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Mature microRNAs (miRNAs) are single-stranded RNAs of 18–24 nucleotides that repress post-transcriptional gene expression. However, it is unknown whether the functions of mature miRNAs can be regulated. Here we report that expression of versican 3′UTR induces organ adhesion in transgenic mice by modulating miR-199a* activities. The study was initiated by the hypothesis that the non-coding 3′UTR plays a role in the regulation of miRNA function. Transgenic mice expressing a construct harboring the 3′UTR of versican exhibits the adhesion of organs. Computational analysis indicated that a large number of microRNAs could bind to this fragment potentially including miR-199a*. Expression of versican and fibronectin, two targets of miR-199a*, are up-regulated in transgenic mice, suggesting that the 3′UTR binds and modulates miR-199a* activities, freeing mRNAs of versican and fibronectin from being repressed by miR-199a*. Confirmation of the binding was performed by PCR using mature miR-199a* as a primer and the targeting was performed by luciferase assays. Enhanced adhesion by expression of the 3′UTR was confirmed by in vitro assays. Our results demonstrated that upon arrival in cytoplasm, miRNA activities can be modulated locally by the 3′UTR. Our assay may be developed as sophisticated approaches for studying the mutual regulation of miRNAs and mRNAs in vitro and in vivo. We anticipate that expression of the 3′UTR may be an approach in the development of gene therapy.
Citation: Lee DY, Shatseva T, Jeyapalan Z, Du WW, Deng Z, Yang BB (2009) A 3′-Untranslated Region (3′UTR) Induces Organ Adhesion by Regulating miR-199a* Functions. PLoS ONE 4(2): e4527. doi:10.1371/journal.pone.0004527
Editor: Stefan Maas, Lehigh University, United States of America
Received: December 17, 2008; Accepted: December 26, 2008; Published: February 18, 2009
Copyright: © 2009 Lee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from Heart and Stroke Foundation of Ontario (NA 6282) and National Sciences and Engineering Research Council of Canada (227937-01) to BBY who is the recipient of a Career Investigator Award (CI 5958) from the Heart and Stroke Foundation of Ontario. DYL is the recipient of a Canada Graduate Scholarship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Human Genome Project identified approximately 25,000 protein-coding genes, occupying 1.9% of total genomic DNA. The remaining DNA has come to be known as "junk" DNA or cellular detritus and was presumed to serve no particular function because it does not code proteins. Pseudogenes are part of these non-functional DNAs since they are the defective copies of the protein-coding genes. In fact, pseudogenes are nearly as abundant as the protein-coding genes and therefore appear to be an important component of the genome. It has been reported that there are approximately 20,000 putative pseudogenes in the human genome [1], [2]. A large number of pseudogenes are found to be transcribed. Analysis of chromosome 22 indicated that approximately 20% of the pseudogenes are potentially transcribed [3]. Pseudogene transcription has also been reported in other species including fly, mouse, cow, and chimp [4]. The assumption that pseudogenes are dysfunctional is based on the fact that pseudogenes do not code for proteins. However, it is unknown whether non-coding RNAs can affect microRNAs (miRNAs) functions.
miRNAs are single-stranded RNA of 18–24 nucleotides in length and are generated by an RNase III-type enzyme from an endogenous transcript that contains a local hairpin structure [5], [6]. miRNA functions as a guide molecule in post-transcriptional gene silencing by partially complementing with the 3′-untranslated region (3′UTR) of the target mRNAs, leading to translational repression [7]–[9]. By silencing various target mRNAs, miRNAs have key roles in diverse regulatory pathways, including controlling development [10]–[15], cell differentiation [16]–[20], apoptosis [21], [22], cell proliferation [23]–[25], division [26], [27], protein secretion [28], [29], immuno-response [30], viral infection [31]–[34], and cancer development [35]–[47]. Functionally, miRNAs are opposite to transcription factors, which turn on genes, while miRNAs function as modulators by down-regulating gene expression [7], [48]. Studies associated with miRNA functions and regulations of gene expression by mature miRNAs are overwhelming. However, it is unknown whether the mature miRNAs can also be regulated. We hypothesize that the mRNA 3′UTRs can bind to and regulate miRNA functions. In this study, we used versican 3′UTR as a model to study the role of the non-coding transcript. Versican is an extracellular proteoglycan that has been shown to mediated varieties of cell activities [49]–[53]. We found that the expression of the versican 3′UTR induced cell, tissue, and organ adhesion by arresting miR-199a* functions.
Results and Discussion
Transgenic expression of versican 3′UTR induces organ adhesion
To study the effect of versican 3′UTR on modulating miRNA functions, we cloned and placed the 3′UTR under the control of the CMV promoter. The 3′UTR was transcribed as expected (Fig 1a). The fragment containing the transcription unit was cropped for the generation of transgenic mice and we obtained four versican 3′UTR transgenic founder animals. All were bred with wildtype mice and we obtained a total of twelve litters of F1 mice. Genotyping was performed by PCR using two different pairs of primers and the result from one litter is shown in Fig 1b. Expression of the transgene in a number of organs was confirmed by RT-PCR (Fig 1c). Real-time PCR analysis indicated that the levels of the 3′UTR were 45-fold higher than that of wild-type (Fig 1d). A number of the positive F1 and wild-type mice were sacrificed and examined. We observed that in some 3′UTR mice, the livers were strongly adhered to the stomach (Fig 1e). In some others, the livers adhered with connective tissues sticking to the internal body wall (Fig 1f, arrows) or directly adhered to it (Fig 1g). In a different transgenic line, stomach adhesion (Fig S1b) and liver adhesion (Fig S1c) were also observed. These adhering organs were subjected to histological analysis by hematoxylin and eosin (H&E) staining. Adhesion of livers with connective tissues could be clearly seen (Fig 1h, arrows).
(a), A fragment of versican 3′UTR (700 bp) immediately after the stop codon containing a small fragment at the 3′ of versican coding sequence was inserted into the pcDNA3.1 plasmid, downstream of the CMV promoter, between XbaI and ApaI sites, producing Ver-UTR construct. Total RNA was prepared from one pooled cell line and two individual clones stably transfected with Ver3′UTR or the empty vector, subjected to RT-PCR, and analyzed on agarose gel electrophoresis. Expression of the 3′UTR was confirmed. (b) Genotyping PCR was performed on tail DNA extracted from the same litter of F1 using two pairs of primers amplifying the promoter region (CMV) and the downstream transcript versican 3′UTR. (c) Expression of the transgene was analyzed by RT-PCR using RNAs isolated from different organs of 3′UTR transgenic mice. (d) The levels of versican 3′UTR were analyzed by real-time PCR in Hek293 cells transfected with the 3′UTR construct or a control vector (detecting endogenous versican). The levels of 3′UTR in the 3′UTR-transfected cells were over 45-fold that of the control. (e) Photograph showing adhesion of the liver to the stomach. (f) Photograph showing organ adhesion occurred between liver and diaphragm. One piece of liver was adhered to the diaphragm. (g) A liver was adhered to the internal body walls of the mouse. (h) Paraffin sections of the adhesion tissues were stained with hematoxylin and eosin (H&E). Both photographs show the linking between the livers and connective tissues.
Expression of versican 3′UTR induces cell adhesion
To study how versican 3′UTR affected organ adhesion, we examined its effects on cell lines stably expressing the 3′UTR. Human astrocytoma cell line U343 was stably transfected with the 3′UTR construct or an empty vector. The cells were cultured in DMEM containing 10% FBS at a cell density of 5×104 cells/ml and examined under a light microscope and photographed. The 3′UTR-transfected cells attached to tissue culture plates slower than the vector-transfected cells (Fig S1d). After cell attachment, the UTR-transfected cells tended to adhere together and appeared less elongated (Fig 2a). Two days after cell inoculation, large groups of the 3′UTR-transfected cells could be detected, forming an island-like morphology (Fig 2b).
Vector- or 3′UTR-transfected cells were inoculated in tissue culture dishes overnight. Cell morphology was examined under a light microscope (a). Two days after cell inoculation, the 3′UTR-expressing cells exhibiting island-like morphology (b). The 3′UTR-transfected cells were transiently transfected with siRNA targeting the 3′UTR or a control sequence, followed by real-time PCR analysis of the 3′UTR levels (c, left). Cell adhesion of the two groups of cells was compared with the vector-transfected cells. Down-regulation of the 3′UTR levels increased cell adhesion (c, right). Typical results of cell adhesion are shown (d, left). Cell morphology was also examined. Down-regulation of the 3′UTR levels reversed the morphology (d, right). Luciferase reporter vector harboring the versican 3′UTR was co-transfected with the versican 3′UTR construct at different amount combined with a control vector in U343 cells. Increase rations of versican 3′UTR bound more endogenous miR199a* and thus freeing the translation of luciferase protein, resulting in higher levels of luciferase activities (e).
To confirm the direct effect of the 3′UTR, 4 different siRNAs complementary to the 3′UTR were synthesized. Down regulation of the 3′UTR was confirmed by real-time PCR amplifying a fragment of the 3′UTR (Fig 2c, left). Cell adhesion assays showed that transfection with the siRNAs partially rescued the reduced adhesion in the 3′UTR-transfected cells (Fig 2c, right). Furthermore, transfection of the siRNA also reversed the altered cell morphology (Fig 2d).
We developed a number of experiments to analyze the effects of the 3′UTR. It was linked with the construct expressing versican G3 domain [54], producing the G3-UTR. Cell lysates were prepared from U343 cells stably transfected with the G3 and G3-UTR constructs and were subjected to Western blot analysis probed with anti-G3 and anti-actin antibodies simultaneously. While actin levels were similar, G3 levels were much lower in cells transfected with the G3-UTR construct (Fig S2a). This result suggests that some endogenous miRNAs targeted the 3′UTR and repressed G3 expression.
The 3′UTR was also linked with the GFP expression unit (Fig S2b, upper). Fluorescent levels of U87 and U343 cells stably transfected with the GFP and GFP-UTR constructs were measured with flow cytometry. Cells transfected with the GFP-UTR construct produced lower levels of fluorescence than that transfected with the GFP construct (Fig S2b, middle and lower). Cells transfected with the GFP and GFP-UTR constructs were also examined under a light and fluorescent microscope. Transfection with the GFP-UTR construct produced lower levels of GFP than the controls (Fig S2c).
Furthermore, the 3′UTR was linked to the luciferase report vector producing the lu-VUTR construct. Cells that were transfected with different concentrations of lu-VUTR always produced lower levels of luciferase activity than cells transfected with a control construct (Fig S3a), suggesting repression of luciferase expression by endogenous miRNAs. Nevertheless, with increased concentrations of lu-VUTR, the relative luciferase activities increased when normalized with the control construct. This suggests that higher concentrations of lu-VUTR would absorb some endogenous miRNAs and free more lu-VUTR to be expressed. To confirm this, the lu-VUTR construct was co-transfected with the 3′UTR construct. Increased 3′UTR concentrations generated higher levels of luciferase activities in U343 cells (Fig 2e) and U87 cells (Fig S3b). These results suggest that versican 3′UTR plays a key role in the induction of adhesion.
Versican 3′UTR interacts with miRNS-199a*
To test the direct interaction of miRNAs with the 3′UTR, we developed a PCR assay to test the potential binding interactions of the miRNAs with the 3′UTR and to validate the target sites in silico. This method assumes that the miRNAs targeting the 3′UTR could serve as a PCR-primer, and the PCR products could be generated by a 5′UTR-specific primer pairing with the miRNA primer. The nucleotide sequence of miRNA-X primer corresponds to the RNA sequence of miRNA-X but with the substitution of uridine for thymidine (Supporting Fig S1a). The versican 3′UTR construct was used as a PCR-template (Fig 3a).
(a) Scheme for PCR method to test the interaction of miRNAs with versican 3′UTR. An oligonucleotide corresponding to miRNA-X is used as a reverse primer. It binds to the potential targeting sites on the antisense strand of the 3′UTR construct, depending on the extent of complementation. One forward primer docked on a different location of the vector was used to pair with the miRNA primer for PCR. (b) PCR products were obtained showing different size of products corresponding to the forward primer and the miRNA sequences. The expected sizes of PCR products are indicated with arrows. The miRNA sequences used were listed in Supporting Fig S1a. (c) Computational analysis showed that miR-199a* potentially targeted both versican and fibronectin 3′UTRs. Overexpression of versican 3′UTR would attract endogenous miR-199a* freeing versican mRNA and fibronectin mRNA for translation.
We analyzed the potential miRNAs that bind to the 3′UTR of the Ver-UTR construct. A number of candidates with low binding energy were detected. We tested 17 different miRNAs selected from the potential candidates for the 3′UTR of versican. As shown in Fig 3b and Supporting Fig S3c, PCR products with expected sizes were generated by using two different annealing temperatures. There was no correlation between positive PCR results and the G/C content or the melting temperature of primers, which indicated the specificity of miRNAs toward the 3′UTR of versican. This method can be used to screen miRNAs that bind to the 3′UTR of interest. It is more efficient than the commonly-used luciferase assay and can be used to confirm the candidate binding sites as identified by other tools. Nevertheless, this is not to replace any existing methods. Rather, it adds an alternative approach to identify miRNAs targeting a 3′UTR of interest. It also produces a shorter list of candidates for validation by luciferase activity assays and transfection experiments.
It is expected that one 3′UTR contains many miRNA binding sites and one miRNA might target many 3′UTRs. This would create a balanced network composing of the synergy and counteraction of miRNA-3′UTR interactions. The homeostatsis of miRNAs and miRNA-binding sites might be disrupted through changes in the expression of transcripts or miRNAs. Over-expression of the versican 3′UTR would affect the levels of free miRNAs through binding the miRNAs, which normally regulate versican expression by targeting its 3′UTRs. The formation of miRNA-3′UTR transcript duplex thus decreases the functional miRNA levels. This interaction would affect protein expression, leading to the functional consequences.
Using the computation algorithm FindTar (http://bio.sz.tsinghua.edu.cn/findtar), we found a great number of miRNAs potentially targeting the versican 3′UTR. One of the positive miRNAs that showed interaction with the versican 3′UTR is miR-199a*. Using the online search engine TargetScan4.0 (www.targetscan.org), we found a great number of genes that are potentially targets of miR-199a* including versican (Genbank access number NM_004385) and fibronectin variant 1-6 (Genbank access number NM_212482, NM_212475, NM_002026, NM_212478, NM_212476, and NM_212474). Fibronectin is an extracellular glycoprotein which binds to integrins and mediates cell adhesion, proliferation, tissue development, and life maintenance [55], [56]. It helps maintain cell shape by lining up cells and organizing their intracellular cytoskeleton. Our previous studies indicated that versican regulates cell proliferation, adhesion, aggregation, blood coagulation, and angiogenesis [57]–[62]. Thus, we hypothesized that the overexpression of versican 3′UTR would attract endogenous miR-199a*, freeing versican and fibronectin mRNA for translation. Increased versican expression would produce the consequences observed in this study.
Transgenic expression of versican 3′UTR increases levels of endogenous versican
To test whether the expression of versican 3′UTR affected versican expression, we prepared protein lysates from the brain, heart, kidneys, lungs, and spleen and analyzed versican expression by western blotting. Our experiments showed that the 3′UTR transgenic mice expressed higher levels of versican compared with the wild-type mice (Fig 4a). The organs were also subjected to histological analysis probed with anti-versican antibody. In the reproductive system, versican levels were much higher in the 3′UTR mice as compared with the wild-type mice (Fig 4b, arrows). As well, versican levels were much higher in the airways and pulmonary blood vessels of the 3′UTR lung (Fig 4c, arrows), in the livers (Fig 4d), the connective tissues (Fig 4e), and the ribs of the 3′UTR mice compared with the wild-type organs (Fig 4f). In the adhering organs of livers and pancreas, versican expression in the 3′UTR pancreas was much higher compared with the wild-type (Fig 4g). Normally, the liver does not adhere to any organ. However, in the 3′UTR mice, the liver adhered tightly to others such as pancreases and connective tissues of the body wall (Fig 4h). In the extreme case, the liver lost its smooth edges (Fig 4h, middle) and even merged with the connective tissue (Fig 4h, bottom). Increased versican expression was also detected in the junction areas of stomach and connective tissues (Fig S3d). The adhesion tissues were also immunostained for type I collagen expression, as type I collagen is an important adhesion molecule [63], [64]. We found that the junction of adhesion tissues expressed high levels of type I collagen (Fig S3e). This result indicated that type I collagen plays a role in versican 3′UTR-induced organ adhesion.
(a) Protein lysates were prepared from different organs and subjected to western blot analysis probed with anti-versican antibody. Detection of β-actin on the same membranes served as a loading control. Increased versican expression was detected in the organs harvested from the transgenic mice. (b–f) Paraffin sections of tissues from reproductive system (b), lung (c), liver (d), connective tissues (e), and rib (f) of the 3′UTR-transgenic and wild-type mice were stained with anti-versican antibody. In all sections shown, versican expression levels in the transgenic mice were higher as compared with the wild-type (arrows). (g) The levels of versican expression were higher in the pancreas that adhered to the liver (arrows). (h) The levels of versican expression were higher in the junctions between liver and the surrounding connective tissues (arrows). In the lower panel identified by the circle, some liver tissues (solid arrow) and connective tissues (open arrow), stained with anti-versican antibody, were mixed up, and no border could be identified between the different tissues. (i) Versican 3′UTR (nucleotides 275–299 of the 3′UTR, Upper) was found to be the potential target of miR-199a*. A versican 3′UTR was cloned and inserted into the luciferase reporter vector pMir-Report. Mutations were generated on the potential target sequence (red color). Lower, U343 cells were co-transfected with the miR-199a construct and the luciferase reporter construct harboring the versican 3′UTR (lu-VUTR) or the mutant construct (lu-Ver-mut). As a negative control, the luciferase reporter construct was engineered with a non-related fragment of cDNA (Ctrl). Luciferase activity assays indicated that the miR-199a construct repressed luciferase activities when it harbored the versican 3′UTR, which was abolished when the potential miR-199a* target site was mutated. Significant differences are indicated by asterisks. ** Error bars, SEM (n = 3), ** p<0.01.
To confirm if versican was a target of miR-199a*, U343 cells were co-transfected with versican 3′UTR-luciferase construct (lu-VUTR) or the mutant lu-Ver-mut, in which the miR-199a* target site was mutated by nucleotide replacement (Fig 4i, upper). Luciferase activity assays showed that while presence of the versican 3′UTR reduced luciferase activities, mutation of the miR-199a* target site partially rescued luciferase activities (Fig 4i, lower). This suggests that miR-199a* repressed versican expression by targeting the 3′UTR. Furthermore, incomplete rescue of luciferase activities suggests that the versican 3′UTR is also targeted by other endogenous miRNAs.
Transgenic expression of versican 3′UTR promotes fibronectin expression
To test whether the expression of versican 3′UTR affected fibronectin levels, we prepared protein lysates from the brain, heart, kidneys, lungs, and spleen and analyzed fibronectin expression by western blotting. Our experiments showed that the 3′UTR transgenic mice expressed higher levels of fibronectin compared with the wild-type mice (Fig 5a). The organs were also subjected to histological analysis probed with anti-fibronectin antibody. In the spleen, fibronectin levels were higher in the connective tissue structures of the 3′UTR mice as compared with the wild-type mice (Fig 5b, arrows). As well, fibronectin levels were higher in the brain (Fig 5c, arrows), connective tissues (Fig 5d), liver (Fig 5e), and ribs (Fig 5f) of the 3′UTR mice compared with the wild-type organs. In the adhering organs of livers and pancreas, fibronectin expression in the 3′UTR pancreas was clearly detected (Fig 5g, upper). In the adhesion organs associated with liver and connective tissue, fibronectin was strongly expressed in the connective tissues adhering to the liver (Fig 5g, middle and lower). In the extreme cases, where the liver lost its smooth edges and mixed up with the connective tissue, fibronectin translation was greatly promoted (Fig 5h). Furthermore, fibronectin levels were much higher in the adhesion areas between liver/liver, liver/stomach, liver/muscle, and pancreas/stomach (Fig 5i).
(a) Protein lysates were prepared from different organs and subjected to western blot analysis probed with anti-fibronectin antibody. Detection of β-actin on the same membranes served as a loading control. Increased fibronectin expression was detected in the organs harvested from the transgenic mice. (b–f) Paraffin sections of tissues from spleen (b), brain (c), connective tissue (d), liver (e), and rib (f) of the 3′UTR-transgenic and wild-type mice were stained with anti-fibronectin antibody. In the transgenic spleen, the connective tissue structures expressed higher levels of fibronectin than the wild-type tissues did (arrows). In the transgenic brain, fibronectin expression was higher in the blood vessels (arrows). In the connective tissues of the transgenic mice, some areas expressed higher levels of fibronectin as compared with the wild-type (arrows). In the transgenic liver, fibronectin expression was higher along the edges of the liver than the wild-type liver. (g) The levels of fibronectin expression were higher in the 3′UTR pancreas that adhered to the liver (arrows). The connective tissues that adhered to the livers also expressed high levels of fibronectin. (h) In a different mouse, the 3′UTR connective tissues, while expressed high levels of fibronectin, were strongly adhered with the liver. Pulling out the connective tissue severely damaged the liver surface (upper panel, arrow). In the areas identified by the circles, some liver tissues (solid arrow) and connective tissues (open arrow, stained with anti-fibronectin antibody) were completely merged. (i) Adhesion of different organs was detected between liver/liver, liver/muscle, liver/stomach, and pancreas/stomach. The junctions between the organs expressed high levels of fibronectin (arrows). (j) Fibronectin 3′UTR (nucleotides 663–683 of the 3′UTR, Upper) was found to be the potential target of miR-199a*. A fibronectin 3′UTR was cloned and inserted into the luciferase reporter vector pMir-Report. Mutations were generated on the potential target sequence (pink color). Lower, U343 cells were co-transfected with the miR-199a construct and the luciferase reporter construct harboring the fibronectin 3′UTR (lu-FNUTR) or the mutant construct (lu-FNmut). Luciferase activity assays indicated that the miR-199a construct repressed luciferase activities when it harbored the fibronectin 3′UTR, which was abolished when the potential miR-199a* target site was mutated. Significant differences are indicated by asterisks. ** Error bars, SEM (n = 3), ** p<0.01.
To confirm that fibronectin was a target of miR-199a*, we cloned the fragment of fibronectin 3′UTR containing the miR-199a* target site. The fragment was inserted into the luciferase reporter vector producing a construct lu-FNUTR. The potential target site of miR-199a* was mutated producing lu-FNmut. U343 cells were co-transfected with fibronectin 3′UTR-luciferase construct (lu-FNUTR) or the mutant lu-FNmut (Fig 5j, upper). Luciferase activity assays showed that insertion of the fibronectin 3′UTR repressed luciferase activities as compared with the control (Fig 5j, lower). Mutation of the miR-199a* target site partially rescued luciferase activities. This suggests that miR-199a* repressed fibronectin expression by targeting the 3′UTR of fibronectin. Incomplete rescue of luciferase activities suggests that the fibronectin 3′UTR is also targeted by other endogenous miRNAs. When lu-FNUTR was co-expressed with Ver-UTR, luciferase activities increased (Fig S4f), suggesting a competition of miRNAs between Ver-UTR and lu-FNUTR.
Our experiments indicated that the functions of miRNAs can be regulated by a fragment of non-coding transcript. Genomic deletion/truncation leading to translational silencing produces mutant phenotypes as the consequence of protein loss/mutation accompanied by the existence of non-coding/mutation transcripts. This strategy has been extensively used to knock-out genes of interest in studying gene functions. After gene knock-out, the protein is no longer expressed, but it is conceivable that the mutated genes are still able to produce non-coding transcripts. Sometimes, no detectable phenotypes are obtained, and it is said that the mutated/lost proteins may be compensated with others. Although compensation by other proteins is possible, the non-coding transcript may play an important role in compensation and balancing the mutant entity. Our results demonstrate a dramatic functional consequence by expressing a non-coding transcript. Exogenous expression of the 3′UTR construct altered the expression of some proteins functionally associated with the 3′UTR. It is possible that the 3′UTR can play more diverse roles than the protein expressed by the same transcript, although proteins are the executants of biological activities. Therefore, while analyzing the results of gene knock-out, one may need to consider the effects of not only the proteins but also the remaining non-coding transcripts. Our results that exogenous expression of the versican 3′UTR promoted versican expression suggest that each mRNA may exert at east two functional roles: through protein translation and miRNA regulation.
The human genome contains a large number of pseudogenes, which are nearly as abundant as the functional genes and therefore appear to be an important component in the genome. It has been reported that there are approximately 20,000 putative pseudogenes in the human genome [2]. A large number of pseudogenes are found to be transcribed. Analysis of chromosome 22 indicated that approximately 20% of the pseudogenes are potentially transcribed [3]. Pseudogene transcription has also been reported in other species including fly, mouse, cow, and chimpanzee [4]. The assumption that pseudogenes are dysfunctional is based on the fact that pseudogenes do not code for proteins. It is possible that these non-coding transcripts of the pseudogenes play important roles as modulators in miRNA functions.
It seemed that expression of the 3′UTR produced a similar functional role as the miRNA inhibitor. However, one 3′UTR has the capacity to modulate multiple miRNAs, while one miRNA inhibitor can only affect one miRNA. In this sense, a 3′UTR may be able to exert diverse biological activities by modulating multiple miRNA functions. As such, an animal gene, with long sequence of the 3′UTR, may have the capacity of exerting complex biological activities. Furthermore, a long fragment of 3′UTR may be more stable, while the miRNA inhibitor may be readily degraded. Thus, expression of a 3′UTR may have great advantage in modulating cell activities. In terms of stability and functionality, the 3′UTR may be better than normal mRNA, in that a mRNA has many tasks to carry out, while a 3′UTR may only exist for miRNA binding. In the former case, binding with multiple factors involved in protein synthesis and formation of secondary structures would decrease the accessibility for miRNAs. In the latter case, a simple 3′UTR fragment would be much more accessible for miRNA binding. This may explain why expression of a non-coding fragment could serve as a vigorous tool and produce potent effects in vitro and in vivo. Furthermore, the 3′UTR may free a group of miRNAs that modulate mRNAs with related biological functions. Changes in miRNA regulation may improve translation of the network of related proteins, providing immediate, effective, and lasting biological effects. One could imagine that the future applications will be benefited by the application of the 3′UTR transcript in gene therapy.
Materials and Methods
Construct generation
To study the effect of versican 3′UTR on cell activities, we have cloned the 3′UTR by RT-PCR using two primers Huver-UTRNXbaI and Huver-UTRCApaI. The PCR product was digested with restriction enzymes XbaI and ApaI and inserted into XbaI- and ApaI-opened pcDNA3.1 vector. After transformation, colony selection, DNA mini-preparation, and restriction enzyme digestion, the correct clones were sequenced to ensure identity of the 3′UTR.
A luciferase reporter vector (pMir-Report; Ambion) was used to generate the luciferase constructs. The 3′UTR of versican was cloned using 2 primers, HuverUTR-NSpeI and HuverUTR-CHindIII, by PCR. The PCR products were then digested with SpeI and HindIII and the fragment was inserted into a SpeI- and HindIII-digested pMir-Report Luciferase plasmid (Ambion), to obtain a luciferase construct, lu-VUTR. Primers used in this study are listed in the Supporting Fig S1a. A mutant construct was generated with two PCRs, one using two primers, HuverUTR-NSpeI and HuverUTR-mu-RXbaI, the other using HuverUTR-mu-FXbaI and HuverUTR-CHindIII. After restriction enzyme digestion, one with SpeI and XbaI, and the other with XbaI and HindIII, both fragments were ligated with pMir-Report vector opened with SpeI and HindIII.
A fragment of the 3′UTR of fibronectin was also cloned using 2 primers, FN1-N3′SacI and FN-199aC3′MluI, by RT-PCR. The PCR products were then digested with SacI and MluI and the fragment was inserted into a SacI- and MluI- digested pMir-Report Luciferase plasmid (Ambion), to obtain a luciferase construct, lu-FNUTR. A mutant construct was generated with two primers FN1-N3′SacI and FN-199aC3′MluI-Mut using similar approach.
To serve as a negative control, a non-related sequence was amplified from the coding sequence of the chicken versican G3 domain using 2 primers, chver10051SpeI and chver10350SacI. It is expected that there is no endogenous miRNA bind to this fragment as it is in the coding region. The PCR product was then inserted into a SpeI- and SacI-digested pMir-Report Luciferase plasmid.
PCR identification for miRNA-UTR interaction
For in vitro binding of microRNAs with versican 3′UTR, a PCR method was developed. The pcDNA3.1 plasmid containing the 3′UTR of versican was used as the template in PCR. The forward primer located at the vector (pcDNA3.1hygro). The reverse primers for different miRNAs are listed in Supporting Fig S1a. In general, the PCR mixture (20 µl) contained: 100 mM KCl, 100 mM (NH4)2SO4, 200 mM Tris HCl, pH 8.75, 1.0% Triton X-100, 1 mg/ml BSA, 200 µM dNTPs, 2 µM forward primer, 2 µM reverse primer, 1 unit Taq DNA polymerase. The parameters for the PCR reaction were: one cycle at 95°C for 5 min; 35 cycles at 95°C for 1 min, 37°C for 1 min, 72°C for 1 min; and a final elongation step at 72°C for 10 min. The PCR products were then visualized with a 1.5% agarose gel stained with ethidium bromide.
Generation and genotyping of the 3′UTR transgenic mice
The transgene was released from the plasmid by digestion with ApaLI and StuI. The digested product was fractionated by agarose gel electrophoresis and the 3 kb transgene fragment was excised from the gel, purified by Elutip mini-column (Schleicher and Schuell, Keene, NH) and then resuspended in injection buffer (10 mM Tris, pH 8.0 and 0.1 mM EDTA) at a concentration of 1 to 2 ng/µl. The transgene was microinjected into the male pronuclei of C57BL/6×CBA F2 mouse zygotes. Injected embryos were implanted into the oviducts of pseudopregnant recipient females using a standard protocol approved by the Animal Use Subcommittee of the University Council on Animal Care, The University of Western Ontario. Transgenic founder lines were maintained by backcrossing with C57BL/6×CBA F1 mice. Genotyping was performed by PCR, using primers EGFP-347F pairing with EGFP-668R (for CMV promoter and huver10861F pairing with Huversican-UTRCApaI (for versican 3′UTR), and tail snip or ear punch DNA as template. GAPDH served as a control using primers mo-Gapdh1F and mo-Gapdh250R. The transgenic mice were then transferred to Sunnybrook Research Institute (Toronto, Ontario). The methods for tissue harvest and analysis have been approved by the Animal Care Committee of Sunnybrook Research Institute, Ontario, Canada.
Cell adhesion assay
Vector- or 3′UTR-transfected cells were plated onto culture dishes at a density of 4×105 cells/ml and incubated for 30 min with DMEM containing 5% FBS. After 30 min, cells were fixed with 3.7% paraformaldehyde. Adhering cells were counted and cell images were captured using a phase-contrast microscope. Ten different fields (100×) were used for cell counting.
Western blot
Organs were weighted and homogenized with lysis buffer containing protease inhibitors (150 mM NaCl, 25 mM Tris-HCl, pH 8.0, 0.5 M EDTA, 20% Triton X-100, 8 M Urea, and 1× protease inhibitor cocktail). Protein concentration was measured by Bio-Rad Protein Assay kit (#5000-0006). The lysates were subjected to SDS-PAGE and then transferred to nitrocellulose membranes probed with a primary antibody against versican (Lifespan Biosciences, LS-C25140), fibronectin (BD, 610078), or β-actin (Sigma-Aldrich) overnight at 4°C. After incubation with corresponding HRP-conjugated secondary antibodies, the membranes were washed, followed by detection with the ECL kit.
Tissue slide preparation and immunohistochemistry
Organs were freshly excised and fixed in formalin overnight, immersed in 70% ethanol, embedded in paraffin, and sectioned by a microtome (Leica RM2255). The sections were de-paraffinized with xylene and ethanol and then boiled in a pressure cooker. After washing with Tris-Buffered-Saline (TBS) containing 0.025% Triton X-100, the sections were blocked with 10% goat serum and incubated with primary antibody against versican, fibronectin, or collagen Iα1 (Santa Cruz, sc-25974) in TBS containing 1% bovine serum albumin (BSA) overnight. The sections were washed and labeled with biotinylated secondary antibody, followed by avidin conjugated horse-radish peroxidase provided by the Vectastain ABC kit (Vector, PK-4000). The staining was developed by DAB kit (Vector, SK-4100). The slides were subsequently stained with Mayer's Hematoxylin for counter staining followed by slide mounting.
Luciferase assay
U343 cells were seeded onto 24-well tissue culture plates at a density of 3×104 cells/well in DMEM containing 10% FBS and maintained at 37°C for 24 hrs following the methods described by us recently [65], [66]. The cells were co-transfected with the luciferase reporter constructs and the 3′UTR construct by using Lipofectamine 2000. The cells were then collected by trypsin and lysed with a luciferase specific lysis buffer from Luciferase Assay Kit (Promega). Cells were centrifuged at 3000 rpm for 5 min. The supernatants were transferred into a black 96-well plate (3×10 µl) for luciferase activity measurement and into a transparent 96-well plate (3×50 µl) for β-gal activity determination. For the luciferase activity measurement, 70 µl of luciferase assay reagent was added into each well and the luciferase activity was detected by using microplate scintillation and luminescence counter (Packard, Perkin Elmer). For the internal control of β-gal activities, 90 µl of assay reagent (4 mg/ml ONPG, 0.5 M MgSO4, β-mercaptoethanol and 0.4 M sodium phosphate buffer) were added into each well. The plate was then incubated at 37°C for 60 min. The absorbance at 410 nm was measured by using a microplate reader (Bio-Tek Instruments, Inc.).
Statistical Analysis
The results (mean values±SD) of all the experiments were subjected to statistical analysis by t-test. The level of significance was set at p<0.05.
Supporting Information
Figure S1.
(a) Primers used in this study. (b–c) Photographs showing organ adhesion occurred between liver and stomach (b), between liver and body (c) in a different transgenic line of mice. (d) Vector- or the 3′UTR-transfected cells were inoculated in tissue culture dishes for 2.5 hours. Cell adhesion was examined under a light microscope and photographed.
Figure S2.
a, Upper, to test the effect of the versican 3′UTR, the versican G3 domain was linked with or without the 3′UTR producing G3 and G3-UTR constructs. Lower, cell lysates prepared from U343 cells stably transfected with the G3 and G3-UTR constructs were subjected to Western blot analysis probed with anti-G3 and anti-actin antibodies simultaneously. While actin levels were similar, G3 levels were much lower in cells transfected with the G3-UTR construct. Fig S2b, the GFP coding sequence was linked with or without the 3′UTR producing GFP and GFP-UTR constructs (Upper). Cells transfected with the GFP-UTR construct produced lower levels of GFP activities than that transfected with the GFP construct. The levels of fluorescent cells were quantified (Middle). Typical fluorescent levels of U87 and U343 cells transiently transfected with the GFP and GFP-UTR constructs were shown (Lower). Fig S2c, Cells transfected with the GFP and GFP-UTR constructs were also examined under a light and fluorescent microscope. Typical results are shown.
Figure S3.
(a) U343 cells were transiently transfected with luciferase reporter vector harboring the versican 3′UTR (lu-VUTR) or a control sequence (ctrl). Luciferase activities were normalized using the control as 100%. The luciferase activities of lu-VUTR never reached the levels of the control, suggesting endogenous miRNAs targeting the versican 3′UTR. Nevertheless, the activities increased with higher does of plasmids, suggesting that increased supplies of versican 3′UTR absorbed some endogenous miRNAs freeing luciferase translation. (b) Luciferase reporter vector harboring the versican 3′UTR was co-transfected with the versican 3′UTR construct at different amount combined with a control vector in U87 cells. Increase amounts of versican 3′UTR bound more endogenous miR199a* and freeing the translation of luciferase protein, resulting in higher levels of luciferase activities. (c) PCR was performed using one forward primer docked on the vector and one of the mature miRNAs as indicated at a different temperature (35°C). PCR products were obtained showing different sizes of products corresponding to the forward primer and the miRNA sequences. (d) Photographs showing organ adhesion occurred between stomach and connective tissues. The sections were immunostained with anti-versican antibody showing that versican was deposited in the adhesion junction areas. (e) The adhesion tissues were sectioned and immunostainined with anti-type I collagen that normally deposits in wound healing areas. Collagen was expressed at high levels in the areas of tissue adhesion.
Figure S4.
Paraffin sections of adhesion organs from a different transgenic line of mice were stained with anti-fibronectin antibody. The levels of fibronectin expression were higher in the adhesion junctions between liver and pancreas (a), between liver and connective tissue (b–e), and between liver and liver (e, right). Luciferase reporter vector harboring the fibronectin 3′UTR was co-transfected with the versican 3′UTR construct at different amount combined with a control vector in U343 cells. Increased ratios of versican 3′UTR bound more endogenous miR199a* and thus freeing the translation of luciferase protein, resulting in higher levels of luciferase activities (f).
We thank Dr. Jennifer Ma at the Core Facilities of Sunnybrook Research Institute for her assistance in real-time PCR experiments. We thank Dr. Siu-Pok Yee and Dr. Sara Gatchell for their assistance in the generation of transgenic mice.
Author Contributions
Conceived and designed the experiments: BBY. Performed the experiments: DYL TS ZJ WD ZD BBY. Analyzed the data: DYL BBY. Wrote the paper: BBY.
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- Brooks College, San Diego Mesa College, Rancho Cucamonga High School
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Home; Tribes; Members; Reviews; Listings. already a member? log in: remember me, forget your password? Find your community on tribe. Example
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text:
Simona Bovha obtained a doctorate degree in International Trade and Finance at Brandeis University, United States.
She has worked as economist at Harvard University; John F. Kennedy School of Government, and the Financial Management Institute of Eastern and Southern Africa (MEFMI), providing macroeconomic policy training for officials from developing countries.
She was also an assistant professor at the Faculty of Economics, University of Ljubljana, where she taught International Trade and International Business.
In 2005 she joined the European Investment Bank, Development Economics Advisory Service, where she has been performing an evaluation of macroeconomic and financial system soundness of countries outside the EU.
She is currently an Economic Adviser at the Bureau of European Policy Analysis (BEPA) of the European Commission.


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Star Tracks: Monday, May 16, 2016 42 years, 2,191 covers and 55,436 stories from PEOPLE magazine's history for you to enjoy
Latest News!
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- April 17, 1989
- Vol. 31
- No. 15
Jon Bon Jovi Gives His Home to Non-Jovi Judy Frappier
As front man for the rock-and-roll band that bears his name, singer Jon Bon Jovi is many things: sex symbol, lust object, teen dream, hunk o' man. Last week he took on another role, that of surreal estate agent, In one of those only-in-America, only-in-the-age-of-music-videos promotions, Judy Frappier, 26, of Levittown, Pa., won Jon Bon Jovi's boyhood home—a four-bedroom in Sayerville, N.J., worth $200,000-plus—in an MTV contest. The deal also included $10,000 for property taxes, a Kawasaki motorcycle and a personal tour of the premises by their most celebrated former occupant.
Despite having moved out seven years ago, Bon Jovi—who changed his name from Bongiovi for marquee purposes—never faltered when it came to room identification. "This is the kitchen," he said confidently. "And this the burner where every Sunday my father would make spaghetti sauce."
"It's the only burner I'll ever make spaghetti sauce on," Judy promised. After a visit to the family room, still filled with memorabilia, Jon, 27, wandered out to the pool and, growing misty, recalled the joys of youth, "We used to sneak girls in through that cellar window," he said. "Jon, don't tell me that," said Judy's husband, Jay, a 28-year-old systems analyst for New Jersey Bell. "I have three daughters [Michelle, 3, Jaymie, 2, and Courtney, 6 months]."
"They're too young yet," said Jon, reassuringly. "Maybe next year." Jay, an avid golfer, found further comfort in the fact that there is a top-rated course within easy driving range. "I should have time to get in a game or two after work," said Jay, with a faraway look in his eyes. The giveaway was made possible by the fact that the Bongiovis—father John, mom Carol, brothers Anthony, 22, and Matthew, 14—were about to move to a new million-dollar home that Jon bought for them not far from his Rumson, N.J., mansion. MTV heard about the impending relocation and cooked up the contest to promote its channel and Bon Jovi's current album, New Jersey. But if the Bongiovis are losing a house, they are also gaining a place to stop for tea and cookies when they're in the old neighborhood. "I told Mrs. Bongiovi that she was always welcome," says Judy. Adds Jay: "Jon should bring the band with him for pool parties."
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LeT Suspect Arrested in Haryana, Sent to Police Custody
By IANS - NEW DELHI
Published: 17th December 2013 11:16 PM
Last Updated: 17th December 2013 11:16 PM
A suspected Lashkar-e-Taiba (LeT) operative, arrested from Haryana, was Tuesday sent to five days in police custody by a Delhi court.
Hafeez Rashid, 30, was arrested from Nuh district in Haryana Monday night, police said.
His name surfaced after police interrogated Mohammad Shahid, another LeT suspect arrested by a special cell of Delhi Police Dec 9 from Choti Mewli village in Nuh district.
An earlier report said he was arrested Dec 10 but police later corrected the date.
Rashid was presented before the court of Additional Sessions Judge Dharmesh Sharma who allowed Delhi Police to question the LeT suspect till Dec 21.
A police officer said Rashid was providing logistics to Shahid to prepare for a terror attack in north India.
"Rashid introduced Shahid to Pakistan-based LeT commander Javed Baluchi early this year. They travelled to Pakistan to meet Baluchi," a police officer said.
Both Rashid and Shahid were imams of a mosque in Choti Mewli, police said.
Shahid told police that he had joined the LeT about six-seven months ago.
Police said the LeT module was at an advanced stage of planning a terrorist strike in Delhi, Haryana and Rajasthan.
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Holiday Inn Laramie places you within a mile (2 km) of University of Wyoming Art Museum and War Memorial Stadium. This 100-room, 3-star hotel has a restaurant along with conveniences like an indoor pool and free in-room WiFi. Arena Auditorium and University of Wyoming are also close to this Laramie hotel.
You can enjoy a drink at the bar/lounge or visit the onsite restaurant for a bite to eat. Satisfy your hunger from the comfort of your room with room service. For your convenience, a cooked-to-order breakfast is available for a fee.
All 100 air-conditioned rooms provide microwaves, refrigerators, and coffee makers. Guests can expect to find free WiFi and wired Internet, plus flat-screen TVs with cable channels and pay movies. Beds are dressed in pillow menus and bathrooms offer hair dryers and free toiletries. Other amenities include free local calls, safes, and ironing boards.
Property features
At Holiday Inn Laramie, guests enjoy an indoor pool, a fitness center, and a spa tub. If you decide to drive, there's free parking. Multilingual staff at the front desk are standing by 24/7 to help with dry cleaning/laundry, luggage storage, and securing valuables. Additional amenities include free WiFi in public areas, conference space, and a business center.
- Free WiFi and wired Internet in rooms and public areas
- Free parking
- Onsite dining
- Indoor pool
Holiday Inn Laramie204 S 30th St Laramie WY 82070
Hotel Amenities
Hotel Amenities
In addition to an indoor pool, Holiday Inn Laramie provides a spa tub and a fitness center. The hotel offers a restaurant. A bar/lounge is on site where guests can unwind with a drink. High-speed wired and wireless Internet access is complimentary.
This 3-star property offers access to a business center and a meeting room. This Laramie hotel also offers multilingual staff, gift shops/newsstands, and a picnic area. Onsite self parking is complimentary.
Holiday Inn Laramie is a smoke-free property.
- Number of floors - 4
- Bar/lounge
- Breakfast available (surcharge)
- Business center
- Free newspapers in lobby
- Elevator/lift
- Conference space
- Dry cleaning/laundry service
- Fitness facilities
- Front desk (limited hours)
- Gift shops or newsstand
- Free WiFi
- Free wired high-speed Internet
- Shopping on site
- 24-hour front desk
- Laundry facilities
- One meeting room
- Multilingual staff
- Total number of rooms - 100
- Picnic area
- Luggage storage
- Restaurant
- Safe-deposit box at front desk
- Conference space size (feet) - 1900
- Free self parking
- Smoke-free property
- Spa tub
- Indoor pool
- Use of nearby fitness center (discount)
Family Friendly Amenities
- Family Hotel
- Indoor pool
- Free Wi-Fi
- Laundry facilities
- Refrigerator
- Microwave
Available in all rooms: Free WiFi , Free wired high-speed Internet
Available in some public areas: Free WiFi , Free wired high-speed Internet
Free self parking
Room Amenities
- Premium TV channels
- Air conditioning
- Phone
- Free toiletries
- Shower/tub combination
- Cable TV service
- Flat-panel TV
- Desk
- Coffee/tea maker
- Free local calls
- Hair dryer
- Daily housekeeping
- Iron/ironing board
- Free WiFi
- Free wired high-speed Internet
- Microwave
- In-room safe
- Pay movies
- Private bathroom
- Refrigerator
- Room service (limited hours)
- Pillow menu
- Rollaway/extra beds (free)
Where to Eat
Cooked-to-order breakfasts are available for a surcharge.
Holiday Inn Laramie has a restaurant on site.
Room service (during limited hours) is available.
Nearby Things to Do
An indoor pool and a spa tub are on site. Other recreational amenities include a fitness center.
To make special requests for accessibility needs, please note them at check-out when you book your room.
- Accessible bathroom
- Roll-in shower
- In-room accessibility
Hotel Policies
Minimum check-in age is 21
Check-in time ends at 3 AM
Check-in time starts at 3 PM
Check-out time is noon
Payment types
- Pets allowed for an extra charge of USD 30.00 per pet, per stay
You need to know
Extra-person charges may apply and vary depending on hotel policy.
Government-issued photo identification and a credit card or cash deposit are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
No cribs (infant beds) available
Optional extras
- Fee for cooked-to-order breakfast: USD 6 for adults and USD 0 for children (approximately)
- Pet fee: USD 30.00 per pet, per stay
Hotel Name
- Holiday Inn Hotel LARAMIE
- Holiday Inn LARAMIE
- LARAMIE Holiday Inn
- Holiday Inn Laramie Hotel Laramie
We should mention
Up to 2 children 12 years old and younger stay free when occupying the parent or guardian's room, using existing bedding.


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Signature Bank of Arkansas VP Carol Waddle Wins Big on 'Wheel of Fortune'
Carol Waddle, a senior vice president for Signature Bank of Arkansas of Fayetteville, won big in an episode of "Wheel of Fortune" that aired last week.
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- At Conference, Tyson Foods Touts Stable Earnings, Sales Growth
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Most Read
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Devon Alexander to return on November 30 in St Louis?
By Ryan Burton
Golden Boy and Showtime are close to solidifying their fourth quarter schedule. BoxingScene.com has been advised by sources with knowledge of the situation that Devon Alexander will likely return to the ring on November 30th in his hometown of St. Louis, MO. at the Scottrade Center. The fight will be televised by Showtime.
BoxingScene.com had previously reported that a fight between Alexander and Luis Collazo was possible for December 7th at the Barclays Center. While that fight is still on the table, it is unlikely.
Alexander hasn't fought at home since his dominant points victory over Marcos Maidana in February 2012. Undefeated welterweight Shawn Porter is the name being mentioned as the frontrunner to get the fight, according to sources.
Send questions or comments to [email protected] . Follow Ryan on Twitter @ringsidewriter
[QUOTE=kardsufur;13813495]Thurman would do to alexander what matthysse did to pederson[/QUOTE] Hell no Alexander would expose Thurman.Comment by Phatfarm1980 on 10-06-2013
Devon must be devastated he won't be getting Amir khan type money lolComment by HBOFanboy on 10-06-2013
[QUOTE=paulf;13814483]In St Louis? Just kick Boxrec a couple hundred bucks to add a W for the date against TBA so we can all move on.[/QUOTE] Where else you want it? Devon can draw 5,000 in St.Louis that's a lot now…Comment by Ray* on 10-06-2013
Soo glad Khan ducked this fight since he didn't deserve it in the first place.. LolComment by Big Lou on 10-06-2013
Since Khan ducked, Make it happen. Good stay busy fight for Devon, Shawn's tough.Post a Comment - View More User Comments (29)


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I'm just a normal, simple, nice guy, that just likes to enjoy life. I'm a kind, very considerate, polite, respectful, open, and happy person. I like to talk and laugh about everyday things, watch movies, cook, have a glass of wine, and enjoy being together. I'm friendly, relaxed and comfortable with myself. I'm confident and honest, and I like to smile at people and give them positive feedback, even a stranger on the street, beach or boardwalk. You'll find me very easy to get along with, comfortable to be with, and wanting the people around me to be comfortable with me. I like meeting and talking to new people and spending quality time with the special woman in my life.
I love and appreciate nature and all the beauty on our planet: wildlife, mountains, forest, rivers, waterfalls, the coastline, birds and people. It's so much nicer to enjoy it with someone you really care for.
Having grown up in Florida, very close to the Gulf, I love being at the beach. I love being in the water, snorkeling, swimming, floating, or just walking along the shoreline. Being there together for the sunset is always special, even more so with a special person.
I love traveling to new and interesting places, exploring all the beautiful places in this country and meeting the people along the way. I also enjoy taking day trips to new places and seeing and doing new things, or just getting in the car and being spontaneous.
A little live theater, a little art, museums, and some festivals add to the variety. A baseball game, football game or a round of golf once in a while is always fun.
I have also done a bit of wildlife, nature, and landscape photography. I have a photo website with some of my best works of art on it. Ask if you'd like to see it. I really enjoyed the time I spent taking those photos.
I like a laid back lady that can appreciate a nice guy. You eat healthy, but not necessarily all the time. A nice person to be with, can laugh at herself, me, and life with it's quirky twists. You are also kind, considerate, have a big heart, and you have a lot of love to give with the right person.
I love a woman that is open, honest, at least somewhat free-spirited, has a good sense of humor and a quick wit, likes having conversations about the things that interest us, and the world around us. You would be understanding and courteous to others, spontaneous, and sensuous. You like physical contact, touching, back rubs, hugs and kisses, and are openly intimate in a comfortable way once you get to know your man better, in a spiritual, emotional, and physical way.
I like to take the time for our intimacy to develop, and you would want to do the same, because jumping into an intimate relationship too soon may not be the best way to develop a strong lasting relationship. You tend to be strong when needed, but you allow your naturally soft way of being to come through when we're together. You are confident in yourself, mostly because you have the above qualities, but also because you know you are a good person.
Ideal Date:
A cup of coffee or a glass of wine, and nice conversation. Then it would be nice to meet for lunch at the beach, and maybe even watch the sunset. No rushing. Just let it be what it is and see where it goes.
An affectionate, loving partner to share my life with.
Traveling, both locally and around the country.
Access to the internet.
Learning new things.


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Mandatory Information Sessions
Preregistration is required and seating is limited. This Mandatory Nurse Aide Information Session is part of the Registration Requirements even if you were previously a CNA. You will not be able to register until all requirements are met.
To sign up for a session, please email your name and CPCC student ID number (if you have one) along with the date, time, and location of the session you would like to attend to firstname.lastname@example.com. Be sure to put the words Information Session Registration in the subject line. Please sign the attendance roster when you arrive. If all requirements are met, you will receive notification of permission to register via your CPCC email address a few days later. Unattended children are not allowed on campus at any time and you may not bring children to the session with you. Please arrange for daycare prior to coming to the information session.
The sessions are held at Central Campus (1335 Elizabeth Ave. - Belk Building, Room 2107 - Charlotte, NC - 28204) and Levine Campus (2800 Campus Ridge Rd. - Levine I, Room 1206 - Matthews, NC 28105). You may choose which location to attend:
September 2nd
Central 10am-12pm - FULL
October 7th
Central 10am-12pm - FULL
November 4th
Central 10am-12pm - FULL


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A personal documentary revealing what it means to be "Bosnian" through the experience of a Bosnian-American in Portland and Sarajevo.
67 backers pledged $5,963 to help bring this project to life.
BIG NEWS: our film has been completed!!
Dearest Donors,
It is with extreme pleasure that LLAMAMAMA Productions (Ivana Horvat and Adrian Hopffgarten) announce the completion of Finding Bosnia! Four years ago, your donation directly funded the production... Read more
Successfully raised $5,963 with 67 backers
Project launched


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Get an overview of this hotelHotel in San Antonio with free breakfast and free airport shuttle
Situated near the airport, this hotel is 1 mi (1.5 km) from North Star Mall and within 3 mi (5 km) of Quarry Golf Club and Alamo Quarry Market. Marion Koogler McNay Art Museum and San Antonio Zoo and Aquarium are also within 6 mi (10 km).
Hotel Features
This smoke-free hotel features an outdoor pool, a fitness center, and a business center. Free buffet breakfast is provided, as well as free WiFi in public areas, free self parking, and a free airport shuttle. Other amenities include conference rooms, dry cleaning, and laundry facilities.
Room Amenities
All 150 rooms offer free WiFi, coffee makers, and LCD TVs with satellite channels. Free newspapers, free local calls, and hair dryers are among the other amenities that guests will find.Information missing or incorrect? Tell us!Opens in a new window
Holiday Inn Express San Antonio-Airport91 Ne Loop 410 San Antonio TX 78216
Hotel Amenities
Hotel Amenities
Holiday Inn Express San Antonio-Airport features an outdoor pool and a fitness center. Public areas are equipped with complimentary high-speed wireless Internet access. This hotel offers access to a business center and meeting rooms. Guests can enjoy a complimentary breakfast. Event facilities measuring 4136 square feet (372 square meters) include conference space. A roundtrip airport shuttle is complimentary to guests (available on request). This business-friendly hotel also offers multilingual staff, laundry facilities, and dry cleaning/laundry services. Complimentary self parking is available on site.
Holiday Inn Express San Antonio-Airport is a smoke-free property.
- Number of buildings/towers - 1
- Free WiFi
- Conference space size (feet) - 4136
- Conference space size (meters) - 372
- Number of meeting rooms - 3
- Free airport transportation
- Business center
- Free breakfast
- Conference space
- Dry cleaning/laundry service
- Fitness facilities
- Front desk (limited hours)
- Laundry facilities
- Meeting rooms
- Multilingual staff
- Number of floors - 10
- Total number of rooms - 150
- Safe-deposit box at front desk
- Free self parking
- Free long-term parking
- Smoke-free property
- Outdoor pool
- Use of nearby fitness center (discount)
Family Friendly Amenities
- Family Hotel
- Outdoor pool
- Free Wi-Fi
- Laundry facilities
- Cribs/infant beds (complimentary)
Available in all rooms: Free WiFi
Available in some public areas: Free WiFi
Free long-term parking, Free self parking
Room Amenities
- Free cribs/infant beds
- Connecting/adjoining rooms available
- Free WiFi
- Air conditioning
- Free newspaper
- Satellite TV service
- Coffee/tea maker
- Desk
- Free local calls
- Hair dryer
- Daily housekeeping
- Iron/ironing board
- Private bathroom
- Phone
Where to Eat
Guests are offered a complimentary buffet breakfast.
Nearby Things to Do
Recreational amenities at the hotel include an outdoor pool and a fitness center.
To make special requests for accessibility needs, please note them at check-out when you book your room.
- Accessible bathroom
- Roll-in shower
- In-room accessibility
Hotel Policies
Check-in time ends at 6 AM
Check-in time starts at 3 PM
Special check-in instructions:
Check-out time is 11 AM
Payment types
- Pets allowed
- Up to 40 lb
- Restrictions apply
- For more information contact the property at the number on the booking confirmation
You need to know
Extra-person charges may apply and vary depending on hotel policy.
Government-issued photo identification and a credit card or cash deposit are required at check-in for incidental charges.
Special requests are subject to availability upon check-in and may incur additional charges. Special requests cannot be guaranteed.
Hotel Name
- Holiday Inn Express Antonio-Airport
- Holiday Inn Express Antonio-Airport Hotel
- Holiday Inn Express Antonio-Airport Hotel San
- Holiday Inn Express San Antonio-Airport
- San Antonio-Airport Holiday Inn Express
- Holiday Inn Express San Antonio-Airport Hotel San Antonio
- Holiday Inn Express San Antonio-Airport Hotel
- Holiday Inn Express San Antonio-Airport San Antonio
We should mention
This property offers transfers from the airport. Guests must contact the property with arrival details before travel, using the contact information on the booking confirmation. The property has connecting/adjoining rooms, which are subject to availability and can be requested by contacting the property using the number on the booking confirmation. Guests can arrange to bring pets by contacting the property directly, using the contact information on the booking confirmation.


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Law Offices Of Desmond Richard Griswold - Fairview Park, OH
Mesothelioma is a rare form of cancer that is usually caused by exposure to asbestos. Most people, in Fairview Park, who have developed mesothelioma have worked within industries which involved inhaling asbestos and construction particles, or were otherwise exposed to asbestos dust such as workers who helped in the aftermath of the collapse of the World Trade Center on September 11th, 2001.
Mesothelioma usually develops years after actual exposure. Victims are commonly diagnosed with mesothelioma when they show damage to the peritoneal lining of the lungs or abdomen. Asbestos was used in the United States from early 1930 to mid 1970. More than 733,000 buildings in the United States, including a good number of those within the borders of Ohio, are said to still contain asbestos.
Mesothelioma litigation is widespread throughout the Unites States due to large financial settlements, as well as the obvious liability and personal injury involved. Court actions are common in which victims seek compensation for his or her expenses. Lawyers within Fairview Park, Ohio fight specifically for the rights of those effected from this disease.
Keurig Introduces Recyclable K-Cups, But Environmentalists Ain't Buying It
(Long Island, NY) People love coffee. They also love convenience. That's why Keurig Green Mountain's eponymous coffee machine has taken the world by storm…instead of being forced to grind beans, dump them into a paper filter, and run hot water through them in order to get a steaming cup of joe, those looking for a quick caffeine fix can simply..


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Business Law and Management Programme
Graduates of the "Business Law & Management" sector will be destined mainly to business lawyer business or tax attorney, lawyer or business tax in France and abroad. They will benefit from network partners and graduates developed by EDHEC Faculty and Royalty Free, which includes prestigious law firms and legal departments of business.
Assets that make the difference:
- The acquisition of two skills added value increasingly demanded by employers and which responds to changes in the market for "legal, tax and business lawyers."
- A strong program dedicated to the fundamentals of Business Law and Management, coupled with a more professional learning.
- Obtaining a double degree Faculty dpi / EDHEC allowing students to seduce legal departments and business law firms but also to attempt the entrance examination at the École du Barreau.
- Courses in business law and management taught in English.
- Original teaching methods with interactive lessons, interventions practitioners promoting the sharing of experiences, role plays and case studies.
The training will also be irrigated by the original work of the research centers of two institutions: LegalEdhec and the Centre for Research on the relationship between risk and Law (C3RD).
Professional immersion students for operational
The "Business Law and Management" course combines his teaching learning professionalizing aimed at giving students the knowledge and skills required in their future function and make fully operational the output path. This professional immersion means:
- "The law in action" and "One Firm, One File" professionalization these devices allow to expose students to the realities of the field through the study and analysis of real cases offered by legal departments and law firms to lawyers. This work will result in the defense proposed by the students in front of a jury composed of directors and legal counsel involved, and teachers solutions.
- A mandatory 12-month experience in business including 6 months abroad (acquired during the three years of the course during training or gap year).
Obtaining a dual degree in business law and management
- End of first year(L3)
> Obtaining a law degree
> Obtaining a certificate EDHEC "Business Law and Management"
> Possibility for students who wish to pass the "AST2" contest (Master cycle) EDHEC (being exempt written according to their results in L3 events)
- End of second year (M1)
> 1 Obtaining Master in Business Law
> Obtaining an Advanced Certificate EDHEC "Business Law and Management"
- End of third year (M2)
> Obtaining the Master 2 in Business Law Practice
> Obtaining LLM Law and Tax Management
> Obtaining EDHEC Grande Ecole diploma for students who joined the Grande Ecole program through prépas contest or AST2 (Master cycle) EDHEC.
Students can not validate their degree in fine condition as proven experience of minimum 12 months in business (including 6 months abroad) in the last three years of the course or during a gap year between the second and third year.
1st year
Licence 3 / Bachelor
The first year is devoted to the fundamentals of business law, economics and management. A transition module for a period of six weeks adapted the original curriculum allows students to acquire the necessary methodology to understand the course and new disciplines.
1st Semester
- Transition Module (for students Jurists)
- English (compulsory LV1) 45h
- LV2 45h
- General culture 30h
- Methodology (common with students EDEHC) 20h
- General Accounting 36h
- Institutions and markets 18h
- Macroeconomics 36h
- Decisions, analysis and data 18h
- English 18h
- LV2 18h
- Career center (professional project) 8h
- 36h civil law
- 36h commercial law
- Droti 18h of goods
- Total 1st semester student 382H
2nd Semester
- Microeconomics 36h
- Consumption and behavior 18h
- Tax 36h
- Behaviors and organizations 36h
- Introduction to Anglo-Saxon law and 18h Asian countries
- English 18h
- LV2 18h
- Career Center 8h
- Civil Law (security) 36h
- Law 36h companies
- Recovery strategy 18h receivables
- Introduction to european law 18h
- Total H2 296h
- Total student 678h first year
2nd year
Master 1
The second year allows students to address specific areas such as competition law, criminal law, business, banking, strategy, marketing, corporate governance, ethics, ...
50% courses are delivered in English.
1st Semester
- 36h Finance
- 36h Marketing
- Strategy 36h
- Business game 24
- LV2 18h
- Law firms in difficulties 36h
- French law 36h competition
- International private law 18h
- Social law and HRM 36h
- 20h contracts and procurement
- Total 1st semester student 296h
2nd Semester
- Legal environment and business decision making 36h
- Corporate governance 36h
- Business development and innovation 18h
- Business game 24
- GMAT 6h
- Informations contracts 36h
- Right effects 18h trade
- Law 18h funding
- Criminal Law 18h business
- International criminal law 36h
- Technical tax 36h
- Total 2nd semester student 282h
- Total student 578h 2nd year
3rd year
Master 2 / LLM
The third year is dedicated to building knowledge in business law (international contracts, M & A's, IP law, ...) and business ethics, and the development of skills in leadership and legal strategy.
80% of courses are taught in English.
1st Semester
- Legal strategy and legal performance 30h
- Strategies in Litigation, arbitration and ADR 30h
Electives (choose 1 of 6)
- Contract engineering 30h
- 30h Structural Engineering
- 30h tax planning
- Advanced competition law 30h
- Drafting and enforcing international contracts 30h
- Legal intelligence and lobbying 30h
- Risk management and insurance 30h
- 30h financial engineering
- Total 1st semester student 210h
2nd Semester
- Intellectual property law in globalized and digital economies 30h
- International tax law 30h
- Corporate law and capital markets law 30h
- Mergers and acquisitions practice of 15h
- Compliance and risk management 15h
- Economic analysis of law 15h
- Legal practice and research 15h
- Law 20h Action
- Business ethics seminar 30h
- Leadership seminar 30h
- Total 2nd semester student 230h
- Total third year student 440H
Cet établissement propose des programmes en :
- Anglais
- Français
Mis à jour le January 11, 2016


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GMAC Talks Big Wave Tour at Nazare and Teases Biggest News of His Career
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The inside track on the BWT paddle event and a tease that Nov 19 will see announcement of the biggest news of his professional career.
The inside track on the BWT paddle event and a tease that Nov 19 will...
Some of Steph's best surfing in front of the lens of Jon Frank
Matt Cardinal on Friday, September 30
Matt Cardinal on 09/30/16
"Our destination is never a place but a new way of seeing life."
An hour and a bit up the road from the planned Wavegarden
Tyler still leader but battle for world champion headed to France
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Paris designers share their inspirations as the spring collections get under way.
In addition to some tony basics, a llittle Eighties-inspired bad taste made for an audacious collection of racy party clothes.
Amid the luxury malaise, Barneys' ceo says the company is profitable and performing well.
Brands and retailers move up the ranking with strategic growth tactics.
E-commerce sales will surpass 10% of total online sales this season.
Andy Mantis, executive vice president of NPD Group's Checkout Tracking, explains.
The city has not lost its sizzle, once again led by Prada and Gucci.
PMX Agency's Luxury Brands Online report measures social media interactions, among other metrics.
The nation's largest retailers have been hit by a confluence of factors, and no upturn is in sight.
WalletHub expects outstanding balances to surpass the $1 trillion mark this year.
The company's interactive mirror, which debuted at the Polo Ralph Lauren Fifth Avenue flagship in New York last year, has begun a rollout for German retailer Gerry Weber's Taifun brand.
Revenue associated with social media marketing soars as Revolve unlocks the key to influencer engagement.
Ryan Patel, former vice president of global development at Pinkberry, explains.
The ABX Advertising Benchmark Index uses a consumer panel and includes 14 variables.
Michele Dupré, group vice president of Verizon Enterprise Solutions' retail and hospitality practice, explains.


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We may have a pass rush today!!
We may have a pass rush today!!
I stopped fighting my inner demons. We're on the same side now.
oh, you're so bad!!
Eh, he has not been the same this year but neither as anyone on the DL. Even JPP has taken a step back.
JPP was getting doubled last year too. When he is one on one this year he is not beating his man. He has taken a step back this year.