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"She spent 8 years, trying to convince me to get married" – A Story of Unconditional Love to be Featured at the Film Festival
A story of unconditional friendship is one of the many touching stories featured in a documentary about love and relationships. Matchmaker Hellen Chen's real-life stories of helping couples will get showcased again at the Oakland International Film Festival.
National (PRWEB) March 29, 2013
Cecilia H is a successful executive who is both competent and independent. She is well-loved by co-workers and well-liked by her customers. She thought she has everything that she could ever hope for – until she met a nosy matchmaker.
The "nosy matchmaker" is none other than matchmaker extraordinaire Hellen Chen, who has helped more than 100 singles, especially those who had been skeptical about marriage, to nod their heads to say "I do."
"I felt there was nothing wrong about being single. It is not a crime. Besides, I have known too many bad stories of marriage from the people around me. Why would I want to get into it?" Said a skeptical Cecilia, who had thought being single for the rest of her life would be an option she was willing to take.
Chen, with a pure intention of simply wanting Cecilia to experience love which she never have had before and not be closing the doors completely, started to talk as a friend to Cecilia about marriage — only to be met with resistance as strong as armor plates.
What ensued was a lengthy 8-year persuasion on the subject, with rejection at every turn.
Chen's story of helping real-life couples who had skepticism about marriage and couples who are on the verge of separation caught the attention of an award-winning director Wuna Wu.
With a camera in hand – reality-TV style, Wu followed Chen to capture the maverick matchmaker in action. The result is a full-length award-winning documentary — the first-ever documentary about love — played in international film festivals garnering critical reviews and becoming a huge audience favorite.
The story of Cecilia H was one of the many stories featured in this documentary aptly titled "Let's Fall in Love."
The documentary was recently played in Beijing and many audiences wrote personal notes to the matchmaker of how the story affected them.
The next showing will be at the Oakland International Film Festival in California on April 5th.
So did Cecilia eventually tie the knot? The answer is yes, and the groom's story is just as unique, featured as well in the documentary.
This year, Cecilia celebrated her 5th wedding anniversary with her husband, and wrote her
matchmaker, "Thanks. Without you, I would never have known the joy of marriage. "
Never expecting that her work of helping men and women in affairs of the heart would be taken to the big screen, Chen said, "I simply like to see men and women experience the type of relationship they have always dreamed about. Creating a lasting meaningful relationship is never a walk in the park but it is well-worth it – it is one of the most beautiful human endeavors."
Chen is also a relationship expert and bestselling author, having written 20 books on self-improvement and relationship topics. Her recent publication "The Matchmaker of the Century" was launched on Barnes and Noble.
Subsequently striking a chord with readers, it achieved #1 bestselling status in 6 categories: marriage, self-help, relationships, love and romance, self-improvement and parenting and family.
Chen announced that she would be personally helping 30 more couples to get married this year. She called this the "Love You Forever" movement.
For more information, visit http://MatchmakerOfTheCentury.com
For the original version on PRWeb visit: http://www.prweb.com/releases/prweb2013/3/prweb10583695.htm


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Contemporary-style homes for sale on LI
Here is this week's look at Long Island homes with a notable feature. If you want to sell or rent a unique, historic or famous home, or one with a special feature, call Lisa Doll Bruno. Phone: (631) 843-2793, fax: (631) 843-5459, email: firstname.lastname@example.com.
Suffolk Class C final: Babylon vs. Mattituck LI notable deaths Meet 137 of Long Island's salutatorians Way to Go kids Tommy Bahama, Aveda, more new stores on LI See LI landmarks by air LI veterans then and now Nassau Class B final: Manhasset vs. Lynbrook Suffolk girls track county championships Suffolk Class B final: Hauppauge vs. Eastport-South Manor Nassau Class A final: Massapequa vs. Syosset Suffolk Class C final: Mt. Sinai vs. Bayport-Blue Point

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Neuronal regeneration in the adult mammalian central nervous system (CNS) is severely compromised due to the presence of extrinsic inhibitory signals and a reduced intrinsic regenerative capacity. In contrast, the CNS of adult Lymnaea stagnalis (L. stagnalis), a freshwater pond snail, is capable of spontaneous regeneration following neuronal injury. Thus, L. stagnalis has served as an animal model to study the cellular mechanisms underlying neuronal regeneration. However, the usage of this model has been limited due to insufficient molecular tools. We have recently conducted a partial neuronal transcriptome sequencing project and reported over 10,000 EST sequences which allowed us to develop and perform a large-scale high throughput microarray analysis.
To identify genes that are involved in the robust regenerative capacity observed in L. stagnalis, we designed the first gene chip covering ~15, 000 L. stagnalis CNS EST sequences. We conducted microarray analysis to compare the gene expression profiles of sham-operated (control) and crush-operated (regenerative model) central ganglia of adult L. stagnalis. The expression levels of 348 genes were found to be significantly altered (p < 0.05) following nerve injury. From this pool, 67 sequences showed a greater than 2-fold change: 42 of which were up-regulated and 25 down-regulated. Our qPCR analysis confirmed that CCAAT enhancer binding protein (C/EBP) was up-regulated following nerve injury in a time-dependent manner. In order to test the role of C/EBP in regeneration, C/EBP siRNA was applied following axotomy of cultured Lymnaea PeA neurons. Knockdown of C/EBP following axotomy prevented extension of the distal, proximal and intact neurites. In vivo knockdown of C/EBP postponed recovery of locomotory activity following nerve crush. Taken together, our data suggest both somatic and local effects of C/EBP are involved in neuronal regeneration.
This is the first high-throughput microarray study in L. stagnalis, a model of axonal regeneration following CNS injury. We reported that 348 genes were regulated following central nerve injury in adult L. stagnalis and provided the first evidence for the involvement of local C/EBP in neuronal regeneration. Our study demonstrates the usefulness of the large-scale gene profiling approach in this invertebrate model to study the molecular mechanisms underlying the intrinsic regenerative capacity of adult CNS neurons.
Injuries of the central nervous system (CNS) can lead to devastating and irreversible loss of function because adult mammalian CNS neurons have a limited regenerative capacity [1-5]. This is partially due to an age dependent reduction in the intrinsic regenerative potential of CNS neurons [6-9]. In contrast, some adult invertebrate neurons are capable of spontaneous regeneration following injury [10,11]. Lymnaea stagnalis (L. stagnalis) has served as a critical model system to study nerve regeneration because of its ability to spontaneously regenerate and restore function in the adult [12-16]. Specifically, identified adult Lymnaea neurons can be isolated individually and maintained in culture, allowing for axonal outgrowth and functional synapse formation between appropriate synaptic partners [17-19]. L. stagnalis neural genes include homologues of well known transcription factors, genes involved in neurotransmission, axon guidance and signalling pathways [20]. Taking advantage of this model system, a number of groups demonstrated the roles of neurotrophins [14,21], and neurotransmitters [12,22] on neurite outgrowth and regeneration. Moreover, retinoic acid (RA) isomers have been identified in adult Lymnaea neurons, and 9-cis-RA has a novel role in chemoattraction and growth cone guidance [23-25]. Since many molecular mechanisms are conserved across species, the identification of molecules involved in neuronal regeneration in L. stagnalis will aid our understanding of factors which may promote regeneration in the mammalian CNS.
One main limitation of using L. stagnalis in genetic functional studies of neuronal regeneration has been a lack of large-scale screening tools, such as microarray analysis [26,27]. To circumvent this limitation, we have recently sequenced more than 10, 000 ESTs (expression sequence tags) from the CNS transcriptome of L. stagnalis [20] and established the largest neuronal EST database in Lymnaea (http://www.lymnaea.org webcite). In combination with the availability of the microarray technology [28,29], these gene sequences provide us with the opportunity to perform a high-throughput screening for altered gene expression levels following nerve injury in L. stagnalis. In this study, we have designed the first microarray chip covering 10, 333 known L. stagnalis genes to profile the gene expression patterns following CNS injury. We identified 348 genes that were differentially regulated following CNS crush. Using real-time PCR (qPCR) analysis we confirmed that the gene expression level of CCAAT enhancer binding protein (C/EBP), a transcription factor, is up-regulated following nerve injury. Knockdown of C/EBP following axotomy of cultured PeA cells lead to retraction of the distal, proximal and intact neurites. Our data suggest that C/EBP plays a crucial role in neuronal regeneration of Lymnaea neurons via both somatic and local mechanisms.
CNS injury model of L. stagnalis
In order to profile the gene expression pattern following injury of the central nervous system, we modified an established [12,14] nerve injury model by crushing the right parietal (RPa) and right cerebral nerves (RCe) (Figure 1A). In control groups, sham operations were performed without nerve injury.
Figure 1. Experimental model and changes in gene expression following injury. (A) The dorsal surface of L. stagnalis CNS and CNS injury protocol: both right parietal (RPa) nerves and right cerebral nerves (RCe) were crushed with fine forceps. Inset: a schematic diagram of the ganglia and location of injury (black lines). (B) Heat map representing the hierarchical clustering of 67 genes showing significant differential expression (P < 0.05). The expression pattern of each gene in the sham and crashed groups is displayed as a horizontal strip. For each gene, the expression ratio of the crush-operated to the sham-operated experiments is represented by the green and red scale at the bottom of the figure. The genes are numbered on the right, and the experimental groups are labelled on the bottom. (C) Ratio of the regulated EST sequences 3 hrs following CNS injury in L. stagnalis. (C1) The signal intensity of 348 genes or ESTs was differentially regulated. Open pie (80.7%) represents genes/EST's changes < 2.0-fold in signal intensity but significant (P < 0.05); grey pie (19.3%): changes ≥ 2.0-fold (P < 0.05). Dark grey: up-regulated genes (12%), and light grey: down regulated genes (7%). (C2) Ratio of the 67 differentially regulated genes, 16 (23.8%) genes have orthologous with known functions related to development, survival or signal transduction, whereas 51 (76.2%) genes have no orthologue.
Identification of differentially expressed genes
To search for genes that may be involved in neuronal regeneration or degeneration, microarray analyses were carried out to compare the gene expression levels between the sham-operated and crush-operated CNS models (Figure 1A, inset). We used an unpaired t-test for comparison of the 15,000 array signals between two groups: sham- and crush-operated groups. This analysis resulted in the selection of 348 genes or ESTs (See Additional File 1) that were differentially regulated 3 hours following CNS injury (p < 0.05). Further screening for genes with a fold-change > 2 resulted in a final group of 67 differentially expressed genes. Figure 1B shows a heat map representing the hierarchical clustering of the 67 genes with significant differential expression levels between the control and injured groups. Overall, more genes were up-regulated (n = 42) than that of which were down-regulated (n = 25), suggesting that diverse regulatory pathways and biological processes are activated within the first 3 hours following injury (Figure 1C1). Among the 67 regulated genes, 16 have been identified with known functions related to development, survival or signal transduction; the functions of the majority of genes (51) have not yet been described (Figure 1C2).
Additional file 1. The raw microarray data from 4 independent sets of experiments. The original data obtained from microarray analysis of the ganglionic mRNAs obtained from 4 sham-operated and 4 crush-operated snails.. The highlighted line indicates C/EBP mRNA expression levels. Red: indicates the genes with 2 fold changes in expression level in crush-operated over sham-operated snails.
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Gene expression levels of C/EBP change in a time-dependent manner following injury
The microarray analysis indicated that the mRNA level of CCAAT enhancer binding protein, C/EBP, was significantly increased 3 hours following CNS injury, as compared to the sham control group. A protein sequence alignment of C/EBP orthologues from L. stagnalis, Aplysia, rat, and human revealed that C/EBP has a high homology in the DNA-binding domain, suggesting that C/EBP may have similar biological functions in these species (Figure 2). To confirm that C/EBP is indeed up-regulated following nerve injury at 3 hours, we performed conventional RT-PCR analysis. We found that the expression level of C/EBP mRNA significantly increased, compared to the control gene GAPDH, in the injured CNS group (2.35 ± 0.26 fold; n = 5, p < 0.05) over the sham groups (Figure 3A).
Figure 2. Protein sequence alignment of C/EBP. Amino acid alignment between CCAAT enhancer binding protein from L. stagnalis [GenBank accession#: BAD16556], A. kurodia [GenBank#: AAG61258], R. norvegicus [GenBank#: AAI29072] and H. sapian [GenBank#: EAW75629] C/EBP. The high degree of sequence similarity at the DNA-binding domain indicates that C/EBP has a conserved functional domain across species.
Figure 3. Time-dependent changes in C/EBP mRNA expression following nerve injury. Real-time PCR was performed with specific primers for C/EBP between sham-operated and crush-operated L. stagnalis CNS at 1 hr, 3 hrs, and 5 hrs post injury. (A) Relative mRNA levels of C/EBP vs GADPH increased significantly following CNS injury as compared to the sham-operated controls at 3 hrs (2.35 ± 0.26, n = 5) (t = -3.5, df = 8, pP < 0.05). (B) Standard curves of C/EBP and GAPDH from 6 independent samples including sham-operated and crush-operated L. stagnalis CNS at 1 hr, 3 hrs, and 5 hrs post injury. The PCR efficiency of the primers was estimated by the slope (m) = -1/log(efficiency). The expression GAPDH was unchanged following CNS injury, and therefore CAPDH was used as an internal control. (C) Representative Ct-Ct correlation plots between C/EBP and the control gene, GADPH, at three different time points following injury. C1: 1 hr; C2: 3 hrs; C3: 5 hrs. Relative expression ratio between C/EBP and GADPH was estimated as Yintercept = -log(ratio)/log(efficiency C/EBP). (D) Relative gene expression of C/EBP vs. GADPH normalized to corresponding control samples. C/EBP increased in a time-dependent manner. 1 hr: 7.36 ± 0.73 (n = 5); 3 hrs: 2.35 ± 0.26 (n = 5), and 5 hrs: 1.98 ± 0.75 (n = 5). * indicates significant differences: ANOVA: F(2,12) = 17.4, p < 0.05. All data are presented as mean ± S.E.M.
To study its temporal expression pattern, we further measured the gene level of C/EBP at 1 hour, 3 hours and 5 hours post CNS injury using qPCR analysis. The standard curves for C/EBP are shown in Figure 3B with a slope of -3.52, indicating the PCR efficiency for the primers are sufficient (Figure 3B). To visualize the gene expression level between the injured and sham groups, the threshold cycles (Ct) of C/EBP (the target gene) and GAPDH (the control gene) were plotted against each other, as described previously [30]. Figure 3C shows the representatives of Ct-Ct correlation plots between the C/EBP and GADPH gene pair at the three different time points (1 hr, 3 hrs, 5 hrs). The slopes of the plots were consistent in all experimental samples, indicating the equality in amplification efficiencies between the primers of the gene pair. The Y-intercepts (- log(R)/log(Eg)) of these plots were smaller in the injury group (crush) than in the controls (sham) at all the times tested, indicating the relative expression level of C/EBP mRNA increased following injury. Specifically, C/EBP mRNA was initially increased by 7.36 ± 0.73, (n = 5) at 1 hr after CNS injury, but appeared to reduce to 2.35 ± 0.26 (n = 5) by 3 hrs and 1.98 ± 0.75, (n = 5) by 5 hrs, albeit at levels that are still significantly greater than in sham-operated (control) animal (Figure 3D). These results suggest that elevation of C/EBP mRNA expression may be necessary for transcriptional induction of regeneration associated genes, and thus likely to be a direct target of injury induced signalling cascades that promote neuronal repair.
The role of C/EBP in axonal elongation following axotomy
The increased expression of C/EBP mRNA following nerve injury in vivo suggests that this protein may be an upstream gene regulator of either pro-regenerative genes or responsive genes for cell injury. To elucidate the functional significance of C/EBP mRNA following nerve injury, we examined the effect of C/EBP on axonal outgrowth and regeneration of PeA neurons in culture using a siRNA gene silencing approach as previously described [31-33]. Axotomy was performed on cultured PeA neurons after 24 hrs which had undergone adequate neurite outgrowth (Figure 4). Immediately following axotomy, cells were treated with either conditioned medium (CM), control siRNA, C/EBP siRNA#1 or C/EBP siRNA#2. The lengths of the proximal and distal ends of the axotomized neurite, as well as an intact neurite were measured at various times over a period of 36 hrs (Figure 4). The net changes of the neurites following treatments were compared, as previously described [33]. Figure 5A shows that the distal end axons did not show additional growth following treatments with either C/EBP siRNA#1 (10 hrs: -0.3 ± 3.0 μm; 24 hrs: -1.8 ± 2.9 μm; 36 hrs: -5.9 ± 2.8 μm. n = 14), or C/EBP siRNA#2 (10 hrs: 1.2 ± 0.7 μm; 24 hrs: 0.1 ± 1.3 μm; 36 hrs: -3 ± 2.2 μm. n = 14) treated cells, whereas that continuously elongated in control CM (10 hrs: 14.2 ± 2.4 μm; 24 hrs: 21.9 ± 3.1 μm; 36 hrs: 29.1 ± 3.9 μm. n = 14) or control siRNA (10 hrs: 17.6 ± 2.7 μm; 24 hrs: 21.2 ± 3.7 μm; 36 hrs: 24.4 ± 4 μm. n = 14) treated cells following axotomy. The net changes in the distal axons were significant between the C/EBP siRNAs and the control CM or control siRNA groups (p < 0.05, for all three time points). In contrast, the net increases in length of the proximal neurites over 36 hrs are 25.6 ± 4.3 μm (n = 14) in control CM, and 23.3 ± 4.3 μm (n = 14) in control siRNA treated cells, whereas that was significantly reduced in C/EBP siRNA #1 (10.12 ± 3.5 μm, n = 14) or C/EBP siRNA #2 (7.2 ± 4.8 μm, n = 14) treated cells, although no significant difference was found at the early hours (10 or 24 hrs) (Figure 5B). Similarly, the net growth of intact neurites was only significantly reduced in C/EBP siRNA #1 (47.4 ± 4 μm, n = 11) and C/EBP siRNA #2 (42 ± 7.1 μm, n = 11) treated cells as compared to control CM (83.9 ± 13.5 μm, n = 11) and control siRNA treated cells (79.5 ± 5.7 μm, n = 11) 36 hrs after treatment (Figure 5C). These findings suggest that neuronal C/EBP is involved in neurite outgrowth. Sufficient C/EBP levels are required for maintaining the integrity of the distal axon. Due to the application of siRNAs following axotomy, the differences found in the distal axons may be associated with local protein synthesis [33,34].
Figure 4. Knockdown of C/EBP reduced net growth of the distal, proximal, and intact neurites following axotomy. Representatives of transected axons in culture. Immediately following axotomy (t = 0), either CM, control siRNA, C/EBP siRNA #1, or C/EBP siRNA #2 was added into culture medium at a final concentration of 7 nM, immediately after neurite transection. The neurons were observed over additional 36 hours and images were taken at 10, 24 and 36 hours as indicated. Arrows denote the transection site.
Figure 5. Differential effects of C/EBP knockdown on the distal, proximal, and intact neurites following axotomy. The length of the distal, proximal, or intact neurite of injured cells was measured at three time points (10 hrs, 24 hrs, 36 hrs) after control or C/EBP siRNA treatment, which was given immediately after axotomy (t = 0). (A) The distal neurite. The net changes in the C/EBP siRNA #1 or #2 treated cells were significantly reduced as compared to that in the control CM or control siRNA treated cells (n = 14; ANOVA: at 10 hrs, F(3,52) = 14.4, p < 0.05; at 24 hrs, F(3,52) = 20.1, P < 0.05; at 36 hrs, F(3,52) = 29.7, p < 0.05). (B) The proximal neurite. A significant reduction in outgrowth was observed only at 36 hrs after axotomy in the C/EBP siRNA #1 or #2 treated cells as compared to control CM and control siRNA treated cells (n = 14; ANOVA: F(3,52) = 5.1, p < 0.05). (C) The intact neurite. A significant reduction in outgrowth was only observed at 36 hrs in the C/EBP siRNA #1 or #2 treated cells as compared to control CM and control siRNA treated cells (n = 11; ANOVA: F(3,40) = 6.5, p < 0.05). (D) Relative gene expression level of C/EBP vs GAPDH was significantly reduced in the C/EBP siRNA #1 (63.34 ± 8.8%) and #2 (56.25 ± 9.4%) (n = 5, ANOVA: F(2,12) = 5.7, p < 0.05) groups as compared to control siRNA group. All data are presented as mean ± S.E.M.. * indicates p < 0.05.
C/EBP siRNA treatment hinders locomotion recovery in vivo following CNS injury
In order to determine whether C/EBP siRNA treatment has a role in an in vivo model, we compared the locomotory activity of sham-operated and crush-operated snails treated with saline, control siRNA and C/EBP siRNA#1. The distance that the snail crawled in 10 min was measured before and after nerve crush (1, 5 and 10 days post-crush), between all six groups. As shown in Figure 6, all groups had severely movement deficit following the nerve crush operation, but slowly gained their ability to crawl over the course of 10 days. However, after 10 days, the distance (per 10 min) measured from the crush-operated C/EBP siRNA#1 group (4.99 ± 0.71 cm, n = 3) was reduced as compared to that from the sham-operated saline (10.28 ± 1.28 cm, n = 6), control siRNA (12.23 ± 1.36 cm, n = 6) or C/EBP siRNA#1 (11.56 ± 2.75 cm, n = 5), as well as to that from the crush-operated saline (10.65 ± 2.84 cm, n = 4), or control siRNA treated snails (10.17 ± 1.0 cm, n = 5). These data suggest that C/EBP siRNA treatment disrupted the recovery of snails following nerve injury and support the notion that C/EBP knockdown impedes CNS regeneration in vivo, potentially through its effects on neurite regeneration.
Figure 6. C/EBP siRNA treatment hinders recovery of locomotion activity of the snails following CNS injury. Sham-operated or crush-operated snails were injected with either saline, control siRNA or C/EBP siRNA. The distance that the injured snails crawled in 10 min was measured at various time points (1 day, 5 days, and 10 days) following the nerve crush procedure. On the 10th day, the crush-operated C/EBP siRNA group crawled an average of 4.99 ± 0.71 cm (n = 5) per 10 min which was reduced as compared to the sham-operated saline (10.28 ± 1.28 cm, n = 6), control siRNA (12.23 ± 1.36 cm, n = 6), or C/EBP siRNA (11.56 ± 2.75 cm, n = 5), and crush-operated saline (10.65 ± 2.84 cm, n = 4) or control siRNA treated snails (10.17 ± 1.0 cm, n = 5). Data are presented as mean ± S.E.M. * indicates p < 0.05.
In this study, we designed the first Lymnaea gene chip for microarray analysis and profiled the gene expression patterns during the early stages of the CNS repair following injury. We found that 67 genes were significantly regulated during the first three hours post injury, including the conserved gene transcription factor C/EBP. We further described the temporal gene regulation pattern of C/EBP following CNS injury using qPCR analysis. Reduction of C/EBP mRNA level by C/EBP siRNAs following axotomy suppressed outgrowth of the distal and somatic neurite in vitro at different time points, and disrupted the recovery of locomotory function of the snails following nerve injury in vivo. Our data further support the notion that C/EBP is required for nerve regeneration.
L. stagnalis has been used as a simple and reliable model to investigate genes involved in regeneration, due to its ability to functionally recover following CNS injury in vivo. For example, central ganglia cell (CGC) connections to buccal ganglia were crushed and as a result the feeding behaviour in animals was disrupted. Two weeks following the procedure the CGC regenerated, functional synapses formed between proper synaptic partners and the feeding behaviour was again observed [12]. In vivo CNS regeneration was also observed following axotomy of innervating axons to the pneumostome and surrounding areas which prevented the occurrence of lung respiration in 69% of snails. Several weeks following surgery, axonal regeneration leading to the reformation of functional synapses was observed and pneumostome opening returned to normal [13]. In this study, we used the modified CNS injury model in which both the right parietal (RPa) and the right cerebral nerves (RCe) were crushed (Figure 1A). We are interested in the gene regulation occurring at the early stages of CNS trauma, thus our in vivo experiments were completed within 5 hours following injury.
The ability to profile the expression of thousands of genes simultaneously makes microarrays an invaluable biomedical research tool [35]. A previous study used microarray approach to assess the genes involved in anti-inflammatory responses in a spinal cord injury model [28]. The anti-inflammatory response is an important component of secondary tissue damage following spinal cord injury. The microarray study allowed for identification of a conserved novel regulator of regeneration, and provided a new mechanism by which to initiate the regenerative response [29]. However, shortcomings of microarray analysis are associated with poor reliability [35-38] and specificity [35]. Due to the inherent sources of error in microarray technology, it is necessary to confirm the findings using an independent methodology from separate samples. Real-time quantitative PCR is a readily used technique to confirm microarray data [39-41]. In our study, we conducted qPCR analysis using different samples and verified that the expression of C/EBP was indeed up-regulated following nerve injury.
Recovery from nerve injury and neuronal regeneration are time-dependent events, from the initial inflammatory response to the development of the glial scar [42]. Inflammation, neurotransmitter dysfunction, ionic imbalance, cytoskeletal alteration and increased transcription occur as early as 3 hrs following spinal cord injury [41,43,44]. Nerve outgrowth and regeneration can be enhanced by steroids during a critical window within the first 6 hrs after nerve injury [45]. In the C. elegans, axons regenerate following axotomy in vivo and by 24 hrs fully functional synapses are formed [46]. We have recently demonstrated that L. stagnalis regeneration post injury in vitro can be seen as early as 3 hrs, and reformation of connections between the proximal and distal axon is observed by 24 hrs [47]. Deducing the gene regulation during the early response following neuronal injury is important in uncovering the molecular basis of neuronal regeneration. In this study, our microarray gene expression analysis identified 67 genes that were significantly regulated following CNS injury in L. stagnalis. We examined C/EBP gene expression at three different time points (1 hr, 3 hrs, 5 hrs) in order to understand how it is regulated temporally in the early stages of recovery and repair. Our results indicated that the C/EBP gene was up-regulated in a time-dependent manner following CNS injury (Figure 3). We further showed the locomotory activity of the control snails was severely disrupted immediately following nerve injury but recovered gradually over 10 days. C/EBP siRNA application prior to the nerve crush procedure disrupted the recovery of locomotory function of the snails (Figure 6), suggesting that C/EBP is required for nerve regeneration in vivo. Although our in vitro data suggest that sufficient C/EBP levels in neurons are required for axonal outgrowth, it is possible that an increased C/EBP expression in non-neuronal (phagocytic) cells involved in nerve regeneration and inflammatory responses also contributes to the observed recovery of locomotory activity in the injured snails.
C/EBPs belong to the basic leucine zipper DNA binding protein family and are transcription factors with diverse cellular roles, including regulating cell growth, differentiation, learning and memory, and apoptosis [48-50]. C/EBPs are also responsive to brain injury and ischemia [51,52]. Six isoforms of C/EBP have been found in mammals [53]. C/EBPβ is highly homologous to Lymnaea C/EBP (LC/EBP) [54]. C/EBPβ was first identified as a mediator of the inflammatory response and IL-6 signalling through its binding to IL-6 response elements in the promoters of acute phase response genes TNF, IL-8, and G-CSF [55]. C/EBP expression is enriched in neurons. It is up-regulated following brain injury in various neuronal regenerative animal models [56-58]. Its expression is regulated by transcription factors such as CREB and NFIL-3 [59-61]. Aplysia nerve injury activates axoplasmic kinase, RISK-1, which phosphorylates apC/EBPβ and initiates the binding of apC/EBPβ to the ERE enhancer site in vitro. Increases in RISK-1 and apC/EBP were detected in injured neurons [59]. In leech, C/EBP mRNA was increased during neuronal regeneration [60]. In this study, we show that C/EBP mRNA expression is increased following CNS injury in a time-dependent manner. The 7-fold increase in C/EBP mRNA at 1 hr following injury is likely related to the inflammatory response. The sustained 2 fold increase over the 5 hr period suggests that C/EBP may be essential for transcriptional induction of regeneration associated genes. Knockdown of C/EBP mRNA expression following axotomy by target-gene siRNA caused a reduction in neurite outgrowth in both distal and proximal axons at different time points. Due to that siRNAs were applied following axotomy, our data of C/EBP siRNAs causing suppression of distal neurites suggests that local C/EBP mRNA is likely involved in neuronal regeneration/elongation. In contrast to the distal section of neurites which may rely on local protein synthesis to grow, the cell somata may continuously provide complimentary mechanisms to sustain C/EBP levels and maintain neurite properties. The delayed effect of C/EBP siRNAs on the proximal regrowth (Figure 5B) further supports the notion that C/EBP is required for axonal regeneration.
CEBP-1, a member of the C/EBP family in the C. elegans, is a direct effecter of the DLK-1 cascade [34]. CEBP-1 mRNA has been found in axons and presynaptic regions, and is stabilized via its 3'UTR by activation of the dual-leucine zipper Kinase-1 (DLK-1) pathway [34]. The role of C/EBP in axon regeneration is suggested via dual-leucine zipper kinase MAPKKK (DLK-1)-dependent pathway [34]. Neuronal regeneration associated proteins such as α-tubulin and GAP-43, are transcriptional targets of C/EBP [48,56,62]. Following nerve injury, up-regulation of C/EBPβ mRNA and C/EBP phosphoprotein coincides with increases in the transcription of the α-tubulin promoter in the wild-type but not in C/EBPβ-/- mice [56]. DLK-1 acts in a MAPK cascade with MAPMKKK-4 and p38 kinase [63]. Both the DLK-1 pathway and CEBP-1 were necessary for regenerative growth of adult touch neurons [34]. We thus proposed a model by which the DLK-1 pathway regulates C/EBP mRNA stability in both distal and proximal axons. Both somatic and local C/EBP levels are required for the regenerative properties of snail neurons (Figure 7). Local protein synthesis has been shown to increase in injured axons and thus play an important role in the regeneration-degradation process [64-66]. Our study provided the first evidence that not only the somatic but also local regulation of C/EBP mRNA in axons is important for outgrowth and regeneration in L. stagnalis.
Figure 7. Schematic illustration of the potential mechanisms of C/EBP action in maintaining distal axon integrity following axotomy. C/EBP mRNA is expressed in both the soma and axon [34,56]. In the soma, C/EBP functions as a transcription factor and results in the transcription of regenerative-associated genes, such as α-tubulin and GAP-43, via a cAMP/PKA/CREB dependent signalling pathway [56]. Axon injury also activates an ERK-dependent pathway that results in the phosphorylation of C/EBP and further activation of pro-regenerative genes [59]. In both proximal and distal axons, C/EBP mRNA is stabilized by DLK-1 following axotomy [34]. Local protein synthesis of C/EBP in the distal axon may be required for regeneration or to prevent degeneration of distal axons following injury.
This is the first microarray study in L. stagnalis, allowing us to identify genes that are differentially regulated following CNS injury. These genes may be involved in regeneration and provide a new database for the study of specific proteins and genes in response to CNS injury. We also identified the role of the transcription factor C/EBP in neuronal regeneration following axotomy of adult neurons in vitro, and provided new insights into the potential targets for CNS injury.
Fresh water pond snails, L. stagnalis, were obtained from culture at the Free University, Amsterdam, and maintained in the laboratory under standard conditions at the University of Toronto [31,32]. All animals used were kept in water at 20°C under a 12 hour light: dark cycle, and fed green leaf lettuce twice a week. Two-month old animals having a shell length of ~20 mm were used for the experiment.
Surgical procedures and in vivo nerve injury
Snails were anesthetized by injecting 0.5 - 1 ml of 60 mM MgCl2 into the foot. In sterile snail saline containing (in mM): NaCl 51.3; KCl 1.7; CaCl2 4.1; MgCl2 1.5 (pH was adjusted to 7.9 with 1 mM HEPES/NaOH) a small incision was made in the dorsal head region and pinned open, exposing the cerebral and buccal ganglia. In the crush-operated animals, all right parietal and cerebral nerves were crushed using forceps [12]. Sham-operated animals were operated in the same way as the crush-operated groups, except that the right parietal and cerebral nerves were not crushed. Following the operation, both the sham-operated and crush-operated animals were maintained under standard laboratory conditions.
RNA extraction and cDNA synthesis
Snails were anesthetized with 10% (v/v) Listerine for 10 min, following which the CNS including the buccal ganglia were dissected out for total RNA extraction at 1 hr, 3 hrs or 5 hrs post-injury. For the microarray there were 4 groups of sham-operated and 4 groups of crush-operated animals each with 2 snails per group. For RT-PCR 5 new groups of crush-operated and 5 new groups of sham-operated snails were used with 2 snails per group. We used TRIzol reagent and a modified protocol (Invitrogen, USA). 100 μl of Trizol was used per two excised CNS and buccal ganglia, and the final pellet was resuspended in 10 μl of RNase free water. RNA quality was checked on an agarose gel prior to cDNA synthesis and RNA concentration was measured using spectrophotometry. First strand synthesis of cDNA was conducted using SuperScript III reverse transcriptase (Invitrogen, USA) with random hexamer primer (Fermentas, USA) in a reaction volume of 20 μl for 1 μg of total RNA.
cDNA Microarray
A microarray chip was designed based on the L. stagnalis CNS EST database http://www.Lymnaea.org/ webcite. The 15 K gene chip (cat#: G2509F) was made by Agilent Technologies (U.S.A). Briefly, the array contained a total of 15,000 spots, 10,333 probes were from unique EST/nucleotides and 4,425 probes included different sequences from the duplicate genes, and 50 technical replicates with 9 replicates per probe. The hybridization was performed by the University Health Network Microarray Centre (Toronto, Canada). One-Color Microarray-based Gene Expression Analysis (Agilent technologies) with cyanine 3-labeled targets was used to measure gene expression in experimental and control samples. Briefly, 500 μg of total RNA were amplified using a Fluorescent Linear Amplification Kit (Agilent Technologies) and labeled with Cy3-CTP and hybridized to the L. stagnalis cDNA microarray at 65°C for 17 hours. The images were scanned using a Genepix 4000B microarray scanner (Axon Instruments, Foster City, CA, USA). Image analysis, spot quality control and normalization were performed using the Feature Extraction software 9.5.3 from Agilent Technologies. The intensity files were loaded into GeneSpring GX 10.0.2 software (Agilent Technologies), a 75th percentile signal value was used to normalize Agilent one-color microarray signals for comparisons between arrays. The signals were log 2 transformed, and the median of the replicated probes was obtained. An unpaired t-test was performed and p < 0.05 was used as a cutoff; from these results we used a 2.0-fold change in signal intensity as a cutoff line to consider the differential expression of a gene as significant.
Real-time quantitative PCR (qPCR)
Real-time PCR was performed using 5 μl of SYBR Greener Reagent System (Invitrogen), added to 1 μl of the appropriate primer set, and a diluted sample of cDNA that was topped off with DEPC water to a final volume of 10 μl. For the qPCR validation and time-course a new set of RNA samples from sham and crush-operated snails was used. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been used as a common control gene for spinal cord injury models [40,67], and its expression was unchanged following CNS injury in L. stagnalis, therefore, we used it as an internal control (Figure 3B). The cycling parameters were: 95°C/5 min, 40 cycles of 95°C/0.5 min, 55°C/1 min, followed by a melting curve protocol. The primer sequences used in the study are as follows: GAPDH 5'CTGCTGATGCCCCTATGTTTG/5'GTTGGTGGTGCAAGACTGCATT; C/EBP 5'CCTCTCATACATCTCCAAGTGC/5'GAGATGTTAGGGTGTAGGAATGG.
Changes in gene expression levels following CNS injury were determined using Ct-Ct plots [30], this analysis is a modification of the ΔΔCT and efficiency correction methods [68]. As described in our recent report [30], the efficiency of the amplification is assumed to be independent of the sample, and the standard curves from all the samples are linear. Assuming that the PCR efficiencies are independent of the sample, a Ct-Ct plot is created by plotting the Ct (threshold cycle) values of the test gene against the control gene. This yields a linear correlation between the gene pair and the slope (m) of the plot is described as m = log(Ec)/log(Eg). The Y axis of these plots is described by Ctg = m × Ctc - log(R)/log(Eg); where R is the relative expression level between the target gene (Qg) and control gene (Qc); Eg is the amplification efficiency of the target gene; Ctg and Ctc are the threshold cycles of the target gene and control gene, respectively [30]. The Y-intercept, a quotient of - log(R)/log(Eg), is as a measure of R, because Eg is constant. Thus, a small change in the Yint in the injury group over the control group indicates a decrease in the relative target-gene expression level.
siRNA construct
Based on L. stagnalis C/EBP sequence, we designed a 27-mer siRNA using SciTools RNAi Design online software (IDT DNA). When choosing siRNA we selected for sequences with moderate to low G/C content, biased towards the 3'-terminus and purposely avoided sequences encoding the transmembrane domains [69]. The selected sequences were then purchased from Shanghai GenePharma Co. Ltd. The sequences for each siRNA are as follows: C/EBP siRNA#1: 5'CUAACAUCUCCUACUCAU UCCCAAA/5'UUUGGGAAUGAGUAGGAGAUGUUAGGG; C/EBP siRNA#2: 5'GGAUUAUAGUCAACAAAC/5'CUGCCUAGUUUGUUGACU; Control siRNA: 5'UUCUCCGAACGUGUCACGUTT/5'ACGUGACACGUUCGGAGAATT. For whole animal knockdown experiments snails were anaesthetized with 10% (v/v) Listerine, following which 2 μl of 20 μM control siRNA or C/EBP siRNA was injected into the head, above the central ganglia using a microlitre syringe (Hamilton Company, Reno, NV, USA) [70]. To confirm the siRNA knockdown using RT-PCR the ganglia were removed 48 hrs post-injection for RNA extraction (Figure 4C).
Quantification of locomotion activity
Sham or crush operations were performed 48 hrs after injection with either saline, control siRNA, or C/EBP siRNA into the central ganglia region. Individual snails were placed in a large Petri dish (10 cm diameter, 2 cm depth) in 15 ml of water from their home aquaria. The snails were given 5 minutes to acclimate to their environment, and then were recorded for 10 minutes using Virtual Dub 1.9 software. The distance that each snail has crawled in 10 min was analyzed using ImageTool 3.1 software (UTHSCSA, Texas). Locomotion was analyzed prior to the surgery, and at day 1, day 5 and day 10 post operation.
Primary cell culture
Snails were anaesthetized for 10 minutes in 10% (v/v) Listerine, and the central ring and the buccal ganglia were excised in snail saline containing (mM): NaCl 51.3, KCl 1.7, CaCl2 4.1, MgCl2 1.5, HEPES 2 (adjusted to pH 7.9 using 1 mM NaOH). The ganglia was then incubated in 3 mg/ml trypsin (Type III, Sigma, Ontario, Canada) for 24 min. PeA neurons, which are involved in locomotion [71], were identified and isolated from pedal ganglia with gentle suction using a Sigmacote (Sigma, Ontario, Canada)-coated fire-polished pipette (2 mm, WPI, 1B200F) as previously described [72,73]. The neurons were then placed onto a poly-L-lysine coated culture dish and maintained in diluted conditioned medium (CM) at room temperature [72-74].
Axotomy and neurite outgrowth
PeA cells were cultured in CM at room temperature for 24 hours, allowing for sufficient neurite outgrowth. Transection of the neurite was then performed using a glass micropipette, controlled by a micromanipulator, and moving it perpendicularly across the neurite until a complete cut was observed [47,75]. Immediately following axotomy the cells were treated with either the brain conditioned medium (CM) or a final concentration of 7 nM C/EBP siRNA#1, C/EBP siRNA#1 siRNA or control siRNA in a final volume of 2 ml of the CM. Images of neurites were collected at the time of RNAi treatment (t = 0), and over a 36 hr period at three time points (10, 24 or 36 hrs), using an inverted microscope (40× objective) (Olympus CK X41) with a digital camera (Olympus C5050). The lengths of the proximal neurite, the distal neurite and an intact neurite of the axotomized cell were measured at various time points, using ImageTool 3.1 software (UTHSCSA, Texas).
Data are presented as mean ± S.E.M. Data were imported to Microsoft Excel or OriginPro v8 (Silverdale Scientific Ltd., Bucks, HP, USA) for graphical presentation. Statistical significance between mean values of experimental groups was evaluated using a Student's t-test for two groups and ANOVA with the Holm-Sidak post hoc test for multiple comparisons, using SigmaStat 3.0 (SPSS, Chicao, IL, USA). Significance was defined by probability level of lower than p < 0.05.
Authors' contributions
ZPF conceived, designed and directed the study. MA conducted the experiments and dada analysis. ZPF and MA wrote the manuscript. ZPF and MA read and approved the final manuscript.
This work was supported by an operating grant to ZPF from the Canadian Institutes of Health Research (CIHR MOP151437). MA is the recipient of an Ontario Graduate Scholarship. ZPF holds a New Investigator Award from the Heart and Stroke Foundation of Canada. We thank Dr. Chi-Yip Ho (Microarray Laboratory, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada) for his comments on gene chip development.
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The holidays are always a good time to take stock of the year, and to be grateful for the good things that happened. Although the media has labeled this the "do nothing" Congress, the news wasn't all bad. Read More
Celia Wexler
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How to Improve the Chemical Safety Improvement Act
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A Chemical Safety Law That Works for the People
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Our Chemical Safety Law Fails to Protect Us
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It's after Labor Day. You're thinking about getting the kids back to school, resuming your doctoral studies, or just gearing up after vacation for a busy September. The last thing you want to think about is the soup of hazardous chemicals you're exposed to every day. For more than 30 years, the Environmental Protection Agency has largely pursued the "ignorance is bliss" approach. That wasn't the agency's fault. It's been rendered virtually impotent by TSCA. Not the opera, the Toxic Substances Control Act. Read More
When "Sound Science" Isn't
August 16th, 2013
"Freedom is the freedom to say that two plus two make four. If that is granted, all else follows." – George Orwell
When I cited that quotation while speaking to a group of students last year, many of them had a hard time grasping what I was getting at. So let me be clear. Orwell, in his classic dystopian novel, 1984, described what it was like to live under a government that believed it could change facts, and make citizens believe them. It could, for example, proclaim that two plus two equaled five, and that would become the new reality. Read More


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shops around town: Unique Artisanal Gifts
Shops around La Alberca carry local artisanal crafts and goods. These would make very special gifts to bring back from your visit.
What to buy: Look for handmade Salamancan filigree ceramic/pottery work in the form of plates or decanters and containers with the tree of life design.
If you are interested in religious pieces, you can also pick up an image of the Virgin of Pena de Francia, a Black Madonna kept in a monastery on the highest peak in the Las Batuecas area--which is where La Alberca is located.
Do not miss a piece with the special symbolic La Alberca designs on them. The birds, dogs, lions and heart with their sinous vines and flowers are representative of the various stages of a woman's life and were traditionally passed down from mother to daughter. These can be found on embroidered and appliqued products, tiles, and porcelain pieces.
What to pay: Varies. 2 euros to 300 euros or more depending upon the item.
- Romantic Travel and Honeymoons
- Arts and Culture
- Budget Travel
Supermercado Rita: Local Goods in Local Grocer
One of the great things about being someone else is taking the chance to shop like a local. Stop into the Supermercado Rita and peruse the local Salamanca wine selection, not to mention all of the different Spanish foods, beverages, housewares and toiletries. The staff (I think they are family-run) is very nice and friendly.
What to buy: Salamanca wines, honey, candied almonds and other sweet treats.
What to pay: Varies. It is not expensively priced though.
- Budget Travel
- Romantic Travel and Honeymoons
- Arts and Culture
- 1
Instant Answers: La Alberca
Get an instant answer from local experts and frequent travelers


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Next, Samp was named to the Harlon Hill Ballot for Division II player of the year. Given his front-running status for the Heisman Trophy, which is given to the best player in all of college football, it would seem he is a shoe-in. Like many other things, however, there may be some politics involved. Also, there may be some bylaws that prevent a player from winning both trophies. That's okay, Samp has plenty of other awards on his mantel.
Finally, Samp has gained an extreme amount of notoriety on the "internet." As stated in an
article about this website, there is a disproportionately low number of fan sites for Chris Samp. Now, however, if you google "Chris Samp", this site is the first link to come up. In addition, if you google for "Heisman Trophy," this website comes up on only the fourth page of results. That may not sound great, but consider how many people write about the Heisman Trophy for major websites like ESPN and such. So, you might be wondering if there is anything you can do to help move it up even further. Well, there is, and it's completely painless (unless you have some kind of debilitating disease). Just go google "Heisman Trophy," and click on the link to this webpage. This tells google that this is a relevant result to that query and helps to move it up in the list.
Posted by mill1991 at November 5, 2004 10:40 AM


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Incorrect. Response "C" is never the correct response. Providing student information over the phone without requesting any further verification is in violation of FERPA and university policies.
The most conservative and best response is "A"– not to provide the individual the information over the phone. Explain to them that under FERPA guidelines, you cannot release this protected information over the telephone. Grades are defined as non-directory information here at the university; FERPA requires a signed release before providing information to a third party. Any person making a phone request for information is immediately considered to be a third party unless verification is completed to ensure the caller is the person he or she claims to be. FERPA does not prohibit you from releasing non-directory information to students themselves but to third parties.
Response "B" - To tell the student her grades over the phone only after she has accurately provided you with the information that verifies her identity can also be a correct response only if you are able to verify, with certainty, the student's identity by their responses to a combination of questions; such as their:
We recommend you document what information was released, and how it was released, with your name, the date, and time you provided the information to the student. The note should then be filed in the student's folder, which is maintained by Records and Advisement, for further reference and documentation.
Return to FERPA Training


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Modern Orthodox
The Ballad of Daniel Antopolsky
Or how a Jewish American country music genius wound up farming chickens in France
Shabbat Is a Day of Rest—But Does That Mean I Can't Text My Friends?
Some Modern Orthodox teens observe 'half-Shabbat,' using cell phones in private. How widespread is the trend? Is it a crisis?
A Visit With Nachum Segal, Avatar of Modern Orthodox Sincerity
'I reach more Jews every day than the prime minister of Israel,' says the popular radio host
In Tel Aviv, Israel's 'Sin City,' an Unexpected Religious Revival Takes Root
Synagogues are full and kosher restaurants abound as liberal immigrants, Orthodox singles, and secular Jews come together
Festival of Tights
Orthodox Jewish women of all stripes flock to an annual holiday sample sale
Orthodox Yeshiva Set To Ordain Three Women. Just Don't Call Them 'Rabbi.'
The first graduating class at Yeshivat Maharat may not have the title, but they do have jobs at Orthodox synagogues
New York's New Firebrand Rabbi
For Sharon Kleinbaum—friend of Christine Quinn, partner to Randi Weingarten—the personal is political
The New 'Morethodox' Rabbi
Asher Lopatin succeeds Avi Weiss at an influential seminary, offering a pluralistic version of Orthodoxy
Herman Wouk's Last Shot
With The Lawgiver, the best-selling novelist takes another stab at the kind of Hollywood fame he's always coveted
Don't Know Much 'bout Orthodoxy
Reports about a religious woman running for office reveal a media blind spot: Orthodox women


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The Central Gwinnett Black Knights are looking to fill 2 to 3 final roster spots for the Spring Season. We have a solid group of players and are looking for a pitcher, infielder, and outfielder to round out the team.
We will play in 8 to 10 tournaments from March thru June as well as the Grand Slam World Series in Florida.
If you are interested in becoming part of a program that focuses on fundamentals and player development, please attend tryouts on Saturday.
Saturday, Dec 3rd
1PM - Field 6
Rhodes Jordan Park
100 East Crogan Street
Lawrenceville GA 30046
If you can't make it on Saturday, please give me a call to schedule a private work-out.
Danny Barber


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A convenient product at a great price. I keep this handy gadget in my car and it's a life saver.
Expert Service 1-877-289-7664 [Hours]
Expert Service 1-877-289-7664 [Hours]
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For example, a JVC Bluetooth adapter will only work with JVC units. A Pioneer iPod adapter will only work with Pioneer head units.
Some Car stereos are compatible with iPods and/or iPhones. If the headunit can connect to an iPod or iPhone via USB or iPod connection cable, these listed iPods are compatible. This list does not apply if the iPod is being connected through the 3.5mm mini-jack plug. Newer iPods are usually compatible if the appropriate adapters are purchased and used.
Any iPods/iPhones released after the release date of this product have not been checked by the manufacturer and/or Sonic Electronix for compatibility. We therefore cannot guarantee their compatibilities or incompatibilities, including, but not limited to: playback, video, charging, and response time.
Brooklyn, NY
October 17, 2013
A convenient product at a great price. I keep this handy gadget in my car and it's a life saver.
1 of 2 found this helpful
June 17, 2012
Bought this to play my sansaclip mp3 player. Just plug one end into radio and the other into your player and enjoy the music. Love the retractable cord, let's you cycle through the songs while driving without taking your eyes off the road, like a remote.
1 of 2 found this helpful
Portland, OR
July 13, 2007
I have an IPod video and this works great with my head unit in my car. I have tried those fm transmitters but there was always alot of noise. This sounds like it comes straight from the ipod itself. Great product.
1 of 3 found this helpful
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Warning: The image depicted shows the resistance change when wiring multiple subwoofer terminals. Please refer to your subwoofer's owner's manual for the proper wiring of its terminals. Sonic Electronix, Inc. is not responsible for damage caused to your audio system or vehicle due to improper installation. Please call tech support at 1-877-289-7664 if you require additional assistance.
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We offer an unequivocal full refund on the purchase price of any of our exclusive supplement formulas (minus shipping) any time within 3 months of your purchase. We do not require the return of unused product for a refund on the first 30 capsules of any formula purchased. However, for fraud protection, we require the return of product for any refunds beyond the initial 30 capsules. Food items must be returned for a refund, as well as apparel and gear items. Fitness equipment, personal care products, knowledge purchases, and DVDs are not eligible for our money back guarantee. To process a refund, or for more information visit our help page.
We implement a variety of security measures to maintain the safety of your personal information when you place an order or enter, submit, or access any information on our website. We incorporate physical, electronic, and administrative procedures to safeguard the confidentiality of your personal information, including Secure Sockets Layer (SSL) for the encryption of all financial transactions through the website. We use industry-standard, 256bit SSL encryption to protect your personal information online, and we also take several steps to protect your personal information in our facilities. For example, when you visit the website, you access servers that are kept in a secure physical environment, behind a locked cage and a hardware firewall. After a transaction, your credit card information is not stored on our servers.
In thanks to our military service men and women, we're extending a 12% discount on all products purchased at Onnit.com.
Anyone shipping their order to an APO/FPO/DPO address will have a 12% discount automatically applied to their order.
In order to military verify your account, please either send an email from your .mil email address or send in a snap shot of your military ID to our customer service help desk. Blacking out rank and or post is acceptable. In either case, please include the civilian email address or whichever email address you have registered with Onnit.com somewhere in the message body so we know which account to verify. Please setup an account with Onnit.com prior to submitting your request.
* US Military members, please choose United States as your destination country when creating your customer profile — this will ensure that your package ships via USPS and that you are not given international shipping rates.
Joe Rogan is perhaps best known for his work on television for the sitcom News Radio, the reality show Fear Factor, and most recently for the SYFY show Joe Rogan Questions Everything. A decorated kick boxer and jiu jitsu blackbelt, Joe also found a home as the premier color commentator for the Ultimate Fighting Championship. While still an actively touring comedian selling out theaters across the world, Joe Rogan is also the host of one of the most successful podcasts in history. The Joe Rogan Experience podcast receives over 11 million downloads a month, providing both inspiration and comedy, while offering a venue for discussing current events, health and civil liberty.
Alpha BRAIN seems to fire up your brain at a higher RPM level ... I feel like it helps me form sentences better.
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text: Events About Our Story Our Team Homes & Lots Homes Floor Plans Lots Portfolio News and Events Events News Blog Getting Started Services 10.17.2014 Austin Luxury Home Tour 6/15/2015 – 6/21/2015 Luxury Home Magazine has selected The Reserve at Lake Travis for its 2015 Luxury Home Tour which will feature 7 of the finest custom home builders in the Austin area. (more...) 12.27.2012 2013 Spring San Antonio Parade of Homes 5/18/13 – 5/27/13 The Spring San Antonio Parade of Homes is approaching quickly! It will be held from May 18-27 in the Palacios at Cibolo Canyons. (more...) 04.05.2013 Spring Tour of Homes April 6-7,13-14 2013 (more...) 03.02.2013 Austin Home and Garden Show 3/1/13 – 3/3/13 We will be in a booth at the Austin Home and Garden show again this March! Come by to see us and talk about building your new home. (more...) 11.21.2012 San Antonio Home and Garden Show 2/22/13-2/24/13 Come join us at the 2013 Spring San Antonio Home and Garden Show at the Alamo Dome! We will be in a booth and would love to see you there. More information will be provided soon. ← Older posts


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Sport picture of the day: sign here, please, Mr Federer
Roger Federer fans hold out a multitude of differing items for their hero to sign. More than Aus $1m was raised in Melbourne as Federer, Jo-Wilfried Tsonga and a host of Australian tennis stars put on a captivating performance in aid of the Roger Federer Foundation. The fans packed into Rod Laver Arena saw the four-time Australian Open champion defeat Tsonga 6-7(5), 6-3, 7-5 in an exhibition match lasting just over two hours and also witnessed Federer warming up for the match with a hit against tennis legend Rod Laver
Contact the Sport editor
- Report errors or inaccuracies: email@example.com
- Letters for publication should be sent to: email@example.com
- If you need help using the site: email@example.com
Call the main Guardian and Observer switchboard:
+44 (0)20 3353 2000
Michael Dodge/Getty Images


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Naked in her next movie? Yep, says Sharon Stone
TEL AVIV — It may not be what Sharon Stone wants to discuss, but the actress is happy to confirm that, yes, she does indeed get naked in her next movie, the sequel to the 1992 hit "Basic Instinct."
In Israel to promote International Women's Day, Stone was badgered not about the fairer sex, but instead about her role as a sexed-up serial killer in "Basic Instinct 2″ on Wednesday.
"People are just sitting there going, like, 'I don't care what she's saying. I don't care what she's saying. I just want to know is she getting naked? Is she getting naked in that movie? Is she naked? Nude? Nude? Naked? Do I see her boobies?"' the actress, now 48, told reporters, laughingly.
"So let's just get through to that. Yes! And now that I've cleared that up, let's just go to the next question, because nobody cares about anything else, really," she said.
"Basic Instinct 2″ opens March 31 in the United States.


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Your mail
Mobiles that must be on steroids
It may look like a phone, but the ultimate personal digital assistant will also be as good as the best personal organisers and stop you getting lost too. Jack Schofield reports
Technology news & features p2
Inventor's toolkit
Trevor Baylis invented the Freeplay self-powered radio and is hoping to establish an Inventors Academy in the UK
Working the web: Vegans
From recipes to which E numbers are made from sperm, the net is great for vegans, says Sanjida O'Connell
Technology news & features p4
Apple unveils all new iBook
Apple has unveiled a "thinner, lighter and cheaper" version of the iBook, the portable aimed at education and home users.
Phone games: next generation
Star Trek fans are one of the many targets for an embryonic industry, writes Tim Green
What's new
Nokia junior | Tiny view
Technology news & features p5
Web watch
Extra ice | Plane risky
Second sight
Mobile operators might want to consider a mission to Mars, says Dave Birch. It might have a better chance of success than 3G.
Technology news & features p7
Fine tuning
Neil McIntosh shows you how to listen to radio stations around the world - on your PC
Technology news & features p8
Games reviews
Gungriffon Blaze | Silpheed | Virtual Kasparov
Games watch
Game time | Penguin alert
Technology news & features p9
The message is the medium
The Guardian's pioneering text poetry competition attracted nearly 7,500 entries from 4,700 mobile phones. A key part of the exercise, done with Consult Hyperion, was the interactive element. The entries were winnowed down to 100 and given to our judges, Justine Jordan, books editor of Guardian Unlimited, U A Fanthorpe and Peter Sansom. They chose a short list of seven, below, each of which was texted to all participating phones over seven days. Most winners, and entrants, had never written a poem before. This met the main aim - to turn text messaging into a creative force. Sorry for the glitches and thanks to everyone who joined in the fun.
Victor Keegan, editor, Online


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Kurt Cobain Added to Guitar Hero 5
Kurt Cobain Added to Guitar Hero 5
Remembered as one of the most iconic artists in the history of alternative rock, Kurt Cobain's enduring legacy continues with his video game debut as one of five exclusive in-game artists in Guitar Hero 5, coming in September. The former lead singer and guitarist of Nirvana will be featured as a playable character, performing two of the band's hit singles from their 1991 genre-defining album Nevermind: "Smells Like Teen Spirit" and a special, previously unreleased live recording of "Lithium." The band's breakthrough hit and one of the greatest rock songs of all time, "Smells Like Teen Spirit" helped define the 1990s and will speak to a whole new generation in Guitar Hero 5.
"Kurt Cobain is one of the most recognizable frontmen in rock and roll history and it's an honor to have two of Nirvana's masterpieces included in Guitar Hero 5," said Tim Riley, Vice President of Music Affairs for Activision. "This is the first time the original recording of 'Smells Like Teen Spirit' has ever been licensed in an entertainment property and we're thrilled to offer Guitar Hero fans exclusive access to the unreleased live version of 'Lithium.'"
Guitar Hero 5 features the strongest, most varied set list to date comprised of master tracks from more than 80 of the hottest bands of today and the biggest classic acts; including Kings of Leon, The Rolling Stones, The White Stripes, Santana, Vampire Weekend, Tom Petty, Johnny Cash, Bob Dylan, plus more than 25 artists from a variety of music genres that will be making their music video game debut. Among the first-time-ever features of Guitar Hero 5 are: Party Play, where players can jump in or drop out of gameplay seamlessly; RockFest, a comprehensive competitive experience available featuring five new head-to-head modes playable online or in your living room; and the ability to play the entire set-list from the first time the game is turned on.
Guitar Hero 5 refines the player experience, enhances the art style and redesigns core features such as GH Music Studio, making it the most accessible, fun-to-play and authentic experience for seasoned music gamers as well as first-time players. New innovations such as Band Moments, where bands are rewarded for hitting special note streams together and song challenges where gamers are tasked to play through a song a specific way, add a new competitive layer of excitement and accomplishment to the music rhythm genre.
You Might Also Like...
February 27, 2015 at 4:40am
September 01, 2009 at 1:56pm
Well if he did kill himself, too bad for him, he should have stayed alive long enough to defend his stance. If Courtney had a hand in his death and he didn't commit suicide, It's possible that Kurt may may not have given a shit about appearing in a video game, he was very self-contradictory. After all, he did allow wal-mart to change his song "rape me" to "waif me" so that more kids could buy In Utero, although he always hated the corporate elite and their effect on society. I love Kurt, believe me, and I think Guitar Hero has done wonders for bringing older music into the limelight, so I'm excited to see Kurt in the game.
August 29, 2009 at 6:48am
I do not believe for a minute that Kurt Cobain killed himself. I think Courtney Love had her hand in it somehow, as with Kristen Pfaff.
August 28, 2009 at 1:38pm
Kirk is rolling in his grave. This is a huge punch in the face for him. He hated commercializm and isn't that the reason his killed himself?
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Bad Brains
Bad Brains Records
Released: 1980
Producer – Jimi Quidd
H.R. – vocals
Dr. Know – guitar
Darryl Jenifer – bass
Earl Hudson – drums
1. Pay to Cum (1:33)
2. Stay Close To Me (2:29)
When it comes to the Bad Brains I am definitely in the minority here, never been a fan. Whenever they would come to Los Angeles everybody, including my Uncle, would rush to see them. Over the years I've gotten used to them, but other than a few compilations, I never owned anything other than this single.
In December of 1979, the Bad Brains recorded their debut single (Pay To Cum/Stay Close To Me) on their Bad Brains label, in New York, which was later included on the Let Them Eat Jellybeans compilation album. The band would later repress the single, and issue it without the picture sleeve (red and tan labels instead of the brown and white of the first pressing). Pay to Cum was recorded by Jimi Quidd who had heard the band play at a Club in New York.
The "A-side" to the single is Pay to Cum, while the flip side is Stay Close To Me, identified as "Side 1″. The original 7″ had no "B-side" or "Side 2″.
Pay To Cum
"I make decision with precision,
Lost inside this manned collision,
Just to see that what is to be,
Perfectly my fantasy.
I came to know with now dismay,
That in this world we all must pay,
Pay to write, pay to play,
Pay to cum, pay to fight.
And all in time,
With just our minds,
We soon will find,
What's left behind?
Not long ago when things were slow,
We all got by with what we know,
The end is near. Hearts filled with fear,
Don't want to listen to what they hear.
And so it's now we choose to fight,
To stick up for our bloody right,
The right to sing, the right to dance,
The right is ours… We'll take the chance.
A peace together,
A piece apart,
A piece of wisdom,
From our hearts."
Rating: ** * two out of three stars
On to the story . . .
A few months ago my wife was driving home from a class. On the drive she noticed out of the corner of her eye a guy in a suit, driving a BMW, trying to get her attention. She glanced over and he is holding out his cell phone, displaying a photo of his privates (for those of you that are under-age and reading this). My wife cracked the window and hurled a few obscenities. This didn't deter him in the least. He responded with, "Yeah, you like that?"
She sped off, cut a few corners and finally lost him. She walked in a bit frazzled and told me the story. Absolutely nothing I could do, but shake my head and wonder when and how this seemed like a good idea to guys?
I told you all that to tell you this: over the last month or so I've had a lot of new "friends" pop-up on my page. A number of them are women that work as pin-up models (car shows, magazines, etc) and I notice that, at least one will post daily, "I am married, I am not looking to "hook-up" please stop sending pictures of your penises."
This floors me. The first time I saw one of these posts I, kind of, thought it was a joke. Like, "Who the hell would do this?" But now I read it daily.
So, the question is, is this a social media thing? I have a computer/cell phone so it's easier to just show you my junk than attempting conversation? Or have men in general become more primal? The digital age flashing has become the equivalent of the caveman clubbing over the head thing?
I have never been great with the opening line thing with people I didn't know. But it never occurred to me that whipping out a snapshot of the package was the way to go in polite society.
Whatever it is, something about seeing a woman with her hair fixed and a bit of make-up makes these primates lose their shit.
And I'm at a loss for ideas on how to fix a situation like this, short of slapping their moms for raising such vile pieces of crap. And that concludes my rant for the day.
Life Won't Wait is out now, grab a copy today: http://goo.gl/n9ofGb


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Inside Apple's New Grand Central Terminal Store in NYC
Grand Central Apple Store Apple's new store in Grand Central Terminal opens on Dec. 9 at 10am.
1 / 50
the lg g4 is here!Want a chance to win one?
Provide your email address and what carrier you are using (only Verizon, Sprint, T-Mobile and AT&T please) and we'll pick FOUR random winners to receive one of the hottest phones of the year, the LG G4.
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To embed, copy and paste the code into your website or blog:
[author: Amy M. Landwehr]
Worldwide EB-2 Becomes Current in November: The worldwide employment-based second preference category (advanced degree/exceptional ability) will become current again in November, as announced in the Visa Bulletin. The category became oversubscribed in July.
TSP Extended for Haitians: Department of Homeland Security (DHS) has extended the designation of Haiti for temporary protected status (TPS) for 18 months from January 23, 2013 through July 22, 2014. The 60-day re-registration period began October 1, 2012, and will remain in effect until November 30, 2012. DHS also announced the extension of the suspension of regulatory requirements so certain Haitian F-1 students experiencing severe economic hardship may obtain employment authorization.
Taiwan Designated for Visa Waiver Program (VWP): Effective November 1, 2012, eligible Taiwan passport holders will be able to use the streamlined Visa Waiver program. Taiwan joins 36 participants in the VWP, which permits visa-free travel to the United States for ESTA-approved travelers visiting the U.S. for 90 day or less for business or tourism.
Recent Consulate Re-openings/Closings: The Consular Section at the U.S. Embassy in Khartoum, Sudan, was recently closed indefinitely, and consular services in Cairo, Egypt and Tripoli, Libya, also have been suspended. The Consular Section at Tunis, Tunisia, was reopened on October 9, 2012.
Employee Rights Toolbox: USCIS has released an online resource page on its website to increase awareness about employee rights during the employment eligibility process, containing a variety of educational materials.
DISCLAIMER: Because of the generality of this update, the information provided herein may not be applicable in all situations and should not be acted upon without specific legal advice based on particular situations.
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text:
it used to be doom
Zandronum is a free open-source advanced multiplayer sourceport capable of running various games such as Doom, Doom II, Final Doom, Heretic, Hexen, and as well as others online. It originated from Skulltag, which was based on ZDoom and its counterpart, GZDoom.
it used to be doom
until it took OpenGl rendering to the knee.
now it looks better than the Quake 3 engine.
Whoa where can i get this map?