaks after 32 generations. DNA ends adjacent to TG repeats, once generated, are protected from DNA degradation by Cdc13-mediated telomere capping [DNA breaks adjacent to telomeric TG repeats can be readily converted to telomeres by telomerase ,40. It i capping . Thus, R capping . In cont capping . In cont capping . Cells t16 repeat after LacI expression but 0.02% of cells were expected to receive DNA breaks at the LacO16 repeat per generation. The ATR/Mec1 checkpoint pathway has been proposed to prevent chromosome breakage during replication stress [Replication forks in yeast and other organisms move more slowly through telomeric DNA than non-telomeric regions ,66–68. Tn stress ,53. Howen stress . This Ran stress ,68. Inten stress ; howevern stress .hpr1 mutation, which increases recombination between direct repeats, elevates the rate of TRD [33-LacO16-telomeres became shorter independently of Rad52 function or the end-replication problem after LacI-Rap1 expression.Over-elongated telomeres can be shortened to normal length through a mechanism termed telomeric rapid deletion (TRD) . TRD appe of TRD . Similare of TRD –13. While of TRD . Since De of TRD , therebyIn summary, we have provided evidence indicating that binding of multiple Rap1 proteins promotes DNA break induction during DNA replication. Since Rap1 binds extensively at telomeric DNA regions , it seem0-URA3, TG81-URA3, TG250-URA3 or LacO16-URA3 cassette was generated by the pNO-URA3, pT81-URA3, pT250-URA3 or pO16-URA3 plasmid after digestion with NotI and SalI, respectively. The strain carrying the LacO0-ura3-ΔC, LacO4-ura3-ΔC, LacO8-ura3-ΔC or LacO16-ura3-ΔC or TetO8-ura3-ΔC cassette was generated by the pUN-O0, pUN-O4, pUN-O8, pUN-O16 or pUN-tetO8 plasmid after digestion with EcoRI and SalI, respectively. The strain containing the LacO4-telomere, TG33-LacO16-telomere, or TG-telomere was generated by the pO4-TG-HO, pTG33-O16-TG-HO or pTG81-HO plasmid after digestion with EcoRI and SalI, respectively. The ura3-ΔN-Hph cassette was introduced into the YER186 locus by a PCR-based method [rap1-(Δ)) construct has been described [rap1-ΔN) has been described [81-HO cells after HO expression [mec1Δ, rad52Δ or sml1Δ mutation has been described [mec1-81 or tel1Δ mutation has been published [est1Δ mutation has been described [rap1 truncation (rap1-ΔC), rif1Δ and rif2Δ mutation have been described [rap1-ΔC allele encodes the same truncated Rap1 proteins as the rap1-17 mutation does [SIR3 was performed as described [SIR4 disruption plasmid was obtained from Masayasu Nomura. The strains used in this study are listed in The strain carrying the TGd method . The copescribed . The N-tescribed . TG-telopression  or the Random Primer DNA Labeling Kit (Clontech), respectively. Genomic DNA was purified using a MasterPure yeast DNA purification kit (Epicentre).The probe that detects the  plasmid . The proescribed ,78. DNA 81-URA3 or TG250-URA3 are Ura- cells. The promoter of URA3Kl was replaced with the strong ADH1 promoter  cells on 5-FOA plates were tested by replica-plating to uracil drop-out plates. None of them grew on uracil-drop out plates.Cells from a single colony were fully grown in uracil dropout medium. The culture was then diluted 1000-fold and grown in rich medium for 24 hr. Aliquots of the cultures were diluted and plated on plates containing 5-FOA or non-selectable rich medium. Rates per generation were calculated using the FALCOR program based on the Luria-Delbruck fluctuation analysis . We confoter see . More thTRP1-marked plasmids and grown in tryptophan-dropout medium containing 2% sucrose overnight. The culture was then diluted and grown in tryptophan-dropout medium containing 2% galactose or 2% sucrose and 0.5% glucose. Aliquots of the cultures were diluted and plated on uracil-dropout or rich medium to estimate the URA3 recombination frequency after expression of LacI or LacI-fusion protein. Rates per generation were calculated using the FALCOR program [Cells were transformed with  program .Purified genomic DNA (100 ng) was incubated with one unit of terminal deoxynucleotidyl-transferase  in a supplied reaction buffer supplemented with 0.2 mM dCTP at 37°C for 60 min, followed by PCR using a poly(dG)-oligonucleotide with either the TdT forward or reverse primer. The PCR condition was 33 cycles of denaturation at 94°C for 30 s, annealing at 62°C for 30 s, and elongation at 72°C for 60 s. Sequences of PCR primers are described in Immunoblotting or DNA flow cytometric analysis was performed as described ,78. Rap1S1 Fig81 or TG250 cassette were treated as in 81-HO cells after HO expression [Cells containing the TGpression . HO endo(TIFF)Click here for additional data file.S2 Fig0 or TG250 cassette were cultured as in 250 repeat is located 3.2 kb from the SacI site and 1.9 kb from the PvuII site. RFP represents replication fork pausing. The arrow indicates the direction of replication fork movement.Cells containing the TG(TIFF)Click here for additional data file.S3 Figrap1Δ cells carrying YCp-RAP1 (URA3) were transformed with YEp-RAP1, pGAL-LacI-RAP1 or the control vector (YCplac22). Transformants were streaked on plates containing 2% galactose, 0.5% glucose with (+) or without 5-FOA (-).(TIFF)Click here for additional data file.S4 Fig(A) Expression level of LacI-Rap1 and Rap1. Cells carrying pGAL-LacI-RAP1 or the control vector were grown in 2% galactose and 0.5% glucose and analyzed by immunoblotting with anti-Rap1 antibodies. Tubulin was detected as a loading control. (B) Expression level of LacI-Rap1 and LacI. Cells carrying pGAL-LacI-RAP1 or pGAL-LacI were grown in 2% galactose and 0.5% glucose and analyzed by immunoblotting with anti-LacI antibodies. Tubulin was detected as a loading control. The asterisk indicates cross-reactive proteins. (C) Expression level of LacI-Rap1 and LacI-Rap1 (224–663). Cells carrying pGAL-LacI-RAP1 or pGAL-LacI-RAP1 (224–663) were analyzed as in (B). (D) Expression level of LacI-Rap1 and LacI-GAL4. Cells carrying pGAL-LacI-RAP1 or pGAL-LacI-GAL4 were analyzed as in (B). (E) Expression level of LacI**-Rap1. Cells carrying pGAL-LacI-RAP1 or pGAL-LacI**-RAP1 were analyzed as in (B).(TIFF)Click here for additional data file.S5 FigKanMX-ura3-ΔN-TetO8, HphMX-ura3-ΔC) carrying YCpT-TetR, YCp-TetR-RAP1 or the control vector were cultured in medium selectable for the TRP1 marker and examined as in 8 cassette and carrying the control vector. The ADH4 locus on chromosome VII was replaced with the KanMX-marked cassette containing the ura3-ΔCKl truncated gene and eight copies of the tetO sequence.Cells ((TIFF)Click here for additional data file.S6 Fig16 cassette. Cells carrying pGAL-LacI, pGAL-LacI-RAP1, or its derivatives were cultured and examined as in 16 cassette and carrying the control vector. (C) Expression level of fusion proteins. Cells carrying pGAL-LacI-RAP1 or various deletion constructs were grown in 2% galactose and 0.5% glucose and analyzed by immunoblotting with anti-LacI antibodies. Tubulin was detected as a loading control.(A) Deletion of Myb or TA domain in the LacI-RAP1 construct. Rap1 contains a BRCT domain, two Myb domains, and a transcription activation (TA) domain and a C-terminal RCT domain. Fusion proteins contain the DNA-binding domain of LacI (black square) with a nuclear localization signal. (B) Effect of Myb domain or TA domain deletion on homologous recombination near the LacO(TIFF)Click here for additional data file.S7 Fig16-URA3 cassette were transformed with pGAL-LacI-RAP1 (R) or pGAL-LacI (I). Transformants were initially grown in 2% sucrose and then incubated with 2% galactose and 0.5% glucose for 4 hr. Genomic DNA from cells expressing LacI or LacI-Rap1 was digested with NcoI (Lane 1–2). The LacO16 repeat is cloned between the BamHI and EcoRI sites Click here for additional data file.S8 Fig16-URA3 cells carrying pGAL-LacI-RAP1 were treated as in 16-URA3 cells received DNA breaks with the LacO16 repeat sequence after LacI-Rap1 expression.LacO(TIFF)Click here for additional data file.S9 Fig16 repeat is located 3.2 kb from the SacI site and 1.9 kb from the PvuII site. RFP represents replication fork pausing. Note that some parts of RFP signal are smearing (ST). The number (%) below each panel denotes the ratio of the signal of RFP to that of total replication intermediates. The arrow indicates the direction of replication fork movement.CsCl gradient purified DNA was digested with SacI and PvuII and analyzed by two-dimensional gel electrophoresis using the indicated probe. The probe detects a 5.5 kb SacI-PvuII fragment. The LacO(TIFF)Click here for additional data file.S10 Fig33-LacO16-tel cells and TG33-LacO16-tel est1Δ cells carrying the URA3-marked EST1 plasmid were transformed with pGAL-LacI-RAP1. Transformants were treated and examined as in TG(TIFF)Click here for additional data file.S1 TableAll the strains are isogenic and the detailed construction is described in Materials and Methods.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 TextDetails of plasmid construction and plasmid information are described.(DOCX)Click here for additional data file.


By using Drosophila PD model expressing α-synuclein A30P, we examined brain miRNA expression with high-throughput small RNA sequencing technology. We found that five miRNAs  were upregulated in PD flies. Among them, miR-13b, miR-133, miR-137 are brain enriched and highly conserved from Drosophila to humans. KEGG pathway analysis using DIANA miR-Path demonstrated that neuroactive-ligand receptor interaction pathway was most likely affected by these miRNAs. Interestingly, miR-137 was predicted to regulate most of the identified targets in this pathway, including dopamine receptor , γ-aminobutyric acid (GABA) receptor  and N-methyl-D-aspartate (NMDA) receptor (Nmdar2). The validation experiments showed that the expression of miR-137 and its targets was negatively correlated in PD flies. Further experiments using luciferase reporter assay confirmed that miR-137 could act on specific sites in 3’ UTR region of D2R, Nmdar2 and GABA-B-R3, which downregulated significantly in PD flies. Collectively, our findings indicate that α-synuclein could induce the dysregulation of miRNAs, which target neuroactive ligand-receptor interaction pathway in vivo. We believe it will help us further understand the contribution of miRNAs to α-synuclein neurotoxicity and provide new insights into the pathogenesis driving PD.Parkinson’s disease (PD) is a prevalent neurodegenerative disorder with pathological features including death of dopaminergic neurons in the substantia nigra and intraneuronal accumulations of Lewy bodies. As the main component of Lewy bodies, α-synuclein is implicated in PD pathogenesis by aggregation into insoluble filaments. However, the detailed mechanisms underlying α-synuclein induced neurotoxicity in PD are still elusive. MicroRNAs are ~20nt small RNA molecules that fine-tune gene expression at posttranscriptional level. A plethora of miRNAs have been found to be dysregulated in the brain and blood cells of PD patients. Nevertheless, the detailed mechanisms and their  Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder affecting the elderly population . Its preCaenorhabditis elegans, Drosophila melanogaster, rat, mouse, and non-human primates [Drosophila models have been widely used to study neurodegenerative diseases including Alzheimer’s disease (AD), Huntington’s disease (HD) and PD [Drosophila conceives complicated central and peripheral nervous systems which are analogous to those of human. Panneuronal expression of human wild type and mutant α-synuclein (A53T and A30P) demonstrate adult onset PD pathological features including DA neuronal loss, decreased dopamine level, impaired locomotive ability and shortened lifespan [Drosophila models provide efficient tools for screening genes participate in PD and potential drugs against PD.PD animal models have been established by ectopic expression of human α-synuclein in yeast, primates –11. Dros) and PD , 12, 13.lifespan , 14, 15.MicroRNAs are ~20nt small RNA molecules that fine-tune gene expression at posttranscriptional level . They usDrosophila model expressing α-synuclein by high throughput small RNA sequencing technology. We found that five miRNAs  were upregulated in PD flies. Among them, miR-13b, miR-133, miR-137 are brain enriched and highly conserved from Drosophila to Homo sapiens. Validation experiment using qRT-PCR confirmed that these miRNAs were elevated in PD flies. KEGG pathway analysis indicated that neuroactive-ligand receptor interaction pathway was most likely affected by these miRNAs. Further studies showed miR-137 targeted multiple molecules in this pathway as predicted, including dopamine receptor , GABA receptor  and NMDA receptor (Nmdar2). The mRNA levels of these molecules were significantly decreased in PD flies. Our findings indicated that α-synuclein could induce the dysregulation of miRNAs, which target neuroactive ligand receptor interaction pathway in vivo.In the present study, we examined the expression of miRNAs in a PD elav-C155 and UAS- α-synuclein flies were obtained from Bloomington Stock Center . Flies were raised in standard yeast agar food at 25°C with a 12/12 hours light/dark cycle. After backcrossing with w118 flies for 6 generations, elav-C155 virgin flies were crossed with w118 or UAS- α-synuclein A30P males. The F1 generation offspring expressed α-synuclein in panneuronal manner and were used for further experiments.The Two days after the eclosion, mated males and females were discriminated and transferred to different vials. Each vial contained 10 flies and at least 100 in total for each group. The vials were changed 3 times a week and deaths were recorded. Data was presented as survival curves and analysis was performed using log-rank tests to compare between groups.In order to characterize behavior defects in PD flies, climbing assay was performed as described previously . BrieflyTotal RNA of each sample  was used to prepare the miRNA sequencing library through following steps: 1) 3'-adapter ligation with T4 RNA ligase; 2) 5'-adapter ligation with T4 RNA ligase; 3) cDNA synthesis with RT primer; 4) PCR amplification; 5) extraction and purification of ~125–145 bp PCR amplified fragments  from the PAGE gel. After the completed libraries were quantified with Agilent 2100 Bioanalyzer, the DNA fragments in the libraries were denatured with 0.1M NaOH to generate single-stranded DNA molecules, captured on Illumina flow cells, amplified in situ and finally sequenced for 36 cycles on Illumina HiSeq2000 according to the manufacturer’s instruction. Raw sequences were generated as clean reads from Illumina HiSeq by real-time base calling and quality filtering. Subsequently, the 3’ adapter sequence was trimmed from the clean reads and the reads with lengths shorter than 15 nt were discarded. As the 5’-adaptor was also used as the sequencing primer site, the 5’-adaptor sequence is not present in the sequencing reads. The trimmed reads (length ≥ 15 nt) were aligned to the fly pre-miRNA in miRBase 21, using novoalign software. The miRNA expression levels were measured and normalized as transcripts per million of total aligned miRNA reads (TPM). When comparing profile differences two groups of samples (PD and Control), the “fold change” (i.e. the ratio of the group averages) and p-value were caculated. miRNAs having fold changes ≥ 1.2, P-value ≤0.05 or fold change ≥ 2.0 were selected as the differentially expressed miRNAs.Quantitative real-time PCR (qRT-PCR) analysis was performed to validate the differently expressed mRNA in PD flies. Fist strand cDNA was sysnthesized using M-MLV reverse transcriptase (Epicentre) according to manufacture’s instructions. The sequences of RT primers are: 5’-GATTTTGCGTGTCATCCTTG-3’ (U6); 5’-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACACAGCTG-3’ (dme-miR-133-3p); 5’-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACCTACGT-3’ (dme-miR-137-3p); 5’-GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACACTCGT-3’(dme-miR-13b-3p). qPCR reaction was performed with 2×SYBR Green PCR Master Mix (Arraystar) and ViiA 7 Real-time PCR System (Applied Biosystems) with the program: 95°C for 10 min to denature DNA templates, followed by 40 cycles of 95°C for 15 s, 60°C for 60s.PCR primers for miRNAs and U6 were listed in The target genes of differentially expressed miRNAs were predicted by miRanda-mirSVR algorithm and then subjected to GO analysis using Database for Annotation, Visualization and Integrated Discovery (DAVID) . The significantly affected GO terms (p<0.05) in biological process, cellular component and molecular function were identified.DIANA-miRPath is an efficient tool for analyzing the combinatorial effect of microRNAs on target pathways. We uploaded the dysregulated miRNAs and predicted potential target pathways using DIANA-microT-CDS algorithm. The significantly influenced pathways (p<0.05) were identified.In order to validate the expression of predicted targets for dysregulated miRNAs, qRT-PCR was performed according to previously reported methods as mentioned before. PCR primers for mRNAs were listed in Drosophila cDNA library and inserted into pGL3-promoter vectors respectively. Each of these vectors was co-transfected with Renilla plasmid pRL-TK and dme-miR-137-3p mimics  into HEK 293 cells in 12-well plates using Lipofactamine 2000 (Invitrogen). 24 hours post-transfection, luciferase activity was measured with Dual-Glo (Promega) according to the manufacturer's instructions. PCR primers for amplification were listed in The 3’UTR fragments flanking miR-137 targeting sites of Nmdar2, D2R and GABA-B-R3 were cloned from t test using GraphPad Prism software, and p <0.05 were considered statistically significant.Log-rank tests were performed to compare lifespan between groups. For other experiments, the significance of the difference was analyzed with Student’s elav-Gal4 flies were crossedwith UAS-α synuclein (A30P) to ectopically express human α synuclein in nervous systems. As reported previously, we found that PD flies exhibited shorter lifespan  in The high-throughput sequencing results showed that 154 miRNAs  were coexpressed in both control and PD flies . In contAs four of the dysregulated miRNAs in PD flies including dme-miR-133-3p, dme-miR-137-3p, dme-miR-13b-3p and dme-miR-932-5p were brain enriched, we predicted targets of them and then submit to DAVID for Gene Ontology analysis . GO enriDIANA miRPath is a powerful tool to analyze the combinational effects of miRNA on signaling pathways . Using tDrosophila to humans.We examined the transcriptional levels of miR-137 targets in neuroactive ligand-receptor interaction pathway. Within five predicted targets, Nmdar2, GABA-B-R3, GABA-B-R1 and D2R were confirmed to be downregulated in PD flies . ParticuIn order to further confirm dme-miR-137 could directly regulate targets in neuroactive-ligand receptor interaction pathway, we constructed luciferase reporter plasmids carrying Nmdar2, D2R and GABA-B-R3 3’UTR fragments containing miR-137 binding sites . Luciferin vivo functions are still elusive. Drosophila models provide powerful tools to investigate etiology and intervention methods for PD. Using high throughput small RNA sequenceing technology, we measured miRNA expression profiles of early stage PD flies and identified five dysregulated mature miRNAs . KEGG functional annotation analysis showed that neuroactive-ligand receptors to be potentially affected by these miRNAs, which were confirmed by qRT-PCR analysis and luciferase reporter assay. Our study proposed miRNAs as potential biomarker for early stage PD and their dysregulaton subsequently participates in PD pathogenesis by interruption of neuroactive-ligand receptor interaction pathway.As regulatory molecules that fine-tune gene expression at posttrascriptional level, miRNAs have been estimated to exert important roles in PD. However, their detailed Ocimum sanctum leaf extract extract, E. citriodora extract and Regrapex-R have been proved to ameliorate PD pathogenesis [PD is a neurodegenerative disorder characterized by intraneuronal accumulation of filamentous inclusions known as Lewy body in substantia nigra. Feany and Bender established PD fly models by panneuronal expression normal and mutant forms of human α-synuclein, the main components accumulated in Lewy body , 14, 15.ogenesis , 39–52. in vitro studies. Further experiments using genetic modified animal models are required to clarify detailed miRNA functions in PD. With advantages discussed previously, Drosophila PD models could contribute to elucidation PD related miRNA functions in vivo.MiRNAs regulate gene expression at posttranscriptional level, which plays important roles in neurodegenerative diseases. Expression profiling analysis has identified a variety of miRNAs dysregulated in brain regions and blood samples from PD patients and animal models –20. As αDrosophila to H. sapiens and their expression was validated by qRT-PCR. MiR-13b’s human homologue is miR-499 [Our study using high throughput sequencing of miRNAs identified miR-13b, miR-133, miR-137, miR-932 and miR-1008 consistently upregulated in early stage PD flies. Among the dysregulated miRNAs, miR-13b, miR-133 and miR-137 were highly conserved from  miR-499  that exp miR-499 . Previou miR-499 . These r miR-499 . Morphin miR-499 . In addi miR-499 . By targ miR-499 . MiR-137 miR-499 . MiR-137 miR-499 . Recentl miR-499 . In addi miR-499 . miR-137 miR-499 –69. The  miR-499 , 70. In  miR-499 .in vitro could be explained in various mechanisms. Firstly, α-synuclein overexpression and aggregation in neuronal cells may influence signaling pathways and transcription factors that mediate miRNA expression. α-synuclein expression could influence signaling pathways including IRS-1/insulin/Akt, mTOR/S6K, MAPK, p53, GSK3β, PKC, synaptic transmission, ubiquitin protein pathway (UPS) and the autophagy pathway [in vitro. Detailed experiments are required to clarify this problemThe reason for α-synuclein induced miRNA dysregulation  pathway –79. Thes pathway , 80–86.  pathway –90. Thes pathway –94. ExtrHomo sapiens, which have been validated in human SY-SH5Y cells [Drosophila to humans. Interestingly, Genome-wide gene-environment study identifies glutamate receptor gene GRIN2A as a Parkinson's disease modifier gene via interaction with coffee [In order to elucidate which signaling pathways potentially affected by these dysregulated miRNAs in PD flies, DIANA-miRPath analysis was performed and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway neuroactive-ligand receptor interaction was identified. Consistent with our findings, Huang et al. reported that when applying to a genome-wide association study (GWAS) dataset for Parkinson disease, extended Bayesian lasso (EBLasso) identified three significant pathways including the neuroactive-ligand receptor interaction, the primary bile acid biosynthesis pathway, and the mitogen-activated protein kinase (MAPK) signaling pathway . Our val5Y cells . Lucifer5Y cells , suggesth coffee . Activath coffee . Hillmanh coffee . More spin vivo. We believe it will contribute to understanding miRNA functions in mediating α-synuclein toxicity and provide new insights into the pathogenesis driving PD.Our findings indicated that α-synuclein could induce the dysregulation of highly conserved and brain enriched miRNAs, which target neuroactive ligand-receptor interaction pathway S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigThe targets predicted by DIANA miRPath v.2.0 in neuroactive ligand-receptor interaction pathway were shown in red square.(TIF)Click here for additional data file.S4 FigThe hsa-miR-137-3p targets predicted by DIANA miRPath v.2.0 in Glutamatergic synapse pathway were shown in red square. NMDA receptor GRIN2A was identifies as potential target.(TIF)Click here for additional data file.S5 FigThe hsa-miR-137-3p targets predicted by DIANA miRPath v.2.0 in GABAergic synapse pathway were shown in red square. GABA receptors including GABRA1, GABRA6 and GABBR2 were identifies as potential targets.(TIF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.S3 Table(XLS)Click here for additional data file.S4 Table(XLS)Click here for additional data file.S5 Table(XLS)Click here for additional data file.S6 Table(XLS)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.


Here, we examine the relationship between HER2 antibody response (AR) and clinical recurrence (CR).We are conducting a Phase II clinical trial of the HER2 peptide vaccine AE37+GM-CSF for prevention of breast cancer recurrence in disease-free, node-positive or high-risk node-negative patients, who have completed standard of care therapy. AE37, an Ii-Key hybrid of the HER2/Patients with any level of HER2 expression (IHC1-3+) were randomized to receive six monthly intradermal inoculations of AE37+GM-CSF or GM-CSF alone (controls) during the primary vaccination series (PVS) then four booster vaccinations administered every six months. Prior to vaccination (R0) and after the PVS (R6), serum samples were tested for AR against 178 overlapping 17-mer peptides spanning the entire HER2 protein molecule. Using flow cytometry, the percentage of anti-HER2 specific antibodies was measured and expressed as mean fluorescent intensity (MFI). Sub group analysis was completed on HER2 over-expressers (OE) (IHC3+) versus non over-expressers (nOE) (IHC1-2+) and hormone receptor positive (HR+) (ER or PR+) versus HR- patients. Data are means and compared using a chi-square and students t test as appropriate.Of 298 enrolled patients, 153 were vaccinated while 145 were randomized to the control arm. Ten nonrecurrent (NR) and five recurrent (R) vaccinated patients were selected and matched for clinico-pathologic factors. HER2 AR was less in NR versus R at R0  and R6 . ΔR6 (R6-R0), AR change post-PVS, was increased in NR compared to R patients . AR was greater in HER2 OE compared to nOE . nOE exhibited larger ΔR6 HER2 AR . AR was greater in HR+ compared to HR- at R0  and R6  with HR+ patients exhibiting larger ΔR6 HER2 AR .In a prospective Phase II trial of a CD4+ -eliciting HER2 peptide vaccine, AE37+GM-CSF, patients who recur have higher levels of pre-existing antibodies and lower levels of antibody induction. This suggests that induction, not amplification, of an immune response may be important. Significantly higher AR change post-vaccination in HER2 nOE and HR+ patients suggests benefit of vaccination in less aggressive disease.http://clinicaltrials.gov: Vaccine Therapy in Treating Patients With Breast Cancer. Unique Identifying Number: NCT00524277Trial Registry at