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The serotonin 2C receptor regulates food uptake, and its activity is regulated by alternative pre‐mRNA splicing. Alternative exon skipping is predicted to generate a truncated receptor protein isoform, whose existence was confirmed with a new antiserum. The truncated receptor sequesters the full‐length receptor in intracellular membranes. We developed an oligonucleotide that promotes exon inclusion, which increases the ratio of the full‐length to truncated receptor protein. Decreasing the amount of truncated receptor results in the accumulation of full‐length, constitutively active receptor at the cell surface. After injection into the third ventricle of mice, the oligonucleotide accumulates in the arcuate nucleus, where it changes alternative splicing of the serotonin 2C receptor and increases pro‐opiomelanocortin expression. Oligonucleotide injection reduced food intake in both wild‐type and ob/ob mice. Unexpectedly, the oligonucleotide crossed the blood–brain barrier and its systemic delivery reduced food intake in wild‐type mice. The physiological effect of the oligonucleotide suggests that a truncated splice variant regulates the activity of the serotonin 2C receptor, indicating that therapies aimed to change pre‐mRNA processing could be useful to treat hyperphagia, characteristic for disorders like Prader–Willi syndrome.
The serotonin 2C receptor regulates food uptake and undergoes alternative pre‐mRNA splicing; the ratio of the two spliced isoforms (truncated versus full length) controls the activity of serotonergic neurons. An oligonucleotide promotes the formation of the full‐length receptor and reduces food intake.
The ratio of serotonin 2C receptor splicing isoforms controls food intake.
This ratio can be changed using a synthetic oligonucleotide.
The oligonucleotide mimics a naturally trans‐acting RNA, SNORD115, that is not expressed in Prader–Willi syndrome, a genetic form of obesity.
Through heterodimerization with other receptors, the isoform ratio could impact on various receptor systems contributing to Prader–Willi syndrome.
Obesity is a growing health problem causing an estimated 100–400,000 deaths per year in the United States. Currently, more than two‐thirds of the adult population in the USA is either overweight or obese (Ogden et al, 2014). The FDA approved drug locaserin is a serotonin 2C receptor (5HT2C) agonist, validating the 5HT2C as an anti‐obesity drug target (Miller, 2005).
Various hormonal systems signal nutritional status to the brain that ultimately regulates feeding behavior. An important brain area integrating hormonal signals is the arcuate nucleus, a structure located in the mediobasal hypothalamus. The arcuate nucleus contains about 1,000 POMC/CART (pro‐opiomelanocortin/cocaine‐ and amphetamine‐regulated transcript) neurons that express insulin, leptin, and serotonin receptors, allowing POMC/CART neurons to sense nutritional status (McNay et al, 2012). Activation of these receptors results in a reduction of food intake due to POMC/CART neurons stimulating anorexigenic neurons in the paraventricular nucleus (PVN) and inhibiting orexigenic neurons in the lateral hypothalamic area (Cone, 2005).
Central to food control in the arcuate nucleus is the 2C‐subtype of serotonin receptor (5HT2C) (Iwamoto et al, 2009). After stimulation with serotonin or other synthetic agonists, the receptor activates phospholipase C and induces expression of the POMC gene. This leads to an increase in expression of the POMC precursor peptide that is further processed to α‐MSH (melanocyte‐stimulating hormone), which causes an anorexic response by activating neurons in the paraventricular nucleus via the melanocortin 4 receptor (MC4R) (Williams & Elmquist, 2012; Berglund et al, 2014).
The pre‐mRNA that generates 5HT2C undergoes extensive processing that includes alternative splicing of exon Vb and adenosine to inosine editing at five sites (Stam et al, 1994). The 5HT2C gene has one promoter but contains two polyadenylation sites (Zhang et al, 2013). The longest transcript contains six exons and undergoes both pre‐mRNA editing and splicing affecting exon Vb. Exon V forms an extended secondary structure in vivo allowing RNA editing by adenosine deaminases acting on RNA (ADAR1 and ADAR2) that require an RNA duplex as a substrate. The RNA secondary structure undergoes conformational changes after binding to synthetic ligands, which regulates the use of the distal alternative splice site (Shen et al, 2013).
RNA editing creates at least 24 full‐length proteins by affecting three amino acids located in the second intracellular loop of the receptor that couples to the effector Gq protein. The non‐edited receptor features the amino acids I, N, and I (isoleucine, asparagine, and isoleucine) at positions 156, 158 and 160 of the receptor protein. The INI isoform shows the greatest coupling efficacy to the G protein in response to serotonin and is constitutively active (Labasque et al, 2010) whereas the highly edited isoforms show a reduced response to serotonin binding and little or no constitutive activation (Wang et al, 2000; Werry et al, 2008). Sole expression of the fully‐edited isoform of the receptor causes hyperphagia in mice, demonstrating that altered 5HT2C signaling is critical for maintaining appropriate energy balance (Morabito et al, 2010).
Skipping of exon Vb creates a mRNA predicted to encode a shorter protein isoform containing only three transmembrane domains. Tagged proteins encoded by cDNAs for the shorter protein localize to the endoplasmic reticulum (ER). These proteins can dimerize with the full‐length serotonin receptor, which likely inhibits serotonin signaling as a result of ER retention (Martin et al, 2013).
The importance of proper hypothalamic food regulation becomes apparent in Prader–Willi syndrome (PWS), a frequent syndromic form of obesity, characterized by hyperphagia (Butler et al, 2006; Cassidy et al, 2012). The syndrome is caused by the loss of gene expression from a maternally imprinted region on chromosome 15q11.2. This region contains several proteins and two clusters of C/D box small nucleolar RNAs (snoRNA). Recent genetic studies showed that the loss of these snoRNAs strongly contributes to PWS (Sahoo et al, 2008; de Smith et al, 2009; Duker et al, 2010). One of these snoRNAs, SNORD115, regulates the processing of the 5HT2C pre‐mRNA (Kishore & Stamm, 2006; Kishore et al, 2010; Shen et al, 2011), showing functions of C/D box snoRNAs different from the 2′‐O‐RNA methylation normally attributed to these non‐coding RNAs (Falaleeva et al, 2016), and suggesting that a dysregulation of 5HT2C function contributes to the hyperphagia observed in PWS.
Since pre‐mRNA processing of the 5HT2C has a strong effect on food intake, we searched for molecules that could compensate for the loss of SNORD115. We identified an RNA oligonucleotide that similar to SNORD115 promotes exon Vb inclusion. This oligonucleotide reduces food intake in mice when delivered to the arcuate nucleus, either through ICV or systemic injections, most likely by increasing full‐length 5HT2C density at the cell surface of neurons in the arcuate nucleus.
The 5HT2C gene expresses both a full‐length and a truncated protein isoform
The 5HT2C pre‐mRNA contains two alternative 5′ splice sites termed the proximal (PS) and distal (DS) splice site that define exons Va and Vb, respectively. Inclusion of exon Vb generates RNA2, which encodes a full‐length seven‐transmembrane receptor. Skipping of exon Vb introduces a frameshift, resulting in an early stop codon in exon VI. The resulting RNA1 thus encodes a truncated receptor isoform containing only three predicted transmembrane domains (Fig 1A). Whereas the full‐length 5HT2C receptor is 90% identical between mouse and human and differs by only one amino acid in length, the C‐terminus of the truncated receptor contains 96 amino acids in humans, but only 19 amino acids in mouse (Fig EV1A) due to differential codon usage. Since the stop codon generating the truncated receptor is located in the last exon, its mRNA is likely not subject to nonsense‐mediated decay (Fatscher et al, 2015). To test whether RNA1 is expressed as an endogenous protein, we generated an antiserum against a peptide common to mouse and human RNA1 (Fig EV1B). After affinity purification, this antiserum detected truncated 5HT2C protein from HEK293 cells transfected with a cDNA encoding the truncated receptor, as well as the endogenous protein from mouse brain (Fig 1B), which shows the existence of a truncated 5HT2C in vivo.
Source Data for Figure 1 [emmm201506030-sup-0002-SDataFig1.pdf]
Identification of oligonucleotides that change exon Vb inclusion
To study alternative pre‐mRNA splicing of serotonin 2C receptor exon Vb, we created a series of minigene constructs that contain the alternative exon V. To test the effect of oligonucleotides on this construct, we omitted a start codon, which limits interference from protein‐coding requirements. The minigene contained exon IV, intron IV, exon Va, exon VI, and intron V that was shorted from about 59 to 6 kb, (Fig 1C) (Kishore & Stamm, 2006). We performed an oligonucleotide walk (Singh et al, 2012) to identify oligonucleotides that promote exon Vb inclusion. We used adjacent 18‐mers showing complementarity against exon Vb and intron V (Fig 1C). The oligonucleotides were 2′‐O‐methyl phosphothioates, where each ribose and each phosphate group was modified by a 2′‐O‐methyl modification or a single sulfur, respectively. Previous studies using oligonucleotides with this chemistry found that cells take up the oligonucleotide directly without adjuvants (Heemskerk et al, 2009) and related 2′‐O‐2‐methoxyethyl oligonucleotides are taken up by neurons in the brain (Passini et al, 2011), which we also observed (Fig EV2). As shown in Fig 1D, oligo#5: 5′‐AGUAUUGAGCAUAGCCGC‐3′ strongly activates exon Vb usage, as determined by RT–PCR. Thus, we identified an oligonucleotide that enters cells and strongly promotes exon Vb inclusion, similar to SNORD115.
Oligo#5 acts at nanomolar concentration in cell culture and can be modified at its 5′ end
We next determined the working concentration of oligo#5 by testing a concentration range from 5 to 100 nM, using the reporter minigene transfected into HEK293 cells. Exon Vb inclusion starts increasing at concentrations higher than 10 nM (Fig 2A and B), showing that oligo#5 can exert an effect in the low nM concentration range; the EC50 (half maximal effect) was 13 nM.
Source Data for Figure 2 [emmm201506030-sup-0003-SDataFig2.pdf]
We then shortened oligo#5 from 18 nt to 12 nt and 10 nt, removing 6 or 8 nt from the 5′ end, respectively (oligo#5‐3; oligo#5‐10, Fig 1C). The shorter oligo#5‐3 retained some activity, which was abolished in oligo#5‐10 (Fig 2C and D). Next, we explored possible modifications and tested oligo#5 derivatives that were modified at the 5′ and 3′ end by cyanine 3. The cyanine dyes were attached to the phosphate via an ester moiety. A modification at the 5′ end had no effect on activity, whereas modification on the 3′ end abolished activity (Fig 2E and F). The data show that oligo#5 works in a low nM concentration and can be modified at its 5′ end.
Oligo#5 promotes exon Vb inclusion without RNA editing
Exon Vb undergoes RNA editing that likely changes the receptor pre‐mRNA structure, as it weakens the central double‐stranded RNA stem by replacing A:U interactions with thermodynamically less stable I:U interactions (Serra et al, 2004). Mutagenesis studies that changed the edited adenosines into guanosine residues showed that editing activated exon Vb inclusion (Flomen et al, 2004). We therefore tested the effect of oligo#5 on RNA editing by directly sequencing the RT–PCR product generated from transfecting the 5HT2C reporter gene with oligo#5. As shown in Fig 3A, none of the editing sites showed a change from adenosine to guanosine, indicating the absence of pre‐mRNA editing. Similarly, editing was absent in exon Vb when we analyzed the minor exon Vb containing PCR product which is generated in the absence of oligo#5 (Fig 3B). Thus, the protein product generated by oligo#5 action encodes the non‐edited receptor that shows the strongest response to serotonin, as well as the greatest constitutive activity.
Source Data for Figure 3 [emmm201506030-sup-0004-SDataFig3.pdf]
Oligo#5 acts directly on the 5HT2C pre‐mRNA
Alternative pre‐mRNA splicing is linked to other steps of gene expression, such as RNA polymerase speed and chromatin structure (de Almeida & Carmo‐Fonseca, 2012). Thus, oligo#5 could promote exon Vb inclusion indirectly. To test how oligo#5 interacts with the pre‐mRNA, we constructed an in vitro splicing minigene that contains exons Va, Vb, and 115 nt of the downstream intron, fused to the adenovirus major late‐transcription unit‐derived MINX exon that contains a strong splicing acceptor (Zillmann et al, 1988). This construct differs from the endogenous gene by the lack of upstream sequences. Most importantly, the length of the downstream intron that is at least 57,969 nt in the endogenous gene was shortened to 190 nt (Fig 3C). Furthermore, we included the strong MINX acceptor splice site to obtain splicing in vitro. Uniformly 32P‐labeled, 7‐methyl guanosine‐capped RNA made from this construct was incubated under standard splicing conditions in HeLa nuclear extract. We then tested the effect of oligonucleotides in Fig 1C on the processing in vitro. In the absence of any oligonucleotide, the proximal splice site is exclusively used, resulting in a 151 nt long fragment. This is similar to transfection experiments, where exon Vb skipping is the default mode. Addition of all oligonucleotides to a final concentration of 25 nM resulted in the release of exon Va (105 nt) and formation of a lariat containing exon Vb (L1). However, the formation of the exon Va‐MINX product (151nt) was suppressed. This indicates that the oligonucleotides promote cleavage at the proximal splice site as the first step of splicing, but inhibit the second step that would result in exon ligation.
Oligo#5, #5‐10 and 5‐3, and #6, 7, 8, 9 also promote cleavage at the distal splice site, allowing for the first step in splicing that leads to exon Vb inclusion (201 nt). Importantly, similar to the cell‐based experiments, oligo#5‐3 promotes formation of the exon Vb‐MINX product (247 nt) that reflects RNA2 formation. The quantification of four experiments shows the doubling of the exon Vb‐MINX splice product, when the inactive oligo#3 is compared with oligo#5‐3 (Fig 3E). The presence of these oligonucleotides also resulted in an accumulation of a lariat LII that lacks exon Vb. It is possible that the oligonucleotides stabilize this structure, which will favor exon Vb inclusion, leading to the formation of the correct spliced product Va–Vb‐MINX. However, oligo#5‐3 had the strongest effect in vitro, whereas oligo#5 had only a small effect, contrasting the findings in cell culture. Since the in vitro reactions are performed at 30°C, it is possible that oligo#5 binds too tightly to the substrate RNA, which inhibits the second step of splicing.
Together, the data suggest that oligo#5 promotes cleavage at the distal splice sites, while still allowing ligation to the downstream exon in the second step of splicing, possibly by binding to the lariat LI.
Oligo#5 changes cellular localization of the 5HT2C protein
To determine the effect of oligo#5 on 5HT2C protein, we generated a reporter construct that expresses a full‐length receptor–GFP fusion protein when exon Vb is included. The construct (Fig 4A) contains the human serotonin 2C receptor protein‐coding cDNA and a shortened intron V between exon Vb and VI containing the natural splice sites. We removed the 5HT2C stop codon and fused it in‐frame with GFP. The start codon is located in exon III in its natural sequence context. Similar to the wild‐type system, inclusion of the alternative exon Vb creates a protein with seven‐transmembrane domains that are now linked to GFP. The GFP domain is predicted to be on the inside of the cell, joined to the last transmembrane domain. Skipping of exon Vb creates a frameshift resulting in a truncated protein without GFP (Fig 4A). Thus, the system can detect the effect of alternative exon usage on a GFP reporter protein. The effect of oligo#5 on the protein‐expressing construct was first tested by RT–PCR. There is a higher basal inclusion level of exon Vb without oligo#5, which is likely the result of the upstream exonic sequences in this minigene. We observed strong inclusion of exon Vb after oligo#5 addition (Fig 4B).
Source Data for Figure 4 [emmm201506030-sup-0005-SDataFig4.pdf]
For further protein studies, we generated stable HeLa cell lines expressing this construct after integration into a single specific flipase recognition target (FRT) site at a transcriptionally active genomic locus. Previous studies showed that the truncated 5HT2C receptor localizes to the endoplasmic reticulum, whereas the full‐length receptor is found both intracellularly and at the surface (Martin et al, 2013). We therefore fractionated cells into a membrane‐enriched and cytosolic fraction (Mancia et al, 2008) and detected the truncated 5HT2C using our antiserum and the full‐length 5HT2C using a polyclonal anti‐GFP antiserum. The truncated receptor is found both in the membrane‐containing pellet and cytosolic fractions. Oligo#5 decreases the amount of the truncated 5HT2C receptor protein found in cytosolic fractions (Fig 4C and E). The full‐length receptor is found only in the membrane associated fraction, and its expression is increased about 2.5‐fold after oligo#5 treatment, reflecting the change in splice site selection (Fig 4D and E). The 5HT2C–GFP fusion protein migrates as several bands on Western blot, which likely reflects differential states of glycosylation (Backstrom et al, 1995). The data indicate that changes at the RNA level are reflected at the splicing level. Furthermore, a fraction of the truncated receptor appears to be only loosely associated with membranes, as it is found in the membrane‐free supernatant, which could be due to the lack of four transmembrane domains.
Next, we analyzed the effect of oligo#5 on the localization of the full‐length receptor using stable cell lines. Transfection of 5 nM oligo#5 resulted in an accumulation of GFP signal on the cell surface, whereas the control oligo had no effect (Fig 4F and G). The GFP signal on the cell surface rose from about 35 to 55% in the presence of oligo#5 (Fig 4H). Western blotting analysis showed a relative increase of the protein ratios of full‐length to truncated receptor (Fig 4C–E). Thus, in agreement with earlier findings using cDNAs (Martin et al, 2013), the data show that oligo#5 causes an accumulation of the full‐length receptor on the cell surface likely by reducing the amount of the truncated receptor isoform that sequesters the receptor intracellularly.
Oligo#5 enters neurons after intracerebroventricular injection
To test the effect of oligo#5 in vivo, we first determined whether the oligonucleotide is taken up by neurons in mouse brain. To circumvent the blood–brain barrier, we performed intracerebroventricular (ICV) injections of oligo#5. We injected 2 μg oligo#5 5′ end‐labeled with Cy3 into the 3rd ventricle of male C57BL/6 mice (Fig EV3). Oligo#5 uptake was determined by confocal microscopy in coronal brain sections. We observed accumulation of oligo#5 in areas adjacent of the 3rd ventricle (Fig 5A), including the arcuate nucleus (Fig 5B). Similar to other oligonucleotides with a 2′‐O‐methyl‐phosphothioate chemistry, oligo#5 was taken up by the cells without adjuvant, which we also observed in cell culture (Fig EV2). Immunohistochemisty with the neuronal marker NeuN showed that the oligonucleotide was detectable in neurons, as well as NeuN‐negative cells, which are likely to be glia (Fig 5C). To determine the uptake of oligo#5 in glia we stained brain sections with GFAP (glial fibrillary acidic protein) after oligo#5 injection and detected oligo#5 staining in some, but not all glial cells (Fig 5D). It is thus likely that oligo#5 enters most cells in the CNS after injection.
Our transfection data indicate that oligo#5 changes the alternative splicing of the 5HT2C (Figs 1C and 4B), and we therefore tested its effect on the endogenous 5HT2C after injection. We microdissected the area in the ventral part of the 3rd ventricle that contained the arcuate nucleus (Fig 5A and B), and performed qRT–PCR on the isolated RNA. Using primers against the 5HT2C, we determined the expression of RNA1 and RNA2 and observed a strong increase of RNA2 after 3 and 6 h (Fig 5E). In contrast, there was no effect on the serotonin 4 receptor (Htr4), showing the selectivity of oligo#5 (Fig EV4). In all injection experiments, we used an oligonucleotide against human SMN2 as a control (Seo et al, 2014). The SMN2‐20mer oligonucleotide had the same 2′‐O‐methyl monophosphothioate chemistry as oligo#5. Oligonucleotides with this chemistry are taken up by cell without adjuvants (Heemskerk et al, 2009), and SMN2 oligonucleotides with the related 2′‐O‐(2‐methoxyethyl) phosphorothioate chemistry were previously shown to enter spinal cord neurons after epidural injection in mice (Hua et al, 2010). SMN2 is not expressed in mice, and thus, the SMN2 oligonucleotide has no specific target and controls for unspecific effects of injection.
To analyze the editing patterns of 5HT2C transcripts, we analyzed cDNA corresponding to exon Vb inclusion using deep sequencing (Fig 5F). Similar to the situation in cell culture, we did not observe a change in editing of 5HT2C RNAs (Fig 3A and B), suggesting that oligo#5 acts mainly on the RNA2/RNA1 ratio (Fig 5E and F).
These data suggest that oligo#5 changes alternative splicing of the endogenous 5HT2C and promotes inclusion of exon Vb, as expected from the in vitro experiments. These changes increase the amount of non‐edited 5HT2C, which encodes a serotonin receptor that is active without ligand, both in cell culture (Wang et al, 2000; Werry et al, 2008; Labasque et al, 2010) and in vivo in spinal cord neurons (Murray et al, 2011). Activation of the serotonin receptor in the arcuate nucleus results in an increase of POMC expression (Valassi et al, 2008; Xu et al, 2008). We therefore measured POMC RNA levels in the ventral part of the 3rd ventricle and found a significant increase in POMC RNA expression after three to 6 h post‐injection (Fig 5G).
Oligo#5 decreases food intake in mice
An increase in POMC expression is expected to decrease food intake through the activation of MC4R receptors (Cone, 2005). We thus investigated the effect of oligo#5 on food consumption by measuring total food intake (Fig 5H). We used C57BL/6 mice that had no access to food for 16 h as a model for hyperphagia. After surgical implantation of a guide cannula and a recovery period, the mice had no access to food for 16 h. Then, we injected the oligonucleotide with the mice having free access to food, and their food consumption was measured. Oligo#5 reduced food intake by about 70% when compared to animals that just received the cannula surgery or both the cannula surgery and a control oligonucleotide against human SMN2 (Fig 5H).
To account for possible changes resulting from food withdrawal, we investigated ob/ob mice as a genetic model for hyperphagia. Ob/ob mice lack leptin and are hyperphagic (Zhang et al, 1994). Throughout the experiments, the mice had free access to food. Similar to fasted, wild‐type mice, oligo#5 injection reduced food uptake by about 75% in these mice (Fig 5I).
To further test the involvement of the melanocortin system in oligo#5 action, we injected oligo#5 into MC4R receptor knockout mice (Srisai et al, 2011). POMC is processed into α‐MSH, which acts on the MC4R receptor, leading to an anorexic response. Using these mice, we did not see a significant reduction in food intake (Fig EV5), further suggesting that oligo#5 works by activating POMC neurons (Fig 5G).
In summary, oligo#5 reduces food intake in fasted (i.e. hungry) mice and in genetically altered, hyperphagic mice and likely acts by stimulating POMC expression.
Oligo#5 reduces food intake after systemic injection
We next tested the effect of oligo#5 after systemic delivery into the blood. Previous studies showed that, in general, oligonucleotides do not cross the blood–brain barrier (Banks, 2009). However, the highest expression of the 5HT2C is in the choroid plexus (Esterle & Sanders‐Bush, 1992), the structure that generates cerebrospinal fluid from blood. In the choroid plexus, almost exclusively RNA1 is expressed (Kishore & Stamm, 2006). To bypass possible liver clearance, we injected the Cy3‐tagged oligonucleotide via a carotid artery catheter directed toward the brain. Unexpectedly, we saw an accumulation of the oligonucleotide in the brain after 1 h of injection (Fig 6A). As expected, the strongest staining was in the choroid plexus that has the greatest exposure to the systemic circulation and showed the highest 5HT2C expression. In addition, we saw staining in the epithelium surrounding the ventricles and unexpectedly in the retrosplenial granular cortex. Delivery through carotid injection results in a more widespread distribution of oligo#5 in the brain than ICV injection into the third ventricle. This is likely due to the 50‐ to 100‐fold higher amount of oligo#5 injected and could reflect oligo entry through each of the four choroid plexuses. To determine a possible effect on 5HT2C processing, we microdissected the region around the 3rd ventricle and analyzed 5HT2C splicing using real‐time PCR (Fig 6B). Again, we found an increase of the RNA2/RNA1 ratio, suggesting that oligo#5 influences 5HT2C splicing after crossing the blood–brain barrier.
We next tested the effect of oligonucleotide injection on food uptake in metabolic cages using mice that had free access to food all the time. We found an about 80% reduction compared to a control SMN2 oligonucleotide after 12 h (Fig 6B). There was no difference in water uptake, the mobility and activity between mice that received control oligonucleotide or oligo#5 (Fig EV6). The effect of oligo#5 injection persisted over 3 days (Fig 6D). Together, the data show that oligo#5 reduces food uptake after systemic injection, likely after crossing the blood–brain barrier and changing serotonin receptor splicing in the brain.
The 5HT2C protein is a validated anti‐obesity drug target that is exploited by the approved agonist locaserin (Miller, 2005). Two major isoforms of 5HT2C‐mRNA are formed as a result of alternative splicing: RNA1 encoding a truncated receptor isoform and RNA2 containing the alternative exon Vb that encodes a full‐length receptor (Fig 6E). RNA1 encodes a truncated receptor that is associated with intercellular membranes. This truncated receptor sequesters the full‐length receptor inside the cell, which strongly decreases the serotonin response (Martin et al, 2013). The loss of a trans‐acting splicing regulatory RNA, SNORD115, that promotes exon Vb inclusion contributes to Prader–Willi syndrome (PWS) (Kishore & Stamm, 2006), suggesting that a dysregulation of 5HT2C pre‐mRNA processing could result in obesity. To identify therapeutic approaches that might substitute for SNORD115, we performed an oligo walk using RNA‐based oligonucleotides containing 2′O‐methyl and monophosphothioate modifications as they are RNAse resistant, are taken up by cells without adjuvants, and show, in contrast to DNA oligonucleotides, no RNAseH activity (Bennett & Swayze, 2010; Hua et al, 2010).
We identified oligo#5 located in the intron downstream of the alternatively spliced exon Vb. Oligo#5 promotes exon inclusion without RNA editing, suggesting that the pre‐mRNA can undergo one of three choices: (i) either include exon Vb in an unedited form or (ii) edit exon Vb, which results in weakening an RNA structure that blocks the proximal splice site to produce an edited mRNA; or (iii) skip exon Vb. In the cell, all pathways are present and likely compete for the pre‐mRNA. Oligo#5 favors the first pathway and increases exon Vb inclusion without editing to promote full‐length receptor formation, but decreases the truncated 5HT2C isoform formation.
Skipping of exon Vb generates a truncated protein isoform that contains only three transmembrane regions. Using a novel antiserum directed against the truncated protein, we demonstrate that this receptor isoform is expressed in the brain, as predicted from its cDNA analysis. A potential physiological role for the truncated receptor isoform emerged only recently. The truncated isoform is localized to internal membranes, and through heterodimerization, it sequesters the full‐length receptor in intracellular sites, reducing serotonin receptor signaling (Martin et al, 2013). Using cell fractionation, we provide additional evidence for this model, as we found that the full‐length receptor is only detectable in membrane fractions, whereas the truncated receptor also is present in the cytosol, indicating a weaker association with membranes. Through heterodimerization, the short splicing variant acts like a dominant negative modulator of signaling through the 5HT2C receptor. Regulating the balance between truncated and full‐length isoforms therefore changes the physiological response of the receptor. Oligo#5 reduces the amount of the truncated isoform and increases the amount of the full‐length receptor protein. The oligo thus changes the relative ratio of the two proteins, without completely abolishing the production of the truncated receptor isoform. Using an EGFP‐tagged splicing reporter resembling the physiological system, we observed accumulation of full‐length receptor on the cell surface in cultured cells after oligo#5 treatment, which likely increase serotonin signaling.
We further tested this model in mice by injecting oligo#5 into the third ventricle. The arcuate nucleus that harbors POMC‐positive neurons expressing the 5HT2C receptor is naturally open toward the cerebrospinal fluid and allows, for example, entry of peptide hormones, such as insulin and ghrelin (Rodriguez et al, 2010). As we observe accumulation of oligo#5 in the arcuate nucleus, it is likely that oligo#5 follows the same uptake route as peptide hormones. Oligo#5 injection increased RNA2, as predicted from the transfection experiments and increased POMC expression. This could explain the observed reduction in food intake, as POMC is processed to α‐melanocyte‐stimulating hormone (α‐MSH), resulting in an anorexic response by mainly activating melanocortin 4 receptor‐containing neurons (MCR4) in the paraventricular nucleus. In agreement with this model, oligo#5 had no statistically significant effect in MCR4 knockout mice (Srisai et al, 2011), (Fig EV5). However, since the full‐length 5HT2C receptor heterodimerizes with other seven‐transmembrane receptors (Schellekens et al, 2013, 2015), it is possible that oligo#5 affects the surface localization of unknown receptor systems, which could contribute to the anorexic response. Oligo#5 had no effect on water uptake or general activity of the animals (Fig EV6), suggesting that the oligo modifies a specific pathway, rather than causing a general sickness that stops food intake.
Finally, we tested oligo#5 by systemic injection, using mice with a catheter in the common carotid artery. Low dosages of oligo#5 showed no statistically significant effect on food uptake (data not shown), but injection of 50 or 100 μg reduced food intake. As the effect on food uptake was unexpected, we repeated the experiments using Cy3‐labeled oligonucleotide and observed rapid accumulation of the oligonucleotide in the brain. The strongest accumulation was in the choroid plexus, a structure that generates cerebrospinal fluid from blood (Mortazavi et al, 2013). Since the choroid plexus expresses high levels of RNA1, it is possible that oligo#5 binds to 5HT2C pre‐mRNA expressed in ependymal cells and follows the secreted CSF. This transport would follow the concentration gradient between blood and CSF and is facilitated by the ability of oligo#5 to enter cells without adjuvants. In this model, the 5HT2C pre‐mRNA acts like a receptor for oligo#5. It is possible that the known in vivo secondary structure of the receptor helps in this interaction, as it is likely more stable than unstructured pre‐mRNAs.
Similar to oligo#5, SNORD115 promotes inclusion of exon Vb into the pre‐mRNA, reducing the amount of the truncated receptor isoform. SNORD115 is absent in subjects with PWS, suggesting that a deregulation of the 5HT2C pre‐mRNA splicing, in particular, an increase of the truncated receptor isoform, contributes to PWS. As the full‐length 5HT2C heterodimerizes with other receptors, such as the ghrelin receptor (Schellekens et al, 2013), it could indirectly regulate other receptors involved in food uptake, which would amplify the effect of the truncated isoform. Thus, a deregulation of 5HT2C splicing caused by the loss of SNORD115 could contribute to a hormonal syndrome seen in PWS. The basis of this regulation is a stable double‐stranded RNA structure in the 5HT2C pre‐mRNA. This structure can be targeted by small‐molecular‐weight substances like pyrvinium pamoate (Shen et al, 2013) or as we showed here, an oligonucleotide. The 5HT2C pre‐mRNA thus forms a promising drug target to treat hyperphagia seen in PWS and possibly to treat non‐genetic forms of obesity.
Materials and Methods
Cell culture and transfection
HEK293T cells (ATCC) were cultured in DMEM containing 10% (v/v) fetal bovine serum (Invitrogen). RNA oligonucleotides and plasmid DNA were transfected into cells with calcium phosphate as described previously (Kishore et al, 2010). About 50 ng of RNA nucleotide and 100 ng of DNA plasmid were transfected into 1 × 106 cells. Serotonin 2C receptor pre‐mRNA splicing analysis was performed as described (Kishore & Stamm, 2006).
Generation of stable cell lines
An established Flp‐in HeLa cell line was a gift from Dr. Jørgen Kjems (Aarhus University, Denmark). The generation of a Flp‐in HeLa cell line stably expresses GFP‐5HT2C as described in Damgaard et al (2012). The stable cells were maintained in medium as described previously with 600 μg/ml hygromycin.
Construction of plasmids
The serotonin 2C receptor (5HT2C) splicing reporter construct used in transfecting cultured cells was described before (Kishore & Stamm, 2006). To generate a reporter construct for in vitro splicing, a DNA fragment including exon V (201nt) and 5′ end of intron V (177 nt) from the above construct was PCR amplified with primers 5′‐Exon5(kpnI) and 3′‐Intron5(BamH1). The fragment was then used to replace the first exon in MINX splicing reporter gene.
To generate 5HT2C fused with a C‐terminal GFP construct, the first DNA fragment containing exon 3, exon 4, and partial exon 5 was PCR amplified from human 5HT2C cDNA construct with primers 5′‐Exon3(NheI) and 3′‐Exon5(BglII). The fragment was then subcloned into pEGFPN3. The second DNA fragment includes the rest of exon 5 and partial intron 5 (1.5 kb from 5′ end). It was PCR amplified from HTR2C splicing reporter construct (Kishore & Stamm, 2006) with primers 5′‐Exon5(BamH1) and 3′‐Exon6. The PCR product was then subcloned with BamH1 and BglII following the first fragment into pEGFPN3. The third fragment includes 3′ end of intron V (1.6 kb) and the encoding sequence in exon VI. It was PCR amplified with primers 5′‐Intron5(Bglll) and 3′Exon6(BamH1). The third fragment was inserted into pEGFP3 after the second fragment.
For stable cell line generation with Flp‐in system, the 5HT2C–GFP fusion fragment described above was subcloned between NheI and NotI into the pcDNA5_FRT construct (Invitrogen Flp‐in system. DNA constructs are available from Addgene, Cambridge, MA, Deposit number 73069.
In vitro splicing assay
32P‐labeled pre‐mRNAs were in vitro transcribed using an SP6 polymerase after linearizing the HTR2C‐MINX construct. The substrates were incubated at 30°C with 40% HeLa nuclear extracts under standard splicing conditions as described in (Mayeda & Krainer, 2012). The RNA splicing products and intermediates were separated on 5% denatured polyacrylamide gels and visualized with a phosphorImager (GE Typhoon). The sequence of the in vitro splicing construct was as follows: Exons are in capital letters, the SP6 promoter is in italic, the transcriptional start site is in capital and bold, and the intronic and exonic part of the adenovirus derived MINX construct is underlined. The oligo#5 binding site is double underlined.
Development of an antiserum against RNA1
Rabbits were immunized using Ac‐SSYPCDWTEGRRKGV‐Ahx‐[K‐HZ]‐amide; Ac‐SSYPCDWTEGRKQSV‐Ahx‐[K‐HZ]‐amide; HZ‐Ahx‐SSYPCDWTEGRRKGV‐amide and HZ‐Ahx‐SSYPCDWTEGRKQSV‐amide. The underlined sequence SSYPCDWTEGR is common to both mouse and human, and the remaining RKGV are specific for human and KQSV are specific for mouse. After six immunizations, the last three boosts containing complete Freund's adjuvants, an antiserum specific for RNA1 was obtained, which as affinity purified using the immunization peptide.
Antibodies used
Anti‐GFP was from Santa Cruz, sc‐8334 (rabbit polyclonal), NeuN from Millipore, MAB377, clone A60. All antibodies were used at a 1:1,000 dilution in PBS/0.1% Tween‐20.
Detection of serotonin 2C receptor isoforms using Western Blot
About 5 × 105 HeLa cells that stably express GFP‐5HT2C were seeded into 6‐well plates. The next day, control oligo#3 and oligo#5 were added to the cells at a 5 nM final concentration. The cell fractionation into membrane and cytosolic fractions was according to Mancia et al (2008). Forty‐eight hours after transfection, the cells were washed with cold PBS and were lysed in CHS buffer (20 mM Hepes pH 7.5, 150 mM NaCl, and 0.1% w/v cholesteryl hemisuccinate in 1% w/v n‐dodecyl‐β‐d‐maltopyranoside, Sigma) for 30 min on ice. The soluble fraction, enriched in cytosol, was transferred to a fresh tube after centrifugation of 10 min at 13,500 g, 4°C. The insoluble fraction enriched in membranes was dissolved in 1× SDS loading buffer. About 20 μg of protein was loaded on a 12% SDS–PAGE gel.
Cell imaging
Stable GFP‐HTR2c cells were seeded onto sterilized cover slips placed to the bottom of tissue culture plates. Oligo#5 was then added to final concentration of 5 nM. Oligo#3 was used as control. After 48 h, the cells were washed with PBS and fixed with 4% paraformaldehyde for 5 min. The cells were then incubated for 10 min with PBS containing 0.25% Triton X‐100 and stained with DAPI. The cells on cover slips were analyzed by confocal microscopy (Nikon A1R‐A1 Confocal AQ5 Microscope System).
Mouse injection and food uptake measurement
Adult male mice (9–12 weeks old) were acquired from Jackson Laboratory. All mice were maintained on a 14/10‐h light/dark cycle with food and water provided ad libitum. Animal work was approved by the IACUC from the University of Kentucky Protocol Number: 2011‐0841.
The mice were stereotaxically implanted with a guide cannula (Plastics One) to the coordinates of 1.82 mm posterior to the Bregma, 4.5 mm below the surface of the skull, and 0 mm lateral to midline. The cannula tip is located above the 3rd ventricle. After the surgery, the mice were individually housed and deprived of food with continuous water supply. The injection was performed 24 h after cannula implant. An infusion cannula with 1.0 mm projection (Plastics One) was inserted through the guide cannula and used to infuse 2 μl (1 μg/μl) of RNA nucleotides over 2 min. Feeding was resumed after the injection. The body mass and food intake were recorded at the designated time points.
Nine‐week‐old C57BL/6 mice with a polyurethane/polyethylene carotid artery catheter were purchased from Charles River, (Charles River, INC). The catheter was inserted into the common carotid artery with the catheter tip directed toward the brain. About 50–100 μg oligonucleotide (10 μg/μl) was infused over 3–5 min. Dosages lower than 31 μg showed no significant effect (1, 2, 10, 20, and 30 μg were tested).
Ob/ob mice B6.Cg‐Lepob/J were acquired from the Jackson Laboratory, Bar Harbor, Maine.
Statistical analysis
All the data obtained were from at least three independent experiments. Data were analyzed with Microsoft Excel version 14.0.7166.5000 (32‐bit) and expressed as the mean ± SD (standard deviation based on the entire population given as arguments, STDEVP function in Excel). The statistical differences were calculated using Student's t‐test, and the difference was considered to be statistically significant at values of P ≤ 0.05. The number of samples analyzed and the P‐value are indicated in the legends of each figure.
Three micrograms of total RNA, 2 μM oligo‐dT, 1 mM dNTPs, and H2O were mixed and heated to 65°C for 5 min and quickly chilled on ice. 1× Reverse transcription buffer and 20 mM of DTT were added to the reaction. The reaction was incubated 42°C for 2 min and 200 U of SuperScript II reverse transcriptase was subsequently added to the reaction, in a total volume of 20 μl. The reverse transcription was performed under the following conditions: 50 min at 42°C and 15 min at 70°C. One‐tenth of the RT reaction was used for the qPCR reaction. The reaction was performed in 20 μl that contained 1× SYBR Green SuperMix (Quanta Bioscience) and 0.5 mM of gene‐specific primers. The amplification was carried out in a Stratagene Mx3005P Thermocycler (Agilent Technologies) using the following conditions: initial denaturation for 10 min at 95°C, 40 cycles for 15 s at 95°C, and an extension of 1 min at 60°C. The relative expression was estimated as follows: 2Ct(reference) − Ct(sample), where Ct (reference) and Ct (sample) were GAPDH and target genes from same RT reaction, respectively. For each experiment, at least three animals were analyzed.
qPCR primers
mGAPDHFor1: TCGTCCCGTAGACAAAATGG
mGAPDHRev1: TTGAGGTCAATGAAGGGGTC
Htr2cEx4 For1: ACTTGTCATGCCCCTGTCTC
mHtr2cExVb Rev1: CCTTAGTCCGCGAATTGAAC
mHtr2cEx4 F: CCATTGCTGATATGCTGGTG
mRNA1junc R: ACTGAAACTCCCGGTCCAG
POMC primers:
mPOMC‐F1: GAGTTCAAGAGGGAGCTGGA
mPOMC‐R1: CTTGATGATGGCGTTCTTGA
Serotonin receptor Htr4
mHtr4‐F1: TGCTATCACCTGCTCTGTGG
mHtr4‐R1: CTGCCTTGGTCTCTGTCCTC
RNA antisense oligonucleotide synthesis
Complementary 18‐base RNA antisense oligonucleotides targeting 5HT2C gene exon 5 and intron 5 junction region for the RNA oligo walk were synthesized and HPLC‐purified from Trilink BioTechnologies (San Diego, CA). All the nucleotides were 2′‐O‐methyl‐modified bases with a monophosphothioate backbone, containing one sulfur atom per phosphate group. The oligonucleotide sequences were as follows:
List of primers for PCR
To clone of HTR2C‐MINX construct
5′‐Exon5(kpnI): CCTCGGTACCATTATGTCTGGCCACTACC
To clone the pEGFPN3‐HTR2C construct
5′‐Exon3(NheI): CATACGGCTAGCCACCATGGTGAACCTGAGG
3′‐Exon5(BglII): CTTGGCAGATCTAGGTAGTGGCCAGAC
5′‐Exon5(BamH1): CATACGGGATCCTTGTGCCCCGTCTGGATTTC
3′‐Exon6: CGTCCCTCAGTCCAATCACAG
5′‐Intron5(BglII): CTAGAGCGGCCGCATCAAGATCCC
3′‐Exon6(BamH1): CTTAGCGGATCCCACACTGCTAATCCTTTCGC
The paper explained
Obesity is a growing health problem, caused by a behavior resulting in caloric intake that exceeds caloric expenditure over longer time periods. Prader–Willi syndrome is a genetic form of obesity, caused by hyperphagia and the inability to reach satiety. The serotonin 2C receptor in proopiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus plays an important role in food uptake behavior. Serotonin 2C receptor activation results in an induction of POMC, which is processed into α‐melanocyte‐stimulating hormones (α‐MSH, member of the melanocortin peptide family). α‐MSH activates the melanocortin 4 receptor (MC4R) in the paraventricular nucleus, leading to an inhibition of food uptake. The serotonin 2C receptor undergoes pre‐mRNA splicing resulting in a full‐length receptor and a truncated receptor, depending on the usage of the alternative exon Vb. In contrast to the full‐length receptor that is present on the cell surface, the truncated receptor is found only intracellularly and can sequester the full‐length serotonin 2C receptor. In addition to alternative splicing, the serotonin 2C receptor pre‐mRNA undergoes RNA editing, which changes the protein sequence of the full‐length receptors. The non‐edited receptor shows constitutive activity, which is strongly reduced by RNA editing. The inclusion of exon Vb resulting in a non‐edited full‐length receptor is promoted by a neuron‐specific trans‐acting RNA, SNORD115. SNORD115 is not expressed in subjects with Prader–Willi syndrome. As a possible therapeutic approach, we sought to find a substitution strategy for SNORD115 and tested its effect on food uptake in mice.
Using oligonucleotides complementary to the regulated part of the serotonin 2C receptor pre‐mRNA, we identified an oligonucleotide (oligo#5) that promoted inclusion of exon Vb into the pre‐mRNA, thereby increasing the full‐length, non‐edited receptor isoform. Oligo#5 binds to an intronic region and will thus not interfere with translation. Injection of oligo#5 into mouse brain increased exon Vb usage and POMC mRNA concentration in the arcuate nucleus, which likely caused the subsequent reduction in food uptake. Using a new antiserum, we showed that the truncated serotonin 2C receptor is expressed as a protein and that oligo#5 increases the ratio of full‐length to truncated receptor proteins, leading to an accumulation of the constitutively active full‐length receptor at the cell surface. Unexpectedly, delivery of oligo#5 through the carotid artery resulted in its accumulation in the brain, an increase of the full‐length receptor isoform and a reduction in food uptake that persisted for 3 days after injection.
The serotonin 2C receptor is a validated anti‐obesity drug target, but similarities with other serotonin receptors on the protein level prevented widespread use of its protein agonists as appetite suppressors. Targeting its RNA through oligonucleotides could be a viable alternative, as the oligo#5 sequence is unique in the genome and thus will be more selective. The oligo#5‐induced changes in serotonin 2C receptor splicing isoforms had a profound effect on food intake, which underscores the role of alternative splicing variants in food regulation. Since the serotonin 2C receptor heterodimerizes with other G‐coupled protein receptors, the regulation of its surface concentration through alternative splicing could impact on other receptors, which could help in integrating food uptake with other behavioral programs. Finally, oligo#5 mimics the action of the natural exon Vb activator, SNORD115, which is not expressed in Prader–Willi syndrome. This points to a deregulation of the serotonin receptor isoforms in Prader–Willi syndrome caused by SNORD115 loss as an underlying cause of the characteristic hyperphagia.
For more information
Author contributions
ZZ and MS performed the cloning experiments and injections; PJG, AGR, and MGL assisted in the mouse work; RBE and SS planned the experiments.
Conflict of Interest
The authors declared that they have no conflict of interest.
Expanded View
Expanded View Figures PDF [emmm201506030-sup-0001-EVFigs.pdf]
Source Data for Expanded View [emmm201506030-sup-0006-SDataEV.pdf]
We are very grateful to the NIMH/Vanderbilt Silvio O. Conte Center for support of these studies (P50 MH096972). This work also was supported by National Institutes of Health RO1 GM083187, R21HD080035, and P20RR020171 to S. S and the Foundation for Prader–Willi Research, and by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health (8 P20 GM103527‐06).
FundingNIHhttp://dx.doi.org/10.13039/100000002 P20RR020171
This is an open access article under the terms of the Creative Commons Attribution 4.0 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
- © 2016 The Authors. Published under the terms of the CC BY 4.0 license


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Iraq's ministry of transport is asking Boeing to accelerate the scheduled delivery of 55 new aircraft the country has on order.
The country needs those aircraft to restore links with the outside world as the nation returns to normalcy, government officials told Reuters.
The country's airline, Iraqi Airways, lost nearly all of its aircraft in the 2003 war, and it is seeking modern replacements.
The country has three Boeing aircraft models, the 737, 777 and 787, on order. The first of those is now scheduled for delivery in 2013. Iraqi Airways now owns four older Boeing 737s.
The airline wants to increase its schedule to connect Iraq with Europe, Asia and North America.
Source: The News Tribune

subset: commoncrawl


text:
Stock price quote for Zacks Dividend Fund Investor Cla, symbol ZDIVX, Fund.
Quote data is updated continously during trading hours.
and [Monthly] stock price charts are available for Zacks Dividend Fund Investor Cla
Market data delivered to TradingCharts.com, Inc. by DDF Plus.
Although it is believed that information provided is accurate, no guarantee is made.


text:
First Drive Mercedes Benz S400 Hybrid HybridCars com
Photo Gallery
Sorry are no photos!
The Mercedes Benz Hybrid is the world's first produced car with a lithium ion It's the first Mercedes a hybrid drive. And, a price tag likely to approach or $100,000, it's going to be an vehicle. The S400 Hybrid arrive in US showrooms beginning in 2009.
Its competition in the nascent green vehicle market be the $109,000 two-seat all-electric Roadster ; the $87,000 four-door Karma plug-in hybrid yet out); and the $104,000 Lexus LS L —a V8 hybrid averaging 21 miles to the Lexus annually sells 1,000 units of the LS 600h L.
We achieved 29.3 miles per in the Mercedes S400 Hybrid, a 150-mile mixed driving though Southern Germany.
estimates fuel economy for the will be 23 in the city and 33 on the highway. EPA are not yet official, but HybridCars.com did our own mileage of this big luxury sedan on the of Southern Germany. Our drive took us from Stuttgart—the of Mercedes Benz—to the town of and back, a round trip of 150 miles. The route was evenly of high-speed driving on the Autobahn—where we ran at, greater than 100 mph—as as moderately paced rural and narrow streets through hamlets. We achieved an impressive miles per gallon—not bad for a car that almost 5,000 pounds. (By the the V6 of the S400 is plenty strong for the
With much stricter limits in the US, it's reasonable fuel economy results be even better. But saving and money is not the first concern for of this car. The buyer of the hybrid is probably just as in pairing the Mercedes three-pointed emblem with a hybrid to demonstrate a combined interest in refinement, and green status. claims that the S400 is the champion in the luxury class," a relatively low 186 grams of carbon per
As the hybrid version of the Benz S-Class sedan, the S400 is by a mild hybrid system a 3.5-liter V6 gas engine. The hybrid allows Mercedes to maintain its profile, while using a V6 of a V8 or V12. The electric-gas combo a total output of 295 horsepower, through a seven-speed automatic Benz is particularly proud of it refers to as "compact" batter Engineers made this system as well-packaged and light as (Mercedes-Benz was able to fit the entire into the same space, at the base of the windshield, that housed the car's standard 12-Volt starter battery.) The weighs only about 120 more than the standard
Driving Manners
The S400 much smaller than it is. through the tight twisties of Black Forest, this cornered flatly and with It is as nimble and agile as its gas-powered Stability comes from Body Control, an active that continually adapts the tuning to the current driving The S400 features Torque Brake, as well as Crosswind both enhancing the sure-footedness of the Other high-tech advancements a pre-collision system, adaptive beam assist, lane assist, and radar-based cruise
The cabin of the car is as plush as the standard It shares all the same luxury including a new split-view driver which allows the front to watch a movie without the driver. There's also a View Assist" feature detects pedestrians on the road The only interior difference is the hybrid gauge showing the of power between engine, and regenerative braking.
The S400 the look of the S-Class. Signs of its facelift include a more grille, revised LED lighting at the and back, and new mirrors, tailpipes, and
The Mercedes Benz S400 is a luxurious, fuel-efficient saloon lots of driving appeal. its steep purchase price, obviously not for everyone—but it is one more for uncompromising affluent buyers a slightly greener ride.
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Sponsored By
News Now: Mosquito Warning, Peeps BeerSpring has arrived, but health officials are warning that mosquito season is here too. Plus, a Fort Worth brewery is offering beer infused with Peeps candy.
Health Officials Warn That Warmer Weather Means Mosquito SeasonIt's that time of year again. The temperatures are getting warmer and more people are heading outside. But health officials have a warning for North Texans. It's also mosquito season.
Dallas County Reports Second Human Case Of West Nile VirusThe patient is a resident of the 75214 zip code in Dallas.
To Help Reduce Mosquito Population, Scientists Unleashing MillionsWhile it may sound counter-intuitive, the scientists' plan will hopefully reduce the number of disease-carrying mosquitos, according to NPR's report.
Dallas County Reports First Human Case Of West Nile Virus This YearThe patient diagnosed with West Nile neuroinvasive disease, lives in the 75248 zip code of Dallas.
Dallas County Reports First Imported Case Of DengueThe 54-year-old resident of Dallas was infected during recent travel to India.
North Texas Mosquito Season Means Battling West Nile & ZikaIt's the first week of May and the start of mosquito season in North Texas. According to health officials, there are different safety concern this year because doctors are dealing with two dangerous viruses – West Nile and Zika.
New Mosquito Trap Smart Enough To Keep Just The Bad BugsA new high-techmosquito trap is promising to catch the bloodsuckers while letting friendlier insects escape — and even record the exact weather conditions when different species emerge to bite.
First Zika Case Transmitted By Mosquito In Texas ConfirmedThe Texas Department of State Health Services said Monday the patient is a resident of Cameron County, located on the border the state shares with Mexico.
Dallas County Health Reports 3rd West Nile Related DeathThe Dallas County Health department is reporting that a third victim has died from West Nile this year.
Spraying For West Nile Virus After 2nd Death In Dallas CountyA second person in Dallas County has died from the West Nile Virus. DCHHS officials say the victim was in his or her late 50s and had been previously diagnosed with West Nile Neuroinvasive Disease.
Lewisville Mosquito Trap Tests Positive For West NileA Lewisville mosquito trap has tested positive for West Nile Virus, it's the 17th positive sample collected in the city this year.


text:
Health and Social Care Sales Representative - Flexible
- Recruiter
- Strive Training
- Location
- West Midlands Region
- Salary
- The salary for this role is £20,000 per annum + commissions + travel expenses. OTE £45,000+
- Posted
- 29 Mar 2018
- Closes
- 29 Apr 2018
- Category
- Healthcare and Care
- Function
- Sales / Marketing
- Contract Type
- Permanent
- Hours
- Flexible
StriveCare makes Health and Social Care training simpler, faster, less disruptive and more productive.
With over 20 years of experience in health and social care training, we specialise in developing, running and managing quality training programmes that allow clients to completely focus on their business's operations and development. StriveCare draws on this extraordinary heritage to provide a portfolio of different training programmes into the Health and Social Care sector including the newly launched FastTrack NVQ Diploma in Health and Social Care.
We are looking for dynamic and passionate Sales Representatives who will be involved in the continuous growth of StriveCare and the provision of our Health and Social Care offerings. The salary for this role is £20,000 per annum + commissions + travel expenses. OTE £45,000+
This position is offered on a full time or part time basis.
It is a work from home and field based role.
You will undertake the following duties as part of this role:
- Responsible for generating leads and selling our Health and Social Care courses into care homes.
- Creating new and building on existing relationships with care homes to maximise both initial sales and repeats sales.
- Arranging meetings with care home managers, managing all correspondence effectively and efficiently until a sales proposal has been agreed.
- Responsible for achieving sales targets set.
- Ensuring that a detailed and accurate record is kept up to date for all sales prospects in our data management system.
- Working closely with the administration team to ensure that sales leads are handed over effectively for sign up.
- Manage your diary efficiently, to ensure regular contact and on-site visits with care home managers.
- To understand, use and promote our Health and Social Care courses.
Some experience in a care capacity would be ideal but not essential.
Proven track record of sales is required.


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For developing along with a JDBC, we must setup a environment of JDBC for example we are operating on windows platform.
Install J2SE 5.0 (JDK 5.0) from official website of java
We should the following environment variables to set as below
e.g. C:\Program Files\Java\jdk1.5.0_20\jre\lib.
e.g. C:\Program Files\Java\jre1.5.0_20\bin.
For a course running a database with a table is a query and solve.
Install a database which is better for you
CLASSPATH: variable to C:\Program Files\MySQL\mysql-connector-java- 5.1.8\mysql-connector-java-5.1.8-bin.jar.
The JDK involves a JDBC-ODBC Bridge driver which makes a Open Database Connectivity (ODBC) drivers accessible to programmers using a JDBC API.
There are 5 steps involved to connect java application with data base using a JDBC
The forName( ) method is used to register a driver class this technique is utilized to powerfully stack the driver class.
Syntax of forNAME( ) Method :
Public void static forName(String className)
throws ClassNotFoundException
The getConnection( )strategy for Driver Manager class is utilized to build up association with the database.
public static Connection getConnection(String url)throws SQLException
public static Connection getConnection(String url,String name,String password)
throws SQLException
The createStatement() technique for Connection interface is utilized to make explanation, the question of proclamation is mindful to execute inquiries with the database.
Syntax of createStatement() method:
public Statement createStatement()throws SQLException
The executeQuery() technique for Statement interface is utilized to execute questions to the database. This strategy restores the protest of ResultSet that can be utilized to get every one of the records of a table .
public ResultSet executeQuery(String sql)throws SQLException
The executeQuery() technique for Statement interface is utilized to execute questions to the database. This technique restores the protest of ResultSet that can be utilized to get every one of the records of a table.
Syntax of close() method:
public void close()throws SQLException
Types of JDBC Driver
JDBC Driver is a software module which enables java application to relate with the database. There are 4 types of JDBC drivers.
1. Bridge driver of JDBC-ODBC
2. Driver Native-API
3. Driver Network Protocol
4. Thin driver
The JDBC-ODBC connect driver utilizes ODBC driver to associate with the database. The JDBC-ODBC connects driver changes over JDBC technique calls into the ODBC work calls. This is presently demoralized in light of thin driver.
For connection of data base is easily associated. JDBC –ODBC Bridge DriverHindrances
2) Native-API driver
The Native API driver utilizes the customer side libraries of the database. The driver changes over JDBC technique calls into local calls of the database API. It is not composed completely in java.
Execution updated than JDBC-ODBC connect driver.
3) Network Protocol driver
The Network Protocol driver utilizes middle ware (application server) that proselytes JDBC calls straightforwardly or by implication into the merchant particular database convention. It is completely composed in java.
No customer side library is required in light of use server that can perform many errands like reviewing, stack adjusting, logging and so on.
4) Thin driver
The thin driver changes over JDBC calls straightforwardly into the merchant particular database convention. That is the reason it is known as thin driver. It is completely composed in Java dialect.
Driver Manager:
The DriverManager example of true excellence as an interface among client and drivers. It monitors the drivers that are accessible and handles setting up an association between a database and the proper driver. The DriverManager class keeps up a rundown of Driver classes that have enlisted themselves by calling the strategy DriverManager.
DriverManager Methods:
Connection interface:
A Connection is the session between java application and database. The Connection interface is a processing plant of Statement, Prepared Statement, and DatabaseMetaData i.e. protest of Connection can be utilized to get question of Statement and DatabaseMetaData.
The Connection interface give numerous strategies to exchange administration like submit() rollback().
Methods of Connection Interface:
Days were moving very quickly. With days, technology is moving simultaneously. Today technology has bot the advantages and disadvantages. Moreover, today data is the heart of any company.
Today many people were enthusiastic, to know the exact details of things happening around him. This can get the proper knowledge on Blockchain.
Python is a dynamic interrupted language which is used in wide varieties of applications. It is very interactive object oriented and high-level programming language.
Tableau is a Software company that caters interactive data visualization products that provide Business Intelligence services. The company's Head Quarters is in Seattle, USA.
Pega Systems Inc. is a Cambridge, Massachusetts based Software Company. It is known for developing software for Customer Relationship Management (CRM) and Business process Management (BPM).
Workday specializes providing Human Capital Management, Financial Management and payroll in online domain.It is a major web based ERP software vendor.


text:
At our first Council meeting of the year on 19 February I successfully moved a resolution requesting the CEO to initiate an audit of all City of Sydney parking signage with the aim of identifying clutter and complexity. Following the audit Council will, as soon as practicably possible, remove all unnecessary parking signage and simply remaining signage.
This initiative is designed to cut Sydney's unnecessary street signage clutter and I look forward to sharing the results of the audit with you as soon as they become available.
Check out the final motion that was carried at the meeting:


text:
A new generation of the popular crossover is close. Fans are excitedly awaiting the news about every detail. Nevertheless, it seems like managers decided that won't make them so happy. The 2019 Jeep Grand Cherokee will get an only minor refresh, and major upgrade comes probably with 2021 model. However, enthusiasts will have a chance to obtain some clues what to anticipate from future SUV. Some parts will be tested on upcoming model, while the majority of other features and details stay the same. Lastly, Jeep can make a surprise with some new trims and higher-end models, to compete with major competitors-- Infiniti QX70 and VW Touareg.
2019 Jeep Grand Wagoneer Review, Interior, Release Date, Price and Photos
American carmaker Jeep is well-known of its off-road vehicles. Beside Wagoneer and Grand Cherokee, this carmaker is planning 2019 Jeep Grand Wagoneer as a mid-cycle refresh. There are currently few info about it. Inning accordance with early reports, traditional appearance of this SUV will get some styling options from other models. With it, we will see modern crossover. As for specs, we don't have doubts. Jeep is a leader in lots of segments, and they are holding position since of the quality and innovations. New Grand Wagoneer is going to update this credibility with new features and devices.
2018 Jeep Truck Review, Design, Features, Release Date, Price and Photos
We expect to see many brand-new models introduced this year, but 2018 Jeep Truck will definitely be a few of the most interesting ones. This will be an entirely new model, various than other models from Jeep brand name. It will be a pickup. This is something we didn't see for a very long time. Last pickup from Jeep remained in production several decades ago. New one will come based upon the brand-new Wrangler, which is likewise being available in future. Besides platform, these two models will share numerous other attributes. We anticipate to see very same design language, engines, interior and so on. As we already pointed out, new model should come later on this year, as 2018 Jeep Truck.
2018 Jeep Wagoneer Review, Design, Engine, Release Date and Photos
Jeep is chosen to make a brand-new intro through the market and there is no much better way than launching a new car. The brand-new 2018 Jeep Wagoneer is coming as a brighten vision of a reliable SUV which every devoted fan can get. This model is simply a start among the other competitive models on a market. With new redesigned parts as well as updated platform, This vehicle will close the retail SUV quality for a mow. Some critics state that new Jeep will have some resemblances with predecessor Grand Cherokee.
2018 Jeep Gladiator Review, Engine, Changes and Photos
Based on Wrangler, brand-new 2018 Jeep Gladiator adds excitement to new truck lineup. Upcoming season is going to be one of the most fascinating ever. Not just Jeep is coming out with numerous brand-new models, however likewise competition. That indicates only one-- buyers will be in advantage. Nevertheless, business must take care in market analysis. This could be decisively when the truck strikes the salons. New Gladiator is elegant and outstanding entertainer. This readies start, but purchasers got utilized to it. However, company is going to offer some brand-new features and option for 2018. Nevertheless, details are not offered yet, however pickup will share some parts with other models.
2018 Jeep Wrangler JT Review, Design, Engine, Price and Photos
If will be luck to discover a fan of truck section that is not delighted with news about 2018 Jeep Wrangler JT. Vehicle is getting updates and upgrades, and there is no doubt we can see something unique in a year or two. Comparing to current vehicle, there will be numerous new components. For example, more aluminum is now in the body of the truck, which results in better fuel economy, managing and total efficiency. Because we can currently see screening mules on streets, there is no doubt vehicle is coming soon. Nevertheless, lots of details are unidentified at this minute.
2018 Jeep Cherokee Review, Redesign, Engine, Release Date and Photos
Jeep engineers are preparing big changes for new Cherokee. Newest reports suggest this. Nonetheless, it will still be a small crossover all set to offer excellent performance in city area. Although big, adjustments become part of mid-cycle refreshment. However, new generation will bring total redesign. Till then, we are going to see outside refreshment, interior with more hi-tech features, and intro of brand-new engine. And on top of all that, cost is still just like existing model. With all this modifications, 2018 Jeep Cherokee will be more attractive. Company hopes outcome could be greater sales numbers in future.


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Photoshop 3D Text Retro Tutorial
List of tutorials for Photoshop 3D Text Retro
Create a Retro 3D Halftone Text Effect in Photoshop Photoshop
supercolortuts.com,53 Images, 866 words
In this Tutorial I will show you how to create a 3D Halftone Text Effect in Photoshop that can be used as a cool rock band cover using some cool flash shapes, the Text Tool and a lot of different layer styles. Let's get started! Open up Photoshop and..


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An exceedingly happy otter, bears caught in the act and a shocked seal feature in this year's Comedy Wildlife Photography Awards.
These are the amazing crisp shots of animals doing things you don't often see… and they're hilarious.
There were only five official winners across the categories, but many worthy competitors.
After 3,500 entries it was an owl photographed trying to get back onto its branch that scooped the top prize.
Winner Tibor Kercz was given a trophy handmade by disabled men and women in Tanzania and some more camera gear.
He also took home first prize in the Amazing Internet Portfolio category as well as a safari to Kenya, not bad.
Before winning the On the Land award, photographer Andrea Zampatti's picture of a laughing dormouse went viral on social media earlier this year.
And while the sea is a pretty big place, the winning photo in the Under the Sea category shows things can still get a little cramped down there.
The In the Air award was nabbed by photographer John Threlfal for his picture of a duck flying in front of a plane trail over the skies of Preston, Lancashire.
Here are pictures from some of the other finalists.


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Garden Easy Daybed
Leisure deluxe
Aspiring to create exclusive outdoor furniture for beautiful moments, we present our Garden Easy series. The range is distinguished by its generous size, slim lines and evident form. A Garden Easy lounge is luxurious, yet laid-back.
Röshults Garden Easy Daybed provides a perfect place to sunbathe, read, think, gather energy and take power naps. It creates a relaxing outdoor ambience while trendy and modern. Our popular daybed is a must for the conscious mind.
Designer: Broberg & Ridderstråle


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Drossinis Museum
|Agion Theodoron & D. Kyriakou St.,
Kifisia, Athens, Greece
|Public transit access
|Athens Metro (green line) station:
Kifisia metro station
|Official site
Drossinis Museum is in the center of Kifisia, a northern suburb of Athens, and it is housed in "Amaryllis" villa, where Georgios Drossinis[1] lived in his last years and which is named after a central character of one of his earliest and most popular works. The museum was founded in 1997 with the aim to preserve and promote Drossinis' (1859-1951) multidimensional work. Drossinis, along with Costis Palamas and Nikos Kampas co-founded the New Athenian School,[2] the Greek literary '1880s Generation', a movement which renewed Greek literature and fought for the establishment of modern Greek language ("Demotic")[3] The museum includes three halls, which are on the first floor, while the ground floor houses the Municipal Library of Kifisia, according to Drossinis's wish.
The museum[edit]
Going upstairs, the first hall on the right is the "Intellectual" Hall. Here lies Drossinis's intellectual treasure. Numerous exhibits demonstrate his significant presence in letters and his important contribution to education in Greece. Here one can find archived all issues of Estia newspaper, of which Drossinis was editor-in-chief and director and which circulated daily since 1894 and had a determining role in Greek life. In the same hall, the visitor can see a sample of Drossinis's work as a publisher: Asty, To Imerologion Tis Megalis Elladas ("Journal of Great Greece"), Nea Hellas ("New Greece"), Ethnike Agoge ("National Education") are some of the magazines and journals published by Drossinis, which aimed towards the intellectual rebirth of Greek people. In addition, there is a great number of books exhibited here which were either edited by Drossinis or published by the "Society for the Propagation of Beneficial Books" (Σ.Ω.Β.), of which Drossinis was a co-founder (along with Demetrios Vikelas) and a lifelong secretary. The poet's collaborations in Greek publications (Athinai, O Noumas, Scrip, Romios) or foreign ones – Cypriot, French, German and American – can also be found in this hall. Manuscripts, correspondence with Greek and foreign intellectual people, a collection of newspapers with articles about Drossinis and personal belongings, all complete the poet's intellectual personality.
The hall on the left is the "Emotional" Hall. It is Drossinis' bedroom, where the visitor has the feeling that the poet is constantly present. Photos of Mesolonghi, Drossinis's birthplace, on the walls and others of his friends and relatives, all complete the picture of the poet amongst his close ones. Fishing and hunting equipment reveal his great love of nature – Drossinis also wrote books about fishing and hunting – while religious icons express his deep faith. Children's reading books from all around the world – as he himself was an author of school reading books – show his care for education, to which he offered a lot from his high position in the Greek Ministry of Education. These objects, together with sculptures by Tombros and Vitsaris, surround the poet's figure as portrayed by the sculptor Angeliki Vlachopoulou sitting in his armchair at old age.
The third hall is the 'Folk' Hall. It is the largest part of the museum and the most interesting one, especially for the younger visitors. The heroes of his writings and his life are here: clay figures in real-life size – most works are by A. Vlachopoulou – represent vividly the world of his prose.[4] Idyllic, interested in folk tradition, rich in emotions, full of sensitive female characters: Amaryllis and Ersie – the heroines of his homonymous works – Morfoula – a tenant farmer's daughter in his family farm in Evia – whom he first met very young and for whom he composed his favourite poem, To Moiroloi tis Omorfis ("The Beauty's Lament"), and his cousin Drosina, who inspired his most popular poem, which was immediately set to music and is still sung today, Anthismeni Amygdalia ("Blooming Almond Tree"). Athanasios Diakos (the Greek Independence fighter) represents Drossinis's patriotic ideals, a grandfather sitting in the center of the hall, surrounded by his grandchildren listening to him reading fairy tales, shows his love for children – he himself also wrote fairy tales with educational content – while the small sofa on which he used to sit with Kate Manou, his last muse, signifies his passion for life until his latest years.
The "Friends of Drossinis Museum" Society[edit]
The 'Friends of Drossinis Museum' society, founded on the same year as the Museum itself, caters for the continuous renewal of the exhibits and the smooth functioning of the Museum, in collaboration with the Municipality of Kifisia.[5] The members of the association keep Drossinis's memory alive by organising events, publishing books,[6] which they send to all Greek libraries for free – especially school libraries – running competitions and being in charge of Museum tours. Finally, the association organises conferences on topics related to special needs, in accordance with Drossinis's sensitivity to these issues.
- ^ About Drossinis' life, see: Logotheti, F. (1960). Georgios Drossinis. Athens: Σ.Ω.Β. Publications.
- ^ See Garantoudis, E. (2007). 'The 1880s Generation', in Lexico Logotechnikon Oron [Dictionary of Literary Terms], Athens: Patakis Editions, pp. 365-367.
- ^ About the language issue, see: Beaton, R. (1996). Eisagogi sti Neoteri Neoelliniki Logotechnia [An Introduction to Modern Greek Literature]. Athens: Nefeli Publications, pp.369-449.
- ^ For Drossinis' complete list of works see: Meraklis, M. and Paradisi, E. (2007). Lexico Logotechnikon Oron [Dictionary of Literary Terms], Athens: Patakis Editions, pp. 561-562.
- ^ About the Association 'Friends of Drossinis' Museum', see: Five Authors Talk About G. Drossinis (collective work) (2001). Athens: Kameiros Εditions, pp. 108-120.
- ^ Some of the publications of the 'Friends of Drossinis Museum' Society are: Chronika [Chronicles] Vol. A –Z (2000 – 2009), Kairofylas, G. Georgios Drossinis, Konstandinidis, L. Mneme Georgiou Drossini [Memory of Georgios Drossinis], Anoiksi sto Mouseio [Spring in the Museum] (collective work), Pende Sygrafeis Miloun gia ton G. Drossini [Five Authors Talk About G. Drossinis] (collective work). For the complete catalogue of the Association, see: Anoiksi sto Mouseio [Spring in the Museum] (2012)


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Tuesday, August 10, 2010
Wordless Wednesday - "Can't I sleep a little longer, Momma?!"
12 comments:
this is so cute!!! and don't forget, if you need any help tweaking your blog or easy HTML tips, you know where to come!ReplyDelete
Check out today's trick;) Happy Wednesday!
Following from Welcome Wednesday. Would you please return the favor and follow me as well too? Thanks and have a lovely day!ReplyDelete
I love the idea of "Wordless Wednesday". and such a cute pic!!ReplyDelete
Ah, I feel the same way.ReplyDelete
- AnonymousAugust 11, 2010 at 5:09 PM
So cute!!ReplyDelete
I'm your newest follower from follow Me Wednesday.
Life of This SAHM
Hello we're your newest followers here with the log hops! You're puppy is absolutely adorable! And I loved those baby booties you had a picture of! So Cute! :)ReplyDelete
Feel free to drop by our blog anytime, we would love to have you! :)
Hello. I am a new follower here from Welcome Wednesday Blog Hop.ReplyDelete
Cute pic of you puppy =0)
Okay now he's just toooo cute!ReplyDelete
Great blog! Came across your blog from one of the blog hops, and looking forward to reading more of your stuff. I am now following ...feel free to follow back if you like!!!!
All the best,
Loving the blog! I design too and I found you thru NFF! I'd love for you to come check out what I've been designing too!ReplyDelete
xo - jami
i m a g i n e
What a cute dog! Check us out atReplyDelete
Super cute doggie :) I'm in FL too!ReplyDelete
I'm following you from Follow Me Chickadee Friday!
Hi Jackie,ReplyDelete
Your cake and outfits are adorable! I even love your dog!
I am a new follower through Mingle Monday.
- Shelli


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A guide to successful Design Handoffs
Mohammed Bilal shares some of his techniques and suggestions for a successful design handoff.
Intuitive Design vs. Shareable Design
While many consider Snapchat's UI unintuitive, it's definitely shareable. So what's the difference between intuitive and shareable design?
We also enjoyed...
Purposeful Design for User Permission
How to use purposeful design to encourage users to accept that pesky iOS notification permissions.
Interfaces On Demand
How platform changes have created entirely new businesses and business models.
How Channels, Devices, and Touchpoints Impact the Customer Journey
A look into the ecosystem of the customer journey and how having a full understanding plays an important role in the ability to make strategic improvements.
Tinder cofounder reveals how he came up with the app's game-changing 'swipe' feature
A behind the scenes look into the ideation of the infamous swipe gesture.
Complete your workflow
Atomic's plugin for Sketch lets you send your Sketch designs easily into Atomic and quickly turn them into an interactive prototype.
Thanks for reading! We love hearing from you – you can tweet us with your feedback, or recommend a product design story. Up for more? Browse the PDW archives.
"Product Design Weekly is my new favorite inbox treat."
— @jesseross


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Day 2 of filmingLonging to fly/Longing to fallPosted by Erika Janunger Sat, February 01, 2014 14:12:46
Today was also a gorgeous day! The fog came rolling in from the Baltic sea and it was a light frost. Not too cold at least, good since we were to perform outdoors today. Weather forcast for the day says sunny and cool untill noon and after that a light rain and slightly higher temperatures.
When we arrived on set, we were all amazed. The light and the fog was magical!
The location today was a wood chip factory located right by the water. SCA Bio norr. Standing on top of one enormous pile of sawdust, the view of the foggy bay was breathtaking, and the frost was really hightlighting the textures of the material. I could not believe our luck. Neither could photographer Johan.
Only thing was that the temperatures kept raising and by the time the dancers arrived, most of the frost was gone. However some of the fog remained and we decided to keep a lot of the landscape in view, even though the original plan was to only depict the strange cream coloured moon-landscape of the piles. It was just too damn pretty to leave out. Even the factory itself got some of the glory as it fit right into the playful mood we set for the filming today.
César and Stacy were fantastic to work with also on this set. They followed our playful instructions and just kept going even when we asked them to throw themselves down the sawdust pile. And they kept goinging even though a light drizzle came and turned the costumes into a smudgy wood/wool mashup and the temperature still wasent more than a few degrees above freezingpoint. Amazing, amazing people!
After a well deserved lunch break, the warmed- and dried up film crew continued to shoot indoors.
Last night we had changed the scenography from a tidy strict room into a messy one. It is intressting how long it can take to create a believable mess, especially when each seperate item has to be attatched to the wall or floor with a number of screws. It took us well over 4 hours.
With this new set the mood was still a cheerful one. For me, an untidy room represents something deliberating, a sidestep from the ordinary life and playfulness. It is a place where you are loosing control - and loving it.
The shooting this afternoon was good, no major incidents and the mood was slightly higher than yesterday due to the music we got to use for this set. Even though Im always in for a bit of melancholy, there are only so many shoots where you want to cry…


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How To Clean A Silk Bouquet
A Silk bouquet will last considerably longer than a bouquet made of real flowers, but, even so, it will still eventually droop,
and get dusty. With a little little bit of preventive care though, you can keep your bouquet looking like new.
Fortunately, caring for your flowers wont take long. 5 to 10 minutes spent caring for your bouquet every 3-6 months will keep
your bouquet looking as good as it did they day you made (or bought) it.
There are 3 simple steps to cleaning your bouquet:
Step 1: Rice Cleaning
Place your bouquet in a plastic bag, along with about half a cup of white rice. Twist the bag closed, and then shake the bag
for about a minute.
This works because the rice in the plastic bag will get statically charged, and the dust will be attracted to the rice. You
may have read that you can use salt instead of rice, but this is a fallacy! Salt is too hard, and will scratch your flowers.
Stick with white rice, which is softer, and won't damage the flowers.
Step 2: Fluffing
Once you have rice-cleaned your silk flowers, hold the bouquet by the handle and then turn it upside down and give it a few
good shakes, dropping the rice back into the bag. This will loosen any rice caught in the flowers and will also clear away
any left over dust. It will also "fluff" the flowers, giving them back their original shape.
Step 3: Plumping And Teasing
After fluffing the bouquet, it is time to move the flowers back into their correct positions. Using the back of your hand,
go around the back of the bouquet and lightly push the flowers back into position, to "plump up" the bouquet. Once you are
happy with the shape, then tease the flowers into the correct position by lightly pulling and pushing on the individual flowers
and petals until they are correctly placed.
Simple eh? I told you it wouldnt take long! Now, you may have read that you can lightly spray your bouquet with soapy water
to get it clean. Regardless of what you may have read, don't ever do this! Never spray your bouquet with water because the
wet dust will run and stain your flowers. And stained flowers are not very attractive!
If you follow these simple steps every 3-6 months, your bouquet will stay beautiful, and will be around in decades to come,
for future generations to enjoy.


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Writopia writer, Jessica Zalph (left), 14, developed a provoking blog post on vegetarianism (with a "dish of feminism") during her summer workshop with Writopia teacher, author, and popular blogger, Deborah Siegel (below, left). Jessica's piece is the first this summer to be posted on Deborah's blog. Check it out on Girl W/Pen and below!
October is Vegetarian Awareness Month. If only people knew about it.
By Jessical Zalph, 14
"Among men [vegetarianism is] regarded as, if not a girl thing, then at least a girlie thing — an anemic regimen for sensitive souls subsisting on rabbit food and tofurkey," says Holly Brubach in her recent New York Times article "Real Men Eat Meat." If the male gender sees vegetarianism as a "girl thing," then that's got to be our hardest obstacle to overcome. Whenever compassion and eating "rabbit food" became a girl thing, it became taboo for boys, because sexism is rooted so deeply in our society that girls are seen as weaker overall. But maybe making a harder decision wouldn't be weak at all. Maybe it'd be more macho, if that's what you're after, to overcome the stereotypes. Overcoming the expectations society has of you could be "manly," no?
I've been a vegetarian for the fourteen years of my existence – my parents stopped eating meat four years before I was born. They were told by a number of smug acquaintances that, just wait, I would become all "teenager-y" and start eating meat once I became obsessed with fitting in and defying my parents out of spite. We're still waiting.
Probably the reason I've stuck with vegetarianism and animal rights is because it's not just an arbitrary ritual I inherited, but is based on the unfortunate reality that the thing on the plate is the same as the cute little thing on the farm. I know I must have adopted this concept at an early age, because I recall feeling appalled fury at a boy in my preschool class who took the unsuspecting snails out of their tank and stepped on them.
Most of the attitudes I've encountered haven't seemed to change much over time.
"Vegetarians are stupid" is the bluntest of the accusations I've received – this one coming just recently in our eighth grade hallway from a guy flaunting an anti-Wendy's flyer, sparking the debate that flares up every now and again at school. It's only in hindsight that I realize that these heated I-wish-they-were-discussions-not-shouting-matches are generally divided by gender. Girls my age tend to be considerably more tolerant, even if they don't adopt the practice of not eating meat themselves, because boys, in general, have macho stereotypes driven into their heads from babyhood.
The anti-Wendy's flyer is waved tauntingly. "Meat is good," comes the challenge, which lingers in the air. Whatever futile hope has caused me to take this bait all these years rises in me again. And so it begins. Detailed description – the cruelty the animals face, the fact that they can feel emotions and pain, even if they don't have your intellect, thank-you-very-much. Wild rebuttal – ending with "Vegetarians are stupid," and exasperated disappointment from me. It's not worth it.
And yet, in a grasping-at-straws way, it is. It's a success any time that you can make someone confront the cruelty involved in butchering animals, because getting people to face the truth is the hardest thing you can make someone do, and possibly the first step toward creating a change.
I'm not sure when vegetarianism became seen as a sign of weakness. Maybe it always has been. "It's human nature to eat meat. The food chain and all that," says my friend. And maybe it is human nature to eat meat, but it's also human nature to use violence to get and keep political power, and yet many countries have incorporated democracy to overcome this problem. If we can overcome our natural tendency to physically fight for power, surely this October we can overcome the meat-eating part of our omnivore selves as well.
Chew on that.


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...With Mark Williams - from The Fast Show..
The Fast Show comedian, Mark Williams, indulges his secret passion for the engineering of the industrial revolution as he presents Industrial Revelations - a fascinating, 10 part documentary series examining how inventive advances in engineering and technology during this period changed Britain forever.
Mark travels across the country to discover the technology at the heart of the industrial revolution in the 18th and 19th Centuries, bringing his own unique wit and style to the series as he dons his overalls to fire-up 200 year old, huge steam engines, pilot early canal boats, descend into ancient mines and operate early industrial machinery.
Along the way he visits Bridgwater's Worsley Mine - the birthplace of Britain's canal system, Arkwright's Cotton Mills, the Leeds-Liverpool canal, the Menai Straits Bridge and Ironbridge, the steel yards of Sheffield and the dockyards of Scotland as well as revealing the development of the first steam locomotive, Birmingham's industrial heritage and the achievements of Josiah Wedgwood in the pottery business.
All ten episodes from the highly acclaimed First Series are included here on this special 2-disc DVD.
- Exempt From Certification


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Circle of Tapawingo celebrates 10 years
By Lisa Williams Ackley
Staff Writer
SWEDEN — Ten years ago this week, right after the horror of 9/11, Sandi Lando Welch had an inspirational idea to help grieving young girls ages eight to 12 who have lost a parent or sibling — bring them to the spot she came to love as a camper herself in the 1950s and 1960s — Camp Tapawingo on the shores of Keyes Pond — to be at an overnight camp for one week with other kids who are living with the same sadness, so they can share their feelings with one another while having fun at the same time.
Sandi named the program "Circle of Tapawingo" and said at that time she hoped the experience of being with other children who have gone through the life-altering trauma of losing a loved one would, hopefully, be a cathartic one for all of them.
The headline on the front page of The Bridgton News in August, 2002, as the first Circle of Tapawingo took place, was: "Circle of Tapawingo Rescues Hearts." A total of 32 girls from all over New England took part in the first Circle of Tapawingo.
Last month, 119 girls came together at Camp Tapawingo, offered graciously each year by owner Jane Lichtman, to experience, as Sandi says, "yet another special week where young girls learned that they are not alone in their grief, that it is okay to smile while holding fast to their memories."
"The days at Circle are filled with swimming and tubing and canoeing, and softball and basketball and soccer, and arts and crafts and hip-hop and team building," said Sandi. "We take a few minutes for morning cleanup (a very few minutes) and squeeze in a rest period. Evening activities include campfires and the annual Circle birthday party and Circle of the Stars Talent Show."
Everyone gathers in the massive dining hall for meals, where even more fun erupts.
"Meals are among the best times of our week," Sandi said. "It isn't the food, but the order-followed-by-chaos that fills the dining room. We start out acting respectably. We sing grace, and serve and clear our tables in an orderly way. Then the singing begins and order breaks down as we all chant, 'Hello, my name is Joe, I have a wife, a dog, and a family, I work in a button factory...' The words are meaningless, and are accompanied by wild gyrations. Campers and counselors become one goofy group, as we sing and stomp our way through silly songs."
Why would adults who have full lives of their own want to do this every summer?
"This program is exceptional and unusual in that every counselor at Circle is a volunteer," said Sandi. "We range in age from our early 20s to our late 60s. We all love children, and we all love camp. Why else would we sleep on army cots with plastic mattresses, brush our teeth with cold water, and shower only once every three days? Does it matter that the temperature often dips into the low 40s and we are sleeping in open bunks? No! We go to bed in our clothes and then wear them to breakfast the next morning. We are happy, and just a little smug at being so cheerful about our discomfort. I'm in Heaven! Our happiness surrounds our campers and invades the inner places where many of them have locked away their smiles. As one camper wrote on the rock she placed in the Circle Memory Garden:
'Dear Daddy, I came here to cry for you but ended up laughing instead.'"
"We couldn't have asked for a better endorsement of our program," stated Sandi. "Circle of Tapawingo has changed the lives of countless grieving young girls. We are very, very proud of what we've created."
"The Counselor in Training program is a huge part of our program," said Sandi, "and next year we expect a 96% return rate of this year's 64 counselors."
"Our lifeguards from Bridgton — Wendy Bretton and her daughter, Sara — have been an integral part of our Circle family," Sandi said. "They are just as important members of our family as any counselor." Another lifeguard at Circle of Tapawingo is Francis J. LaRosa III, said Sandi.
New goal will help more grieving children
Sandi said her new goal is to encourage other summer camps — their owners, current and former campers and staff — to also offer a one-week program like Circle of Tapawingo for grieving boys and girls throughout the United States.
"I realized my first dream," Sandi said on Monday, "and I have a whole team ready to move with me to start 'Circle' at other camps. We could never grow larger than 120 campers at Camp Tapawingo. The program is so well scripted, and I'm the best cheerleader. If someone is interested, we'll help them in whatever way we can, because this is too good to keep at one camp."
Other summer camp owners wanting to also help grieving kids through the sharing of their emotions and laughter with other kids just like them must have unique qualities that make them special, however, said Sandi.
"It has to be someone like Jane who has a heart of gold — and the goodness she's done by allowing us to come here every summer for one week," Sandi said. "Without our particular angel, we never would have had the opportunity to help so many kids."
Why is Sandi willing to take more of her time away from her family and other endeavors she may want to try in order to expand the Circle program to other camps?
"You have to want to pay it forward," said Sandi. "When you have good fortune in the world, you want to pay it back. There are so many children who need what we do."
Those wanting more information about Circle of Tapawingo may contact Sandi Lando Welch at: 5124 Holyrood Road, Pittsburgh, Pa., 15213, or contact her at (412) 491-8151 or e-mail her at firstname.lastname@example.com
Camper referrals should be forwarded to: Cathy Spear, Director of Camper Services, 2251 Commonwealth Avenue, Auburndale, Mass., 02466, or phone her at (781) 820-3388. Cathy's e-mail is: email@example.com