Long non-coding RNAs (lncRNA) play an important role in carcinogenesis; knowledge on lncRNA expression in renal cell carcinoma is rudimental. As a basis for biomarker development, we aimed to explore the lncRNA expression profile in clear cell renal cell carcinoma (ccRCC) tissue.Microarray experiments were performed to determine the expression of 32,183 lncRNA transcripts belonging to 17,512 lncRNAs in 15 corresponding normal and malignant renal tissues. Validation was performed using quantitative real-time PCR in 55 ccRCC and 52 normal renal specimens. Computational analysis was performed to determine lncRNA-microRNA (MiRTarget2) and lncRNA-protein (catRAPID omics) interactions. We identified 1,308 dysregulated transcripts  in ccRCC tissue. Among these, aberrant expression was validated using PCR: lnc-BMP2-2 (mean expression change: 37-fold), lnc-CPN2-1 (13-fold), lnc-FZD1-2 (9-fold), lnc-ITPR2-3 (15-fold), lnc-SLC30A4-1 (15-fold), and lnc-SPAM1-6 (10-fold) were highly overexpressed in ccRCC, whereas lnc-ACACA-1 (135-fold), lnc-FOXG1-2 (19-fold), lnc-LCP2-2 (2-fold), lnc-RP3-368B9 (19-fold), and lnc-TTC34-3 (314-fold) were downregulated. There was no correlation between lncRNA expression with clinical-pathological parameters. Computational analyses revealed that these lncRNAs are involved in RNA-protein networks related to splicing, binding, transport, localization, and processing of RNA. Small interfering RNA (siRNA)-mediated knockdown of lnc-BMP2-2 and lnc-CPN2-1 did not influence cell proliferation.We identified many novel lncRNA transcripts dysregulated in ccRCC which may be useful for novel diagnostic biomarkers.The online version of this article (doi:10.1186/s13148-015-0047-7) contains supplementary material, which is available to authorized users. Renal cell carcinoma (RCC) is one of the most common malignancies; its incidence is varying substantially worldwide: RCC incidence is high in Europe and North America and low in Asia and South America . Today, Nucleic acids are under discussion as potential biomarkers for patients with RCC . Long noA gene expression microarray was used to identify dysregulated lncRNAs in 15 corresponding tumor and normal renal tissue samples as a discovery cohort. Among the 32,183 analyzed lncRNA transcripts, we observed differential expression (fold change >2) in 1,308 transcripts: 568 lncRNA transcripts were upregulated and 740 were downregulated in ccRCC samples. The 20 most differentially expressed lncRNAs in ccRCC and normal renal tissue are listed in Table n = 55) and normal (n = 52) renal tissue samples. The selection of representative lncRNAs for validation was based on the degree of dysregulation in the microarray . As expected, lnc-BMP2-2 (mean: 37-fold), lnc-CPN2-1 (13-fold), lnc-FZD1-2 (9-fold), lnc-ITPR2-3 (15-fold), lnc-SLC30A4-1 (15-fold), and lnc-SPAM1-6  were highly overexpressed in RCC, whereas lnc-ACACA-1 (135-fold), lnc-FOXG1-2 (19-fold), lnc-LCP2-2 (2-fold), lnc-RP3-368B9 (19-fold), and lnc-TTC34-3 (314-fold) were downregulated . Both, lnc-ERCC5-1 (p = 0.401) and lnc-RP11-480I12.4.1-1 (p = 0.731) were—as observed in the microarray studies—not dysregulated in tumor samples. The lncRNA expression levels allowed highly sensitive and specific discrimination between RCC and control subjects as determined by using ROC analyses: the area under the curve was >0.9 for all dysregulated samples, especially lnc-CPN2-1 overexpression allowed molecular identification of RCC tissue . See Figure In order to confirm aberrant lncRNA expression, we determined the expression of 13 lncRNAs in an independent validation cohort of ccRCC . None of the lncRNAs was significantly correlated with staging nor grading. However, there was a tendency towards lower lnc-ERCC5-1 (p = 0.034) and RP3-368B9 (p = 0.016) levels in Fuhrman grade 3/4 tumors, lower lnc-ERCC5-1 (p = 0.026) expression in vascular invasive tumors, and advanced AJCC stage in samples with low lnc-RP3-368B9 (p = 0.011). lncRNAs were not associated with progression-free survival, overall survival or cancer-specific survival , as determined using Cox regression analysis.We also analyzed whether lncRNA expression levels were correlated with poor prognostic parameters; the Bonferroni method was applied to correct for multiple hypothesis testing  decreased in siRNA-treated Caki-1 and Caki-2 (only siRNA #2) cells; it was basically unexpressed in A-498 cells. lnc-CPN2-1 was also decreased in Caki-2 cells (p < 0.05). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression was significantly reduced in all cell lines (not shown); the negative controls did not change gene expression. However, the EZ4U test did not show any changes in cellular proliferative activity after siRNA-mediated lncRNA knockdown. See Figure We next treated Caki-1, Caki-2, and A-498 RCC cell lines with small interfering RNA (siRNA) targeting lnc-BMP2-2 and lnc-CPN2-1; see Figure p > 0.1). MicroRNA expression was similar in RCC and normal renal tissue .The MiRTarget2 algorithm  was usedn = 91) are predicted for all transcripts . Thereby, they are involved in carcinogenesis of many human malignancies , but so n = 4 ; n = 6 [ [n = 11 ), thereb [n = 11 . During  [n = 11  re-analy [n = 11 . EarlierSERPINC1 ,14; aHIFSERPINC1 ; MALAT1 SERPINC1 ); we coulncRNAs could serve as diagnostic and prognostic biomarkers: The distinct overexpression of lncRNAs in RCC could be used for the development of a non-invasive diagnostic biomarker. We demonstrated distinct (>10-fold) expression level differences for a number of lncRNAs including lnc-ACACA-1, lnc-BMP2-2, lnc-CPN2-1, lnc-FOXG1-2, lnc-FZD1-2, lnc-ITPR2-3, lnc-RP3-368B9, lnc-SLC30A4-1, lnc-SPAM1-6, and lnc-TTC34-3; if expression differences are validated by independent researchers, these lncRNAs could represent diagnostic biomarkers. lncRNAs are detectable in bodily fluids and may thereby serve for cancer diagnosis: The PROGENSA® PCA3 Assay utilizes the detection of the lncRNA PCA3 in urine as diagnostic biomarker for prostate cancer . Furthern = 16) and the lack of a validation cohort limit the informative value of the study [i) a microarray study not powered for the identification of expression differences in localized (n = 5) and advanced RCC (n = 10), (ii) the selection of lncRNAs for validation based on the differential expression of tumor and normal tissue samples, and (iii) the high number of patients with localized ccRCC (56% AJCC stage 1 or 2) in the validation cohort. We expect that future studies designed to identify prognostic relevant lncRNAs in RCC patients will be able to present some candidates, because altered expression of specific lncRNAs was predictive of cancer-specific survival in former studies on other human malignancies; i.e. upregulation of HOTAIR in breast cancer [In addition, lncRNAs may be of prognostic relevance: Fachel et al.  reportedhe study . Malouf he study  identifit cancer  and SChLt cancer  was assoin silico analyses to point out putative functions of dysregulated lncRNAs: (i) lncRNA may act as miRNA sponge (e.g. miR-372 expression is downregulated by the lncRNA HULC [ii) Furthermore, lncRNA may function via lncRNA-protein interactions [in silico predictions is necessary in future studies.As interest in lncRNA research has increased a short time ago, the function of lncRNAs remains largely unknown, but involvement in many fundamental cellular processes is assumed . The shoRNA HULC ). Howeveractions . Using cractions , we idenSomewhat surprisingly, neither siRNA-mediated knockdown of lncRNA-CPN2-2 nor lnc-BMP2-2 resulted in a significant reduction of cell proliferation. However, the lncRNA knockdown was of moderate success , and thus the toxicity of the transfection reagent may inhibit determining the functional relevance of both lncRNAs. Many lncRNAs regulate nuclear events and must therefore be localized in the nucleus . As is kIn summary, our study reveals that dysregulation of approximately 4% of the lncRNA transcripts occurs in ccRCC, and altered lncRNA expression may modulate fundamental cellular processes. lncRNA profiles allow to accurately distinguish ccRCC and normal renal tissue. Thus, lncRNAs may be used as non-invasive biomarker for RCC patients.Biomaterials including fresh-frozen tissues are prospectively collected in the Biobank at the CIO Köln Bonn at the Universitätsklinikum Bonn according to standard operating procedures. Tissue from patients undergoing radical nephrectomy or nephron-sparing surgery was snap frozen, and samples from tumor and normal renal tissue were stored at liquid nitrogen. Hematoxylin- and eosin-stained sections were performed to confirm the histology of the samples. The final histological diagnosis was made on the formalin-fixed paraffin-embedded tissue samples. All cases used in this study were reviewed by an experienced uropathologist; the TNM classification (7th edition from 2009) was applied. We used a discovery cohort with 15 patients  for screening 32,183 lncRNA transcripts; the independent validation cohort consisted of 55 ccRCC and 52 normal renal tissue samples. The detailed clinical-pathological parameters are reported in Table 2 in RPMI 1640 culture medium, supplemented with 10% heat-inactivated fetal calf serum and 2 mM of glutamine . The knockdown of lnc-BMP2-2  and lnc-CPN2-1  was performed with individually designed Ambion Silencer Select siRNAs ; two different siRNAs were designed for each target. In addition, negative controls  and a positive control (Silencer GAPDH Positive Control siRNA) were used. The cell lines were seeded at 2.5 × 105 cells/well (6-well plate), and a forward transfection using the Screenfect Transfection Kit  with a siRNA concentration of 20 pmol was performed according to the manufacturer’s recommendations. The transfection complex was replaced after 24 h with RPMI medium and incubated for up to 72 h. siRNA experiments were confirmed in three independent experiments.The cell lines Caki-1, Caki-2, and A-498 were obtained from the DSMZ . All cell lines were maintained at 37°C, 5% CO4 cells per well into a 96-well plate. After forward transfection (siRNA concentration 1.2 pmol), the cells were cultured up to 72 h to determine the proliferative activity after siRNA-mediated knockdown of lnc-BMP2-2 and lnc-CPN2-1. The EZ4U assay  was used to determine cell viability using a 340 ATTC Spectra Thermo SLT photometer  according to the manufacturer’s protocol.The cell lines were seeded at a concentration of 1.5 × 10Fresh-frozen tissue samples (approximately 50 mg) were cut on liquid nitrogen and homogenized with Yttrium stabilized Zirkonoxid-Beads  using a Precellys®24 tissue homogenizer . Total RNA was then isolated using the mirVana miRNA Isolation Kit  according to the manufacturer’s recommendation. To remove residual DNA fragments, we treated the isolate twice with DNase . RNA quantity was determined with a NanoDrop 2000 spectrophotometer . RNA integrity was measured in samples used for microarray experiments using the Bioanalyzer 2100 with a RNA 6000 Nano Kit ; only samples with a RIN >6 were used. RNA degradation was excluded by agarose gel electrophoresis in samples used for PCR.The microarray experiments were performed by Biogazelle  as a contract service. RNA isolates from 15 corresponding normal renal and ccRCC tissues  were sent on dry ice to Biogazelle. A custom microarray (Agilent SurePrint G3 technology) based on LNCipedia 2.1 [U test, Cox regression analysis).Quantitative real-time PCR was performed to validate the microarray experiments using an independent cohort of 55 ccRCC and 52 normal renal tissue samples and to confirm the success of siRNA-mediated lncRNA knockdown. Complementary DNA (cDNA) was synthesized with 1 μg RNA using the PrimeScript RT Reagent Kit with gDNA Eraser. Real-time PCR was performed with 5 ng cDNA template using the 1× SYBR Premix Ex Taq II with ROX Plus and 10 pmol/μl PCR primers : 1,000 ng total RNA was reverse transcribed and real-time PCR was performed with 5 ng cDNA template. Pre-designed miScript Primer Assays for miR-519a, miR-519b, miR-519c, miR-548, miR-3120, miR-4284, miR-4658, and SNORD43 were used. PCR was performed using the ABIPrism 7900 HT Fast Real-Time PCR System. Data analysis was performed using Qbase + with SNORD43 as reference genes in the 2-∆∆CT algorithm. IBM SPSS Statistics v21 was used for statistical analyses (Pearson correlation test).The MiRTarget2 algorithm  was usedThe microarray data sets supporting the results of this article are available in the Gene Expression Omnibus repository (data entry: GSE61763).


To this end, a DC-SIGN binding ELISA, a gp140 trimer competition ELISA and HIV-1 capture/ transfer assays were utilized. Subsequently we aimed to identify the DC-SIGN binding component through biochemical characterization and mass spectrometry analysis. CM was shown to bind DC-SIGN and competes with HIV-1 gp140 trimer for binding. Pre-incubation of Raji-DC-SIGN cells or immature dendritic cells (iDCs) with CM potently inhibits DC-SIGN mediated trans-infection of CD4+ T-lymphocytes with CCR5 and CXCR4 using HIV-1 strains, while no effect on direct infection is observed. Preliminary biochemical characterization demonstrates that the component seems to be large (>100kDa), heat and proteinase K resistant, binds in a α1–3 mannose independent manner and is highly variant between individuals. Immunoprecipitation using DC-SIGN-Fc coated agarose beads followed by mass spectrometry indicated lactoferrin (fragments) and its receptor (intelectin-1) as candidates. Using ELISA we showed that lactoferrin levels within CM correlate with DC-SIGN binding capacity. In conclusion, CM can bind the C-type lectin DC-SIGN and block HIV-1 trans-infection of both CCR5 and CXCR4 using HIV-1 strains. Furthermore, our data indicate that lactoferrin is a DC-SIGN binding component of CM. These results indicate that CM has the potential to interfere with pathogen transmission and modulate immune responses at the colorectal mucosa.Bodily secretions, including breast milk and semen, contain factors that modulate HIV-1 infection. Since anal intercourse caries one of the highest risks for HIV-1 transmission, our aim was to determine whether colorectal mucus (CM) also contains factors interfering with HIV-1 infection and replication. CM from a number of individuals was collected and tested for the capacity to bind DC-SIGN and inhibit HIV-1  Sexual contact is the major route of transmission, yet the risk of transmission is predicted to be 10 times higher when comparing receptive anal intercourse with vaginal intercourse [ the gut . AdditioSchistosomes, which have been associated with enhanced HIV-1 transmission [trans-infection, a mechanism where DCs capture HIV-1 through C-type lectins, mainly Dendritic Cell-Specific Intracellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN), and transfer the virus to CD4+ T-lymphocytes. DCs found below mucosal surfaces can form dendrites which protrude through the epithelial barrier and thereby may facilitate HIV-1 trans-infection or where such cells can be exposed to virus through tears and breaches in the mucosal layer [In both routes, HIV-1 is introduced via semen and successful transmission requires the virus to cross a mucosal barrier, either through ruptures, transcytosis or dendritic cell (DC) uptake. Disruption of the mucosal layer can occur during intercourse or result from infections with pathogens such as smission ,4. Howeval layer ,6.DC-SIGN binds mannosylated as well as fucosylated glycans and is thus able to bind an array of pathogens ,8. The p+ T-lymphocytes [trans-infection, including BSSL, varies between individuals [trans-infection and identified human lactoferrin from CM as being a molecule with such binding activity.Recently a number of host glycoproteins, bile-salt stimulated lipase (BSSL) and mucin (MUC) 1 from human milk as well as MUC6 and clusterin from seminal plasma have been shown to bind DC-SIGN and thereby interfere with HIV-1 capture and transfer to CD4phocytes –13. Simiphocytes . Strikinividuals . These sMucus was collected by gentle washing with small volumes of PBS of surgically-resected colorectal tissue from HIV-1 negative patients undergoing rectocele repair and colectomy for colorectal cancer (n = 2). CM was collected from healthy tissue located approximately 10 to 15 cm from the tumour. The procedure was performed at St George’s Hospital, London, UK with signed informed consent from the patients . Cells i3 (pH9.2). After o/n incubation at 4°C the plate was blocked with TSM 5% BSA after which 333ng/ml DC-SIGN-Fc (R&D systems) was added. Subsequently, DC-SIGN was detected by a secondary goat-anti-human-Fc HRP labelled antibody (Jackson Immunology) (1:1000) using standard ELISA procedures. The same set up was used for detecting lactoferrin and intelectin-1 in CM, only instead of DC-SIGN-Fc, 333ng/ml polyclonal anti-lactoferrin  and 333ng/ml anti-intelectin-1  was used. In the DC-SIGN blocking ELISA, 10μg/ml anti-HIV-1 gp120 antibody, D7324  in 0.1M NaHCO3 (pH8.6) was coated on an ELISA plate. After overnight incubation at 4°C the plate was blocked with TSM 5%BSA after which trimeric HIV-1 gp140 (JR-FL SOSIP.R6-IZ-D7324) was added to the plate and which has been previously described [Escherichia coli-expressed recombinant HIV-1-p24  was analyzed.The DC-SIGN binding ELISA was performed as described . In shorescribed ,18. Meanescribed . The Capology) 1:00 using + T-lymphocytes were enriched by removing the CD8+ cells using CD8 dynabeads (Life Technologies) according to manufacturer’s protocol. Monocytes were isolated from buffy coats by ficoll-hypaque density centrifugation followed by selection for CD14+ cells via MACS (Miltenyi Biotec). Monocytes were cultured in RPMI containing 10% FCS supplemented with 500U/ml IL4 and 800U/ml GM-CSF (Schering-Plough) for 6 days which differentiated them into iDCs [TZM-bl cells were cultured in DMEM (Invitrogen) containing 10% FCS, MEM non-essential amino acids , penicillin and streptavidin (pen/strep), both 100U/ml. Raji DC-SIGN cells were cultured in RPMI 1640 (Invitrogen) supplemented with 10% FCS, 100U/ml pen/ strep. The peripheral blood mononuclear cells (PBMC) were isolated from buffy coats (Sanquin) of three healthy CCR5 wild-type homozygous donors using ficoll-hypaque density centrifugation. The cells were pooled and kept at −150°C until required. After thawing PBMCs were cultured in RPMI supplemented with 10% FCS, 100U/ml pen/strep and 100U/ml IL2. 3μg/ml phytohemaglutinin was used to activate the cells and CD4nto iDCs .+ T-lymphocytes [50) was determined by limiting dilutions on CD4+ T-lymphocytes according to the Reed and Muench method, previously described [Replication-competent HIV-1 subtype B NSI-18 (R5) and subtype B LAI (X4) were passaged on CD4phocytes . NSI-18 escribed + T-lymphocytes was determined by pre-incubating these cells with different dilutions of CM for 30min after which LAI (1000 TCID50) was added. Viral growth was monitored by measuring capsid p24 levels in culture supernatants using ELISA.Different CM dilutions were pre-incubated, 30min. on ice, with NSI-18 or LAI (5ng/ml p24) in DMEM supplemented with 10% FCS, 100U/ml pen/strep and 40μg/ml (end concentration) DEAE-dextran (Sigma). Subsequently, this mixture was added to TZM-bl cells (70–80% confluent) that were washed with PBS. Two days post infection the cells were washed again with PBS after which they were lysed with Reporter Lysis Buffer (Promega). After at least 30min. at −80°C the luciferase activity was measured on the Glomax luminometer (Turner BioSystems) using the Luciferase Assay kit (Promega) according to manufacturer’s protocol. Direct infection of CD44 cells/well) were pre-incubated with CM for 30min after which 500 TCID50/ml HIV-1 isolate NSI-18 or LAI was added. After 1h the cells were washed thoroughly and co-cultured with 2x105 CD4+ T-lymphocytes/well. Viral outgrowth was measured by capsid p24 ELISA. HIV-1 capture/transfer by iDCs and FACS analysis of the infection was performed as described [5 iDCs were pre-incubated with CM for 30min, 1x103 TCID50/ml virus (end concentration) was added, and after 2h the iDCs were washed and co-cultured with 2x105 CD4+ T-lymphocytes. After 48h the medium was replaced by fresh RPMI containing rIL-2 (2μg/ml) and indinavir . For FACS analysis the cells were fixed in 3.7% formaldehyde and subsequently permeabilized in PBS containing 1% BSA, 50mM NH4Cl and 0.1% saponin . Using anti-human CD3-APC  and anti-CA-p24-FITC  the number of infected cells per 1x105 CD3+ T-lymphocytes was determined.In Raji DC-SIGN mediated capture/transfer experiments Raji DC-SIGN cells  according to the manufacturer’s protocol. In short, the samples were incubated with the beads for 1h at RT after which the beads were removed from the sample via centrifugation.Size fractionation of CM was performed with YM-30 and YM-100 Microcon centrifugal filter devices (Millipore) according to manufacturer instructions. Heat treated samples were placed in a heat block for 10min at 95°C and proteinase K (Promega) treated samples were incubated for 30–60min at 56°C after which the enzyme was inactivated (10 min at 95°C). The depletion of (α1–3) mannose containing glycans was performed with p values of less than 0.05 are considered significant.To prepare samples for mass spectrometry analysis, CM of 6 DC-SIGN high binding individuals were pooled and incubated on a Protein A/G (Pierce Biotechnology) column overnight. An immunoprecipitationcarboxy using DC-SIGN-Fc (R&D systems) coated agarose beads (Sigma) was performed on the throughput, beads and CM were incubated together O/N. After two wash steps the beads were taken up in DTT containing sample buffer, placed at 99°C for 10min and run on a 4–12% SDS PAGE gel . Protein bands of interest were excised, alkylated and subjected to tryptic digestion according to standard protocols. Digests were analysed using a Bruker Ion Trap (Amazon Speed) upon separation by a 30min LC run using a C-18 column controlled by an EasyLC system (Bruker-Protana). Acquired data files were processed by the Bruker Compass DataAnalysis software  and exported MGF files (Mascot generic) were used to search with Mascot. Search parameters for Mascot were: ESI-trap; charge states: 1+, 2+, 3+; tolerance: 0.3; ms/ms tolerance: 0.4; trypsin; one missed cleavage; fixed modification: carbamidomethylcysteine; variable modification: oxidation of methionine. Probabilistic Mascot scoring evaluates peptide identifications and This approach resulted in identification of human lactoferrin with a total score of 211 -15% sequence coverage- (in band 2), and 302 -16% coverage- (in band 3), as well as human intelectin-1 (band 4) with a total score of 148 and 18% coverage. Band 2 does not contain higher scoring human proteins than lactoferrin (apart from some immunoglobulin and keratin contaminants); at much lower confidence levels we find the polymeric immunoglobulin receptor (score 66), intelectin-1 (score 46), IgGFc-binding protein (score 39), and Mucin-2 (score 30). Band 3 does not contain higher scoring human proteins than lactoferrin (apart from some immunoglobulin and keratin contaminants); at much lower confidence levels we find IgGFc-binding protein (score 98), Krev interaction trapped protein I (score 48), and the polymeric immunoglobulin receptor (score 43). Band 4 does not contain higher scoring human proteins than intelectin-1 (apart from some immunoglobulin and keratin contaminants); at lower confidence levels we find lactoferrin (score 102), serum albumin (score 44), and IgGFc-binding protein (score 31).Two tailed unpaired t-tests were performed for data sets except when comparing DC-SIGN binding to CM (OD450) with DC-SIGN binding to gp140 (OD450) where a Spearman’s rank correlation was used. P values <0.05 were considered statistically significant.2+ ions are required for specific DC-SIGN binding, incubation of DC-SIGN-Fc in the presence of the calcium chelator EGTA served as a negative control. We observed that CM bound DC-SIGN-Fc in comparison to the EGTA inactivated DC-SIGN-Fc  or LAI (X4) HIV-1 . Additiophocytes . To dete300 fold . Since Ctrans-infection of HIV-1 by iDCs.Next, we repeated these experiments using the physiologically more relevant immature monocyte derived DCs (iDC). These were pre-incubated with CM or medium, 20μg/ml DC-SIGN blocking antibody AZN or 50μg/ml mannan which served as controls. The result demonstrates that 100 fold diluted CM inhibits viral capture and transfer of LAI by iDCs and this effect was lost when CM was diluted 500 times . A repreInitially CM was size-fractionated using 30- and 100-kDa centrifugal filter devices. Utilizing the DC-SIGN binding ELISA we found that the fraction above 100 kDa contained factor(s) with strong DC-SIGN binding properties whereas weak binding was observed in the 30–100 kDa fraction and the below 30 kDa fraction did not bind DC-SIGN-Fc . Non-fraTo evaluate donor variation, 21 additional samples were analyzed using a serial dilution from 750μg/ml to 11.4ng/ml protein input in the DC-SIGN binding ELISA. Four samples did not bind DC-SIGN, whereas the remaining samples reached a binding plateau at 11.7μg/ml input. We observed clear differences in the DC-SIGN binding capacity of CM between individuals which could be divided in high, intermediate and low/no DC-SIGN binding . To deteIn order to identify the DC-SIGN binding component within CM we performed an immunoprecipitation on pooled CM from 6 samples showing high DC-SIGN binding and which had been depleted of antibodies, using DC-SIGN-Fc coated agarose beads. Subsequently, the DC-SIGN-Fc coated agarose beads with the component(s) of CM were ran on an SDS PAGE gel resulting in the identification of three bands  can bind HIV-1 and promote infection of target cells [trans-infection [To gain better insight into mechanisms involved in HIV-1 transmission deciphering the role specific bodily fluids can play in modulating infection is crucial. Here we demonstrate that although CM is unable to protect against direct infection it does block HIV-1 capture and subsequent transfer by both Raji-DC-SIGN cells and iDCs to CD4et cells ,22, whilnfection ,13,23. Snfection ,24. ThusAnalysis of the DC-SIGN binding component in CM revealed it binds DC-SIGN in a α1–3 mannose independent manner, which implies that the factor is likely fucosylated as DC-SIGN recognizes either of these two carbohydrate structures . Mass sptrans-infection one might have expected another member of this family to be expressed in CM and binding DC-SIGN [trans-infection, while certain allele combinations are correlated with a lower risk of HIV-1 infection, indicating BSSL potentially protects against transmission [Mass spectrometric analysis revealed no indication for MUC proteins binding DC-SIGN. Given that MUC1 from human milk and MUC6 from seminal plasma are known to inhibit DC-SIGN mediated HIV-1  DC-SIGN ,27. Prevsmission ,10,28. Fsmission . Howevertrans-infection, with human lactoferrin contributing to the binding. These results indicate that CM has the potential to interfere with HIV-1 transmission whilst simultaneously restricting antigen capture at the anal mucosa and potentially skewing mucosal immune responses in the rectum.In conclusion, as with other bodily fluids, CM contains a DC-SIGN binding component with the ability to block HIV-1


Caenorhabditis elegans. Unregulated EFF-1 expression causes lethality due to ectopic fusion between cells not developmentally programmed to fuse, highlighting the necessity of tight fusogen regulation for proper development. Identifying factors that regulate EFF-1 and its paralog AFF-1 could lead to discovery of molecular mechanisms that control cell fusion upstream of the action of a membrane fusogen. Bioinformatic analysis of the EFF-1A isoform’s predicted cytoplasmic domain (endodomain) previously revealed two motifs that have high probabilities of interacting with 14-3-3 proteins when phosphorylated. Mutation of predicted phosphorylation sites within these motifs caused measurable loss of eff-1 gene function in cell fusion in vivo. Moreover, a human 14-3-3 isoform bound to EFF-1::GFP in vitro. We hypothesized that the two 14-3-3 proteins in C. elegans, PAR-5 and FTT-2, may regulate either localization or fusion-inducing activity of EFF-1.Regulatory and biophysical mechanisms of cell-cell fusion are largely unknown despite the fundamental requirement for fused cells in eukaryotic development. Only two cellular fusogens that are not of clear recent viral origin have been identified to date, both in nematodes. One of these, EFF-1, is necessary for most cell fusions in eff-1-dependent epidermal cell fusions, reduced levels of FTT-2 and PAR-5 did not visibly affect the function of wild-type EFF-1 in the hypodermis.Timing of fusion events was slightly but significantly delayed in animals unable to produce full-length EFF-1A. Yet, mutagenesis and live imaging showed that phosphoserines in putative 14-3-3 binding sites are not essential for EFF-1::GFP accumulation at the membrane contact between fusion partner cells. Moreover, although the EFF-1A endodomain was required for normal rates of in vivo. However, prohibiting phosphorylation of candidate 14-3-3-binding sites does not impact localization of the fusogen. Hypodermal membrane fusion activity persists when 14-3-3 expression levels are reduced.Deletion of the EFF-1A endodomain noticeably affects the timing of hypodermal cell fusions  Intercellular fusion is a crucial biological process in the development of many organisms. In humans, it allows for fertilization and the functional development of muscle, bone, placental tissue, and the lens of the eye –8. OngoiDespite its prevalence and significance, understanding the molecular, biochemical, and biophysical mechanisms of developmental cell fusions is in its infancy ,5,7,26. Caenorhabditis elegans (C. elegans) is an ideal organism in which to study cell fusion because of its nearly invariant sequence of embryonic and postembryonic fusion events, wherein over one-third of somatic cells fuse to develop multiple syncytial cells in major tissues such as the vulva, pharynx and hypodermis (epidermis)  IV): A kind gift from Jeff Simske  IV) hermaphrodites and the subsequent F1 generation was screened for transheterozygote progeny  with Eff mutant phenotypes. Worms were made homozygous by selecting for the mIs12 marker and backcrossed three times to N2 (Bristol) ) [FC183: (1006))]) unc-119 (e2498) III; zzEx98): Site-directed mutagenesis  in EFF-1::GFP (pJdC41) was used to mutate three predicted phosphorylatable serine residues in both 14-3-3-consensus sites to alanines. Mutations were introduced in two consecutive steps. First, primers were designed to mutate S654 to S654A in pJdC41 . The resulting construct, EFF-1(S654A)::GFP (pJHS16), was used as a template to create EFF-1(S632/634/654A)::GFP (pJHS18) by mutating the remaining serines to alanine, S632/634A . By traditional C. elegans microinjection techniques, pJHS18 was co-injected with an unc-119(+) rescuing transgene  into a severely paralyzed Unc strain, CB4845 (unc-119 (e2498) III, obtained from David Pilgram). Transgenic worms were maintained by identifying non-Unc worms that expressed EFF-1(S632/634/654A)::GFP.FC254 (unc-119 (e2498) III; qtIs19: Expresses a cytoplasmic fluorescent protein reporter in a subset of hypodermal precursor cells in the developing embryo ftt-2 (n4426Δ) X; unc-119 (e2498) III; qtIs19): A C. elegans strain that contains a null allele of ftt-2, MT14355 (ftt-2 (n4426Δ) X), was generously provided by Robert Horvitz’s lab. This deletion allele removes the ftt-2 promoter and start codon. MT14355 is characterized by a “bagging” phenotype in which embryos are retained and hatch in utero (a few embryos are layed before bagging is observed) . Worms homozygous for ftt-2 (n4426Δ) and elt-3p::yfp were isolated by identifying “bagging” non-Unc hermaphrodites that produced 100% elt-3p::yfp-expressing progeny. This new strain was genotyped to confirm homozygosity of the ftt-2 deletion allele using a primer set  that amplifies only the wild-type allele. Individual worms were lysed in 3 μl of single-worm lysis buffer  by freezing the worms in lysis buffer at -80°C for 1 hour, heating to 65°C for 1.5 hours, and finishing with a 15 minute incubation at 95°C. The presence or absence of the wild-type allele in ftt-2 was detected by PCR using Taq DNA polymerase . No wild-type allele was detected in strain FC275.FC275 (bserved) . MT14355ftt-2 (n4426Δ) X; jcIs1 IV): MT14355 (ftt-2 (n4426Δ) X) heterozygous males were crossed with SU93 (jcIs1 IV) hermaphrodites. Worms homozygous for both loci were obtained and genotyped in a manner similar to FC275.FC276 (par-5(it55) unc-22(e66) IV/nT1[unc-?(n754dm) let-?] ; zzEx116): Homozygous loss-of-function par-5 mutants are maternal-effect lethal and therefore cannot be propagated [par-5(it55) unc-22(e66) IV/nT1[unc-?(n754dm) let-?] ), containing a balanced strong loss-of-function par-5 allele was obtained from the Caenorhabditis Genetics Center (CGC) ): FC142 (eff-1(zz10) II) hermaphrodites were crossed with HC396 heterozygous males (unc-119 (e2498) III; qtIs19). “Dumpy” worms (a phenotype of homozygous eff-1(zz10) animals) that produced 100% elt-3p::yfp positive progeny were isolated to create the homozygous strain.FC280 (par-5 RNAi feeding construct was generously provided by Sieu Sylvia Lee’s lab ) males were mated with FC75 (eff-1(zz10) II; jcIs1 IV) hermaphrodites. A strain carrying zzEx78 and homozygous for both eff-1(zz10), and jcIs1 was identified by observing that all worms not carrying zzEx78 exhibited 100% fusion-defective phenotypes (homozygous eff-1(zz10)), while progeny carrying zzEx78 were rescued for larval tail-whip defects and disappearance of AJM-1::GFP junctions in the hypodermis. Imaging: Loss of the extrachromosomal array zzEx78 expressing eff-1+and DsRed2 during embryonic cell division results in a mosaic pattern of red fluorescence. Mosaic animals were identified in which eff-1-rescued cells expressing DsRed2 lay adjacent to non-red (eff-1 null) cells. Larvae were paralyzed with 1M sodium azide and confocal image stacks were acquired on either a Perkin Elmer Ultraview RS5 or a Zeiss LSM 510 Meta confocal scanning microscope. Laser excitation used was at 488nm for GFP excitation and either at 568nm or at 543nm for DsRed2. GFP and DsRed2 channels were separated using linear unmixing software (Zeiss). Confocal z-stacks were converted to TIFF format and rendered as projections using Image J software [Cells without DsRed2 have lost a rescuing  animals , and the pTG96.2 . Worms wsoftware .(TIF)Click here for additional data file.S1 Movieeff-1(zz1) embryo expressing an adherens junction marker (AJM-1::GFP) imaged by 4-dimensional confocal microscopy. Arrows denote fused junctions and arrowheads indicate unfused cell borders, with intact junctions still observed before the embryo begins muscular movement. Anterior is left, dorsal is up. Time shown is approximate age since fertilization. Scalebar = 10 μm. Early cytoplasmic fluorescence seen in gut-fated cells (no longer visible at the time of adherens junctions phenotyping) is expressed from the mIs12 transgene, which was included in the background in which we screened for the zz1 mutation and is tightly linked to eff-1 on chromosome II.Maximum-intensity projection of an (MOV)Click here for additional data file.S2 MovieTime-lapse maximum-intensity projection of the ventral surface of the embryo shown in (MOV)Click here for additional data file.S3 MovieTime-lapse maximum-intensity projection of the dorsal surface of the embryo. Arrow indicates EFF-1(S632/634/654A)::GFP accumulation at a cell contact. One-micron-spaced image stacks were captured every 2.5 minutes using widefield microscopy, and maximum intensity Z-projections of the dorsal surface were rendered. In 100% of the mutant embryos (n = 4), the same pattern of junctional localization is seen as for wild-type EFF-1::GFP . Scaleba(MOV)Click here for additional data file.S4 MovieTime-lapse maximum-intensity projection of the wild-type embryo shown in (MOV)Click here for additional data file.S5 MovieTime-lapse maximum-intensity projection of the embryo shown in (MOV)Click here for additional data file.S6 MovieTime-lapse maximum-intensity projection of the embryo shown in (MOV)Click here for additional data file.S7 MovieTime-lapse maximum-intensity projection of the embryo in (MOV)Click here for additional data file.S1 SUPPORTING INFORMATION(DOCX)Click here for additional data file.


Colorectal cancer (CRC) ranks third among the estimated cancer cases and cancer related mortalities in the Western world. Early detection and efficient therapy of CRC remains a major health challenge. Therefore, there is a need to identify novel tumor markers for early diagnosis and treatment of CRC.in vitro and in human xenograft mouse model.A-kinase anchor protein 4 (AKAP4) gene and protein expression was monitored by quantitative polymerase chain reaction (qPCR), reverse transcription (RT)-PCR and Western blotting in normal colon tissue lysate, normal colon epithelial cells and in colon cancer cell lines viz., Caco-2, COLO205, COLO320DM, HCT-15, HCT116, HT-29, SW480, and SW620. The effect of AKAP4 on cellular growth, migration and invasion abilities was studied using gene silencing approach. The role of AKAP4 in various pathways involved in cell cycle, apoptosis, senescence was investigated in in vitro assays and tumor growth in human xenograft mouse. Human colon xenograft studies showed a significant decrease in the levels of cyclins B1, D and E and cyclin dependent kinases such as CDK1, CDK2, CDK4 and CDK6. Interestingly, an up-regulation in the levels of p16 and p21 was also observed. Besides, an increase in the levels of pro-apoptotic molecules AIF, APAF1, BAD, BID, BAK, BAX, PARP1, NOXA, PUMA and cyt-C and Caspase 3, 7, 8 and 9 was also found in cancer cells as well as in xenograft tissue sections. However, anti-apoptotic molecules BCL2, Bcl-xL, cIAP2, XIAP, Axin2 and Survivin were down regulated in these samples. Our data also revealed elevated expression of epithelial marker E-cadherin and down regulation of EMT markers N-cadherin, P-cadherin, SLUG, α-SMA, SNAIL, TWIST and Vimentin. Further ablation of AKAP4 resulted in the down regulation of invasion molecules matrix metalloproteinase MMP2, MMP3 and MMP9.Our studies showed that AKAP4 gene and protein expression was expressed in all colon cancer cells while no expression was detectable in normal colon cells. Ablation of AKAP4 led to reduced cellular growth, migration, invasion and increased apoptosis and senescence of CRC cells in AKAP4 appears to be a novel CRC-associated antigen with a potential for developing as a new clinical therapeutic target.The online version of this article (doi:10.1186/s13046-015-0258-y) contains supplementary material, which is available to authorized users. Colorectal cancer (CRC) is the third common cancer and a common cause of cancer-related death among US men and women . CRC proOur previous studies have demonstrated an association of a novel CT antigen A-kinase anchor protein 4 (AKAP4) expression in breast cancer , cervicain vitro and in vivo in human CRC xenograft mouse model. We show that ablation of AKAP4 lead to the down regulation of cyclins  along with their CDK-partners  and upregulation of cyclin dependent kinase inhibitors (CKIs), p16, p21 and retinoblastoma. Further, we investigated its role in cellular proliferation, migration, invasion, wound healing, colony forming abilities and tumor growth which suggested that AKAP4 could be used as a novel therapeutic target for CRC treatment.In the present study, we analyzed the expression of AKAP4 gene and protein in CRC cell lines and its potential role in cellular growth, proliferation, migration and invasion at molecular level in 2 incubator and were checked for mycoplasma contamination by mycoplasma PCR detection kit . Human normal colon epithelial cell NCM460 was procured and maintained according to manufacturer’s directions . Transient transfection was carried out by seeding 1 × 105COLO 205 or HCT 116 cells in 6-well plate using Lipofectamine reagent  according to the manufacturer’s instructions.Human colon cancer cell lines COLO 205 and HCT 116 were procured from the American Type Culture Collection  and were maintained according to standard procedures. Human colon cancer cell lines CaCo-2, COLO320 DM, HCT-15, HT-29, SW480 and SW620 were procured from National Centre for Cell Sciences , and were used within 8 weeks by growing in DMEM medium  supplemented with 10 % fetal bovine serum (FBS) maintained in a humidified 37 °C and 5 % COL, mouse anti-cytochrome-C, mouse anti-NOXA, rabbit anti-p53 upregulated modulator of apoptosis (PUMA), mouse anti-poly ADP ribose Polymerase 1 (PARP1), mouse anti-Caspase 7, mouse anti-Caspase 8, mouse anti-Caspase9, rabbit anti-cIAP2, Cyclin-dependent kinases (CDKs): mouse anti-CDK4 and mouse anti-CDK6, mouse anti-Cyclin B1, mouse anti-Cyclin D1, mouse anti-Cyclin E, anti-cyclin-dependent kinase inhibitor (CKI), mouse anti-p21, mouse anti-p16, and mouse anti-Retinoblastoma (Rb) were procured from Santa Cruz Biotechnology. Mouse anti-B-cell lymphoma 2 (BCL-2) was procured from Cell Signaling Technology, USA.Western blot and immunohistochemistry analysis was carried out using following antibodies; mouse anti-AKAP4 antibody was procured from Sigma-Aldrich , mouse anti-proliferating cell nuclear antigen (PCNA), mouse anti-calnexin (endoplasmic reticulum maker), mouse anti-GM130 (Golgi body marker) and mouse anti-lamin A/C (nuclear envelope marker) were purchased from Santa Cruz Biotechnology, USA. Horseradish peroxidase-conjugated anti-rat IgG, FITC-conjugated anti-rat IgG, and Texas Red-conjugated anti-mouse IgG were procured from Jackson ImmunoResearch Laboratories, West Grove, PA, USA. Mouse anti-beta actin, anti-MTCO2 , mouse anti-E-cadherin, mouse anti-N-cadherin, mouse anti-P-cadherin, Matrix metalloproteinases (MMP’s): rabbit anti-MMP2, rabbit anti-MMP3, mouse anti-MMP9, rabbit anti-SNAIL, mouse anti-SLUG, mouse anti-TWIST, mouse anti-alpha smooth muscle actin (αSMA), rabbit anti-Vimentin, mouse anti-Caspase 3, mouse anti-AIF, rabbit anti-APAF1, rabbit anti-XIAP, rabbit anti-Survivin, rabbit anti-DCR2, mouse anti-CDK1,rabbit anti-CDK2, and rabbit anti-phosphoRb were procured from Abcam, Cambridge, UK. Mouse anti-BCL-2-associated death promoter (BAD), rabbit anti-BCL-2 homologous antagonist/killer (BAK), mouse anti-BCL-2-associated X Protein (BAX), rabbit anti-BID, rabbit anti-Bcl-xAKAP4 Forward primer 5′-TGATACTACAATGATGTCTGATGAT-3′, AKAP4 Reverse primer 5′-GGAACTAGCAGCATCCTTGTAATCTTTATC-3′, β-actin was used as an internal control to check the quality of cDNA synthesis with following primers: β-actin Forward primer 5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3′, β-actin Reverse primer 5′-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3′. The PCR products were electrophoresed on 2 % agarose gel and photographed under UV light in EC3 Imaging System . The amplicons of AKAP4 thus obtained were sub-cloned into TOPO vector  to confirm the sequence of the AKAP4. β-Actin mRNA expression was checked as an internal control. Quantitative PCR was done using 5 ng of cDNA from normal colon epithelial cells and eight colon cancer cell lines in triplicate with Brilliant III Ultra-Fast SYBR QPCR MM  in iCycleriQmulticolor real time PCR detection system  according to manufacturer’s instructions. β-Actin was used as an internal control in all the reactions. AKAP4 mRNA expression was also checked in normal colon epithelial cells as a negative control. AKAP4 gene expression levels was subsequently normalized using expression levels of endogenous control β-Actinin the same mRNA samples in each colon cancer cell lines.Total RNA from all cancer cell lines and normal colon cells was isolated using RNeasy Mini kit  as per manufacturer’s protocol. The RNA was reverse transcribed using a set of primers and High-Capacity cDNA Reverse Transcription Kit  as described earlier . FollowiProteins from colorectal cancer cell extracts (10 μg/lane) and from normal colon tissue were resolved on 10 % sodium dodecylsulphate-10 % Polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting was carried out as described earlier . The proFlow cytometeric analysis was carried by culturing CRC cells. Subsequently cells were harvested and processed for AKAP4 surface localization as described earlier  using anIndirect immunofluorescence was carried in CRC cells by probing with anti-AKAP4 antibody or a control IgG as described earlier . The celAKAP4: 5′-TCTATGTTCACTTGATCGG-3′ ; 5′-CAAGCGAACGGGCAATTTA-3′ (AKAP4 shRNA2 Clone ID V2LHS-53113); 5′-TTACCAGAGAAGATAGTCG-3′ (AKAP4 shRNA3 Clone ID V2LHS-53116) and 5′-ATCTCGCTTGGGCGAGAGTAAG-3′ . The shRNA plasmids were prepared and transfected in COLO 205 and HCT 116 cells using lipofectamine and plus reagent . Further, qPCR was carried out to find out the knockdown of AKAP4 mRNA with various targets under investigation. Total RNA was extracted using RNeasy mini kit  and subsequently subjected to synthesize cDNA using High Capacity cDNA Reverse Transcription Kits . Quantification of AKAP4 mRNA was done using 5 ng of cDNA employing following AKAP4 specific primers (Forward primer 5′-TGATACTACAATGATGTCTGATGAT-3′ and Reverse primer 5′-GGAACTAGCAGCATCCTTGTAATCTTTATC-3′) and iQ SYBR Green Supermix . Cell lysates post 48 h post transfection were prepared for Western blotting as described earlier [Plasmid driven short hairpin RNA (shRNA) constructs and NC shRNA (scrambled shRNA) were procured from Super Array . The following target sequences were used in this study: Viability assay was carried out by transfecting AKAP4 shRNA3 in COLO 205 and HCT 116 cells and were visualized using chromogenic dye 3--2, 5-diphenyltetrazolium bromide . Absorbance at 570 nm-650 nm was recorded on ELISA plate reader . In addition, cellular growth analysis was also carried out as described earlier . Cells aBoth COLO 205 and HCT 116 cells after transfection with AKAP4 shRNA3 or NC shRNA targets were seeded in 6-well plates at three different cell densities in duplicates . Ten days post-seeding, the cells were fixed with 5 % glutaraldehyde in phosphate buffered saline (PBS) and stained with 0.5 % toluidine blue . The colonies were manually counted after washing cells with PBS. Images of representative fields were also captured using Nikon Eclipse E 400 microscope . Each experiment was repeated twice in triplicates.in-situ DNA fragmentation assay kit . AKAP4 shRNA3or NC shRNA transfected COLO 205 and HCT 116 cells were harvested by trypsinization and processed as per manufacturer’s instructions. The cells were analyzed at 576 nm using BD-FACS VERSA. .The effect of shRNA treatment in cancer cells on DNA fragmentation was assessed using Apo-BrdU- Red To study the effect of AKAP4 shRNA3or NC shRNA on apoptosis, cells transfected with shRNA were stained by annexin V using annexinV-PerCP-Cy5-5-A staining kit (Biovision). Staining was performed according to the manufacturer’s instructions. The cells were analyzed with a flow cytometer . Data acquisition and analysis was done using Cell QuestPro software.5each were transfected with AKAP4 shRNA3 or NC shRNA in 6-well plate. Post 48 h transfection, senescence assay was carried out using Senescence kit  as per manufacturer’s protocol. The images were captured using Nikon Eclipse E 400 microscope .Both COLO 205 and HCT 116 cells at a density of 3 x 10To investigate the potential role of AKAP4 in cellular migration and invasion assay were performed as described earlier .COLO 205 and HCT 116 cells were transfected with AKAP4 shRNA3or NC shRNA. Cells were seeded onto 12 mm coverslip and were fixed with 2.5 % glutaraldehyde in 4 % paraformaldehyde solution in 0.2 M sodium cacodylate at different time intervals (24 h and 48 h). Subsequently, cancer cells were processed by washing with sodium cacodylate solution, followed by staining with osmium tetraoxide. Coverslip having cells were washed with deionised water and dehydrated using different gradients of methanol (25–100 %). Critical point drying was done with hexa methyl disilane (HMDS) and coverslips were mounted on aluminium stubs. Gold and palladium coating was done using electron sputter coater under vacuum by Argon based Thermionic emission. The images were captured using electron microscope  at 20 kV using SmartSEM software in central microscopic facility.3 as described earlier [Human tumor xenografts were established in 6–8 weeks old athymic nude mice  as described earlier [2O2 (9:1) for 45 min to block and remove all traces of endogenous peroxidase. Subsequently, tissue sections were blocked with 5 % normal goat serum for 1 h at RT and probed with various antibodies  for overnight at 4 °C. After three washes with PBS, sections were incubated with secondary antibody . After incubation sections were subjected to three washings with PBS and the color was developed using 3,3′-Diaminobenzidine  as a substrate. Slides were counter stained with hematoxylin solution, mounted and observed under a Nikon Eclipse E400 microscope .Immunohistochemical analysis was performed on 4-μm thick sections of tumor tissue excised from AKAP4 shRNA3or NC shRNA treated mice as described earlier . Brieflyin-vitro assays and in in-vivo colorectal human xenograft model. Initially, we examined COLO 205 and HCT 116 cells for the AKAP4 gene expression by RT-PCR using AKAP4 specific primers. As shown in Fig. AKAP4 gene expression in both CRC cell lines. We further assessed AKAP4 mRNA expression in normal colon epithelial cells, CaCo-2, COLO 205, COLO320DM, HCT-15, HT-29, SW480 and SW620 colon cancer cell lines by quantitative PCR (qPCR). All cancer cell lines showed higher levels of AKAP4 expression compared to normal colon epithelial cells  and cyclin dependent kinases  in both CRC cell lines Fig. . NotablyL, cIAP2, XIAP, Axin2 and Survivin were down regulated  in tumor growth in AKAP4 shRNA3 treated mice as compared to NC shRNA treated mice as shown in Fig. To further validate our observations on malignant properties of colorectal cancer, following L, cIAP2, XIAP and Survivin . Interestingly, ablation of AKAP4 resulted in down regulation of pro-EMT molecules including N-cadherin, P-cadherin, α-SMA, SLUG, SNAIL, TWIST, Vimentin, MMP2, MMP3 and MMP9 protein. Cell migration and invasion potential and colony forming ability were also significantly reduced due to AKAP4 down regulation. We further assessed and validated our In conclusion, the present study shows that AKAP4 is over expressed in CRC cell lines. Ablation of AKAP4 apparently has multiple effects at molecular level in various malignant properties of the cancer cells including reduction in colony formation ability, expression of EMT molecules and growth arrest of cells (senescence). We strongly believe that AKAP4 may be used as a potential therapeutic target for the development of better CRC treatment management.


Caenorhabditis elegans Fic protein FIC-1 in vitro and in vivo. FIC-1 is an AMPylase that localizes to the nuclear surface and modifies core histones H2 and H3 as well as heat shock protein 70 family members and translation elongation factors. The three-dimensional structure of FIC-1 is similar to that of its human ortholog, HYPE, with 38% sequence identity. We identify a link between FIC-1-mediated AMPylation and susceptibility to the pathogen Pseudomonas aeruginosa, establishing a connection between AMPylation and innate immunity in C. elegans.Protein AMPylation by Fic domain-containing proteins (Fic proteins) is an ancient and conserved post-translational modification of mostly unexplored significance. Here we characterize the  C. elegans Fic protein FIC-1 on a biochemical, structural and functional level in vitro as well as in vivo. We solve the 3-dimensional structure of FIC-1 and identify novel FIC-1 substrates belonging to the translation elongation as well as heat-shock protein families. Investigating the consequence of diminished (fic-1(n5823)) or increased (FIC-1[E274G](nIs733)) AMPylation levels in vivo, we find a link between AMPylation and the innate immune response to the bacterial pathogen P. aeruginosa, describing a novel in vivo phenotype associated with Fic protein mediated target AMPylation.Eukaryotic Fic domain containing proteins (Fic proteins) AMPylate target proteins at the expense of a single ATP molecule. Previous studies have established a first link between target protein AMPylation and the unfolded protein response (UPR) in the endoplasmic reticulum. Yet, the consequences of target AMPylation remain poorly understood. Here, we take a multi-faceted approach to investigate the role of the  How post-translational modifications regulate protein activity is a fundamental question in biology. Phosphorylation, methylation or acetylation reversibly control cellular signaling pathways, the misregulation of which is often associated with pathologies, including cancer or autoimmune diseases , 2. AMPyCaenorhabditis elegans, Drosophila melanogaster, Mus musculus and Homo sapiens—carry only a single gene that specifies a Fic family member. All Fic proteins share a conserved sequence motif (HxFx(D/E)GN(G/K)R) found in their respective fic domains, including an invariant histidine required for catalysis (nIs733) constitutively active animals or wild type controls at 20°C or 25°C  mutants displayed increased susceptibility to killing by P. aeruginosa as compared to wild type (fic-1(n5823) mutant animals appeared to be unaffected in avoidance assays (fic-1(n5823) mutants by expressing wild type fic-1 in the mutant animals. Together, these results establishing a role for Fic proteins in innate immunity.op codon . We also or 25°C . When grild type . This wae assays . ExpressD. melanogaster as well as in human cells suggested a connection between Fic proteins and the regulation of the unfolded protein response (UPR) (nIs733)  loss of function nor the constitutively active form (nIs733) affected the outcomes  mutants or constitutive active (nIs733) animals and their respective controls  , using Eoutcomes . Thus, wruginosa . As C. eches are . None ofcontrols . fic-1 iin vivo, we applied a single molecule fluorescence in situ hybridization assay (smFISH) to detect endogenous fic-1 mRNA  V), RB1104 (hsp-3(ok1083) X), MT22519 , MT22798 , MT22849 (n5823 IV [fic-1 KO]), MT23529 , MT23188 , MT23262 , MT23307 , MT23265 , MT23494  X), MT23495 ), MT23497 (xbp-1(tm2482) III; n5823 IV), MT23498 (xbp-1(tm2482) III; nEx2237), MT23503 , MT23506 , MT23508 , MT23527 (daf-2(e1370ts) III; n5823 IV), MT23528 (daf-2(e1370ts) III; nIs733), MT23530 , CB1370 (daf-2(e1370ts)) III, ZD418(xbp-1(tm2482) III, RB1104 (hsp-3(ok1083)), SJ4005 ]), SJ4100 (zcIs13 V [Phsp-6::GFP])SJ4005  was cloned by replacing the fic-1 gene with a gfp orf. For inducible protein expression, the fic-1 gene was cloned into pPD49.78 (Fire lab vector kit) with an additional C-terminal HA-tag introduced as part of the primer Recombinant FIC-1 protein purification for in vitro AMPylation assays was based on fic-1258−508 cloned into a single orf of a pET-DUET1 vector. Primer sequences are available upon request. For fic-1(n5823) rescue, a 4.5 kbps linear DNA fragment was amplified using genomic DNA as template.Pfic-1134−508 was cloned with a non-cleavable N-terminal 6x His tag into an Ampicillin resistant T7 based bacterial expression plasmid. The transmembrane helix and membrane proximal domain of FIC-1 were excluded from both the wild type and the E274G construct.For crystallization, eft-3::Cas9::tbb-2–3′ UTR , zcIs4;fic-1(n5823) and zcIs4;FIC-1[E274G](nIs733) animals were exposed to either tunicamycin or ethidium bromide. GFP-reporter expression was scored 24 hours post exposure scale bar equals 500 μm. (B) FIC-1 does not control the onset of the mitochondrial stress response: zcIs13[hsp-6::GFP], zcIs13;fic-1(n5823) and zcIs13;FIC-1[E274G](nIs733) animals were exposed to either tunicamycin or ethidium bromide. GFP-reporter expression was scored 72 hours post exposure scale bar equals 500 μm. (C) and (D) nematode development under acute ER stress: eggs of indicated lines were transferred to OP50 plates containing 5 μg/ml tunicamycin to induce acute ER stress. Embryo development was scored.(A) FIC-1 does not control the onset of the ER stress response: zcIs4[(TIF)Click here for additional data file.S3 Figfic-1::GFP]; scale bar equals 40 μm. (B) Assessment of inducible FIC-1 expression. nEx2396 animals were heat-shocked for 2 hours at 34°C and FIC-1 protein level was assessed by Western blotting using anti-HA antibodies. (C) Characterization of FIC-1-specific mouse serum: Indicated recombinant proteins were probed with FIC-1-specific serum. (D) FIC-1 E274G localizes to the nuclear envelope: Staining of uninduced (-Dox) and induced (+Dox) Hela cells inducibly expressing GFP-tagged FIC-1 E274G or GFP-tagged HYPE E234G and its localization analyzed by confocal microscopy; scale bar equals 10 μm.(A) FIC-1 is enriched in nematode embryos: representative images of embryos containing a promotor-trap construct nEx2219(nIs733) animals were kept at either 20°C and survival was scored every other day. Depicted n refers to number of animals at experiment initiation; number in brackets represents total counted dead events. P-values (Gehan-Breslow-Wilcoxon test) as compared to N2 wild type control: (A) not significant; (B) not significant; (C) N2. vs fic-1: 0.006; all others not significant.(A)—(C) wild type, (TIF)Click here for additional data file.S9 Figfic-1(n5823), fic-1 rescue and FIC-1[E274G](nIs733) L4 animals of indicted lines were place in the center of a P. aeruginosa loan and nematode survival was scored once per day until last animal vanished. Depicted n refers to number of animals at experiment initiation; number in brackets represents total counted dead events. P-values (Gehan-Breslow-Wilcoxon test) are as compared to N2 wild type control.(A)—(G) wild type, (TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


In the murine model of Concanavalin A-induced T cell-mediated hepatitis, we showed that hepatic expression of the inflammatory CXC chemokine ligands (CXCL)9 and CXCL10 strongly increased whereas homeostatic CXCL12 significantly decreased. Consistently, CD4+ T cells expressing the CXC chemokine receptor (CXCR)3 accumulated within the inflamed liver tissue. In histology, CXCL9 was associated with liver sinusoidal endothelial cells (LSEC) which represent the first contact site for T-cell immigration into the liver. LSEC actively transferred basolaterally internalized CXCL12, CXCL9 and CXCL10 via clathrin-coated vesicles to CD4+ T cells leading to enhanced transmigration of CXCR4+ total CD4+ T cells and CXCR3+ effector/memory CD4+ T cells, respectively in vitro. LSEC-expressed CXCR4 mediated CXCL12 transport and blockage of endothelial CXCR4 inhibited CXCL12-dependent CD4+ T-cell transmigration. In contrast, CXCR3 was not involved in the endothelial transport of its ligands CXCL9 and CXCL10. The clathrin-specific inhibitor chlorpromazine blocked endothelial chemokine internalization and CD4+ T-cell transmigration in vitro as well as migration of CD4+ T cells into the inflamed liver in vivo. Moreover, hepatic accumulation of CXCR3+ CD4+ T cells during T cell-mediated hepatitis was strongly reduced after administration of chlorpromazine. These data demonstrate that LSEC actively provide perivascularly expressed homeostatic and inflammatory chemokines by CXCR4- and clathrin-dependent intracellular transport mechanisms thereby contributing to the hepatic recruitment of CD4+ T-cell populations during immune surveillance and liver inflammation.Leukocyte adhesion and transmigration are central features governing immune surveillance and inflammatory reactions in body tissues. Within the liver sinusoids, chemokines initiate the first crucial step of T-cell migration into the hepatic tissue. We studied molecular mechanisms involved in endothelial chemokine supply during hepatic immune surveillance and liver inflammation and their impact on the recruitment of CD4 Migration of leukocytes into tissues is central for immune surveillance and pathogen defence as well as for the pathogenesis of autoimmune diseases. Leukocyte recruitment from the circulation is a tightly regulated multistep process depending on specific interactions between adhesion molecules and chemokine receptors expressed by the leukocytes and their respective ligands on the vascular endothelial cells ,2. The mChemokines can be generally classified into inflammatory and homeostatic chemokines. Constitutively expressed homeostatic chemokines like the CXC chemokine ligand (CXCL)12 are involved in the control of physiological leukocyte recirculation and in immune surveillance. Inflammatory chemokine expression is induced in inflamed tissues by pro-inflammatory cytokines like interferon (IFN)-γ and consistently controls recruitment of effector cells determining the composition of inflammatory cell infiltrates and the outcome of local inflammation ,8. CXCL9Circulating T cells pass the liver via the sinusoids, a network of small capillaries lined by liver sinusoidal endothelial cells (LSEC). The sinusoidal endothelium is a major route for T-cell entry into the liver parenchyma from where recruited cells can subsequently migrate to sites of inflammation ,15. T-cein vitro [We previously showed that LSEC promote chemokine-dependent CD4+ T-cell transmigration in vitro . In the C57BL/6 mice were obtained from the Charité animal facility  or Charles River . CXCR3-/- mice  were kinin situ with digestion medium containing collagenase IV  injected into the portal vein, excised and further incubated in the digestion medium. To eliminate parenchymal cells, the single-cell suspension was subjected to a one-step density gradient centrifugation with 26% Nycodenz . LSEC were isolated from NPC by magnetic cell sorting using anti-CD146 antibody  as previously described [To isolate non-parenchymal cells (NPC), livers were perfused escribed . LSEC adescribed . For traNPC were stained with anti-CD4 (RM4-5), anti-CD90.2 (53–2.1), anti-CD69  or anti-CXCR3 antibody . For detection of cytokine-expressing cells, NPC were re-stimulated with phorbol myristate acetate (10 ng/ml) and ionomycin (500 ng/ml) for 4 h with the addition of brefeldin A  after 60 min. Cells were fixed with 2% paraformaldehyde. After permeabilization using 0.5% saponin, NPC were stained with anti-IFN-γ antibody . Unspecific binding was blocked with rat immunoglobulin  and anti-CD16/32 antibody . LSEC were incubated with AlexaFluor 647-labeled CXCL10 or CXCL12 . LSEC were treated with chlorpromazine , nystatin (10 μM) or filipin (15 μM) for 10 min and AMD3100  for 60 min, washed and further incubated with the chemokines for 60 min. Data were acquired using a FACS Canto II (BD Biosciences) and analyzed by the FlowJo software .Ex vivo isolated LSEC were cultured in the presence of tumor necrosis factor (TNF)-α  and IFN-γ  for 24 h. Adhered cells were detached by accutase treatment (Sigma-Aldrich).Ex vivo isolated LSEC (1x106) were re-suspended in 100 μl electroporation buffer  before 4 μg plasmid DNA was added. Using an electroporation cuvette  in the ELPorator 1000 device [The expression vector Clathrin-LCa-EYFP (Addgene plasmid 21741) was provided by Chen Chen  . Ex vivo0 device , LSEC weTotal RNA was isolated from liver samples or LSEC by single-phase organic extraction  and 100 ng RNA was reversely transcribed into cDNA using the High-Capacity cDNA Reverse Transcription Kit . Quantitative PCR was performed using a TaqMan Universal Master Kit and exon-spanning, gene-specific assays  in a StepOne Plus real-time PCR system (Applied Biosystems). Specific chemokine and chemokine receptor mRNA expression was quantified in relation to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the housekeeping gene using the ΔΔCT method [LSEC were cultured overnight on gelatine-coated transwell membranes with a 5 μm pore size  to form confluent cell layers . LSEC weLSEC were cultured overnight on transwell inserts. AlexaFluor 647-labeled CXCL12 and CXCL10 (both 50 nM) or AlexaFluor 488-labeled acetylated-low density lipoprotein  were added to the lower chamber of the transwell for indicated times. Subsequently, LSEC were fixed with 4% paraformaldehyde (Sigma-Aldrich) and stained with mouse anti-clathrin (3F133), mouse anti-caveolin-1 , rabbit anti-early endosome antigen (EEA)1 , rabbit anti-green fluorescent protein (GFP), rabbit anti-transferrin receptor (TfR) and rat anti-lysosomal-associated membrane protein (LAMP)-1  antibody followed by AlexaFluor 488-conjugated anti-mouse, AlexaFluor 647-labeled anti-rat, AlexaFluor 488-conjugated anti-rabbit or anti-rat  secondary antibody. Negative controls were performed by omitting the primary antibodies. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) . LSEC were treated with CPZ (30 μM), filipin (15 nM), nystatin (10 nM) or AMD3100 (10 μM) added to the lower chamber for 10 min  or 60 min (AMD3100). LSEC were washed, followed by chemokine incubation (60 min) as described above. Images were taken with an LSM 510 Meta confocal laser scanning microscope .To quantify T-cell infiltration, paraffin-embedded liver sections were stained with rabbit anti-CD3 antibody  followed by anti-rabbit secondary antibody  and the Streptavidin-Alkaline Phosphatase Kit (Dako) using Fast Red as chromogen. All CD3+ cells per high power field (hpf) and three hpf per liver sample were counted. For immunofluorescence, shock-frozen liver tissue was cut into 4 μm sections and stained with rat anti-CD146 antibody followed by AlexaFluor 488-labeled anti-rat secondary antibody (Dianova). Subsequently, liver sections were stained with rabbit anti-CXCL9 (Santa Cruz Biotechnology) followed by AlexaFluor 455-labeled anti-rabbit secondary antibody (Dianova). Negative controls were performed by omitting the primary antibodies. Images were taken with an AxioImager Z1 microscope and processed with Axiovision software (Carl Zeiss MicroImaging).Concanavalin (Con) A  was injected intravenously to induce T cell-mediated hepatitis in C57BL/6 mice. CPZ (3 mg/kg) was intraperitoneally administered 60 min after Con A-treatment. Blood was drawn from individual mice to monitor progression of hepatitis. Liver injury was quantified by automated measurement of plasma activities of alanin transaminase (ALT) using a Roche modular analyzer.Mice received CPZ 7 h after Con A treatment. Radioactively labeled CD4+ T cells  were intravenously injected 120 min after administration of CPZ. After 60 min migration time, the radioactivity of the liver and the remaining body was counted using a Wizard gamma counter . The percentage of organ-specific radioactivity in relation to the total recovered radioactivity reflected the percentage of cells that have migrated into the respective organ .Data were analyzed and statistical significance was determined using the GraphPad Prism software . Statistical comparison was carried out using the nonparametric two-tailed Mann-Whitney test.In situ production, transport and presentation of chemokines in the liver are crucial for the hepatic recruitment of circulating T cells [ T cells ,29,30. Iin vitro, LSEC were activated with TNF-α and IFN-γ. Activated LSEC markedly down-regulated CXCL12 whereas CXCL9 and CXCL10 expression was strongly increased  Percentages of CD90.2+ and CD4+ T cells expressing IFN-γ were detected. Representative plots and mean values ± SD of 2–4 independent experiments with three mice per group are shown. * p< 0.05; ns, not significant.Mice were treated with Con A and received CPZ 60 min after hepatitis induction. NPC isolated 6 h after Con A treatment and stained for CD4, CD90.2, CD69 and IFN-γ were assessed by flow cytometry. (A) GMFI of CD69 was determined on gated CD90.2(TIF)Click here for additional data file.


Caenorhabditis elegans. Whereas PMK-2 functions redundantly with PMK-1 in the nervous system to regulate neuronal development and behavioral responses to pathogenic bacteria, the miR-58, miR-80, miR-81, and miR-82 microRNAs function redundantly to destabilize pmk-2 mRNA in non-neuronal cells with switch-like potency. Our data suggest a role for the miR-58 family in the establishment of neuronal-specific gene expression in C. elegans, and support a more general role for microRNAs in the establishment of tissue-specific gene expression.Analyses of gene expression profiles in evolutionarily diverse organisms have revealed a role for microRNAs in tuning tissue-specific gene expression. Here, we show that the relatively abundant and constitutively expressed miR-58 family of microRNAs sharply defines the tissue-specific expression of the broadly transcribed gene encoding PMK-2 p38 MAPK in  C. elegans microRNAs at all developmental stages, defines the spatial expression pattern of PMK-2 p38 MAPK. While the pmk-2 gene is broadly transcribed, its tissue-specific expression is established by the redundant activities of miR-58, miR-80, miR-81, and miR-82, which switch off expression of PMK-2 through destabilization of pmk-2 mRNA in non-neuronal tissues. Our data suggest a housekeeping role for the miR-58/80-82 family in establishing and maintaining neuronal patterns of gene expression in C. elegans, and supports a more general role for microRNAs in establishing patterns of tissue expression.MicroRNAs are small, noncoding RNAs that act post-transcriptionally to inhibit expression of their target mRNAs. Gene expression studies of microRNAs and their target transcripts in diverse organisms have suggested that microRNAs may function to shape patterns of tissue expression. In this paper, we show that the miR-58/80-82 family of microRNAs, which accounts for roughly half of all  Caenorhabditis elegans LGI: T24B8.5agIs219, tir-1(qd4), mir-80(nDf53)LGIII: mir-58(n4640), pmk-2(qd284), pmk-2(qd307), pmk-2(qd287), pmk-2(qd279), pmk-2(qd280), pmk-2(qd305), pmk-2(qd171), pmk-1(km25)LGIV: mir-81-82(nDf54), sek-1(km4), nIs145LGX: qdEx101[Poperon::islo-1::pmk-3::pmk-2::GFP::pmk-1::mCherry], qdEx102[Poperon::islo-1::pmk-3::pmk-2mut::GFP::pmk-1::mCherry]Extrachromosomal arrays: A list of all strains used in this study is provided in the Supporting Information .P. aeruginosa PA14 were grown in Luria-Bertani (LB) broth overnight at 37°C. Five microliters of the overnight culture was used to seed 35-mm slow-kill assay plates  containing 50 μg/ml 5-fluorodeoxyuridine (FUdR), used to prevent eggs from hatching. The culture was seeded in the middle of the plates and was not spread to the edges, meaning the resulting lawn would be “small,” allowing for behavioral avoidance. The seeded plates were incubated overnight at 37°C and then overnight at room temperature. Roughly 40 L4 larval stage worms were placed onto a plate containing the prepared P. aeruginosa bacterial lawn with four plates per strain. The assay was carried out at 25°C. Plates were checked at the indicated times and worms that did not respond to a gentle prod from a platinum wire were scored as dead. Worms that crawled off of the plate or burrowed were censored.Cultures of E. coli OP50 or pathogenic P. aeruginosa PA14 and incubated at 25°C for 6 hr at which time the worms were immobilized with 50 mM sodium azide and mounted on 2% agarose pads. Immobilized worms were viewed using an AxioImager Z1 fluorescence microscope  with an EC Plan-Neofluar 40x/1.3 Oil DIC objective and the focal plane with the strongest GFP signal in the ADF neuron was used to take a 30 ms exposure picture with an AxioCam HRm camera. The images were analyzed in Fiji . Screening for mutations was performed as outlined . Screening for deletions was performed by PCR analysis of F1 transgenic animals.The eviously . Three seviously  using thqd279 and qd280 alleles of pmk-2 were isolated from a screen for suppressors of the pmk-2(qd171) suppressor phenotype of the pmk-1(km25) loss-of-function phenotype. pmk-2(qd171) pmk-1(km25) mutant L4 larvae carrying the agIs219 reporter transgene for p38 activity in the intestine were mutagenized with EMS as described previously [2 progeny from roughly 14,000 mutagenized genomes were screened for a decrease in expression of GFP from the agIs219 reporter transgene using a dissecting microscope equipped to detect GFP fluorescence. The sequence of pmk-2 was determined in mutants with diminished GFP expression from the agIs219 transgene. We identified three alleles of pmk-2: qd279 and qd280 (which are missense mutations in conserved residues and were utilized in this study), as well as qd281.The eviously . Synchromir-58,80-82, grew slower than the other strains and therefore was harvested ~10 hr after the wild type strain. For 3’ RACE experiments, cDNA was prepared using the FirstChoice RLM-RACE Kit (Ambion). Two successive rounds of PCR were performed using nested primers specific to pmk-2 and an adaptor at the 3’-end. Sequence determination was performed by direct Sanger sequencing of 3’ RACE products. miR-58/80-82 seed match sites were identified using TargetScan [pmk-1, pmk-2, and pmk-3 mRNA levels were normalized to the control gene snb-1. Fold change relative to wild type was determined using the Pfaffl method [pmk-1, tgaatgatgatgtaagggcaga and cttcctcttcgtcagcaaatg; pmk-2, caagtgttacgtgggctcaa and cgagaatcttgacttcgcatc; pmk-3, gtatcgaagcaacgggaaac and tggaccacatggttttgaga; snb-1, ccggataagaccatcttgacg and gacgacttcatcaacctgagc.Hypochlorite-synchronized populations of L4 larval stage worms were flash-frozen in liquid nitrogen and stored at -80°C until RNA extraction using TRI reagent . Strain MT15563, carrying mutations in rgetScan . For qual method . Sequencpmk-1(km25), pmk-2(qd171) pmk-1(km25), and pmk-2(qd279 qd171) pmk-1(km25) strains with RNAi treatment (mir-58/80-82 family microRNA deletion strains (pmk-2(qd305)  was added to the worm pellets, which were then boiled for 15 minutes with occasional vortexing and then centrifuged to pellet the debris. The protein concentration of the lysates (supernatant from the previous step) was determined using the BCA Protein Assay Kit . For each sample, 50 μg of total protein was separated on a 10% SDS-PAGE gel  and then transferred to a nitrocellulose membrane . Blots were blocked with TBST supplemented with 5% skim milk power and then probed with either a 1:1,000 dilution of rabbit anti-ACTIVE p38 MAPK pAb , which recognizes the dually phosphorylated TGY motif of activated p38 MAPK, or a 1:10,000 dilution of mouse anti-β-tubulin  in TBST with 5% skim milk powder. Horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse IgG secondary antibodies  were used followed by detection with ECL reagents .For the experiments with reatment , mixed s strains  and pmk-2(qd305) , hypochlE. coli HT115 bacterial cultures carrying the control plasmid pPD129.36 (Ligation number L4440) or carrying specific plasmids derived from pPD129.36 designed to target either sek-1 or pmk-2 for RNAi. For pathogenesis assays and visualization of GFP expression from the agIs219 reporter, 3–6 L4 larval stage worms were placed onto seeded RNAi plates and their progeny were assayed. For Western analysis, ~10 L4 larval stage worms were placed onto seeded RNAi plates and the progeny of these worms were harvested before being deprived of food.Feeding RNAi was performed as previously described . NGM agapmk operon translational reporter was constructed through ligation of overlapping PCR amplicons by homologous recombination in Saccharomyces cerevisiae strain FY2 as previously described [pmk operon and ~4.4 kb of upstream sequence was amplified from fosmid 34bC01 in adjacent fragments with at least 50 bp of overlap between fragments. For the pmk operon translational reporter carrying mutations in the 3’UTR of pmk-2, 90 bp reverse complement primers containing the desired mutations were designed and used to amplify the 3’UTR of pmk-2. The gene encoding GFP was amplified from pPD95.75 . The gene encoding mCherry was amplified from pCFJ90 (Addgene). The PCR amplicons were transformed into S. cerevisiae strain FY2 along with destination vector pNP30  digested with XhoI and AvrII . Yeast DNA was extracted with phenol-chloroform and transformed into DH5-α electrocompetent cells . Plasmids were prepped using a Miniprep Kit  and their sequences were verified. Germline transformations were performed as described previously [The escribed . A ~20.3eviously .agIs219 reporter transgene, adult worms  were picked over to an unseeded NGM agar plate and immobilized with 50 mM sodium azide. Worms were viewed using a Stereo V12 fluorescence microscope (Zeiss) and pictures were taken with an AxioCam MRc camera. To visualize expression of GFP and mCherry from the pmk operon translational reporter, L4 larval stage transgenic animals were immobilized with 50 mM sodium azide and mounted on 2% agarose pads. Worms were viewed using a LSM510 confocal microscope (Zeiss). Stacks of confocal images were acquired and processed in Fiji to obtain maximum projections [To visualize expression of GFP from the jections . All imaS1 Figpmk-2 3’UTR (release WS245). Uppercase, last exon of pmk-2 and first exon of pmk-1, respectively. Underline, polyadenylation signal. Bold font, miR-58/80-82 family seed match site. Gray highlight, qd305 deletion. (B) qRT-PCR analysis of pmk-1, pmk-2, and pmk-3 mRNA levels in L4 larval stage wild type worms and the pmk-2(qd305) mutant. Levels of pmk-1, pmk-2, and pmk-3 mRNA are normalized to the levels of snb-1 mRNA. Values plotted are the fold changes relative to wild type. Shown is the mean ± SEM . (C) Immunoblot analysis of lysates from L4 larval stage wild type worms, pmk-2(qd305) mutant animals, and pmk-2(qd307 qd305) intragenic suppressor mutant animals using antibodies that recognize activated p38 MAPK and β-tubulin.(A) DNA sequence of the (PDF)Click here for additional data file.S2 Figmir-80; mir-58; mir-81-82 mutant animals, and mir-80; mir-58 pmk-2(qd284); mir-81-82 mutant animals using antibodies that recognize activated p38 MAPK and β-tubulin.Immunoblot analysis of lysates from L4 larval stage wild type worms, (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Cine DENSE (Displacement Encoding with Stimulated Echoes) provides accurate and high-resolution displacement and strain imaging of the heart; however, image acquisition times are relatively long and, due to properties inherent to stimulated echoes, signal-to-noise ratio (SNR) is relatively low. Accelerated cine DENSE could substantially shorten scan times and/or provide improved spatial and/or temporal resolution; however, the low SNR, requirement to preserve phase information, and cardiac motion present technical challenges. We aimed to develop acceleration methods that overcome these challenges.A variable-density spiral cine DENSE sequence was implemented on a 1.5T scanner  where the center of k-space was fully sampled and the outer portion of k-space was undersampled. Spiral interleaves were distributed uniformly within each cardiac phase and rotated by the golden angle through different cardiac phases. Cine DENSE datasets were collected from 5 healthy volunteers (age 25-28) using a 5-channel RF coil. To make comparisons, fully-sampled cine DENSE datasets with 6 to 8 spiral interleaves per image were acquired within one breathhold (20 to 26 heartbeats), and these datasets were retrospectively rate-2 undersampled. Also, prospectively rate-2 undersampled data with 4 spiral interleaves per image were acquired within a shorter breathhold (14 heartbeats). Images had spatial resolution of 1.8-2.2 × 1.8-2.2 × 8 mm and temporal resolution of 19.6 ms. All undersampled data were reconstructed using a recently-developed compressed sensing (CS) method called Block LOw-rank Sparsity with Motion-guidance (BLOSM), combined with SENSE parallel imaging. The BLOSM-SENSE algorithm exploits matrix low-rank sparsity within motion-tracked regions of SENSE-combined images. Complex-valued images were reconstructed to preserve the phase information used for displacement encoding.In Figure Rate-2 accelerated spiral cine DENSE with the BLOSM-SENSE reconstruction method provides high-quality complex images. Retrospective undersampling of fully-sampled datasets demonstrated accurate displacement and strain measurements. Prospectively undersampled cine DENSE demonstrated typical myocardial strain measurements, using approximately half the data acquisition time of conventional cine DENSE.R01 EB 001763, R01 HL 115225, AHA Award 12PRE12040059 and Siemens Healthcare.


The activation, or maturation, of dendritic cells (DCs) is crucial for the initiation of adaptive T-cell mediated immune responses. Research on the molecular mechanisms implicated in DC maturation has focused primarily on inducible gene-expression events promoting the acquisition of new functions, such as cytokine production and enhanced T-cell-stimulatory capacity. In contrast, mechanisms that modulate DC function by inducing widespread gene-silencing remain poorly understood. Yet the termination of key functions is known to be critical for the function of activated DCs. Genome-wide analysis of activation-induced histone deacetylation, combined with genome-wide quantification of activation-induced silencing of nascent transcription, led us to identify a novel inducible transcriptional-repression pathway that makes major contributions to the DC-maturation process. This silencing response is a rapid primary event distinct from repression mechanisms known to operate at later stages of DC maturation. The repressed genes function in pivotal processes—including antigen-presentation, extracellular signal detection, intracellular signal transduction and lipid-mediator biosynthesis—underscoring the central contribution of the silencing mechanism to rapid reshaping of DC function. Interestingly, promoters of the repressed genes exhibit a surprisingly high frequency of PU.1-occupied sites, suggesting a novel role for this lineage-specific transcription factor in marking genes poised for inducible repression. Dendritic cells (DCs) are professional antigen (Ag) presenting cells playing central roles in the initiation, regulation and implementation of Ag-specific immune responses ,2. They DCs detect PAMPs and DAMPs via pattern recognition receptors (PRRs), including Toll-like receptors (TLRs), Nod-like receptors (NLRs), C-type lectin receptors (CLRs) and Rig-I-like receptors (RLRs) ,5. PRR-eCompared to current knowledge on TF networks governing transcriptional activation in DCs, little is known about mechanisms controlling maturation-induced transcriptional silencing. Certain class II TFs—such as ATF3, BCL6 and PRDM1—function as repressors that dampen or terminate transcriptional responses in activated DCs . Howeverde novo MHCII synthesis , CLEC10A (Abnova), SOCS5 (GeneTex), CLEC4A (GeneTex) and tubulin (SIGMA-ALDRICH).ChIP experiments were performed as described  using anCIITA locus. DNA was purified, amplified by LPMCR as described , 0.5 mM Ethylenediaminetetraacetic acid (EDTA), 0.15 mM Spermine, 0.5 mM Spermidine, 0.5 mM PMSF, 1 mM Dithiothreitol (DTT). Cells were lysed for 10 min on ice after adding one volume of the same solution containing 0.8% NP40. Nuclei were pelleted through a 1 ml cushion of the same solution containing 0.9 M sucrose, resuspended in 75 mM NaCl, 20 mM Tris-HCL (pH 7.9), 0.5 mM EDTA 0.125 mM PMSF 0.85 mM DTT, 50% glycerol and lysed for 10 min on ice after adding 8 volumes of 0.3 M NaCl, 20 mM HEPES (pH 7.6), 0.2 mM EDTA, 7.5 mM MgCl2, 1 M Urea, 1 mM DTT, 1% NP-40. Chromatin was pelleted by centrifugation and resuspended in 500 μl of 50 mM NaAc (pH5), 50 mM NaCl, 0.5% sodium dodecyl sulphate (SDS). Nascent RNA was purified by three extractions with hot phenol  and precipitated with 0.15 M NaCl and ethanol. RNA was resuspended in 50 μl 10 mM Tris-HCL (pH 7.6) and treated with DNaseI for 15 min at 37°C in 10 mM MgCl2, 1 mM DTT. DNase activity was stopped by adding 50 mM EDTA, 1.5 M NaAc, 1% SDS and RNA was purified by phenol-chloroform extraction and ethanol precipitation. Nascent RNA samples were depleted of ribosomal RNA (rRNA) using the Ribominus kit (Invitrogen). Nascent RNA samples corresponding to three independent experiments were subjected to quality controls by qRT-PCR. DNA contamination was excluded by omitting reverse transcriptase. Maturation was assessed by measuring IL12B, IL1B and TNF mRNAs induction. Enrichment of primary transcripts was verified using primers specific for selected unspliced precursor RNAs. rRNA depletion  was verified using primers for 18 and 28S RNAs.Nascent RNA was isolated essentially as described ,25. 10–2http://www.ncbi.nlm.nih.gov/blast/html/megablast.html). Paired information was used to generate alignments of each read on the genomic region where hits were found by previous steps using SIBsim4 http://sibsim4.sourceforge.net/. Alignments were analyzed using Tromer (cDNA libraries were generated from three independent nascent RNA or mRNA samples using Illumina specifications. Illumina technology was used to generate paired-end reads (80 bp) for nascent RNA or single-end reads (50 bp) for mRNA. Data analysis consisted of a filtering step to eliminate reads consisting mono- and di-nucleotide repeats followed by the identification of exact unique matches using fetchGWI . Megablag Tromer .http://bowtie-bio.sourceforge.net/index.shtml). Only uniquely mapped reads were considered for further analysis. Peaks were called using MACS software version 2.0.10.20130520 (http://liulab.dfci.harvard.edu/MACS/). Input-DNA was used as background control. Peaks were considered significant if their P-values were <10−5 and if present (overlapping by >1 nt) in two biological repeats.10 ng of immunoprecipitated-DNA or chromatin-input-DNA were used to prepare sequencing libraries using Illumina specifications. 50-nucleotide single-end reads were generated using Illumina technology. Reads were aligned to the reference human genome GRCh37.71 using Bowtie version 0.12.7 (http://159.149.160.51/pscan/). TFBSs with P-values <10−3 were considered to be significantly overrepresented.Pscan was used to identify potential TF-binding-sites (TFBSs) (JASPAR database) that are overrepresented between nucleotides −450 and +50 relative to the TSS . CIITA-silencing was induced by diverse stimuli—including LPS, tumor necrosis factor (TNFα), peptidoglycan (PGN), Pam3CysSerLys4 (PAM), polyinosinic-polycytidylic acid (pIC) and flagellin (Flag)—indicating that it is a general feature of Mo-DC maturation , and H3-lysine-4 trimethylation (H3K4trim). Repressed-chromatin marks examined were H3-lysine-9 dimethylation and H3-lysine-27 trimethylation. Deposition of heterochromatin protein (HP1) was also examined. These chromatin features were assessed in LPS-treated Mo-DCs at strategic positions in the regulatory region of CIITA. Strong reductions in H4Ac were observed at all four CIITA promoters (pI–pIV) and at a position situated 4 kb downstream of pIV  experiments were performed to analyze H4-deacetylation at the ol genes . Input DA Figure . Deacetye Figure .CIITA silencing was investigated by using the histone-deacetylase (HDAC) inhibitor Trichostatin A (TSA). TSA impaired LPS-induced CIITA mRNA down-regulation, suggesting that CIITA silencing requires histone deacetylation , p38 (SB202190) and extracellular signal-regulated kinases ERK (U0126) MAPK pathways also had no impact when added individually. However, CIITA silencing was completely abrogated by blocking both the p38 and ERK pathways  and blocked by combined inhibition of the ERK and p38 pathways .Key features of the silencing mechanism documented for e Figure . DownregCIITA, MARCH1 and CD1A  of the genes subjected to rapid histone-deacetylation were expressed in immature Mo-DCs above a baseline level of 1 read per kb per million (RPKM) Figure . 590 70, indicatInsight into the silencing mechanism was gained by analyzing predicted TFBSs. TFBS-enrichment was investigated in genes that are induced, unchanged in their expression, or deacetylated and silenced Figure . InducedPU.1 is the most strongly expressed ETS-family member in Mo-DCs Figure . The proCIITA, MARCH1 and CD36 , CLEC4A  and CLEC12A  is likely to impair Ag-uptake in mature DCs. These CLRs bind glycans displayed on pathogen or self-derived proteins, mediating their internalization and delivery to Ag-loading compartments, and promoting Ag-presentation to CD4+ T cells and cross-presentation to CD8+ T cells  allowing mature DCs to exit peripheral tissues and home to lymphoid tissues  synthesis. Silencing of arachidonate-15-lipoxygenase (ALOX15) expression will reduce the synthesis of several bioactive lipids, including lipoxins. Reduced expression of these enzymes should have a dramatic impact on lipid-mediator synthesis. The functional consequences of this alteration are difficult to predict, as lipid-biosynthesis pathways are complex and lipid mediators can exert distinct pro-inflammatory and immunosuppressive effects on diverse cell types. Knowledge on the patterns of lipid-mediator production by DCs remains fragmentary. Our results suggest that this aspect of DC function has been neglected and requires investigation.A third process affected by the silencing mechanism is lipid mediator production. Reduced prostaglandin-endoperoxidase-synthesase-1 (PTGS1) expression will impair the production of prostanoids. Decreased leukotriene-A4-hydrolase (LTA4H) expression will impair leukotriene-B4 .Supplementary Data are available at NAR Online.


Ad5-GUCY2C-PADRE is a replication-deficient human type 5 recombinant adenovirus (Ad5) vaccine encoding guanylyl cyclase C (GUCY2C) fused to the PAn DR Epitope (PADRE). GUCY2C, a paracrine hormone receptor producing the second messenger cyclic GMP (cGMP), is selectively expressed by intestinal epithelial cells and a subset of hypothalamic neurons, but not other tissues. Importantly, GUCY2C is over-expressed in nearly all primary and metastatic human colorectal tumors. Preclinical studies in mice demonstrated selective tolerance of GUCY2C-specific CD4+ T cells, but not CD8+ T or B cells, necessitating inclusion of the exogenous CD4+ T helper cell epitope PADRE to maximize GUCY2C-specific CD8+ T cell and antibody responses and antitumor efficacy, without autoimmunity.11 Ad5-GUCY2C-PADRE viral particles. Safety and immunomonitoring where examined at 30, 90 and 180 days following vaccination. Primary objectives were to determine the safety, tolerability and toxicity of Ad5-GUCY2C-PADRE and to determine whether Ad5-GUCY2C-PADRE induces GUCY2C-specific immune responses. The study employed a joint efficacy-toxicity design and included stopping rules for either efficacy or toxicity. Results here were obtained during the planned interim analysis following accrual of 10 subjects.This is an open-label, single arm “proof-of-concept” study evaluating a single dose level of Ad5-GUCY2C-PADRE as a vaccine for surgically-treated, node-negative colon cancer subjects (NCT01972737). Patients received a single intramuscular administration of 10The vaccine was well tolerated, producing only mild adverse events (AEs). Short-lived injection site pain/swelling, body aches and chills were the most commonly observed AEs and occurred in 30-40% of subjects. GUCY2C-specific antibody and T-cell responses were observed in a subset of subjects. Consistent with preclinical mouse data, T-cell responses were composed of CD8+, but not CD4+, T cells. Importantly, GUCY2C-specific responses occurred only in subjects with low Ad5 neutralizing antibody (NAb) titers at the time of vaccination, suggesting that pre-existing Ad5 immunity limits Ad5-GUCY2C-PADRE immunogenicity.Interim analysis of 10 subjects receiving Ad5-GUCY2C-PADRE demonstrates proof-of-concept that GUCY2C is immunogenic in humans and that GUCY2C-directed vaccination is safe. Moreover, the presence of GUCY2C-specific antibody and CD8+ T-cell, but not CD4+ T-cell, responses is consistent with selective CD4+ T-cell tolerance observed in mouse models. These data establish GUCY2C as a safe and immunogenic target for immunotherapy in cancer patients.ClincialTrials.gov identifier NCT01972737.