In CAV1−/− mice fasting caused loss of adipose tissue mass despite a lack of hormone-sensitive lipase (HSL) phosphorylation. In addition, fasting resulted in increased macrophage infiltration, enhanced deposition of collagen, and a reduction in the level of the lipid droplet protein perilipin A (PLIN1a). Explant cultures of CAV1−/− adipose tissue also showed a loss of PLIN1a during culture, enhanced secretion of IL-6, increased release of lactate dehydrogenase, and demonstrated increased susceptibility to cell death upon collagenase treatment. Attenuated PKA-mediated signaling to HSL, loss of PLIN1a and increased secretion of IL-6 were also observed in adipose tissue explants of CAV1+/+ mice with diet-induced obesity. Together these results suggest that while alterations in adipocyte lipid droplet biology support adipose tissue metabolism in the absence of PKA-mediated pro-lipolytic signaling in CAV1−/− mice, the tissue is intrinsically unstable resulting in increased susceptibility to cell death, which we suggest underlies the development of fibrosis and inflammation during periods of metabolic stress.Caveolin-1 (CAV1) is an important regulator of adipose tissue homeostasis. In the present study we examined the impact of CAV1 deficiency on the properties of mouse adipose tissue both  Dysregulation of systemic lipid levels plays an important role in the development of numerous metabolic disorders including obesity and lipodystrophy in vivo is regulated by specific mechanisms (reviewed in in vitro and in vivoThe mobilization of fatty acids from adipose tissue −/−) show impaired catecholamine-stimulated lipolysis and PKA signaling in adipose tissue −/− mice show enhanced stimulated lipolysis and normal pro-lipolytic signaling in vivo might be secondary effects, rather than primary defects, due to loss of the proposed CAV1 scaffold −/− mice are resistant to diet-induced obesity and show a mild lipodystrophy −/− mice is metabolized normally for thermogenesis −/− mice, concomitant with an increase in serum free fatty acids, suggesting a normal metabolic response to fasting Caveolin-1 (CAV1) is an integral membrane protein that together with members of the Cavin family of proteins is essential for the formation of caveolae at the cell surface −/− mice both during fasting and following maintenance on a high fat diet. Fasting caused loss of adipose tissue, despite a loss of PKA-mediated site-specific HSL phosphorylation, increased macrophage infiltration into adipose tissue, enhanced deposition of collagen, and a reduction in the level of the lipid droplet protein PLIN1a. Loss of PLIN1a could be recapitulated by ex vivo culture of CAV1−/− adipose tissue, which correlated with enhanced secretion of IL-6 and release of lactate dehydrogenase. Consistent with structural fragility of CAV1−/− adipocytes, collagenase treatment of adipose tissue resulted in significantly increased rates of cell death relative to tissue from control mice. Together these results suggest that CAV1 loss from adipose tissue affects adipocyte robustness, resulting in increased collagen deposition and eliciting an inflammatory response. Intriguingly, the phenotype of adipose tissue in CAV1−/− mice closely mirrored that of wild type mice maintained on a high fat diet, but without the increase in obesity, suggesting that the inflammatory state observed in obesity could be directly associated with alterations in the adipocyte cell surface. Intrinsic tissue instability could impact on both the development of an inflammatory state and, in the case of CAV1 deficiency, the development of lipodystrophy.In the current study we have examined adipose tissue from CAV1Ser563 (#4139), P-HSLSer660 (#4126) and HSL (#4107) were obtained from Cell Signaling Technology, rabbit anti-PLIN1a (P1998) was from Sigma , mouse anti-beta actin antibody (#MAB1501) was from Chemicon, rat anti-mouse F4/80 was from Serotec , mouse anti-PKAcat (#610980) was from BD Biosciences and mouse anti-collagen-1 was from Abcam (ab34710). Forskolin was obtained from Merck . Glycerol release was measured using free glycerol reagent . The release of non-esterified fatty acid (NEFA) was measured using the NEFA kit from Wako Pure Chemicals Industries Ltd . Collagenase I was obtained from Worthington Biochemical Corp. . Remaining reagents were obtained from Sigma unless stated otherwise.Rabbit anti-Phospho PKA Substrate (RRxS*/T*) (#9624), P-HSL−/− mice were developed in the Kurzchalia laboratory −/− and CAV1+/+ mice were kept under a controlled humidity and lighting schedule with a 12 h dark period. All animals received care in strict compliance with institutional guidelines regulated by the Australian Government. All animal experiments were performed with the approval of the University of Queensland Animal Ethics Committee. Food and water were available ad libitum. Fasting. CAV1−/− and CAV1+/+ mice  were used. Food withdrawal was initiated at the start of the light period and fasting continued for the times shown in the results section. High fat diet. CAV1−/− and CAV1+/+ mice  were fasted for 24 hours then fed ad libitum with control diet  or a high fat diet  for 12 weeks. At the end of the diet period mice were sacrificed, samples of plasma obtained and adipose tissue immediately frozen in liquid nitrogen and stored at −80°C.CAV1+/+ and CAV1−/− mice between the ages of 12–21 weeks. Explants were cultured as described previously Adipose tissue was obtained from the epididymal fat pads of CAV1Blood was extracted by cardiac puncture and collected in BD microcontainer PST LH (#365987). Serum was collected after centrifugation of blood samples at 6000 rpm for 10 min at RT. Explant media samples were collected directly from the culture vessels. Cytokine levels were measured in serum and media samples using Flex Set bead array kits and a FACSCanto II (BD Biosciences), according to the manufacturers instructions.+/+ and CAV1−/− mice. Body fat was measured using DXA. Mice were anaesthetized with Ketamine (80 mg/kg) and Xylazine (10 mg/kg) administered ip and scanned within a PIXImus Densitometer  to measure % body fat prior to and immediately following a 24 h fast.To study the effect of fasting on body and adipose tissue mass we measured both the weight and the % body fat of CAV1Tissue or explants were homogenized in TNE  containing protease  and phosphatase  inhibitors using an Ultra-Turrax homogenizer . SDS-PAGE and Western blot analysis was carried out as described previously −/− and CAV1+/+ mice was fixed in 4% PFA in PBS. Tissue was processed and embedded in paraffin by the QIMR Histology service. Sections (4 µm) were stained with Sirius red to detect collagen (QIMR Histology Service). To detect macrophages sections were cleared and hydrated using standard methods, digested using the Carezyme-1 Trypsin  and labelled for F4/80 using the R.T.U. Vectastain Elite ABC kit according to manufacturers instructions . Reaction product was visualized by incubating with ImmPACT DAB substrate (Vector laboratories). Images were taken using a DP-70 12 Mp colour camera mounted on an Olympus BX-51 microscope.Epididymal adipose tissue from ∼14 week old CAV1−/− mice to respond to the acute activation of lipolysis by catecholamines is blunted, and that adipocytes from CAV1−/− mice show impaired prolipolytic PKA signalling and PLIN1a phosphorylation, despite elevated levels of PKA activity against other substrates and normal levels of the ß3-adrenergic receptor −/− mouse strains have shown that the genetic background can impact on some consequences of CAV1 deficiency et al. (2004) we found that acute in vivo stimulation of lipolysis using the beta-3 adrenergic receptor agonist CL316, 243, in an independent strain of CAV1−/− mice , we found that the secretion of IL-6 from CAV1−/− adipose tissue was significantly higher than from CAV1+/+ tissue (−/− and CAV1+/+ mice IL-6 serum levels following a 24 h fast (+/+ and CAV1−/− mice that had been maintained on a high-fat diet (HFD) or a control diet for 12 weeks. Consistent with previously published data, CAV1+/+ mice showed a significant increase in weight and adipose tissue mass on the HFD compared to the control diet, whereas CAV1−/− mice were resistant to diet-induced weight gain +/+ mice on the HFD, and from CAV1−/− mice on either diet , a well-known marker of acute or chronic tissue damage. Consistent with decreased integrity of the plasma membrane in adipose tissue explants from CAV1 culture . Intrigu+ tissue . This ef+/+ and CAV1−/− mice. CAV1−/− adipose tissue showed increased collagen deposition . Together these data show an increased fragility of adipose tissue of CAV1−/− mice in vitro. The strikingly increased susceptibility to cell lysis after collagen digestion suggests that increased collagen deposition in the CAV1−/− tissue may serve to partially compensate for increased fragility of adipocytes.Intriguingly, genetic loss of adipose collagen has been reported to increase caveola density in adipocytes position  and a hiposition . This wa−/− mice shows increased cell lysis when isolated and cultured as tissue explants, and this susceptibility to damage is even more pronounced upon removal of the collagen matrix. These studies suggest that the previously proposed roles of CAV1 in mechano-protection −/− mice and patients.In this study we have examined the effect of CAV1 deficiency on mouse adipose tissue as a model of human lipodystrophy. CAV1-deficient mice are resistant to diet-induced obesity and have defects in adipose tissue lipolysis −/− mice include an increase in macrophage infiltration, increased capacity for IL-6 production and secretion in vitro, increased collagen deposition and loss of PLIN1a protein. Adipose tissue is one of the major tissue sources of inflammatory cytokines including TNF and IL-6 −/− mice. However, we found no significant change in systemic IL-6 during fasting, recombinant IL-6 failed to elicit an increase in glycerol release in 3T3-L1 adipocytes, and adipose tissue explants of CAV1−/− mice had suppressed basal lipolysis despite the high levels of IL-6 present in the culture media. We therefore consider it unlikely that IL-6 is directly responsible for adipose tissue lipid metabolism during fasting in CAV1−/− mice. However, in several pathological conditions associated with increased fibrosis, such as systemic sclerosis −/− adipose tissue could be related to the elevated levels of IL-6.The striking phenotypic changes in the adipose tissue of CAV1−/− mice to HSL, which predominantly catalyses the conversion of diacylglycerol (DAG) to monoacylglycerol, the loss of PLIN1a could potentially have a direct effect upon the activity of the predominant TAG to DAG lipase, adipose triglyceride lipase (ATGL). ATGL activity depends upon a co-activator protein, CGI-58, which binds to PLIN1a in unstimulated cells, thereby preventing ATGL activity. During normal activation of lipolysis the phosphorylation of PLIN1a by PKA results in its dissociation from CGI-58, freeing CGI-58 to interact with and activate ATGL. One interesting possibility that requires further investigation is that ATGL activity could be directly elevated by the reduction in PLIN1a levels observed in CAV1−/− adipose tissue during fasting, allowing partial mobilisation of TAG-derived fatty acids and DAG.Adipose tissue lipolysis is catalysed by a sequential series of lipase enzymes, the activities of which are tightly controlled by a variety of mechanisms including accessory proteins/co-activators and post-translational modifications , as indicated by increased collagen deposition. Recent studies have implicated caveolae in protection of the plasma membrane in response to membrane stress −/− mice by standard collagenase digestion techniques, despite this being a well-established procedure for culture of primary adipocytes. Release from the collagen matrix is sufficient to trigger cell lysis.In addition to an inflammatory response and macrophage infiltration, CAV1in vitro-differentiated adipocytes derived from CAV1−/− MEFs show a higher sensitivity to lipolytic flux than CAV1+/+ MEF-derived adipocytes −/− tissue would partially compensate for this in vivo. This model suggests that the proposed role for caveolae in adrenergic signaling could be coupled to a protective effect monitoring the flux of NEFAs. As in vitro-differentiated adipocytes derived from CAV1−/− MEFs do not show the loss of prolipolytic signalling observed in adipose tissue of CAV1−/− mice Recent work has shown that


UAUUUAU. Recognition sites for some of these RBPs tended to localize at the end of long 3′UTRs. A specific group of miRNA recognition sites were enriched within 50 nts from the RBP recognition sites for PUM and UAUUUAU. The presence of both a PUM recognition site and a recognition site for preferentially co-occurring miRNAs was associated with faster decay of the associated transcripts. For PUM and its co-occurring miRNAs, binding of the RBP to its recognition sites was predicted to release nearby miRNA recognition sites from RNA secondary structures. The mammalian miRNAs that preferentially co-occur with PUM binding sites have recognition seeds that are reverse complements to the PUM recognition motif. Their binding sites have the potential to form hairpin secondary structures with proximal PUM binding sites that would normally limit RISC accessibility, but would be more accessible to miRNAs in response to the binding of PUM. In sum, our computational analyses suggest that a specific set of RBPs and miRNAs work together to affect transcript decay, with the rescue of miRNA recognition sites via RBP binding as one possible mechanism of cooperativity.Transcript degradation is a widespread and important mechanism for regulating protein abundance. Two major regulators of transcript degradation are RNA Binding Proteins (RBPs) and microRNAs (miRNAs). We computationally explored whether RBPs and miRNAs cooperate to promote transcript decay. We defined five RBP motifs based on the evolutionary conservation of their recognition sites in 3′UTRs as the binding motifs for Pumilio (PUM), U1A, Fox-1, Nova, and  Transcript degradation represents an important mechanism of regulation used in diverse biological processes, including during development to eliminate maternally inherited transcripts, in adult tissues to define cell lineages, and as part of signaling pathways to down-regulate unneeded transcripts. RNA binding proteins (RBPs) and microRNAs are two major classes of molecules utilized to degrade transcripts. Using computational methods, we analyzed the genomewide cooperativity between microRNA and RBP recognition sites. We observed cooperativity between Pumilio (PUM) and specific microRNAs that impacts transcript decay. Our analysis suggests that approximately seven mammalian microRNAs preferentially co-localize with PUM binding sites, and these microRNAs have recognition motifs that are reverse complements to the PUM recognition motif. Their binding sites are more likely to form RNA hairpin structures with proximal PUM recognition sites that would limit microRNA efficiency, but would be more accessible to microRNAs in response to the binding of PUM. These results indicate that rescuing microRNA recognition sites from hairpin structures may be an important role for PUM. Transcript degradation is an important mechanism for regulating the levels of proteins in a time or space-dependent manner miRNAs generally bind their targets through complementary pairing in a short 7 bp seed sequence Transcript degradation can also be regulated by RNA binding proteins (RBPs). These proteins can affect transcript stability by binding to recognition sequences within 3′UTRs. Some RBPs, for instance, AU rich element (ARE) binding proteins or Pumilio (PUM), increase the degradation of target transcripts let-7 repress c-MYC expression though a mechanism that requires both HuR and let-7C. elegans PUM homolog puf-9 is required for 3′UTR-mediated repression of the let-7 target hbl-1 Drosophila, an association between the RBP dFXR and RISC is required for efficient RNA interference Specific instances in which RBPs enhance or inhibit the effectiveness of miRNAs have been experimentally verified. Competition between miRNAs and RBPs for the same sequence has been reported Kip1 has been reported to cause a local change in structure that promotes p27Kip1 repression by miR-221/miR-222 One specific mechanism through which the Pumilio RNA binding protein has been proposed to modulate miRNA function is by binding to sequences that can hybridize with miRNA recognition sites and thereby make them more accessible for the RISC complex. Binding of PUM to the 3′UTR of the cyclin-dependent kinase inhibitor p27We hypothesized that miRNAs and RBPs might cooperate to facilitate transcript decay more extensively than had been realized. Using computational models, we systematically explored RBP-miRNA interactions within human and mouse 3′UTRs and discovered that RBP recognition sites co-occur with subsets of miRNA recognition sites. Our analyses revealed that PUM is likely to cooperate with specific miRNAs to promote decay. Moreover, we found that a subset of miRNAs that co-occur with PUM recognition sites have recognition seed sequences that are the reverse complements of the PUM recognition motif, and thus, may form hairpin secondary structures that would be disrupted by PUM binding. Based on our computational analysis, we discovered seven miRNAs in human and five in mouse that followed this pattern. Approximately 4% of the target sites for these miRNAs colocalize with PUM sites in a pattern that would have the potential for miRNA binding site rescue.UGUANAUA) UGCAUGU) AUUGCAC)(a component of the snRNP complex) YCAUUUCAY) UAUUUAU, which is bound by many different ARE binding proteins We performed a literature search and identified 15 instances in which an RBP and its putative recognition motif were reported AUUGCAGFor the PUM recognition motif, for instance, there is a large increase in the number of observed recognition sites compared with the number expected based on shuffled controls . In contUGCAUGU), which represents a family of RBPs that are well-conserved in metazoans. In mammals, there are three members of the Fox-1 family, Fox-1, Fox-2 and Fox-3 UGCAUGUUGCAUGU, so we compared the density of Fox-1 family motifs within the immunoprecipitated 3′UTR sequences with the density in 3′UTRs outside the immunoprecipitated sequences. The enrichment for the Fox-1 family motif increased monotonically with an increasing conservation threshold, from twice as frequent for all binding sites to 4 times more frequent when requiring perfect conservation through all placental mammals  and UAUUUAU  were significantly more frequent at the very 3′ end of 3′UTRs, even after correcting for the high AU content in this region of 3′UTRs . However, UAUUUAU was present in a similar percentage of 3′UTRs compared to shuffled control motifs with the same AU content  less than 0.05 as determined by the Benjamini Hochberg procedure We then asked whether the recognition sites of RBPs and miRNAs tend to be present close to each other on the same transcripts, as previous studies have reported that RBPs and miRNAs that functionally interact are often located close to each other UAUUUAU have miRNAs that are more abundant than expected within 50 nts of the RBP recognition site using this procedure  We also recognized that not all miRNAs are expressed in all cells. To address this issue, the interacting miRNAs were further classified based on the sequence read abundance in HEK293T cells UAUUUAU and their co-localizing miRNA recognition seeds tend to be AU-rich , real RBP recognition sites and real recognition sites for interacting miRNAs within 50 nts; (miR_control), real RBP recognition sites and sites for shuffled interacting miRNA motifs within 50 nts; and (RBP_control), shuffled RBP motif sites and real interacting miRNA sites within 50 nts. We observed that transcripts with real PUM and interacting miRNA recognition sites have consistently shorter half-lives compared to transcripts in the two other control groups . Thus, tUAUUUAU, transcripts with both UAUUUAU sites and recognition sites of its interacting miRNAs in close proximity tend to have shorter mRNA half lives than transcripts in other groups . We also performed a control in which, for interacting miRNAs, the sequence of the miRNA and RBP recognition sites were shuffled while preserving mono and di-nucleotide frequency p-value<1E-10 for both human and mouse). For UAUUUAU, we did not observe any enrichment of high rescue counts for interacting miRNAs compared with controls . Multiple genome alignments for the human genome (hg19) aligned with 32 placental mammals and mouse genome (mm9) with 29 vertebrates were also downloaded from the UCSC genome browser. 3′UTR regions were extracted for further analysis. Branch lengths for the associated phylogenetic tree were also downloaded.Gene annotations for human, mouse, fly and worm were downloaded from the UCSC genome browser  by defining the minimum phylogenetic sub-tree that includes all conserved instances of the motif. The BLS is the branch length of this sub-tree as a fraction of the entire tree. Using this method, the BLS of individual motif hits can be inflated by a single hit to a distant species. To avoid this, we assigned no score to the most distant hit if there was a gap to that species that included more than one-third of the number of aligned genomes, and if the evolutionary distance from the most distant genome to the reference genome was more than twice as large as the distance to the second-most distant genome.p-values among the canonical and shuffled recognition motifs p-values and determined the 10% threshold for the pairs with the most similar p-values. We eliminated shuffled motifs if they fell within this range and thus were considered too similar to any previously generated shuffled motif or the canonical motif. From the remaining motifs, we selected ten or the maximum possible number of shuffled motifs. When possible, we selected shuffled motifs that had a similar number of hits (±20%) to the canonical motif from the reference genome. Through these criteria, we largely corrected for differences in the frequencies of di and tri-nucleotides In order to assess the extent of conservation for each RBP motif, we generated 200 shuffled motifs by randomly swapping the nucleotides within the recognition motif. To remove redundant shuffled motifs, we profiled the similarity of each pair of motifs using Tomtom to generate We compared the conservation BLS scores for the canonical motif and each shuffled motif within the genome. We then determined the number of occurrences of hits to the genome for the canonical motifs and the average among shuffled motifs for 100 different BLS thresholds from 0 to 1 with increments of 0.01. For each BLS threshold, we determined the precision as 1 – (the average number of matches of shuffled motifs)/. We selected for further analysis RBPs for which the recognition motif contained more than 10 motif hits above a precision threshold of 0.6.http://targetscan.org). Two types of miRNA seeds were used: miRNA nucleotides 2–8 (m8) and nucleotides 2–7 with an adenosine opposite miRNA position 1 (1A) Mature human, mouse, fly and worm miRNA annotations were downloaded from Targetscan  of 3′UTRs were swapped exclusively with other miRNA sites located at the very end (or beginning) of 3′UTRs.Both RBPs and miRNAs tend to have recognition sites located at the beginning or end of 3′UTRs  [48], [4p-values and Benjamini-Hochberg correction were performed as described above.To control for AU content, miRNA recognition seeds of 1A and m8 were classified into 3 groups based on the number of nucleotides that are an A or U out of the seven base pairs in the seed sequence. Category 1 contained miRNAs with a high (6 or 7) number of A/U nucleotides; category 2 contained miRNAs with a medium (3–5) number of A/U nucleotides; and category 3 contained miRNAs with a low (0–2) number of A/U nucleotides. The identities of miRNAs were shuffled with other miRNAs within the same category. In this way, AU-rich miRNA recognition sites were swapped exclusively with other AU rich miRNA sites. Empirical We only accepted miRNAs identified by the intersection of all three methods as interacting miRNAs for each RBP in each window. We found the window of 50 nts closest to the RBP site contained the largest number of interacting miRNAs, while windows that were more distant contained fewer or none . To comphttp://cat.princeton.edu/miRNA_RBP/.Detailed statistics for all possible pairs of RBP and miRNA recognition motifs are available on the webpage When defining interacting miRNAs for each RBP, we explicitly accounted for the AU content of miRNAs by only shuffling across miRNAs with similar recognition seed AU content. In order to test the impact of the AU content of the RBP motifs, we generated window plots comparing the distribution of true RBP motifs to the distribution of shuffled RBP motifs (generated in the RBP recognition motif selection section). We created ten 50 nt windows upstream and downstream of each RBP motif and shuffled RBP motifs and counted the number of real miRNA recognition sites. For each window, the enrichment ratio was defined as  normalized by an overall ratio of . For each RBP and its interacting miRNAs, enrichment ratios were visualized with heatmaps .For the cell lines included in our analysis, we identified companion, published small RNA sequencing experiments. For mRNA half-life measurements in human B cells (BL41) For each sequencing experiment, all conserved miRNAs annotated in TargetScan were ranked by the number of their mapped sequence reads. The most highly expressed 25% of the miRNAs were defined as expressed and the rest were defined as non-expressed.To test whether the binding of RBPs makes miRNA recognition sites more accessible, we analyzed sequences that contain RBP and miRNA recognition sites within 50 nts of each other, and included an extra 5 nts upstream of the 5′ most site and 5 nts downstream of the 3′ most site. We computationally folded these sequences using RNAfold In order to generate a background distribution of rescue counts, for each pair of neighboring miRNA and RBP sites localized within 50 nts, we randomized the sequence itself, but preserved the relative positions of the miRNA and RBP recognition sites. For this randomization, we preserved the mono and di-nucleotide frequency To score the ability of miRNA recognition seed sequences to hybridize with the reverse RBP recognition motif, we used a simple scoring scheme in which A-U, G-C and G-U base pair matches were scored as 2, mismatches were scored as 0, and insertions and deletions were penalized with −1. The Smith-Waterman algorithm was then applied to find the best alignment Figure S1RBP recognition motifs. The positional weight matrix logos are plotted for the 15 RBP motifs that were evaluated.(PDF)Click here for additional data file.Figure S2RBP recognition motif selection. The number of instances of each RBP was plotted for different Branch Length Scores (BLS) as in UAUUUAU. (B) U1A. (C) Nova. (D) U2B.(PDF)Click here for additional data file.Figure S3AU-content is high at the end of 3′UTRs. Each 3′UTR longer than 500 nts was equally divided into ten deciles. For each decile, AU-content was calculated and box-plots across all genes are shown. (A) Human. (B) Mouse. (C) Fruit fly. (D) Worm.(PDF)Click here for additional data file.Figure S4RBP localization compared with shuffled control motifs. Each 3′UTR longer than 2000 nts was equally divided into ten bins, numbered from 0.0 to 0.9. The percentage of RBP recognition sites in each bin was compared with its shuffled control motifs, which have the same AU-content. For each 3′UTR bin, asterisks represent comparisons of percentages between real and random motifs using the binomial test with a Bonferroni correction for 10 tests. One asterisk indicates p<0.05, two asterisks indicate p<0.01, and three asterisks indicate p<0.001. (A) PUM localization in human. (B) UAUUUAU localization in human. (C) PUM localization in mouse. (D) UAUUUAU localization in mouse.(PDF)Click here for additional data file.Figure S5Localization of RBP motifs in 3′UTRs across four organisms. For each organism, 3′UTRs with length longer than 500 nts, but shorter than 2000 nts, were considered. Each 3′UTR was equally divided into 10 bins, numbered from 0.0 to 0.9. The percentage of RBP recognition sites in each bin was plotted. (A) PUM localization pattern. (B) UAUUUAU localization pattern.(PDF)Click here for additional data file.Figure S6Percentage of miRNA recognition sites within 50 nts from RBP sites. For each RBP, the percentage of miRNA recognition sites within 50 nts of an RBP recognition site was determined for the set of all interacting miRNAs  Human and (B) Mouse.g miRNAs  and for (PDF)Click here for additional data file.Figure S7PUM co-localizes with its interacting miRNAs in the Par-CLIP region. The recognition sites of PUM and miRNAs were restricted to those experimentally identified by Par-CLIP analysis of PUM2 and AGO binding in the HEK293T cell line p<0.05, two asterisks indicate p<0.01, and three asterisks indicate p<0.001. (B) The PUM-interacting miRNAs were classified as expressed if they were among the 25% of the most frequently sequenced small RNAs in HEK293T cells (PDF)Click here for additional data file.Figure S8miRNA-RBP colocalization is not simply a consequence of AU-content. For RBPs and their interacting miRNAs Click here for additional data file.Figure S9UAUUUAU results in faster transcript decay.The presence of PUM and  Decay rates are shown in box-plots for transcripts with recognition sites for the designated RBP or shuffled RBP motifs. The Wilcoxon-test with a Bonferroni correction was applied to measure the difference between real RBP sites  and shuffled RBP controls (Random). For each RBP, an asterisk designates a significant difference between transcripts with RBP recognition sites and transcripts with shuffled RBP motif sites. One asterisk indicates p<0.05, two asterisks indicate p<0.01, and three asterisks indicate p<0.001.  Half-lives are based on the published dataset (PDF)Click here for additional data file.Figure S10Pumilio recognition sites promote decay more effectively and are better conserved when present with interacting miRNAs. For each RBP, miRNAs were classified into four groups as described for (PDF)Click here for additional data file.Figure S11Expressed miRNAs promote decay more effectively than non-expressed miRNAs. For each of the cell lines used in half-life or decay rate datasets, companion small RNA sequencing datasets were identified from the literature. In each dataset, the reads of miRNAs were ranked and the most frequently expressed 25% of small RNA reads was established as a threshold for classifying interacting miRNAs for each RBP as “Expressed” or “Non-expressed”. Transcripts with proximal miRNA and RBP sites were compared with respect to their half-lives or decay rates. Asterisks represent comparisons of half-lives between two groups determined by Wilcoxon tests. One asterisk indicates p<0.05, two asterisks indicate p<0.01, and three asterisks indicate p<0.001. (A) For mRNA half-lives measured in Human B cells (BL41) (PDF)Click here for additional data file.Figure S12More rapid mRNA decay in transcripts in which Pumilio and interacting miRNA recognition sites colocalize is not only a consequence of AU-content. For each RBP or miRNA recognition motif, the shuffled RBP or miRNA motifs were used as controls for AU content. For each group of transcripts, boxplots of half-lives or decay rates were plotted as described for p<0.05, two asterisks indicate p<0.01, and three asterisks indicate p<0.001.  Half-life data from Friedel and colleagues (PDF)Click here for additional data file.Figure S13UAUUUAU binding.Histograms of rescue counts for miRNA recognition sites upon  Histograms of site rescue were calculated for both UAUUUAU-interacting miRNAs and non-interacting miRNAs as described in (PDF)Click here for additional data file.Figure S14PUM rescues nucleotides in neighboring interacting miRNA recognition sites. The histogram of miRNA seed rescue was compared with rescue values calculated with the background model of (PDF)Click here for additional data file.Figure S15Histograms of alignment scores between miRNA seeds and Pumilio recognition motifs. For all interacting miRNAs of PUM, their seed alignment scores with the reverse PUM motif were determined for real PUM motif and shuffled PUM motifs. Histograms for all shuffled PUM motifs were merged into average values and standard deviations, and are shown for PUM interacting miRNAs identified in (A) Human and (B) Mouse.(PDF)Click here for additional data file.Table S1RBP and miRNA recognition motifs colocalize. For each RBP, the number of predicted interacting miRNAs is shown for each of ten 50-nt-windows from the RBP motif.(PDF)Click here for additional data file.Table S2AU composition of RBP recognition motifs and interacting miRNA recognition seeds. The fraction of nucleotides that are AU was calculated for each RBP recognition motif and the recognition seeds of miRNAs that interact with each RBP. The average value is shown. The average AU fraction for all miRNA seeds is 52.7%.(PDF)Click here for additional data file.Table S3miRNA sites are enriched around RBP sites in human and mouse 3′UTRs. For each organism, the ratio of miRNA recognition site density 50 nts upstream or downstream of the RBP recognition sites to miRNA site density across all 3′UTRs is reported for PUM and UAUUUAU.(PDF)Click here for additional data file.Table S4Expressed RBP-interacting miRNAs and their effects on mRNA decay. For each of the mRNA decay datasets, we considered each RBP and its expressed interacting miRNAs  Results from mRNA half-life datasets of human B cells (PDF)Click here for additional data file.Table S5RBP recognition sites are more conserved when present with interacting miRNAs. For each RBP and its interacting miRNAs, their neighbor recognition sites were classified into either the proximal or distant groups as described for p-values are calculated based on Wilcoxon rank tests as a measure of the difference between the two groups. (A) Human-interacting miRNAs are shown. (B) Mouse-interacting miRNAs are shown.(PDF)Click here for additional data file.Table S6GO enrichments for PUM and its interacting miRNAs. Transcripts were classified into categories as in p-values. We then applied the Benjamini-Hochberg procedure on the p-values, and selected enriched GO terms with FDR< = 0.05. The number of annotated genes and hypergeometric p-values are shown for each significant GO term.(PDF)Click here for additional data file.


The International AIDS Society convened the multi-stakeholder “Towards an HIV Cure” symposium in Kuala Lumpur, Malaysia in 2013 to address the significant research challenges posed by the search for a cure for HIV infection. Current antiretroviral regimens select for a small reservoir of cells that harbour latent HIV provirus, produce few or no HIV virions, and resist detection or clearance by host immunity. The symposium examined basic molecular science and animal model data, and emerging and ongoing clinical trial results to prioritise strategies and determine the viral and immune responses that could lead to HIV remission without ART. Here we review the presentations that scrutinized the molecular mechanisms controlling virus expression from proviral DNA, and the intrinsic cellular restriction and immune mechanisms preventing viral production. Insights from the basic science have translated into new therapeutic strategies seeking HIV remission without ongoing therapy, and much interest was focused on these ongoing trials. We also summarise the emerging ethical issues and patient expectations as concepts move into the clinic. The International AIDS Society (IAS) meetings over the past 25 years have been a focal point for optimising strategies to tackle the HIV epidemic. Antiretroviral therapy (ART) has dramatically improved HIV-associated morbidity and mortality, but is not a cure. Under the leadership of Françoise Barré-Sinoussi, co-discoverer of HIV, the IAS is focussing on achieving a safe, affordable and scalable cure for HIV infection .There have been a number of high profile case reports of apparent HIV cure. The well-publicised “Berlin patient” was treated for acute myeloid leukemia with intensive conditioning to deplete his haematopoietic system ahead of restorative transplantation with homozygous CCR5∆32 bone marrow stem cells, and subsequently became aviraemic  and adulThe IAS “Towards an HIV Cure” annual symposium brings together basic scientists, clinicians, funders, and community advocates to address the key challenges facing the community in bringing about a cure for HIV. In this review, we summarise the key findings from the 2013 symposium, held in Kuala Lumpur, Malaysia on 29 and 30 June. Initially focusing on the basic science underpinning the reservoir, HIV transcription control and immune responses, the report then focuses on clinical trial and ex vivo data, before summarising the key ethical and regulatory issues.Investigations of non-productive HIV infection in reservoir cells have yielded important insights into molecular factors governing HIV latency ,7. Oliviin vitro analyses of chemotherapeutic approaches targeting expression from the latent HIV provirus promoter. Most of these studies compared effects with the pan-HDAC inhibitor (HDACi), vorinostat (SAHA) that targets all four classes of HDAC and has been shown to activate virus transcription in resting CD4+ T-cells in HIV-infected patients on ART in two clinical studies lead by David Margolis [Several presentations detailed Margolis  and SharMargolis . While aDr. Mahmoudi from Erasmus Medical College, Rotterdam explored how the Wnt pathway promotes an accumulation of β-catenin, which in turn evicts repressors from TCF/LEF-1 sites, as found in the HIV LTR promoter to promote transcription . TreatmeSCM), a recently described T cell subset with a naïve-like phenotype, but functional memory characteristics. Although the overall contribution to the CD4 T cell reservoir was low compared with central or effector memory cells, the proportion of TSCM’s infected was higher  and less impacted by ART. Inhibition of the Wnt/ β-catenin pathway - which has been associated with TSCM induction – was used to test impact on the HIV-1 reservoir. The agent PRI-724/C82 (a β-catenin inhibitor) was shown to induce differentiation of TSCM into shorter-lived subsets ex vivo, suggesting a possible role in cure strategies. Preliminary data showed synergy between Panobinostat and PRI-724/C82 for both acetylation and intracellular HIV-1 RNA expression in the HIV latently infected ACH-2 cell line. Taken together, the studies on Wnt/ β-catenin pathway highlight opportunities for developing more effective drug cocktails to activate latent HIV and reduce the lifespan of the TSCM reservoir. However, potential problems from the strong association of Wnt pathway activation with solid tumour malignancies and other broad cellular activities need to be addressed.Mathias Lichterfeld presented data on memory stem cells (TCellular interactions between T-lymphocytes and HIV infected dendritic cells (DC) are known to promote efficient HIV-1 transmission . Bin Su A study from Dr. Ndhlovu from University of Hawaii examined a β-galactoside binding lectin, Galectin-9 , that was proposed to contribute to persistent T-cell activation and HIV entry into resting cells, but also to induce CD4 T cell apoptosis and immunosuppression after binding the Tim-3 receptor ,15. PlasSarah Palmer presented longitudinal analyses of single genome sequences exploring the dynamics of the HIV reservoir during suppressive therapy. Patients recruited in both acute and chronic infection and on ART for between 4 to 12 years were sampled from compartments including peripheral blood, bone marrow, gut associated lymphoid tissue and lymph node. Patients were sampled prior to ART and then twice (6 months apart) after suppressive therapy. Dr Palmer reported a substantially higher frequency of infection in patients treated in chronic versus acute infection in all T cell subsets from both blood and GALT. Interestingly, the central/transitional memory cell compartment contained the majority of the reservoir in the peripheral blood (mean 56.3%), whereas in the GALT most infected cells were in the effector memory compartment (mean 68.4%). Phylogenetic analyses based on a Gag-Pol fragment provided no evidence for on-going viral replication during the years of suppressive therapy in any compartment.in vitro model , the signal from ex vivo index sorted samples was weak making the detection of HIV Env expression using mAbs difficult in these samples.Richard Koup presented an analysis of spliced and unspliced HIV RNA in CD4 T cells sorted into bright, dim and null cells. Cells containing single copies of spliced and unspliced RNA transcripts were identified by qPCR, with spliced tat and rev transcripts used to discriminate active viral RNA production from the genomic RNA present in incoming virions. Full-length HIV RNA transcription was associated with CD4 down-regulation, and the greatest numbers of HIV RNA transcripts were found predominantly in activated cells. Labeled monoclonal antibodies against Env were used to sort HIV infected cells. However, although monoclonal antibodies could be used to identify infected T cells in an Deborah Persaud gave a keynote presentation on the impact of ART on the HIV-1 reservoir in the paediatric setting. She defined 'cure’ in children as permanent disease remission off ART, with normal CD4 counts, preservation of immunity to childhood vaccinations and no evidence for immune activation or inflammation. The case of the Mississippi child first reported at the Conference on Retroviruses and Opportunistic Infections (CROI) 2013 was reviewed, with confirmation that the child – now 33 months old – remains in remission with no virological or immunological evidence of HIV-associated pathogenesis ,17.Whereas it is clear that HIV-related mortality is high in untreated children (36.8% at one year), early ART started within 3 months saves lives, based on data from the CHER Trial , but alt6 PBMC, after initiating ART in the < 200, 200 – 499, ≥ 500 CD4 count strata were 0% (n = 124), 3% (n = 155) and 30% (n = 30), respectively, reinforcing the importance of the nadir CD4 cell count. In contrast, an analysis of treatment commenced in primary HIV infection (PHI) was presented by Antoine Chéret from the ANRS 147 OPTIPRIM study - a randomized study of tenofovir/emtricitabine and ritonavir-boosted darunavir with or without raltegravir and maraviroc [6 PBMC between baseline and 12 months in 67 patients. This reduction negatively correlated with baseline plasma HIV RNA and HIV DNA. In the SPARTAC trial a delay in viral rebound had been reported following treatment with a 48-week course of ART in primary HIV infection [Laurent Hocqueloux presented data on the timing of ART initiation in chronically infected adults ,21. In aaraviroc ,23. Data 0% n = 1, 3%  system  was presented showing that the proportion of samples which were positive increased from 26% pre-treatment to 60% during the first treatment week (p = 0.009). These may represent the first data of an activating agent leading to plasma release of viral particles, although a more comprehensive analysis is needed. In addition, there was clear evidence of rapid acetylation/deacetylation with each cycle of treatment and good tolerability, with no adverse events greater than grade I, and no significant differences in neutrophil or platelet counts. An optional analytical treatment interruption is planned for all participants.Preliminary findings from the CLEAR trial were presented by Dr. Tolstrup from Aarhus University, Denmark. This was a single arm study of panobinostat, a pan-HDAC inhibitor, given as a 20 mg oral dose three times a week, every other week over 8 weeks, to 15 adults on suppressive ART with a CD4 count > 500 cells/mmin vitro from resting CD4 T cell subsets [3 and proviral HIV DNA between 10 and 1000 copies/106 PBMC. Twenty-nine participants were randomized and all have completed 80 weeks of follow up. Baseline characteristics were well matched apart from longer duration of ART (median 13 versus 7 years) and higher median baseline HIV DNA (564 versus 287 copies/106 PBMC) in the IL-7 arm. No participant reached the primary endpoint of at least a 0.5 log decrease in proviral HIV DNA in PBMC at 56 weeks. A large increase in CD4+ and CD8+ cells in blood was seen in the IL-7 arm at week 12, which declined thereafter, but remained significantly higher than baseline at week 56. Most of this increase was in central memory T-cells (TCM). No significant change in HIV DNA concentration was seen at 56 or 80 weeks in either arm when analysed as copies per 106 PBMC or per 106 CD4 cells, but a significant increase was seen at both time points in the IL-7 arm when analysed as copies per mL of whole blood, due to persistence of the CD4 cell count increase. A trend was seen towards lower HIV DNA in the intensification only arm at week 80 (P = 0.064), but not at earlier time points. The increase in the HIV reservoir in the IL-7 arm would seem to preclude the use of this agent to purge the HIV reservoir.IL-7 induces HIV production  subsets . The ERATimothy Henrich presented data from two HIV + ve individuals who had received allogeneic stem cell transplants and had subsequently undertaken a treatment interruption. Both recipients were CCR5Δ32 heterozygotes, underwent reduced-intensity conditioning ahead of allogeneic hematopoietic stem cell transplantation from wild type (WT) CCR5 donors and both had significant chronic graft-versus-host disease . Prior tClinical trials testing therapeutic strategies to reactivate latent virus have highlighted the need to concurrently boost immune responses to clear cells expressing antigens from reawakened provirus. The role of dendritic cells (DC) in enhancing this response received attention in three presentations. Kellie Smith from the University of Pittsburgh developed a DC immunotherapy using autologous DC loaded with inactivated HIV-1 derived from the autologous ART reservoir to efficiently prime residual memory T-cells and naïve CD8+ T cells that jointly kill HIV superinfected CD4+ T-cells. These data demonstrated that chronic, untreated HIV-1 infection did not irreparably damage the ability to prime CTL against autologous virus with DC expressing antigens specific for the autologous HIV-1 reservoir, reinforcing the potential for DC-based immunotherapies.Eric Arts showed that efficient yeast cloning and recombination techniques  could beAn alternative approach developed by Argos Therapeutics uses electroporation to introduce Gag, Nef, Rev and Vpr RNA from autologous HIV into DC, together with RNA expressing CD40L to enhance antigen-specific immune stimulation without CD4+ T-cell help as an immunotherapy concept called AGS-004. Initial clinical studies examining six monthly dosing of AGS-004 gave promising results in reducing viral set point by 1.2 logs and reducing proviral load in some patients.in vivo application of the model, only between 0.1 and 2% of latently infected cells were found to produce Gag, which might be of concern if immunotherapy is to be an integral part of HIV cure.Una O’Doherty from the University of Pennsylvania presented data using a primary CD4 T cell model of latency in which she has previously reported Gag protein expression . CD4 T c3 and a HIV envelope gene sequence encoding the principal neutralizing determinant (V3-tip) corresponding to high KD-247 binding activity. Participants were randomized to one of three dosing cohorts and received active infusion or placebo weekly for three weeks. Mean change from baseline in plasma HIV RNA was in the range of -0.2 and -0.4 log copies/ml for the two larger dose cohorts (8 mg/kg and 16 mg/kg) following the day 8 and day 15 infusions.Dr. Shuzo Matsushita presented data from KD-1002, a phase 1b dose escalation study of KD-247, a neutralizing humanized monoclonal antibody ,30. TwenJ. Victor Garcia took the strategy of targeting the latent reservoir further by using an anti-Env antibody-immunotoxin that delivered Pseudomonas exotoxin to residual HIV infected cells in the humanized mouse model . Adminisa to discuss issues that will accelerate product development and regulatory agency approval for a rapid and safe transition into the clinic of HIV cure therapies having strong end-user acceptance and wide accessibility for all patient populations. Open discussion with relevant regulatory authorities was advised to clarify the endpoints measured and the risk-benefit criteria required for an accelerated approval. Appropriate endpoints are especially important for combination cure therapies, such as a small molecule inhibitor together with a biologic and a vaccine. Although analytical treatment interruption (ATI) was previously considered unacceptable, it is now a valid option for cure strategies in appropriate well-controlled patient populations to minimise the potential risk of viral rebound. At these early stages of clinical cure investigation, it is unlikely that research of blood samples will be sufficient, and tissue sampling will also be necessary, at least for a portion of study participants, to gain insight into biomarkers for long-term control.Two roundtable panel sessions discussed key issues from the diverse perspectives of the different stakeholders. The first panel assembled HIV cure clinical trialists, pharmaceutical industry representatives and community ethicistsb, who included scientists and clinicians involved in cure research, community members, research funders and bioethics specialists, resolved that the scientific excitement for a potential HIV cure should not detract attention from the financial, social, ethical and moral implications of cure research. Many of the ethical considerations for HIV cure research have been recently reviewed [The second roundtable discussion on ethics and patient expectations provided a timely reminder of the need to address the biomedical question of HIV cure in parallel to the social and ethical questions. The panellistsreviewed  and it iThe “Towards a Cure” symposium captured the increasing research momentum based on the growing scientific understanding of the HIV reservoir and the increasing evidence from cases of HIV remission or cure. There is increasing interest in chemotherapeutic interventions promoting viral remission, and the first small steps have been encouraging. Early data from clinical studies with HDACi showed they were safe, well tolerated and successfully stimulated unspliced HIV RNA as the primary endpoint, providing optimism for broadly applicable viral reactivation strategies. However, viral RNA has not yet been linked with viral protein expression, and reduction in proviral load. Other biomarkers are needed that better correlate with elimination of the viral reservoir during curative interventions. Importantly, more HIV-specific activating drugs are following and these offer better specificity for HIV induction, and may potentially team with therapeutic vaccines or other strategies to eliminate cells containing reawakened HIV. But there is clearly a long way to go and key challenges lie in the search for combination interventions that not only lead to activation of latent HIV infection, but also significant decreases in the size of the HIV reservoir.Evidence is mounting that early implementation of ART can assist in achieving viral control off therapy, possibly by preventing the seeding of virus into reservoirs that endure for the life of the patient. It remains to be resolved if the reservoir seeded during chronic infection differs from that laid down in the primary infection, and if it can be eliminated by chemotherapeutic intervention.There are still large gaps in our understanding of the molecular mechanisms that restrict the expression and release of replication competent HIV from the different tissue reservoirs. While restrictive epigenetic and transcriptional programs largely underpin the limited HIV expression from most residual integrated proviruses, it is clear that various other molecular mechanisms can reversibly restrict viral expression, producing a diverse reservoir of residual latent provirus. An increasingly important impediment in the field is access to validated methods to measure the true size of the relevant replication competent latent virus reservoir.The terms “sterilising” and “functional” cure have been used to describe patients who have achieved complete eradication or who control viral replication in the absence of ART, respectively . WhereasHIV cure research has benefited from strong interactions between basic scientists and clinicians, but it is clear that important insights provided by social scientists and members of the HIV community are required to determine the expectations surrounding HIV cure and the acceptability of different curative strategies. A trans-disciplinary approach must include non-HIV experts to bring new insights from the fields of oncology, other chronic infections and inflammatory diseases. Wide consultation is also needed to identify the appropriate conditions for interruption of ART and the protocols for monitoring viral recrudescence during treatment interruptions and the thresholds for reinstating therapy.aRoundtable panelists: David Margolis, University of North Carolina at Chapel Hill (Chair); Guenter Kraus, Tibotec/Janssen; Veronica Miller, The Forum for HIV collaborative research; Bill Whittaker, National Association of People living with HIV Australia; Hiroyu Hatano, University of California San Francisco.bJintanat Ananworanich, The Thai Red Cross AIDS Research Centre (chair), Jeremy Sugarman , Laurindo Garcia (B-Change), Eric Fleutelot (Sidaction), Bruno Spire (Inserm), Rowena Johnston (amfAR), Sharon Lewin .The authors declare that they have no competing interests.All authors contributed to writing these critiques, and all authors read and approved the final manuscript.


Raman and SERS molecular characterization of melanoma tissue is proposed here for the first time. Optical nanosensors based on Ag and Au nanoparticles with chemisorbed cresyl violet molecular species as labels revealed sensitive capability to tissues tagging and local molecular characterization. Sensitive information originating from surrounding native biological molecules is provided by the tissue SERS spectra obtained either with visible or NIR laser line. Labelled nanoparticles introduced systematic differences in tissue response compared with unlabelled ones, suggesting that the label functional groups tag specific tissue components revealed by proteins or nucleic acids bands. Vibrational data collected from tissue are presented in conjunction with the immunohistochemical analysis. The results obtained here open perspectives in applied plasmonic nanoparticles and SERS for the early cancer diagnostic based on the appropriate spectral databank.An interdisciplinary approach employing functionalized nanoparticles and ultrasensitive spectroscopic techniques is reported here to track the molecular changes in early stage of malignancy. Melanoma tissue tracking at molecular level using both labelled and unlabelled silver and gold nanoparticles has been achieved using surface enhanced Raman scattering (SERS) technique. We used skin tissue from  Raman spectroscopy has currently become a powerful vibrational technique largely used to probe the molecular composition of biological tissues –4. RamanA critical review recently provided the latest methods for the metabolic fingerprinting using vibrational techniques as potential tools for rapid disease diagnosis, detection of dysfunction, the early intervention of therapeutic strategies, highlighting their overall conclusions that vibrational techniques could be successfully applied in such medical areas . Owing t2, TiO2 or a polymer, a core-shell complex is formed in which the outer shell may be decorated with capture molecules such as antibodies, peptides or small molecules. When conjugated with biomolecular targeting ligands, these shell-coated nanoparticles can be used to target malignant tumours with high specificity and affinity. Thus, specimens may be captured and detected via a sandwich structure. This extrinsic SERS detection method has been successfully used for in vivo tumour targeting or rare cancer cells imaging  anthracene (DMBA) solution (0.025%–100 μl) in the first week of experiment (induction) followed by repeated applications of a pro-inflammatory phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) solution (5 nM–100 μl twice a week) for 13 weeks (promotion) [C57BL/6J and Balb/c mice of 8 weeks were purchased from Charles River . Animals were maintained on tap water and diets omotion) . The solB16 melanoma 4A5  cells were grown in DME media, supplemented with 10% foetal bovine serum, penicillin (100 U/ml) and streptomycin (100 mg/ml) in specific cells culture plates. The cells were prepared in three steps (three passages respectively).2. For the subcutaneous injection with B16 cells on mice were used 0.5 ml of cells without media, in saline solution [Cells were incubated with 1 ml of media at 37°C in an incubator for cell culture with 95% air and 5% COsolution , 30.Animals were killed at 14 and 25 days after implantation of cells because at this time tumours were enough in over 90% of animals . Sections of tumours were formalin fixed. For this experiment, only the healthy mice in the incipient phase of the disease (treated with B16 melanoma cells) and one treated with 7-12 dimethylbenzantracen (DMBA) and 12-O-tetradecanoilforbol-13-acetate (TPA) cancer inductor and promoter, respectively, were used.Colloidal Ag and Au nanoparticles have been prepared according to classical procedures , 32. In 3) with hydroxylamine hydrochloride (NH2OH.HCl) [4) to doubled distilled water and trisodium citrate solution drop by drop while stirring. The resulting mixture was boiled and the colloidal solution obtained was of dark red colour (pH 7).Briefly, Ag colloidal nanoparticles were prepared by reduction in silver nitrate (AgNO2OH.HCl) . The fin2OH.HCl)  by addinversus damaged tissue for reasons of minimizing the number of killed animals relative to the aim of the present work.Tissues from normal, early stage melanoma (C57BL/6J mouse with B16 cells injected) and advanced melanoma  have been selected. Detailed experimental protocol in inducing melanoma has been reported elsewhere . The SER−4 M aqueous solution to 1500 μl colloid, resulting in a final concentration of about 0.6 μM, whereas the nanoparticles concentration corresponding to the absorption maxima is about 2.9 × 1010 particles/ml.The SERS labels were obtained by adding 10 μl cresyl violet perchlorate (CV) 10Formalin-fixed tumour sections were H & E stained and analysed as histology samples and by application of S-100 and melan-A methods. Balb/c skin week 14, A revealeanova analysis. All P values were considered significant at the <0.05 level.Data are presented as mean and standard deviation and were analysed using one-way The extinction spectra of the colloidal nanoparticles were recorded with a Lambda 25 double-beam scanning UV-Vis spectrometer.−1, respectively. The laser spot size was about 3 μm.The Raman and SERS spectra were acquired using two dispersive spectrometers, a DeltaNu Advantage Raman spectrometer with a USB microscope attachment, equipped with He-Ne laser operating at 4 mW, 632.8 nm, and a Bruker Senterra dispersive Raman spectrometer using the 785 nm line of a diode laser, resolution 3 cmTransmission electron microscopy (TEM) images were recorded with a JEOL JEM 1010 electron microscope .The obtained melanoma model described in the histology and immunohistochemistry section is presented in the The optical properties of the metal nanoparticles were investigated using UV-Vis spectroscopy and the tunnelling electron microscopy (TEM). The Ag colloid A presentThe UV-VS absorption spectrum of the Au nanoparticles B presentet al. [in vitro nanoparticle skin permeation is possible and that silver nanoparticles as small as 25 nm stable for 24 h were located in the stratum corneum and the upper layers of the epidermis. The same passive fluid-phase uptake was accounted for the absorption of gold nanoparticles into cultured eukaryotic cells [We evaluated the nanoparticles uptake ability of the skin tissues using transmission electron microscopy  and several characteristic reproducible SERS bands located at 1618, 1455, 1359, 1283, 1119, 976, 899, 798 and 752 cm−1. According to Kneipp et al. [Both labelled and unlabelled noble metal nanoparticles were employed to record and compare tissue SERS signal. p et al.  such ban−1 was assigned to in plane skeletal quadrant stretching mode.The CV labelled nanoparticles allowed high-quality SERS spectra, as shown in −1 bands, we note different features compared to that from the unlabelled tissue. The amide I spectral shape band clearly shows two distinct peaks at 1618 and 1639 cm−1 and none of them is attributable to the label. The unlabelled tissue spectra showed one predominant band located at 1618 cm−1. This results suggest that the amino terminal of the label could target and bind to specific sites of some tissue components.The spectral signature of the labelled nanoparticles inside the tissue showed the characteristic SERS bands of the label, as well as specific bands from the molecular components from the close vicinity of the label. The label bands are marked in −1, and weaker bands at 1129, 1014, 893, 749, 650 and 486 cm−1 on a strong autofluorescence background which is not completely quenched by the Au nanoparticles. CV-Au labelled tissue showed preserved major bands positions and relative intensities, however, specific differences are noted for the band shapes and relative intensities at 1233, 1286, 1591 and 1687 cm−1. According to Movasaghi et al. [−1 can be assigned to either amide I disordered structure or glutamic and aspartic acid. However, this band is specific for carbonyl bond that could be targeted by the amino functionals of the CV-Au label. This supposition is also supported by the observed spectral differences in the amide III region (1286 cm−1 band). However, further investigations are certainly needed to confirm the binding specificity of the SERS label to a particular tissue component. We also recorded for comparison a series of SERS spectra from the healthy and diseased skin in both cases of Au-CV or Ag-CV labels, respectively. Although the preparation of the colloidal nanoparticles differs, the SERS spectra acquired within just 1 sec. integration time from the same sample type are very similar (spectra not shown here). To interpret the tissue SERS signature, we compared the results with previous SERS on cells and tissue reported so far [Surface enhanced Raman spectroscopy spectra from healthy skin using Au naoparticles are shown in the i et al. , the band so far –39. We nd so far , 36, or d so far . Thus, tex vivo skin samples showed contributions from DNA and nucleic acid bases which makes us believe that some nanoparticles are able to enter the cellular membranes. On the other hand, the disease induced by the inoculation of B16 cells into the mice specimens showed the enhancement of melanin content and the good adaptability and acceptability of skin for this B16 4A5 cell line prepared from C57BL/6J mice [For C57BL/6J melanoma model with B164A5 cells is a clear detection of melanocyte with melanin. These appear because of the mouse strain compatibility with the parent cell line and also this cell line is a melanin containing one. The SERS investigation of the /6J mice .−4 M labels.We supposed that the abundant melanin is responsible for signal background enhancement when visible laser line is employed. Therefore, we recorded SERS signal using a near infrared laser line to evaluate the label efficiency in surface plasmon pre-resonance condition. In NIR-SERS experiment an autopsy tissue collected from a Balb/c mice specimen treated with TPA and DMBA carcinogens was divided into three small samples, by sectioning thin transversal pieces. One sample was formaline fixed, the second was treated with formaline solution mixed with unlabelled Ag nanoparticles and one sample was immersed in formaline solution mixed with the 500 μl CV 10−1 , 1450 cm−1 , 1003 cm−1 , 937 cm−1 and 853 cm−1(collagen type I). This result is in agreement with the previous reports [−1, 1315–1333 cm−1 range). We note the amide I band absence, whereas the region 1200–1400 cm−1 exhibits strong SERS bands.The NIR-Raman signal of the tissue shows vibrational modes characteristic to the main molecular components, whereas the background was substantially lower. The most intense peaks are 1658 cm reports  on confo−1 spectral range, including the band positions, width and relative intensity of the amide III, δCH2, amide II and the CH2, CH3 and OH stretching modes. The main changes were detected for collagen [These correlations can be related to the biological observations that indicate structure changes  which are important to be quantified along the pathology. The previous reports  on FT-Racollagen , 42.The spectra from −1 and wave number shifts more than 3 cm−1 for the 1379, 1424, 1499 and the 1635 cm−1 bands. The band shape at 1635 cm−1 assigned to CV amino groups (3 cm−1 shift) changes and the relative intensity of the band decreases , whereas other CV contributions are hardly spotted because of the bands overlap with those of tissue . Other CV modes are observed at 1145, 1379 and 1574 cm−1. Spectrum c) collected from depth clearly showed the highly enhanced band at 1271 cm−1 ; the 1346 cm−1 is enhanced and becomes broaden, probably encompassing several contributions from the skin tissue, the 1451 cm−1 which was previously seen as a weak shoulder is also enhanced and appears as a broad band and the 1492 cm−1 band of CV is shifted to 1500 cm−1. The amino bending of CV is distorted and a broadband centred at 1660 cm−1 is observed . The CV label was tracked inside the tissue and recognized owing to its fingerprint bands. Besides, SERS signal from the tissue molecular components was also observed. Spectrum b) in ecreases B. The CVobserved C.−1 band assigned to amide III and stretching vibration of C–N bonds [All these results strongly suggest an interaction between the CV amino functional groups of the labelled nanoparticles and certain tissue components. –N bonds .The proof of concept in employing SERS nanosensors in interrogating the melanoma tissue from mice models is demonstrated. Upon tissue uptake of the labelled Ag and Au nanoparticles a characteristic SERS spectral feature could be obtained either in visible or NIR spectral range excitation. The nanoparticles enhanced the Raman scattering signal of the tissue molecular components found in their immediate vicinity. SERS spectra revealed dominant bands characteristic to proteins and DNA constituents indicating that some nanoparticles are located in the close vicinity of the nucleus components. SERS labels based on CV-functionalized nanoparticles proved signal specificity in different skin tissues. Such optical nanosensors can be tracked inside the tissue samples based on the spectral signature of the Raman reporter. The SERS signal reported information on either the label or the tissue molecular components from the vicinity of the label, thus such optical nanosensors hold the potential of chemical probing of tissue environment. The double amino-functionalized CV SERS labels appear to interact through their amino groups with other molecular components inside the tissue.in vitro, ex vivo and in vivo. The signal obtained from the SERS target in tissue environment provided information from the molecular components of tissue located in the close vicinity of the SERS sensor. Such information would be essential in understanding the molecular changes in the early cancer diagnostic, using a rapid, sensitive and non-invasive technique. Further investigations to confirm the binding specificity of the SERS label to particular tissue component as well as the molecular changes along the induced malignancy are in progress.These results create perspectives for further progress in early cancer diagnostic based on isolating and manipulating amino-functionalized nanoparticles as strong Raman scattering composites, for monitoring specific tissue components


Epithelial ovarian cancer has the highest mortality rate of all gynecological malignancies. We have shown that high RAN expression strongly correlates with high-grade and poor patient survival in epithelial ovarian cancer. However, as RAN is a small GTPase involved in two main biological functions, nucleo-cytoplasmic transport and mitosis, it is still unknown which of these functions associate with poor prognosis.To examine the biomarker value of RAN network components in serous epithelial ovarian cancer, protein expression of six specific RAN partners was analyzed by immunohistochemistry using a tissue microarray representing 143 patients associated with clinical parameters. The RAN GDP/GTP cycle was evaluated by the expression of RANBP1 and RCC1, the mitotic function by TPX2 and IMPβ, and the nucleo-cytoplasmic trafficking function by XPO7, XPOT and IMPβ.Based on Kaplan-Meier analyses, RAN, cytoplasmic XPO7 and TPX2 were significantly associated with poor overall patient survival, and RAN and TPX2 were associated with lower disease free survival in patients with high-grade serous carcinoma. Cox regression analysis revealed that RAN and TPX2 expression were independent prognostic factors for both overall and disease free survival, and that cytoplasmic XPO7 expression was a prognostic factor for overall patient survival.In this systematic study, we show that RAN and two protein partners involved in its nucleo-cytoplasmic and mitotic functions  can be used as biomarkers to stratify patients based on prognosis. In particular, we reported for the first time the clinical relevance of the exportin XPO7 and showed that TPX2 expression had the strongest prognostic value. These findings suggest that protein partners in each of RAN’s functions can discriminate between different outcomes in high-grade serous epithelial ovarian cancer patients. Furthermore, these proteins point to cellular processes that may ultimately be targeted to improve the survival in serous epithelial ovarian cancer. Epithelial ovarian cancer (EOC) is the most lethal of all gynecologic malignancies in North America RAs-related Nuclear protein), in EOC, is over expressed as tumor grade increases and is strongly associated with poor patient survival In vitro, the use of a short hairpin RNA (shRNA) targeting RAN expression prevents EOC cell proliferation, suggesting RAN as a potential amenable therapeutic target for the treatment of this disease in vivo mouse xenograft experiments resulted in the arrest of EOC tumor growth In previous studies, we have demonstrated that RAN . In proximity to chromosomes, TPX2 is subsequently released facilitating the regulation of microtubule organization and dynamics Although RAN uses the same regulators for GTP/GDP exchange, it utilizes unique partner proteins during interphase and mitosis to perform different physiological functions (transport vs. spindle assembly). During interphase, the enrichment of RAN-GTP in the nucleus is coupled to exportins such as exportin-t (XPOT) versus the other, the expression of molecular partners, specific to nucleo-cytoplasmic transport and mitosis, were analyzed by immunohistochemistry on a tissue microarray of 143 serous carcinomas comprising 131 cases of HG and 12 of LG tumors. We evaluated the expression of individual proteins in correlation with clinical parameters and determined their association with EOC progression and outcome. Promising results were obtained for two RAN partners, XPO7 and TPX2, with potential to be clinically relevant in stratifying HG serous EOC patients.The objective of the present study was to investigate which function of RAN would be more relevantly implicated in HG serous EOC malignancy and poor patient survival. To verify the impact of one function The CHUM institutional ethics committee  approved this study and written consent was obtained from patients prior to sample collection.Tumor samples were obtained from patients who underwent surgery for ovarian cancer at the Department of Gynecologic Oncology - Centre hospitalier de l’Université de Montréal (CHUM). A gynecologist-oncologist determined disease stage as defined by the Federation International of Gynecology and Obstetrics (FIGO). An independent pathologist reviewed histopathology and tumor grade. Tissue selection criteria for this study were based on independent confirmation of serous histopathology in samples from chemotherapy-naïve patients. Samples were collected between 1990 and 2006. Patient overall survival was defined as the time from surgery to death from ovarian cancer or last follow-up. Patient disease free survival was calculated from the time of surgery until the first progression. Therefore only patients that progressed were included in our analysis. Clinical data on progression-free interval were defined according to level of blood CA125 and tumor size assessed by imaging. Patients known to be still alive at time of analysis were censored at time of their last follow-up. The ages of diagnosis of patients with LG tumors ranged from 27 to 71 years (average = 48.5 years) and they were followed 50.6 months on average. The ages of patients with HG tumors ranged from 34 to 87 years (average = 62.6 years) and their average follow-up was 37.2 months. The cohort is described in All cases were reviewed by a gynecologic pathologist. The grade and type of ovarian carcinoma The quality and specificity of individual antibodies where verified by Western blot analysis. Total protein extracts (30 µg) were loaded and electrophoresed on SDS-polyacrylamide gel then transferred onto a nitrocellulose membrane. The membranes were blocked with 5% milk-PBS (1 hr) and subsequently probed with primary antibodies (2 hrs at room temperature) at the optimal dilutions . Each primary antibody was detected with an HRP-conjugated secondary antibody (Santa Cruz Biotechnology Inc.) and visualized by the enhanced chemiluminescence (ECL) method . Beta-actin was used as a loading control  .For the manual staining method, the TMA block was sectioned at 4 µm on superfrost+ glass microscope slides . Slides were heated at 60°C for 15 min, deparaffinized in xylene, rehydrated in an ethanol gradient, and washed in phosphate-buffered saline (PBS). To unmask antigens, slides were placed for 20 min at high temperature under high pressure in citrate buffer  for staining with anti-RCC1 , anti-IMPβ  and anti-TPX2 , or in citrate-EDTA buffer  for staining with anti-RANBP1 , anti-XPOT  and anti-XPO7  antibodies. Slides were then incubated with 3% hydrogen peroxide in PBS (to block endogenous peroxidase), and washed in PBS. Tissue sections were blocked with a non-serum protein-blocking reagent  for 15 min at room temperature and incubated with primary antibody for 60 min at room temperature in a humid chamber. Substitution of the primary antibody with PBS served as a negative control. The slides were then washed in PBS, incubated with secondary antibodies conjugated with horseradish peroxidase . Reaction products were developed using diaminobenzidine (DAB) containing 0.3% hydrogen peroxide up to 5 min. Cell nuclei were counterstained with diluted hematoxylin for 1 min.For the automated staining method, sections of formalin fixed paraffin embedded tumors (4 µm) were stained using the BenchMark XT automated stainer . Antigen retrieval for RAN was performed with Cell Conditioning 1  for 30 min. Slides were incubated with anti-RAN antibody  for 40 min, and developed by the iView DAB detection kit . Hematoxylin and bluing reagent were used for counterstaining . TMAs were observed by brightfield microscopy and digitally imaged .Protein expression by IHC was scored according to the subcellular localization and staining intensity of malignant cells. Nuclear and cytoplasmic expression of RAN network proteins in ovarian cancer tissues was observed using digitally imaged scans of each stained TMA and scored according to the intensity of staining. For each RAN network protein in epithelial zones of the tumor cores, the staining intensity of DAB was defined as 0 (no staining), 1 (weak staining), 2 (moderate staining) and 3 (strong staining). All TMAs were analyzed in a blind study by two independent observers. Inter-rating correlation (ICC) was greater than 75% for all assays. The average of all cores with cancer from the same patient was used for analysis. When strong differences in scoring between the two observers occurred the core was re-evaluated to reach a consensus between the two observers.All statistical analyses were performed using SPSS software version 16.0  where p<0.05 were deemed significant. Protein expression patterns of RAN partners were correlated  with the protein expression of RAN and to disease stage (I to IV). Survival curves  were plotted by the Kaplan-Meier estimator, compared using the log rank test and analyzed for significant differences. For each RAN partner, the number of patients in every survival curve is reviewed in To determine if any of the partners of RAN are implicated in the biology of EOC, we performed an IHC analysis using a TMA containing a total of 286 cores of cancer specimens representing 143 patients. RAN and the six RAN-partner proteins , were analyzed using this tissue array. Specificity for each antibody was first evaluated by western blot analysis using cellular extracts of four EOC cell lines  and by IThe expression of RANBP1 (GDP) and RCC1 (GTP), which are general proteins related to the RAN-GTP cycle, were initially investigated. IMPβ expression was analyzed to concomitantly evaluate both of the two main functions of RAN. Then we specifically explored the nucleo-cytoplasmic function of RAN by assessing the expression of two exportins: XPO7 and XPOT. Finally, we examined TPX2 expression in order to evaluate the spindle assembly during mitosis, another important function of RAN. Having established the staining pattern for each of the RAN network proteins, we then assessed the staining intensity according to tumor grade . In a prWe next verified the association between the expression of RAN and each of the RAN network partners using the Pearson correlation test (two-tailed) . As expeThe association between the expression of RAN or any of the RAN network partners with disease stage was also investigated using the Pearson correlation test (two-tailed) . A signiWe investigated the relationship between the expression of RAN partner proteins and overall survival in the cohort of 143 patients. Kaplan–Meier survival curves were derived and compared by the log-rank test for HG cases only –S3. LG cIncreasing levels of cytoplasmic RAN staining in HG serous EOC was significantly associated with a poorer overall survival . In HG tumors, only RAN  and TPX2  were independent variables predicting a high risk of recurrence .Our results indicate that the expression patterns of RAN and its mitotic partner TPX2 in serous EOC act as independent prognostic biomarkers for both overall patient survival and recurrence. We next evaluated the combined impact of these two candidate biomarkers on overall and disease free survival using Kaplan-Meier curve analysis. Our results showed a significant difference between staining patterns of RAN and TPX2 in progression free survival  in HG caHigh RAN expression levels have been reported in different tumors, such as pancreas, colon, lung, nasopharynx, stomach and kidney, when compared to normal tissues Using a larger cohort, we have corroborated our initial findings that patients with high RAN expression in serous EOC are associated with disease progression and poor outcome The exportin XPO7 has only recently been described Apart from XPO7, we also found an association between high TPX2 staining and poor patient outcome in HG serous EOC –4. TPX2 in vitro studies showing that RAN associated nuclear export and mitotic spindle assembly correlate with clinical outcomes in serous ovarian carcinomas. Further dissection of the role of RAN, and its cellular functions, will need to be undertaken to fully understand their role in ovarian cancer.Overall, our findings indicated that the RAN network, including both of RAN’s main functions (nucleo-cytoplasmic transport and mitosis), are deregulated in serous ovarian carcinomas affecting tumor progression and patient survival. However, multivariate analyses demonstrate a higher risk for patients expressing either RAN or TPX2 than for those expressing XPO7 , suggestFigure S1Specificity of antibodies by immunoblotting. Western-blot analysis of whole cell lysate from four epithelial ovarian cancer cell lines *. Extracts were loaded on 8% or 10% SDS/PAGE gel and membranes were hybridized with anti-RAN (A), anti-RANBP1 (B), anti- RCC1 (C), anti-IMPβ (D), anti-XPOT (E), anti-XPO7 (F) and anti-TPX2 (G). Immunoblots were performed on four different proteins extracts from cell lines and representative images are presented. β-actin was used as a loading control. *V. Ouellet et al., BMC Cancer. 2008. D. Provencher et al. In Vitro Cell Dev Biol Anim. 2000.(TIF)Click here for additional data file.Figure S2Specificity of antibodies for RAN protein network on cell pellets. Immunohistochemistry analyses were performed on paraffin-embedded cell pellets* of the four epithelial ovarian cancer cell lines . Images are representative staining patterns for each member of RAN network corresponding to low (left panel) or high (right panel) expression (magnification 20 X). Note that high and low expression correlated between Western blot and immunohistochemistry on cell pellets. * Zietarska M et al., Histopathology. 2010.(TIF)Click here for additional data file.Table S1Clinical parameters of the serous EOC samples in the cohort.(DOCX)Click here for additional data file.Table S2Number of HG serous EOC patients for each RAN partner in every Kaplan-Meier curve.(DOCX)Click here for additional data file.Table S3Kaplan-Meier analysis of RAN network in HG serous EOC.(DOCX)Click here for additional data file.