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Practitioner directory
Directory or practitioners working on business-related impacts on community rights
Access FacilityEnglish
Access Facility focuses on non-judicial mechanisms to resolve conflicts between companies and communities. It provides a database of existing mechanisms, case stories, and profiles of...
Accountability CounselEnglish
Accountability Counsel assists communities around the world to defend their environmental and human rights
Action Contre l'Impunité pour les Droits Humains (ACIDH)
L'Action Contre l'Impunité pour les droits Humains est une organisation non gouvernementale de lutte contre l'impunité des droits humains en République Démocratique du Congo (RDC)
ActionAid English
ActionAid is a global movement of people working together to further human rights and defeat poverty for all.
Advocates Coalition for Development & Environment (ACODE)English
ACODE is an independent public policy research and advocacy think tank registered in Uganda with operations in the Eastern and Southern Africa sub-region.
African Resources Watch (AFREWATCH)English
AFREWATCH is a regional nongovernmental organization promoting equal access to natural resources including land, water and minerals in the Democratic Republic of the Congo
Amnesty Intl. Sénégal
Asia Indigenous Peoples PactEnglish
Asia Indigenous Peoples Pact aims to strengthen the movements of indigenous peoples of Asia for recognition of their collective rights, and protection of traditional knowledge, bio-diversity and...
Asociación Civil por la Igualdad y la Justicia (ACIJ)English
Asociación Civil por la Igualdad y la Justicia is an NGO focused on protecting the rights of the most vulnerable members of society & strengthening democracy in Argentina
Asociación Interétnica de Desarrollo de la Selva PeruanaEnglish
Interethnic Association for the Development of the Peruvian Rainforest


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Awoke to another beautiful Florida day, and realized we could see Epcot (and some of the other parks) from our balcony.
Got showered and out the door to the hotel shuttle to Epcot. Jennifer, Jonas and I had been here before, but it was once again new for Nellie, Tristan, and Zoe.
Based on a recommendation from Alex and Ella, we went right to Soarin' and secured FastPasses for later in the day. We then located Andy via text message and headed over to Test Track. We had Tristan and Zoe surprise Alex and Ella by walking up to them and talking to them. They didn't know that we'd also be in Florida with them. Wearing their best Prizer, they were completely unphased by the surprise turn of events.
We made some tentative plans to meet up later, and headed into Test Track, followed by World Showcase. We skipped Mexico since we can see that every day at home, and went right to Norway.
We rode like Vikings through Norway where Andy and Shelley, being much more Disney-experienced than us, schooled us on some very cool stuff.
(Jennifer getting schooled by Shelley)
First; the kids could collect these paper masks, and get them stamped, decorated, and festooned with a unique paper cutout at each country.
Second; the park had a Kim Possible-themed spy game that included communicators and missions and all kinds of international intrigue. And third: Epcot is the only Disney park that sells booze, and that maybe an alcohol-around-the (Disney) world is a pretty cool adult alternative to collecting masks and stamps. We quickly adopted that new mission, followed by some plum wine from China, and some Danish beer from Norway. (Denmark, Norway. . . all of Scandinavia is exactly the same anyway, right?)
Most of the rest of the afternoon is somewhat of a blur, not due to the alcohol, but rather to the wandering all over creation, hitting countries like China,
as well as completing Kim Possible missions, eating Disney food, and riding on stuff like the aforementioned Soarin' (which the kids absolutely loved) and Mission Space, and meeting a buttload of Disney characters for autographs and pictures.
We had made reservations and plans in advance for everyone to eat dinner together in Morocco. This was kind of my idea, and everyone else was cool to go along with it. I wanted to go to Disney Morocco because a) I really like Moroccan food, and know something about it, and b) having visited the real Morocco six months previous, I was really curious to see how the Disney version compared.
We got there a little early, so there was plenty of time to take pictures. And I must compliment Disney on their architecture. They did a great job of making it look like the real thing, in my opinion.
Disney Minaret (Epcot)
Real Minaret (Koutoubia Mosque, Marrakesh)
Real windows
Fake windows
Pretty close, I'd say.
We even got to meet Aladdin, Jasmine, and Genie, who, not surprisingly, found Morocco the most appropriate of the Epcot World Showcase countries to hang out in.
Dinner was kind of a disappointment. The chef was alleged to have worked for King Hassan II, and the food was just okay. The decor was very realistic, but the food was overpriced, and the service wasn't very good at all. It was still a fun dinner with the ten of us, and it was great to have that opportunity to have that much extended family all in one place.
We got back outside just in time to watch the big multimedia light/sound/fireworks/fire show in the world showcase lagoon. It was impressive, to say the least. Here's Ella, still small enough to watch the show from Andy's shoulders.
We said our goodbyes to the other Roths, and made our way back to the bus parking lot near the entrance.
This spot holds a special place for me. It was here on February 12, 1985 that the Owen J. Roberts High School Marching Unit spent maybe three hours hanging out in this parking lot due to the fact that the charter bus company only sent one of our four buses to pick the 130+ of us after our long day of travel from Pennsylvania, and hanging out at Epcot. How can that be fun, you ask? When you're 15 years old, and on vacation with a hundred of your closest friends in Florida in the middle of winter, even hanging out in a Disney parking lot is better than whatever you would've been doing back in Pennsylvania on a cold February Tuesday night.
Next up, Day 3: The Magic Kingdom
Saturday, February 28, 2009
Florida, Day 2: Epcot
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subset: commoncrawl


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Helen Russell, played by co-star Oona Mekas, is a minor character in the Season 3 episode "You'd Be Surprised". She is Dr. Douglas Mason fiancée.
Biography Edit
Season 3 Edit
Helen comes one night to the women's health clinic in Saint Theresa's to pick up her fiancé. Margaret mistakes her for a new alumn and tells her that the class is over before Mason clarifies their relationship and introduces the women to each other.
Appearances Edit
|Season three appearances
|"Spaghetti and Coffee"
|"Bone for Tuna"
|"Blue Bell Boy"
|"You'd Be Surprised"
|"Ging Gang Goolie"
|"Sunday Best"
|"The Pony"
|"The Milkmaid's Lot"
|"A Man, A Plan..."
|"Two Imposters"
|"Margate Sands"
Community content is available under CC-BY-SA unless otherwise noted.


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In the mouse, three genes that are homologous to the Drosophila Nanos (Nos) gene have been identified. Deletion of one of these genes, Nanos2, results in male sterility, owing to loss of germ cells during fetal life. Before apoptosis, Nanos2-null gonocytes enter meiosis, suggesting that Nanos2 functions as a meiotic repressor. Here, we show that Nanos2 is continuously expressed in male germ cells from fetal gonocytes to postnatal spermatogonial stem cells. We observed that the promeiotic factor AtRA, an analog of retinoic acid (RA), downregulates NANOS2 levels, in both fetal and postnatal gonocytes, while promoting meiosis. Interestingly, FGF9, a growth factor crucial for sex differentiation and survival of fetal gonocytes, upregulates levels of NANOS2 in both male and female primordial germ cells (PGCs) and in premeiotic spermatogonia. This effect was paralleled by an impairment of meiotic entry, suggesting that FGF9 acts as an inhibitor of meiosis through the upregulation of Nanos2. We found that NANOS2 interacts with PUM2, and that these two proteins colocalize in the ribonucleoparticle and polysomal fractions on sucrose gradients, supporting the notion that they bind RNA. Finally, we found that recombinant NANOS2 binds to two spermatogonial mRNAs, Gata2 and Taf7l, which are involved in germ-cell differentiation.
Nanos is an evolutionarily conserved protein that is essential for the survival of primordial germ cells (PGCs). It has been shown to act as a putative RNA-binding protein involved in germ cell development in both vertebrates (Koprunner et al., 2001; Tsuda et al., 2003) and invertebrates (Fujii et al., 2006; Lehmann and Nusslein-Volhard, 1991; Pilon and Weisblat, 1997; Subramaniam and Seydoux, 1999). In the mouse, three genes encoding Nanos have been identified (Haraguchi et al., 2003). Although Nanos1 is predominantly expressed in the neural system (Haraguchi et al., 2003), Nanos2 and Nanos3 are exclusively expressed in fetal and postnatal germ cells (Tsuda et al., 2006; Tsuda et al., 2003). The expression pattern of Nanos2 is restricted to gonocytes within fetal testis undergoing mitotic arrest, and is then maintained in spermatogonia after birth. Deletion of Nanos2 results in male sterility, due to germ-cell loss during fetal life (Tsuda et al., 2003). Nanos2-null male fetal germ cells enter meiosis before undergoing apoptosis at around 15.5 days post coitus (d.p.c.) (Suzuki and Saga, 2008). Furthermore, misexpression of Nanos2 in female fetal germ cells prevents meiotic entry, strongly suggesting that Nanos2 is an antimeiotic gene (Suzuki and Saga, 2008).
The mechanisms that trigger meiosis have been a subject of debate, but it is now clear that retinoic acid (RA) produced by the mesonephros induces meiosis in female PGCs (Bowles et al., 2006; Koubova et al., 2006), whereas the same action in the fetal testis is prevented by the presence of the retinoid-degrading enzyme CYP26B1 (Bowles et al., 2006; MacLean et al., 2007). The role of RA as a meiotic inducer is also conserved postnatally, because it is able to induce the meiotic entry of differentiating, KIT-positive spermatogonia (Pellegrini et al., 2008). An inverse correlation between levels of RA and NANOS2 exists in Cyp26b1-null fetal testis — a condition in which RA is elevated (MacLean et al., 2007), gonocytes enter meiosis and NANOS2 levels are low (Suzuki and Saga, 2008). The crucial role of RA in inducing meiosis in both female and male is played by Stra8 (stimulated by retinoic acid gene 8) (Anderson et al., 2008; Mark et al., 2008); indeed, Stra8-null males and females show impairment of gametogenesis owing to failure of meiotic commitment (Anderson et al., 2008; Mark et al., 2008). However, meiotic commitment is aberrantly induced, not only in Nanos2-null mice, but also in Fgf9 knockouts (Colvin et al., 2001).
Fgf9 is known to be expressed by somatic cells in XX and XY gonads at 11.5 d.p.c., but it becomes XY restricted by 12.5 d.p.c. and is then expressed within the testis cords (Schmahl et al., 2004). Fgf9-null mice die shortly after birth owing to defects in lung formation, but XY embryos show phenotypic male-to-female sex-reversal, and the surviving gonocytes are found in meiosis (Colvin et al., 2001). The consequences of Fgf9 ablation on male germ cells are probably independent of the proliferative and differentiative effect on fetal Sertoli cell, because it has been shown that FGF9 is able to promote survival of male germ cells in vitro (DiNapoli et al., 2006).
We report that NANOS2 is continuously expressed in male germ cells from fetal to postnatal development. In particular, we investigated the role of RA and FGF9 on the regulation of NANOS2 in correlation with their ability to influence meiosis. We found that AtRA represses NANOS2 expression, whereas FGF9 upregulates Nanos2 mRNA levels in female and male germ cells and inhibits meiosis in both sexes. Moreover, we show evidence that NANOS2 associates with ribonucleoparticles (RNPs) and polysomes, both in fetal and postnatal germ cells. Finally, by GST-RNA pull-down assay, we found that NANOS2 shows affinity for two RNA transcripts, Gata2 and Taf7l, which are involved in spermatogonial differentiation, strengthening the hypothesis that NANOS2 can act as a meiotic inhibitor through a post-transcriptional regulation mechanism.
Developmental expression of Nanos2 in the male gonad
It is known that Nanos2 mRNA starts to be expressed in male PGCs at around 12.5 d.p.c. (Suzuki et al., 2007). Furthermore, by immunofluorescence analysis, it has been very recently shown that undifferentiated spermatogonia type Asingle (As) and Apaired (Apr) are the testicular cells that express NANOS2 in the adult testis (Suzuki et al., 2009). However, it is not clear whether NANOS2 levels drop before birth, or remain elevated in the early postnatal testis. To clearly address this question, we performed a western blot analysis on male germ cells isolated at various stages of fetal and postnatal development. As shown in Fig. 1A we found that NANOS2 protein levels were high at 15.5 d.p.c. and then decreased up to birth. Since NANOS2 levels were already high at 15.5 d.p.c., to better define the fetal period when they start to increase, we analyzed protein extracts obtained from germ cells isolated at 12.5, 13.5, 15.5 d.p.c. and 1 day post partum (d.p.p.). As shown in Fig. 1B, we found that NANOS2 levels were low at 12.5 d.p.c., but they consistently increased up to 15.5 d.p.c. We also found that NANOS2 was expressed after birth and its levels peaked at 5 d.p.p. and then decreased at 7 d.p.p., when differentiating premeiotic spermatogonia (KIT positive) and preleptotene spermatocytes are found (Fig. 1A,B). Isolated pachytene spermatocytes and spermatids did not express Nanos2, either at the RNA or protein level (not shown). Thus, to determine which spermatogonial cell type expressed Nanos2 in the prepuberal testis, we isolated pure populations of undifferentiated or differentiating spermatogonia by immunomagnetic cell sorting. KIT-negative (undifferentiated) and KIT-positive (differentiating) spermatogonia were obtained by using anti-CD117 (Kit)-coated beads and analyzed for Nanos2 expression by semiquantitative RT-PCR. As shown in Fig. 1C, Nanos2 amplification was obtained only in KIT-negative spermatogonia, whereas KIT-positive cells did not show any signal. Purity of the cell fractions was assessed by western blot analysis probing extracts for KIT and OCT4 (Fig. 1C) or RT-PCR amplification of PLZF and OCT4 (not shown), as previously reported (Lolicato et al., 2008).
Retinoic acid downregulates Nanos2 expression in fetal gonocytes and postnatal spermatogonia
RA has been proposed as a meiosis-inducing substance, both in fetal female germ cells and in postnatal male germ cells (Bowles et al., 2006; Koubova et al., 2006; Pellegrini et al., 2008). In Cyp26b-null fetal testis, gonocytes enter meiosis and Nanos2 mRNA levels are low (Suzuki and Saga, 2008). This suggests that, apart from Stra8 induction, the promeiotic action of RA might also result from the downregulation of Nanos2 expression. To test this hypothesis, we cultured testes from 12.5 d.p.c. embryos, the developmental stage at which Nanos2 starts to be expressed, in the presence of 0.3 μM AtRA for 48 hours. As shown in Fig. 2A, AtRA completely abolished the expression of Nanos2 mRNA, whereas it upregulated Stra8 mRNA levels. The same effect was observed when we cultured disaggregated 13.5 d.p.c. testes for 24 hours in the presence of AtRA (Fig. 2A, see also Fig. 3A). Using chromosomal spreads stained for SCP3, we found that about 33% of male germ cells were in the early meiotic leptotene stage in the presence of AtRA whereas no meiotic germ cells were present in the control. Using the same culture conditions, in the presence of AtRA about 85% female germ cells were found in leptotene and lepto-zygotene compared with 75% of the control (Fig. 2A, see also Fig. 3B).
We, and others, have previously demonstrated that G0-arrested gonocytes and undifferentiated KIT-negative spermatogonia are not able to enter meiosis in vitro upon stimulation by AtRA (Pellegrini et al., 2008; Trautmann et al., 2008). Since NANOS2 expression is high in these cells (see Fig. 1) we tested whether AtRA was also able to downregulate NANOS2 at these developmental stages. The results showed that 48 hours of AtRA treatment actually downregulated the levels of Nanos2 mRNA and protein in 15.5 d.p.c. and 4 d.p.p. isolated male germ cells (Fig. 2B). This effect was accompanied by upregulation of STRA8 (Fig. 2B) but, as expected, did not result in meiotic induction (Fig. 2C).
FGF9 upregulates Nanos2 expression and represses meiosis in both male and female germ cells
XY Fgf9-null gonocytes enter meiosis before undergoing apoptosis, suggesting that FGF9 prevents meiosis in XY germ cells (DiNapoli et al., 2006). Since this phenotype resembles that of Nanos2-null gonocytes, we hypothesized that FGF9 could directly or indirectly upregulate Nanos2 expression in germ cells. To test this possibility, we chose a developmental stage at which Nanos2 mRNA levels were low in the fetal testis and were obviously absent in the fetal ovary. Thus, 12.5 d.p.c. male or female gonads were cultured for 48 hours in the presence or absence of 25 ng/ml FGF9. By semiquantitative RT-PCR, we found that levels of Nanos2 mRNA were upregulated by treatment with FGF9 in fetal testes and, interestingly, Nanos2 mRNA expression was stimulated also in fetal ovaries (Fig. 3A,B). The same effect was observed when disaggregated 13.5 d.p.c. gonads were cultured for only 24 hours in the presence of FGF9 (Fig. 3A). A control PCR minus RT was run for Nanos2 mRNA to exclude genomic contamination of the cDNA samples (Fig. 3A) Since the expression of NANOS2 in female germ cells was found to be detrimental for meiotic entry (Suzuki and Saga, 2008), we prepared SCP3-stained nuclear spreads from fetal ovaries after 48 hours of culture with FGF9. The results showed that whereas in the control most of the female germ cells were in leptotene and lepto-zygotene (75%), less than one third of them entered meiosis in the presence of FGF9 (22%; Fig. 3B). The nuclear morphology of the non-meiotic nuclei was similar to that of G0-arrested gonocytes (Fig. 3B, compare also with Fig. 2C) and showed faintly stained chromatin using DAPI stain.
Finally, we investigated whether FGF9 was able to affect meiosis also in 7 d.p.p. spermatogonia. We found that FGF9 reduced the number of meiotic cells after 48 hours of culture, as judged by chromosomal spread counts (40% in the control versus 20% in the FGF9 treated cells, Fig. 4A) and by the decrease of SCP3 levels (Fig. 4B). The role of FGF9 in meiotic inhibition was not mediated by the decrease of STRA8 levels, because they did not change after FGF9 stimulation. To understand which cell population was responsive to FGF9, we then isolated KIT-positive and KIT-negative spermatogonia by immunomagnetic cell sorting, and stimulated them with the factor for 24 hours. FGF9 sensitivity was present in both cell types, because it induced upregulation of NANOS2 in KIT-negative and KIT-positive spermatogonia, as shown by RT-PCR and by western blot analyses (Fig. 4C).
NANOS2 associates with RNPs and polysomes and interacts with PUM2
In Drosophila germline progenitor pole cells, NANOS has been shown to interact with pumilio (PUM) to repress Cyclin B (CCNB1) mRNA translation (Kadyrova et al., 2007). To date, none of the mammalian NANOS homologs has been demonstrated to act as a translational repressor, but we have recently shown that NANOS3 is able to interact with PUM2 and associates with the RNPs, in an heterologous system (Lolicato et al., 2008). To test whether NANOS2 might be involved in the control of translation, a Myc-tagged Nanos2-expressing vector was transfected into HEK293T cells. By immunofluorescence analysis using anti-MYC antibodies, we found that NANOS2-MYC was distributed both in the cytoplasmic and the nuclear compartments of transfected cells (Fig. 5A). Cytoplasmic extracts were then fractionated on a sucrose gradient and analyzed by western blot. Fig. 5B shows that NANOS2-MYC was found mostly in the RNP fractions but also in the ribosomal and light polysome fractions. We then followed the distribution pattern of endogenous NANOS2 on sucrose gradients using 16.5 d.p.c. or 7 d.p.p. spermatogonia extracts (Fig. 6A). Similarly to what was observed with transfected cells, NANOS2 colocalized with the RNP fractions, as well as with those containing the ribosomal subunits and light polysomes. The sedimentation pattern of PUM2 on the sucrose gradients mirrored that of NANOS2. However, TIAR, another RNA-binding protein known to be a translational repressor (Mazan-Mamczarz et al., 2006) that is essential for germ-cell survival (Beck et al., 1998), was found to be exclusively present in the RNP fractions (Fig. 6A). The finding that NANOS2 also associates with polysomes was further verified by fractionating 7 d.p.p. spermatogonial extracts on sucrose gradients in the presence of EDTA, to disrupt polysomes. As shown in Fig. 6, association of NANOS2 with the light polysomal fractions was completely reverted toward the ribosomal subunits and the RNPs in the presence of EDTA, as occurs for DAZL, an RNA-binding protein that has been shown to be present both in the RNP and in the polysomal fractions (Tsui et al., 2000).
NANOS2 interacts with PUM2 and binds to target mRNAs
We have previously shown that PUM2 is able to bind not only NANOS3 but also NANOS2 when used as a bait in pull-down experiments (Lolicato et al., 2008). Since the sedimentation profile of NANOS2 and PUM2 suggested that they might associate in vivo (Fig. 6), we tested the ability of NANOS2 to interact with the endogenous PUM2 from germ-cell extracts in a pull-down assay. We prepared protein extracts from fetal gonocytes (16.5 d.p.c.) or (4 d.p.p.) postnatal spermatogonia and incubated them with a GST- or a GST-NANOS2-agarose coupled resin (Fig. 7A). We found that PUM2 from both fetal and postnatal cell extracts was able to interact with GST-NANOS2, whereas a faint background band was observed in the presence of GST (Fig. 7B).
Preliminary data obtained by microarray analysis reported that significant alterations occur in the transcriptome of the developing male gonad of Nanos2-null mice (Saba, 2009). To verify whether murine NANOS2 works as an RNA-binding protein, we undertook a GST-RNA pull-down approach, by probing GST or GST-NANOS2 agarose beads with ribonucleoprotein extracts from postnatal spermatogonia. RNA molecules bound to GST-NANOS2 or to GST were then extracted and reverse transcribed to obtain cDNAs. Using primers that spanned the 3′UTR of the putative target genes, we looked for mRNAs whose expression was altered in the Nanos2 knockouts and/or for transcripts that displayed a sexually dimorphic expression pattern during fetal gonad development. Specifically, we screened the cDNAs obtained from the RNA pull-down for Stra8, Taf7l, Kit, Gcnf1 and Gata2, by PCR analysis. We found that Gata2 and Taf7l mRNA was retained specifically by GST-NANOS2 beads (Fig. 7C), whereas we did not observe any specific signal for Kit, Stra8 or Gcnf1 mRNAs (Fig. 7C). We actually found that perfect consensus binding sites for PUM2 [UGUANAUA(A/G)NNNN(C/G/U)(C/G/U)(C/G/U)(C/G/U)(C/G) CC] (White et al., 2001) are located within the 3′UTR of both Gata2 and Taf7l mRNAs, supporting the notion that a complex of NANOS-pumilio is essential for mRNA binding. When looking in the Geo Profile database for Taf7l and Gata2 expression in germ cells, we found that these two genes were not only differentially regulated in the developing male and female gonads, but their levels were higher in the differentiating type B compared with type A spermatogonia (GDS2223, GDS2390). Interestingly, Gata2 mRNA levels were found to be significantly increased in the postnatal testis in a period in which high levels of Kit are also found (GDS401).
NANOS2 is a potential RNA-binding protein that functions cell autonomously to control germ-cell sexual differentiation during fetal development. It has been proposed to act as an antimeiotic gene, because its deletion in mice is associated with abortive meiosis in fetal gonocytes, before they undergo apoptosis. Furthermore, its misexpression in female PGCs prevents meiosis and makes the nuclear morphology of these cells similar to that of G0-arrested gonocytes (Suzuki and Saga, 2008).
In the present paper, we used fetal germ cells and postnatal spermatogonia as models to understand the function of NANOS2 and its relationship with the beginning of meiosis.
NANOS2 expression and meiotic commitment
By analyzing the developmental profile of NANOS2 expression in the testis during the perinatal period, we found that NANOS2 is continuously expressed from the fetal to the postnatal period and it shows two peaks of expression: one at around 15.5 d.p.c. and another one at around 5 d.p.p. The first peak temporally corresponds to the mitotic block period, which male germ cells undergo prenatally. In this period, gonocytes do not enter meiosis and retain stem cell features, as demonstrated by their ability to complete spermatogenesis when transplanted into adult testes (Ohta et al., 2004). The second peak corresponds to a period in which cells expressing neurogenin-3 (NGN3) (a marker for undifferentiated spermatogonia) (Nakagawa et al., 2007; Yoshida et al., 2004) start differentiation into KIT-positive cells (Pellegrini et al., 2008). During this period, the spermatogonial stem cell factor PLZF (promyelocytic leukemia zinc finger) tightly controls Kit mRNA expression in stem cells (Filipponi et al., 2007), and they are completely lost when Kit is not repressed (Buaas et al., 2004; Costoya et al., 2004). By purifying KIT-positive and KIT-negative spermatogonia, we found that NANOS2-expressing cells were present only within the KIT-negative spermatogonia population, which corresponds to PLZF-expressing spermatogonia at 4-5 d.p.n., indicating that Nanos2 is a spermatogonial stem-cell-expressed gene. In agreement with our results, during the revision of this paper, two studies have shown that NANOS2 is mostly expressed in As and Apr spermatogonia from the adult testis and that its conditional deletion in adulthood leads to spermatogonia stem cell loss (Sada et al., 2009; Suzuki et al., 2009).
Evidence from genetic studies suggests that NANOS2 acts as a suppressor of meiosis in male fetal germ cells (Suzuki and Saga, 2008). We found that AtRA, a derivative of RA and a well-known promeiotic factor (Bowles et al., 2006; Pellegrini et al., 2008), was able to downregulate Nanos2 at both mRNA and protein levels in male PGCs (12.5 d.p.c.) and in undifferentiated spermatogonia. In mitotically arrested gonocytes (15.5 d.p.c.), however, we observed that the strong decrease of Nanos2 mRNA levels induced by AtRA was paralleled only by a less-intense decrease of the protein levels. Since this different behaviour was consistently reproduced, we hypothesize that NANOS2 protein stability is higher in mitotically arrested germ cells than in proliferating germ cells. We found that AtRA increased the percentage of proliferating male PGCs entering meiosis, but it was not effective on mitotically arrested gonocytes or on undifferentiated spermatogonia. For arrested gonocytes, this discrepancy can be explained by the existence of a narrow window for meiotic competence, which corresponds to the period in which NANOS2 starts to be expressed (12.5 d.p.c.) and is needed to repress the meiotic fate (Saga, 2008; Suzuki and Saga, 2008). After this period, high levels of NANOS2, as a result of increased protein stability and/or enhanced synthesis stimulated by factors such as FGF9 (see below), 'masculinize' gonocytes. The evidence that AtRA does not trigger meiosis in undifferentiated spermatogonia, notwithstanding the modulation of NANOS2 and STRA8, suggests that these cells lack other molecular factors required for meiotic entry. One of these factors could be the KIT tyrosine kinase receptor, whose downstream signalling is essential for meiotic entry of differentiating spermatogonia in vitro (Pellegrini et al., 2008). In line with this hypothesis is the observation that Kit is expressed in proliferating fetal germ cells of both sexes up to 12.5-13.5 d.p.c. (Manova and Bachvarova, 1991) when the 'meiotic window' is active, and is then re-expressed in the male only postnatally, in differentiating spermatogonia — the meiosis-competent cells of the testis.
In a search for factors that could upregulate NANOS2 levels and negatively influence meiosis, we found that FGF9 was a good candidate. Fibroblast growth factors (FGFs) have a role in the proliferation and survival of many cell types (Ornitz and Itoh, 2001). Fgf9 is expressed in the undifferentiated gonads of both sexes at 11.5 d.p.c., but it becomes XY restricted by 12.5 d.p.c. and is maintained in the testis cords (Schmahl et al., 2004). Sertoli cell precursors fail to proliferate in Fgf9 mutants and testis differentiation is disrupted (Schmahl et al., 2004). The majority of germ cells are lost at 12.5 d.p.c.; however, the surviving gonocytes within the Fgf9-null gonads are found in meiosis (DiNapoli et al., 2006), suggesting that FGF9 normally promotes survival and prevents meiosis of fetal male germ cells. Indeed, we found that FGF9 upregulated NANOS2 and strongly inhibited meiosis in female PGCs in vitro. This observation is in line with the evidence that Nanos2 misexpression in female germ cells inhibits meiosis and masculinizes germ-cell nuclei (Suzuki and Saga, 2008). FGF9 also upregulated NANOS2 in postnatal spermatogonia, and such an increase was only observed after a long incubation time (not shown), suggesting that a transcriptional mechanism probably mediates this effect. Sensitivity to FGF9 was present in the undifferentiated and in the differentiating spermatogonia, because both these cell types express an FGF9 receptor, as confirmed by microarray analysis (Rossi et al., 2008). We also observed a negative effect of FGF9 on meiotic entry in differentiating spermatogonia, suggesting a common mechanism shared between female and male germ cells in the control of the mitotic-meiotic switch. It has been hypothesized that a possible role for NANOS2 in the prevention of meiosis is to repress STRA8 expression during the period in which CYP26B1 levels decrease in the male gonad and RA is increased in the mesonephros. In our studies we did not observe STRA8 downregulation after FGF9 stimulation of postnatal spermatogonia in vitro, suggesting that NANOS2 might act independently or downstream of STRA8, even if we cannot exclude that other mechanisms are active in vivo.
NANOS2 subcellular localization and molecular interactions
In contrast to what we observed for NANOS3 (Lolicato et al., 2008), NANOS2 was found to be associated not only with RNPs, but also with the ribosomal and polysomal fractions. This localization is uncommon for translational repressors, but it has been recently shown that translational repression can also occur at the polysomal level, during protein synthesis, through a miRNA repressive mechanism (Cannell et al., 2008; Gebauer and Hentze, 2004; Petersen et al., 2006). Furthermore, the possibility cannot be ruled out that NANOS2, and eventually PUM2, might function both as translational repressors and activators, as has been shown for FMRP (Zalfa et al., 2006); they could also function at other levels of post-transcriptional regulation of mRNA expression, such as the control of mRNA stability.
When transfected in HEK93T cells, NANOS3 associates with PUM2 and is found within the RNPs (Lolicato et al., 2008). Since it has been shown that NANOS2 can complement the NANOS3 defect in Nanos3-null mice, we reasoned that it could also recognize the same partners in male germ cells. Indeed, we found that NANOS2 was able to bind PUM2 expressed either in fetal gonocytes or in postnatal spermatogonia, suggesting that PUM2 can interact in vivo with NANOS2, probably both acting in post-transcriptional mRNA regulation. In the attempt to discover potential NANOS2 targets, we looked for genes showing a sexually dimorphic expression pattern during gonadal differentiation and/or that were more abundantly expressed in differentiating spermatogonia. Among several genes tested (Kit, Taf7l, Gata2, Stra8 and Gcnf1), Gata2 and Taf7l mRNAs were found to interact with GST-NANOS2. GATA2 is a zinc finger transcription factor expressed in fetal oocytes up to 15.5 d.p.c., but not in fetal gonocytes (Siggers et al., 2002). There are no studies on Gata2 expression in postnatal gonads; however, its mRNA levels are strongly increased in differentiating spermatogonia, according to the microarray analysis database (Geo Profiles) at NCBI. Although the role of GATA2 in germ cells is unknown, it is well established that in hematopoietic stem cells, this factor controls Kit expression at the transcriptional level (Orkin, 1992). Taf7l is a component of the transcriptional machinery specific to preleptotene spermatocytes and haploid spermatids (Pointud et al., 2003) and its deletion leads to reduced male fertility (Cheng et al., 2007). Thus, the identification of two transcription factors involved in the differentiation of spermatogonia as targets of post-transcriptional control by NANOS2 is in line with the evidence that NANOS2 negatively regulates differentiation of spermatogonia stem cells (Sada et al., 2009). Studies to identify other NANOS2 mRNA targets, whose post-transcriptional regulation is determinant for spermatogonia differentiation, will be important to understand how commitment of stem cell occurs in the initial phase of spermatogenesis.
In conclusion, our results can be interpreted as schematically depicted in Fig. 8. RA and FGF9 produced locally by the somatic cells of the gonads act in opposite ways to regulate Nanos2 expression and meiotic entry both in fetal and postnatal germ cells. In the fetal ovary, FGF9 levels sharply decrease at 11.5 d.p.c. in parallel with a rise of circulating levels of RA, which stimulates Stra8 expression in PGCs and their commitment to meiosis. In the fetal testis, FGF9 levels are kept high, and circulating RA is degraded by CYP26B1 (not depicted) in somatic cells. High levels of FGF9 without RA signalling in male PGCs upregulate Nanos2, which determines their transition into mitotically arrested gonocytes. In the postnatal testis, Nanos2 is expressed exclusively in SSCs and Sertoli cells to safeguard the delicate balance between stem cell self-renewal and meiotic commitment of spermatogonia by modulating the levels of FGF9 and RA.
Materials and Methods
Cell isolation and culture
Postnatal male germ cells (0-7 d.p.p.) were obtained as previously reported, by sequential enzymatic digestion of testes from CD1 albino mice (Pellegrini et al., 2008). After preplating of cell suspension, germ cells were cultured in modified Earle's medium (MEM, Gibco) with 20 mM glutamine (Gibco), 2 mM pyruvic acid (Sigma), 1 mM lactic acid (Sigma), non-essential amino acids (Gibco), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco), without serum supplementation.
To obtain enriched fetal germ-cell suspensions, fetal testes and ovaries were collected at different developmental stages and digested with trypsin (Gibco) and DNaseI (Sigma). Cell suspensions were pre-plated in the same medium as postnatal germ cells supplemented with 10% fetal bovine serum, (FBS) and recovered after 3 hours of culture, to allow most of the somatic cells to adhere to the plastic dishes. In the case of organ culture, fetal gonads were cultured for 48 hours onto 0.8% agarose blocks pre-equilibrated with MEM and 10% FBS.
All-trans-retinoic acid (AtRA, Sigma) was dissolved at a concentration of 10 mM in ethanol and diluted to a final concentration of 0.3 μM (Pellegrini et al., 2008) in culture medium. Control cultures received the corresponding doses of diluted ethanol. FGF9 (from Società Italiana Chimici) was dissolved in PBS 1 mg/ml BSA and used to a final concentration of 25 ng/ml. All the factors were replaced in the culture medium after 24 hours. Separation of KIT-positive spermatogonia from KIT-negative spermatogonia was performed by magnetic-activated cell sorting (MACS) with CD117 conjugated microbeads (Miltenyi Biotech, Germany) as previously described (Pellegrini et al., 2008).
Definition of the germ-cell stages
Fetal male and female germ cells are defined as PGCs as long as they reach the gonads and proliferate. After gonadal colonization, female PGCs start entering meiosis (at around 13.5 d.p.c.) and are then defined as oocytes. Male PGCs, however, enter mitotic block after gonadal colonization and are then defined as gonocytes. This definition is retained up to birth, when gonocytes resume mitosis and spermatogonial stem cells can be identified. KIT-negative (OCT4- and PLZF-positive) spermatogonia represent the stem cell population of the testis whereas KIT-positive spermatogonia are the differentiating mitotic germ cells which are committed to enter meiosis.
Polysome-RNP fractionation
Prepuberal spermatogonia and fetal gonocytes were subjected to polysome-RNP fractionation on sucrose gradients as previously described (Lolicato et al., 2008). In brief, 600 μg cytoplasmic extracts in polysome buffer were loaded onto 15-50% sucrose gradients. Ten fractions were collected and protein precipitation from each fraction was performed with 72% trichloroacetic acid (TCA) and subsequently resuspended in 2× sample buffer and processed for SDS-PAGE. Absorbance at 260 nm was continuously recorded while collecting the fractions. EDTA treatment was performed as previously described (Grivna et al., 2006).
Recombinant proteins, transfection, GST protein and RNA pull-down
Nanos2-Myc was produced by RT-PCR using primers using a proofreading polymerase (Pfu, Stratagene) containing BamHI-EcoRI sites at the extremities. The amplified band was cloned in pcDNA3-Myc (Invitrogen, Milan, Italy) or in pGEX4T1 (GE Healthcare, Milan, Italy). For transient transfections, HEK293T cells were transfected with pcDNA3Myc-Nanos2 by Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.
Pull-down experiments were performed using GST fusion proteins purified from bacterial lysates on glutathione-agarose (Sigma). In the case of protein pull-down, cell extracts (600 μg total proteins) of 16.5 d.p.c. gonocytes or 4 d.p.p. spermatogonia, precleared for 1 hour with glutathione-agarose in lysis buffer, were added to 2 μg GST protein or GST-NANOS2 fusion protein absorbed on glutathione-agarose in lysis buffer supplemented with 0.05% BSA. After incubation for 90 minutes at 4°C under constant shaking, beads were washed three times with the same buffer, absorbed proteins were eluted in SDS sample buffer and resolved on a 12% SDS-PAGE for western blot analysis. Input represents 1/10 of the initial samples.
For RNA pull-down experiments 1 mg cytoplasmic extracts in polysome buffer were obtained from 7 d.p.p. spermatogonia and processed as described (Keene et al., 2006). Extracts containing RNA and protein were incubated with GST or GST-NANOS2 agarose beads for 4 hours at 4°C. RNA-protein complexes were washed four times in NT2 buffer and RNA was eluted from the beads by incubation at 55° for 30 minutes with 30 μg proteinase K. After Trizol extraction (Invitrogen) and DNaseI treatment (Zymo Research, Rome, Italy), RNA was reverse transcribed as described in the following section.
Total RNA from whole fetal gonads and fetal and postnatal germ cells was extracted with Trizol reagent and treated with DNase I to avoid potential contamination by genomic DNA. DNA-free RNA was reverse transcribed using SuperScript First Strand Synthesis kit (Invitrogen) according to the manufacturer's instructions. Primers used for PCR were as follows: Nanos2 F, AGTGCCATGGACCTACCGCCCTTT and Nanos2 R, TCTCAATTATCGCTTGACTCTGC; c-Kit F, GCCACGTCTCAGCCATCTG and c-Kit R, GTCGGGATCAATGCACGTCA; Stra8 F, TAGGATCCATGGCCACCCCTGGAGAAG and Stra8 R, GAATTCTTACAGATCGTCAAAGGTCTCCA; Actin F, GGCTGTATTCCCCTCCATCG and Actin R, CCAGTTGGTAACAATGCCATGT. For semiquantitative RT-PCR 25 cycles were performed for the amplification of Actin and all other genes, 30 cycles for Nanos2 amplification from male germ cells or 40 cycles from female germ cells.
RNA from RNA pull-down experiments was reverse transcribed using SuperScript First Strand Synthesis kit (Invitrogen) according to the manufacturer's instructions. Primers for putative targets were designed, when possible, to span a region between the last two exons. Primers were the following: Gata2 F, CAAGCTGCACAATGTTAACAGGC and Gata2 R, ATTCACAGTAATGGCGGCACAAGG; Taf7l F, GGAGGAGGAAGAGACAGACAATTC and Taf7l R, GTAGAGGGACAGAAGTATGTGGTTCCAC; Stra8 F, TGCCGGACCTCATGGAATTT and Stra8 R, GAATTCTTACAGATCGTCAAAGGTCTCCA; Gcnf1 F, GGATGGAGGTGATTGAACGACT and Gcnf1 R, AGTCTCCATCTTGGTCTCTGGCT. PCR conditions were: 95°C for 20 seconds, 58°C for 30 seconds, 72°C for 30 seconds, for 40 cycles.
Transfected HEK293T cells were adhered onto poly-L-lysine glass slides and fixed for 10 minutes at room temperature in 2% paraformaldehyde. After washing with PBS, cells were permeabilized for 10 minutes with PBS containing 0.1% Triton X-100, and incubated for 30 minutes at room temperature with PBS containing 0.5% BSA. Samples were then incubated overnight at 4°C in a humidified chamber with anti-MYC antibody from Santa Cruz (sc-2048) at a final concentration of 2 μg/ml, then for 1 hour at room temperature with cyanin 3 (AP 180 C; Chemicon). Slides were washed and mounted in 50% glycerol in PBS and immediately examined by fluorescence microscopy. Nuclei were counterstained with 1 μg/ml Hoechst 33342 (Sigma). Control experiments were performed using non-immune immunoglobulins instead of the specific antibody.
Western blotting
For western blot analysis, germ cells were harvested and washed three times with ice-cold PBS. Cell lysis was performed with 10 mM HEPES, pH 7.9, 1% Triton X-100, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT), 10 mM β-glycerophosphate, 0.1 mM sodium vanadate and protease inhibitor cocktail (Sigma). 40 μg protein extracts were separated by SDS-PAGE in 4-20% gradient gels (Serva, Milan, Italy) or uniform gels of 15% polyacrylamide and transferred to nitrocellulose membrane (Amersham, Piscataway, NJ). The membrane was blocked in phosphate saline buffer with 0.1% Triton (PBST) and 5% fat-free milk powder for 1 hour at room temperature. Incubation with primary antibodies was carried out at 4°C overnight in PBST-5% BSA. Appropriate horseradish-peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) was used at a 1:5000 dilution in PBST for 1 hour at room temperature. Anti-NANOS2 serum (in this study we used both one obtained from Abnova, Taipei, Taiwan, and a gift from Yumiko Saga, Sokendai, Shizuoka, Japan; in the figure legends we indicated when the commercial antibody was used) was used at a 1:300 dilution in PBST with 5% BSA. Anti-KIT rabbit polyclonal antibody (Albanesi et al., 1996) was diluted at 1:1000. Anti-PUM2 rabbit polyclonal (Abcam) was diluted at 1:3000. Anti-TIAR mouse monoclonal antibody (a kind gift from P. Anderson, Harvard, MA) was diluted at 1:1000. Anti-S6 rabbit polyclonal antibody (Cell Signaling, Beverly, MA) was diluted 1:1000. Anti-Actin rabbit polyclonal (A2066, Sigma) was diluted 1:1000. Anti-MYC antibody was from Santa Cruz (sc-2048) and diluted 1:1000. Mouse monoclonal antibody anti human DAZL was from AbD Serotec, Milan Italy and used at a 1:1000 dilution. Anti-STRA8 rabbit polyclonal from Abcam (Ab 49602) was used at a 1:1000 dilution. Anti-SCP3 rabbit polyclonal (1:1000) was from Novus. Anti-OCT4 mouse monoclonal (1:1000) was from Santa Cruz (sc-5279). The horseradish peroxidase conjugate was detected by chemioluminescence with an ECL Kit (Santa Cruz) and autofluorography. Densitometric analysis was performed by ImageQuant™ TL software (GE Healthcare).
Cultured spermatogonia or single cell suspensions from cultured fetal gonads were prepared and stained essentially as previously described (Romanienko and Camerini-Otero, 2000). Slides were washed twice in PBS, and incubated overnight at 4°C with anti-SCP3 rabbit polyclonal antibody (Novus) diluted in blocking solution (1:100 in 10% goat serum, 3% BSA, 0.5% Triton X-100 in PBS). After washing, secondary antibody was added for 1 hour at 37°C. The slides were washed and allowed to dry. Vectashield Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA) was added and the slides were viewed using a Leica microscope. Spreads analysis was performed in three independent experiments.
Statistical analysis
Continuous variables were summarized as means ± s.d. All tests were two-sided and were determined by Monte Carlo's significance. A significance value threshold of 0.05 was used for the current analysis. Student's t-test was used to test for differences between two independent groups, whereas one-way ANOVA was used to test for differences among three or more independent groups. For multiple comparisons the Tukey's HSD test was carried out. All statistical tests were carried out using the SPSS statistical analysis software package, version 10.0.
We thank Yumiko Saga (Sokendai, Japan) for anti-NANOS2 antibody, Paul Anderson (Brigham and Women's Hospital, MA) for providing anti-TIAR antibody; S. Pedrotti for technical help; C. Sette and P. Grimaldi for critically reading the manuscript and helpful suggestions. This work was supported by grants from the Italian Ministry of University (Prin 2005059793_005, 200734H7WW_004, and 200788TPYE_002).
- Accepted December 9, 2009.
- © 2010.


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Message-ID: <firstname.lastname@example.com> Subject: Exported From Confluence MIME-Version: 1.0 Content-Type: multipart/related; boundary="----=_Part_18469_1596372184.1597468392499" ------=_Part_18469_1596372184.1597468392499 Content-Type: text/html; charset=UTF-8 Content-Transfer-Encoding: quoted-printable Content-Location: file:///C:/exported.html
Democrats Abroad passed a resolution during the DPCA Internation= al Meeting held in May 2018 in Tokyo to support and help with the passage o= f the Equal Rights Amendment. The resolution was submitted by Shari T= emple and Quaide Williams of DA Germany. It passed unanimously. = Here is the full text of the resolution.
WHEREAS the U.S. Constitution does not explicitly grant equal righ= ts to women;
WHEREAS the Equal Rights Amendment (ERA) to the Constitution was f= irst introduced in Congress in 1923;
WHEREAS Congress passed the ERA in 1972 and, as required by the Co= nstitution, sent it to the States for ratification;
WHEREAS only 35 of the required 38 States ratified the amendment t= o the Constitution by the specified deadline;
WHEREAS a 36th state, Nevada, ratified the amendment on= March 21, 2017 because, according to the bill=E2=80=99s chief sponsor, =E2= =80=9CThe ERA is about equality, period;=E2=80=9D
WHEREAS over the past five years, two other states (Illinois and V= irginia) have come close to ratifying it;
WHEREAS women=E2=80=99s rights, including but not limited to repro= ductive rights, are in jeopardy;
WHEREAS women=E2=80=99s wages are less than 80% of men=E2=80=99s w= ages and =E2=80=9Cequal pay for equal work=E2=80=9D lacks any form of legal= protection;
WHEREAS a Constitutional amendment would make explicit that equal = rights for women are guaranteed and protected by the law of the land;
WHEREAS Democrats should build on the momentum generated following= the election of Trump, the ongoing sexual harassment and abuse scandals hi= ghlighted by the #metoo movement, and recent renewed interest in the ERA;= span>
WHEREAS The members of Democrats Abroad, living in over 190 countr= ies in the world, include women voters in all 50 states;
WHEREAS Democrats Abroad has expressed support in its 2016 Platfor= m for revival of the campaign to ratify the ERA;
WHEREAS the Democratic Party Platform of 2016 similarly expressed = support for revival of the campaign to ratify the ERA;
THEREFORE BE IT RESOLVED THAT Democrats Abroad shall initiate a ca= mpaign among its members to promote the passage of the ERA;
BE IT FURTHER RESOLVED THAT Democrats Abroad will urge the Democra= tic National Committee and the Party as a whole to strive for the enactment= of the Equal Rights Amendment;
BE IT FURTHER RESOLVED THAT Democrats Abroad will work with the DN= C and the State parties to promote and support the campaigns to gain passag= e of the ERA.


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In 2017-18 the Nashville Predators won the Presidents Trophy by setting new franchise records in wins (53) and points (117) while scoring their second-most goals (3.18 GF/PG) and fourth fewest goals against (2.49 GA/PG).
The Predators are mostly the same team that they were last year, so there's no reason not to expect them to finish near the top of a loaded Western Conference.
➕ Dan Hamhuis (Free Agency)
➕ Zac Rinaldo (Free Agency)
➕ Connor Brickley (Free Agency)
➕ Rocco Grimaldi (Free Agency)
Projected Forwards
Up front the Predators are led by the duo of Filip Forsberg and Ryan Johansen, who were often joined by Viktor Arvidsson last year. This year they will have either Arvidsson or Kevin Fiala on their right wing, but it shouldn't matter either way. Forsberg and Johansen spent 618:45 5v5 TOI together last season and carried a 55.06 CorsiFor%, 50.78 ScoringChancesFor% and astounding 68.42 GoalsFor%. They will lead the charge and if Fiala plays meaningful minutes with them this season, he becomes one of the NHL's biggest breakout candidates.
Kyle Turris enters his first full season with Nashville after being acquired from Ottawa early last year. Turris' arrival was massive for Fiala, but moving Arvidsson to his wing will help spread out the Predators depth. Rookie Eeli Tolvanen or Calle Jarnkrok are candidates to play on the left-side of that line. Tolvanen is the more attractive of those two options from a fantasy perspective, but Jarnkrok is coming off the best year of his career—19 goal, 23-assist per 82-game pace.
What separates the Predators from the majority of teams is their depth. Jarnkrok or Tolvanen will join Nick Bonino and Craig Smith on the third line and their fourth-line will likely consist of Ryan Hartman, Frederick Gaudreau and Colton Sissons. A few of them would play in the top-6 of some teams and if gives them the No.15 ranked third line and No.2 ranked fourth line in hockey according to Corsica Players Ratings.
Projected Defensemen
The Predators have arguable the best top-4 defensemen in the NHL, even after the San Jose Sharks acquired Erik Karlsson from the Ottawa Senators. Roman Josi and P.K. Subban are two of the NHL's best defensemen and they have the luxury of splitting them up and playing them over 24 minutes a night.
Josi plays with the offensive minded Ryan Ellis, who missed the start of last season but was on a 17-goal, 43-assist (60 points) per 82-game pace. The duo had a 54.7 CF% and 52.8 SCF%. Subban and Mattias Ekholm make up their other top-pairing and their numbers were even more impressive last year—they had a 55.6 CF% and 55.0 SCF%.
Matt Irwin and Yannick Weber eat up the other 10-to-12 minutes that are available just to give their top-4 a breather. Josi and Subban could both be top-5 fantasy defensemen and Ellis will easily climb into the top-15/20 if he can stay healthy.
Projected Goalies
Pekka Rinne is entering the final-year of his contract but just had one of the best years of his career at age-35. Rinne was 42-13-4 with a 2.31 goals against average, .927 save percentage and led the league in shutouts (8) and goals saved above average (27.49). Despite his recent success, the continued development of Juuse Saros is in the best interest of the Predators. Rinne will likely continue to see a decrease in starts as Nashville limits his workload to keep his fresh and Saros sharp.
The 23-year-old Saros has a career 2.42 GAA and .923 SV% in 48 NHL games and is the future No.1 netminder in Nashville. He is easily one of the best handcuff and backup options in fantasy hockey and has the ability to be a top-10 goalie if Rinne gets hurt.
Projected Scoring Leaders
|Filip Forsberg (30)
|P.K. Subbam (44)
|Filip Forsberg (65)
|Filip Forsberg (8)
|Viktor Arvidsson (30)
|Ryan Johansen (43)
|Ryan Johansen (61)
|Roman Josi (7)
|Kevin Fiala (23)
|Roman Josi (41)
|Viktor Arvidsson (58)
|Kyle Turris (6)
Predators in the DFO Top 300
- 40. Roman Josi — D5
- 41. P.K. Subban — D6
- 43. Filip Forsberg — LW8
- 63. Pekka Rinne — G8
- 88. Ryan Johansen — C25
- 93. Viktor Arvidsson — RW17
- 99. Ryan Ellis — D22
- 165. Kyle Turris — C37
- 172. Juuse Saros — G27
- 192. Kevin Fiala — LW28
- 259. Craig Smith — RW53
- 263. Mattias Ekholm — D67
- 278. Eeli Tolvanen — RW56
2018-19 Outlook
Predicted Central Standings
1. Nashville Predators
The Predators are loaded up front with one of the best bluelines and goaltending duos in the NHL. The Winnipeg Jets are likely the only team in the Central Division who are capable of giving the Predators a run for the Division lead. Nashville should have no problem topping 100 points and could clinch their second straight Presidents Trophy.


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Our team is skilled at providing notary services. We are committed toward achieving client satisfaction each and every time. You can expect our notaries to provide you the notary services you need in fast, affordable manner.
Satisfaction is our primary goal and we are committed toward acquiring your trust as a client. Whether you would like us to oversee loan signings or send business-class faxes, you can rest assured knowing that we will do so with the years of expertise our team brings to the table.


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Internet Marketing:
There is no point in having a website if it does not receive traffic. Therefore, it is important to include your website in all your marketing materials such as business cards, brochures, banners, etc… In addition to offline marketing, you can also choose to promote your website online. One major advantage of online marketing is that you can track your marketing campaign performance quite accurately and detect where site visitors are coming from. The following are various internet marketing strategies you can use to drive more business to your site:
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• Website Statistics Analysis


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In my early days, someone once remarked to me that you really did not have to worry about someone stealing your ideas. Odds were, you would have to metaphorically shove it down someone's throat to get them to use it. The introduction of change always starts with a new idea.
So what is the life of a new business idea and how does it evolve to bring about change?
I see five phases for a new idea. At any one of these phases the idea may die. As an idea progresses through the various phases, it becomes much easier to convince others to change and adopt the new idea.
Idea Phase
Obviously one needs an idea to start. The idea usually comes about to fulfill a need. It may be something new, or the application of an existing practice in a new area, or some combination.
Introducing the idea to others is necessary to leave this phase. Many ideas stop here because of the fortitude, or lack thereof, of the idea's creator.
Quackery Phase
As it is a new idea, it is generally seen as quackery. The quackery phase is where most ideas die. The more contrary to conventional wisdom, the more likely an idea will suffer death by quacking.
Selling an idea includes actually selling it as a product or service, or having an idea adopted internally, or both.
Getting an idea past the quackery phase is the hardest. It is here that leaders of organizations get that dazed look when you are describing the new idea. It is also where resistance to change starts. What would you believe, the tried and true use of leeches or this new snake oil?
Convincing someone else to try the idea is the exit from this phase.
Thought Leadership Phase
Once adopted (assuming it works) a new idea will enter the Thought Leadership phase. This is the sexy phase where it is new and exciting. As a thought leader it is time to face the world of withering criticism. Even with a few successes as supporting evidence, an idea may not spread. The conditions of success will be seen as exceptions rather than the rule.
The adoption of the new idea by others is the key to this phase. If a critical mass of adopters occurs, the new idea becomes the choice of experts.
Expertise Phase
As the creator of the idea you have become THE expert. You are no longer selling the idea, others are coming to you for your expertise. The idea is no longer cutting edge but is now seen as tried and true.
Most successful ideas end their journey here when it becomes the conventional wisdom. The shelf life of conventional wisdom depends upon the rise of new and different ideas.
It is at the end of the phase that as the creator of the idea, a graceful exit may be needed. As new ideas usurp your own, it is better to leave as a respected expert and not a crackpot clinging to former glory.
Some ideas leap from conventional wisdom to dogma. The idea becomes part of the culture and is accepted as a fact – there number zero, the Earth rotates around the sun, assembly lines, etc.
The idea usually outlives its creator.
Dislocation of the idea will be part of a larger cultural change. We are living through an era of significant dislocation as the Industrial age succumbs to the Information Age.
Where are your ideas?


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Last Updated on
Question – I have an LLC that does online retail and I was told I needed a Sellers Permit. I tried to do this with an "online" service but even after I provided the fed tax ID and other information about my business they called and requested my SSN. Is this also required for a Sellers Permit?
Our Answer – The SSN and FEIN are somewhat related and you could use your SSN in place of the FEIN if you were a sole proprietorship with no employees.
To sell items at retail, both online and retail, you need to register with the Taxation and Revenue Department in New Mexico. You will need to register through the Combined Reporting System (CRS) http://www.tax.newmexico.gov/Businesses/register-your-business.aspx to obtain a New Mexico tax identification number. This number will allow you to pay state and local gross receipts tax. This is probably what was meant by the Seller's Permit.
There is no fee to apply for this number.


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Hainan-Manila direct flights eyed under Belt and Road Initiative
HAINAN, China-- The tourism sectors of Hainan, China's famous tropical province, and the Philippines are seen to benefit under economic integration strategy Belt and Road Initiative.
Chen Yibo, deputy director-general of Hainan Provincial Tourism Development Commission, said the Belt and Road initiative could propel a plan to open direct flights between Manila and Hainan.
There is a great possibility that direct flight between Manila and Hainan will materialize soon, Chen told the Philippine News Agency when asked if the Belt and Road Initiative would also spur tourist arrivals between Hainan and the Philippines.
The Belt and Road Initiative, which calls for massive investment infrastructure, trade, and cultural exchanges, was lengthily discussed during the Boao Forum for Asia Annual Conference 2017 held last March 23 to 27 in Boao, Hainan.
Chen said tourism is one sector that will benefit from the Belt and Road, also known as One Belt, One Road which was proposed by Chinese President Xi Jinping in 2013 to integrate the economic development not only in Asia but also part of Europe, Middle East and Africa.
The infrastructures to be built in the countries under this initiative will boost the tourism industry, Chen told the 15 journalists from South Asia and Southeast Asia who covered the Boao Forum for Asia and visited some famous tourist destinations of Hainan.
Chen said representatives from concerned travel groups and airlines are now discussing a proposal to open direct flight between Hainan and the Philippines.
Like the Philippines, Hainan is famous for its tropical climate, white beaches and clean and sound ecological environment that made the province one of the best places for international conferences like Boao Forum for Asia.
Chen said that while the province achieved an impressive 62 million tourist arrivals in 2016, only 750,600 of them were foreigners.
He said direct flights, including the recently opened Hainan-Phnom Penh and Hainan-Singapore as well as future foreign direct flights, would improve the number of the province's visitors from other countries.
Hainan province, according to Chen, is also known for its medical tourism, maritime tourism and countryside tourism highlighted by agriculture villages development aimed at improving the lives of the farmers.
Sanya, located in the southernmost of Hainan, remains the province's top favorite destination primarily due to its well-managed tourism industry and breathtaking tourist spots.
Sanya Tourism Development Commission Kaizhong Ye said Sanya City government has placed massive investments to provide better services particularly security for the tourists as well as environment protection.
Ye said Sanya is called 'Hawaii' of China due to its picturesque scenery, beach and hot spring resorts, world's biggest duty free shopping mall and vast forested land including the famous 15,000 square kilometer Yalong Bay Tropical Rainforest Park.
Sanya received 16 million tourists in 2016 but only 438,083 of them came from other countries, including the Philippines and other Southeast Asian countries which delivered a total of 39,700 tourists.
Sanya Vice Mayor Dai Yuming said opening up more direct flights, including Hainan-Manila route, will improve the tourist arrivals not only in Sanya but the whole Hainan province.
We will not stop exploring best practices to improve and to make our city the best place to visit and spend holiday for the tourists, he said.
The relationship between China and the Philippines has been revived under the leadership of President Rodrigo Duterte.
According the data from the Department of Tourism (DOT) of the Philippines, more than 675,700 Chinese tourists visited the Philippines in 2016 or an increase by 37.7 percent compared to the previous year.
In 2016, China has reportedly replaced Japan to be the third largest source of the Philippines' foreign tourists, next to South Korea and the United States.
Source: Philippines News Agency


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|Latest News
|Feb. 2, 2020 6:28 PM PT7:28 PM MT8:28 PM CT9:28 PM ET2:28 GMT10:28 7:28 PM MST8:28 PM CST9:28 PM EST8:28 PM CT6:28 UAE (+1)21:28 ET0:28 - Raheem Mostert ran for 58 yards and one touchdown on 12 carries Sunday in the San Francisco 49ers' 31-20 loss to the Kansas City Chiefs. He added one reception for two yards.


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- Leslie Street
- Lebovic Avenue
- Little Blvd.
- Mount Pleasant Road
- Oakwood Avenue
- Petman Avenue
- Pharmacy Avenue
- Victoria Park Avenue
- Warden Avenue
- Weston Road
- Wynford Drive
- Yonge Street
Tertiary Entrance Excavation Update at Chaplin Station
Information as of August 22, 2019
Excavation continues at the Chaplin Station Tertiary Entrance site, located on the south side of Eglinton Avenue, east of Chaplin Crescent, beside the former Toronto Fire Hall. Excavators dig and remove soil from the work zone. Excavated material will be transferred into dump trucks and hauled to an off-site facility. Installation of tie-backs will take place from within the site as excavation progresses. Excavation will begin at 6:00 a.m. on weekdays.
DETAILS Read more »
Temporary Traffic Change for Wet Utility Work at Chaplin Station September 16 – 24, 2019
On September 16, a temporary change will be implemented on Eglinton Avenue, east of Chaplin Crescent to accommodate wet utility work for Chaplin Station. The northeast work zone will be extended further east and south of Latimer Avenue. Traffic on Eglinton Ave will be shifted south. No changes to pedestrian routes are anticipated. Once the work is complete, the work zone will return to the current configuration.
Upcoming Traffic and Pedestrian Configuration Read more »
Pedestrian Changes at Chaplin and Eglinton
What Work is Taking Place? Read more »
Pile Installation at Chaplin Station Secondary Entrance
In July and August 2019, crews will be installing micro piles at the Chaplin Station Secondary Entrance site, located on the southwest corner of Chaplin Crescent and Eglinton Avenue West. Piles will form the future supports of the station entrance structure below ground.
Construction Configuration
Details Read more »
Temporary Lane Reduction at Chaplin Station Week of July 29, 2019
A temporary lane reduction will be in at Chaplin Station the week of July 29, 2019, while crews construct a pedestrian walkway and install a dewatering pipe. Eglinton Avenue West will be reduced to one lane only between Gilgorm Road and Latimer Avenue, alternating traffic in each directi Read more »
Main Entrance Excavation at Chaplin Station
What Work is Taking Place?
Beginning the week of July 29, 2019, crews will commence excavation at the Chaplin Station Main Entrance site, located on the north side of Eglinton Avenue, between Chaplin Crescent and Gilgorm Road. Excavators will dig and remove soil from the work zone. The excavated material will be transferred into dump trucks and hauled to an off-site facility. Installation of tie-backs will take place from within the site as excavation progresses.
Current Construction Staging Read more »
Construction Notice: Chaplin Station Traffic Change July 2019
Upcoming Traffic Change at Chaplin Station
July 2019 Read more »
Updated: Road Closures for Traffic Bridge Installation at Chaplin Station
Updated July 4, 2019
Update: Due to various unforseen circumstances the re-opening of Chaplin Cres will be on July 12th, 2019.
Weekend Road Closures at Chaplin Station - Extended June 7-10, 2019
Update: Weekend Road Closures at Chaplin Station - Extended
June 7 – 10, 2019
Construction Notice: Overnight Hydro Utility Work at Chaplin Station
Overnight Hydro Utility Work at Chaplin Station
Week of May 5 – 10, 2019
Permit Number: 81015401
What Work is Taking Place? Read more »


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Jack Herer
Will the real Jack Herer please stand up? The 55/45 sativa hybrid with 23% THC was named after the famous marijuana activist and is one of the most coveted strains in America to date. Staying true to its namesake, just like the real Jack Herer, this hybrid offers a focused, clear-headed and creative experience, sought after in the academic and creative worlds.
Flavors: Earthy, Pine, Woody
Effects: Happy, Uplifted, Energetic, Euphoric, Creative
Medical: Stress, Depression, Pain, Fatigue, Lack of Appetite
Originating in California, the Haze hybrid combined with Northern Lights #5 and Shiva Skunk was developed to offer a heightened creative buzz, ideal for patients suffering from neurological disorders including: bipolar disorder, anxiety, migraines, PTSD and ADHD.
3 reviews for Jack Herer
Best deal on the net ! didn't know you could get smoke this nice for so cheap, you guys are awesome.
Fantastic strain. I'd done lots of research beforehand on Jack Herer and boy, this didn't disappoint. Reminds me a bit of Kratom: great engery booster, anxiety reliever without clouding up the head – provided you go easy on it.


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Re: Accumulating error counts?
- To: mathgroup at smc.vnet.net
- Subject: [mg52204] Re: [mg52171] Accumulating error counts?
- From: jmt <jmt at nerim.net>
- Date: Sun, 14 Nov 2004 04:31:25 -0500 (EST)
- References: <email@example.com>
- Sender: owner-wri-mathgroup at wolfram.com
Try this : test::failure="Failure !"; f[]:=If[Random[Integer]\[Equal]0,0,Message[test::failure]] globalCounter=0; Do[ Check[f[],globalCounter++],{5}]; globalCounter On Saturday 13 November 2004 10:40, AES/newspost wrote: > I'd like to turn off certain error messages like > > NIntegrate::ncvb: NIntegrate failed to converge > NIntegrate::slwcon: Numerical integration converging too slowly > > using Off[], but accumulate a count of the total number of times the > message occurred in a sequence of calculations. How do I get at that > number? > > (Better yet, I'd like to know the number of times it could have > occurred, as well as the number of times it did occur.)
- References:
- Accumulating error counts?
- From: AES/newspost <firstname.lastname@example.com>
- Accumulating error counts?


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Holmes Report 12 Aug 2018 // 7:00PM GMT
WASHINGTON — Digital agency REQ has named Kat Kuhl VP of technology and engineering. Kuhl (pictured) previously served as director of solutions consulting at FFW and director of technology at Chief. She brings deep experience in technical architecture, UX design, and application development, as well as an extensive background in technical project management and business strategy.
WASHINGTON — Signal Group has hired healthcare specialist Michelle Baker to join the firm as an executive VP. Baker , who has more than 20 years of public health communications and public affairs experience, joins from Weber Shandwick/Powell Tate where she led the firm's Washington healthcare practice.
LOS ANGELES — The Pollack PR Marketing Group has named A.J. Hernandez its new digital practice lead. Hernandez most recently oversaw digital communications for the Ronald Reagan Presidential Foundation and Institute. He was the marketing strategist for DP Technology before that.
POUGHKEEPSIE, NY — Kate Wark has been named executive VP of Impact PR & Communications. Wark brings more than 17 years of PR experience, having held positions at Quinn, Mower and Carolyn Izzo Integrated Communications.


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Click here to print &/or download a copy of this policy & the Instructions for the Student
For class sessions that have been video recorded, we offer the video (via Vimeo) to students who have a conflict on the Saturdays when the EEYM classes are held. With this option we require that the student meet with a mentor, pastor, or faith formation committee member(s) from his/her church of employment to view the video. After viewing the video together and participating in the class discussions with each other, the student will discuss two Reflection Questions with the mentor/pastor/committee member(s), and then submit written responses to these questions, which are included in an evaluation form for the session. The mentor/pastor/committee representative will also be asked to submit a brief reflection on this time spent with the student. Upon receipt of both sets of reflections (one from the student and one from the mentor/pastor/committee representative) the student will receive credit for "attending" the class.
Reflection Questions for the Student
Guiding Reflection Questions for the Mentor/Pastor/Committee Representative
Pens or pencils & paper or laptop/tablet for jotting down notes
Markers of various colors to share
[Just one for the group]: an inflatable globe or beachball or playground ball
Morning Worship - "bulletin"
Morning Worship - includes instructions
Prayer Sketching and Scavenger Hunt
Creative Worship with Youth
Biblical Reflection with Youth
Doing Bible study - Jairus' daughter
Doing Bible study - David
Doing Bible study - Miriam
Parts of Worship
Reflection Questions & Evaluation Form (for the student)
Guiding Reflection Questions Form (for the pastor/mentor/committee representative)
Please contact Debbie Gline Allen at firstname.lastname@example.com or call 508-603-6601


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Diynamic Festival reveals the lineup for its first London edition
The Hamburg-based label moves to Britain's capital city.
Diynamic has announced the lineup for its first London festival to take place next September 8th and 9th.
As usual, the label head Solomun will be leading the music gathering, followed by Adriatique, Kollektiv Turmstrasse, HOSH, Karmon, Johannes Brecht, Undercatt, Lehar, Magdalena, Musumeci, Andy Bros and Magit Cacoon.
The festival will be featuring a custom designed main stage with a D&B soundsystem, street food and bars, in order to receive over 5000 dance music enthusiasts.
For more info, click here.


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Welcome! The Vienna Coffee House opened the doors on October 6th, 2010 with a goal to be THE great neighborhood coffeehouse in Maryville, TN. We serve premier espresso drinks, (Mocha, Latte, traditional Cappuccino) crafted by serious Baristas and freshly roasted specialty coffees from the Vienna Coffee Company Roastery, located in the lower level of the coffee house. We strive to DELIGHT the customer with every visit! In addition to great coffee, we serve up a variety teas, chai, smoothies, steamers and lots of fresh baked goods made in house, and other delicious food items. We offer free Wi-Fi and we have live entertainment weekly on many nights of the week, whether it's a local musician or folks playing for open mic night. Check out our Music schedule. We hope you stop by to check out the great selection of treats and cozy atmosphere!
Frequently Asked Questions
Places You Should Consider
People Connected
Add to My Connections
Plug Updates
Say Something or Ask a Question about The Vienna Coffeehouse


text: We can talk about ourselves all we want, but your personal contact with us will mean more than our words ever can. That's why we invite you to visit Amrein Wohnen, to form your own impression of us and our inspiring ideas for your home. Click around our website, or even better: Visit us in person. You will find the first info here at amrein.ch. And in our large showroom in Kriens you will find everything you need to turn your very individual home furnishing ideas into reality.


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The continual increase in aircraft travel, coupled with the increasing drive to reduce the impact of noise on the environment (and on passengers and crew!), has significantly raised the profile of aircraft noise reduction. The ISVR has, for many years, been at the forefront of the analysis and control of sound radiation from vibrating panels, such as in aircraft structures.
Current research in the group, in collaboration with our Dynamics group, is directed at co-locating novel sensors and actuators into flexible panels and in distributing such pairs strategically around the panel to act as local controllers of vibration within their own local regions of interest. This promises to produce improved control of vibration and sound radiation and to overcome the dependency on a centralised controller. With the current thrust to develop micro-fabricated sensor-actuators such technology could then be integrated into panels such as used in an aircraft fuselage and could offer the possibility of further reductions in radiated noise.
Active Control of Sound and Vibration
Active Control of Structure-Borne and Air-Borne Sound Transmission Through Double Walls
Active Control of Vibration Transmission
Active Position Control of Flexible Structures
Active Sound Transmission Control
Active Structural Acoustic Control
Control Strategies for Active Headsets


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CSCC Spring Autocross #5 4/28/19
Event Requirements
Registration closed April 27, 2019 11:00 PM on April 27, 2019 11:00 PM EDT Registration ends April 27, 2019 11:00 PM on April 27, 2019 11:00 PM EDT Registration opens April 22, 2019 12:00 AM on April 22, 2019 12:00 AM EDT Registration runs from April 22, 2019 12:00 AM to April 27, 2019 11:00 PM
NEW! - Membership Validation
- When you register, MotorsportReg will be verifying your CSCC membership status against our current member database. If you do not have a valid membership, you must select the "Non-Member" option before you will be allowed to complete your registration.
Your First Name, Last Name, and Member Number in MotorsportReg must match what is on your CSCC Membership Card:
- You can check your MotorsportReg info by clicking the "My Account" tab, then "Edit Clubs" and editing the information under "CSCC - Clemson Sports Car Club"
- You can check your membership info by contacting CSCC on the forum at clemsonscc.org, e-mailing us at firstname.lastname@example.com, or messaging us on the Official Facebook page.
- If you are having trouble registering due to this new feature, or if you recently joined CSCC and your membership is still pending, please contact us at the above noted email address.
- Thanks for your patience as we implement this new process! This will make our on-site registrars even faster and more efficient, so we appreciate you working with us!
- This event is held under CSCC classing and rules.
- Please review the CSCC Supplemental Rules for specific details on event conduct, car numbering requirements, and event procedures.
- All participants must sign the Release and Waiver form. Minors (under age 18) must have a Minor Release and Waiver form signed by at least one parent in order to be allowed on site.
- Cancelations must be sent to the club at email@example.com immediately. Please do not simply cancel your registration on the site.
- Refunds will be issued for anyone who is unable to attend; however, there is a $5.00 cancellation fee.
Number Format
- We allow up to three digit numbers for entrants (0-999). Numbers are first come first serve for each event. Duplicating numbers such as 001 vs. 1 are not recognized as being different or unique than the number with no leading zeros and you will be asked to change your number accordingly.
Registration closed April 27, 2019 11:00 PM at April 27, 2019 11:00 PM EDT
Registration opens April 22, 2019 12:00 AM at April 22, 2019 12:00 AM EDT
To register, select one of the following options and continue...Sign in Create free account


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There were two weddings these past few months I could not go to because I'm in another state. I'd like to get them gifts, but am wondering if it's in bad taste to give them one of those visa gift cards or something. I'm going back to Texas next month so I could give it to them then.
I don't know. I also don't have a lot of money to give as I am in grad school and yeah some of us should know how that goes.
Let me know what you think.
Edit: For both couples, I know the bride better. For one of them, I've actually never met her husband or then-fiance. So in both cases I really don't know the couple themselves very well at all. I'd say they are my friends, but we are not extremely close.


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Symptoms of sickle cell disease may be noticed as soon as 4 months of age, or it may go undetected until later in the baby's first year of life.
Oxygen deprivation is a result of blood vessels that are blocked by the misshapen red blood cells of sickle cell anemia. Periods of acute oxygen deprivation cause severely painful episodes called pain crises. The location of the pain and the types of symptoms depend on what tissues or organs of the body have been deprived of oxygen.
Symptoms of sickle cell disease include:
Other medical conditions that can result from sickle cell disease include:
Sickle cell crisis can be provoked by certain triggers, including:
Sickle cell disease. Kids Health—Nemours Foundation website. Available at: http://kidshealth.org/en/parents/sickle-cell-anemia.html. Updated September 2012. Accessed July 1, 2013.
Sickle cell disease in adults and adolescents. EBSCO DynaMed Plus website. Available at:http://www.dynamed.com/topics/dmp~AN~T902929/Sickle-cell-disease-in-adults-and-adolescents. Updated October 4, 2016. Accessed October 5, 2016.
Sickle cell disease in infants and children. EBSCO DynaMed Plus website. Available at:http://www.dynamed.com/topics/dmp~AN~T902928/Sickle-cell-disease-in-infants-and-children. Updated September 20, 2016. Accessed October 5, 2016.
What are the signs and symptoms of sickle cell disease? National Heart, Lung, and Blood Institute website. Available at: http://www.nhlbi.nih.gov/health/health-topics/topics/sca/signs. Updated August 2, 2016. Accessed December 13, 2016.
Last reviewed December 2016 by Marcin Chwistek, MD Last Updated: 5/20/2015


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In five thousand years of Chinese history, Sima Clan family produced the best known and most important historians. Sima Qian and Sima Guang top the list.
Sima Qian is considered the father of Chinese historiography for his Records of the Grand Historian. Sima Guang's Zizhi Tongjian is a pioneering reference work in Chinese historiography.
Not only their historical books are well-known, their private lives with remarkable stories are often remembered as moral models in the minds of millions. We have a brief introduction here.
Sima Qian and Records of the Grand Historian
Sima Qian was born at Xiayang in today's Shaanxi Province around 145 BC.
Around 136 BC, his father, Sima Tan, received an appointment to the position of "grand historian". The grand historian's primary duty was to formulate the yearly calendar, identifying which days were ritually auspicious or inauspicious, and present it to the emperor at the end of the year.
Besides these duties, the grand historian was also to travel with the emperor for important rituals and to record the daily events both at the court and within the country.
By the age of ten, Sima Qian, able to read the old writings, was considered a promising scholar. As he grew up in a Confucian environment, Sima always regarded his historical work as an act of Confucian filial piety to his father.
Around the age of twenty, Sima Qian began an extensive tour around the country. He started his journey from the imperial capital of Chang'an to the south of Changsha across the Yangtze River, then to the north of Huaiyin and Qufu,.the hometown of Confucius.
After his travels, Sima became an attendant in the imperial palace. He married young and had one daughter.
In 110 BC, at the age of thirty-five, Sima Qian was sent westward on a military expedition against some "barbarian" tribes. That year, his father fell ill due to the distress of not being invited to attend the Imperial Feng Sacrifice. Suspecting his time was running out, he summoned his son back home to complete the historical work he had begun.
Fueled by his father's inspiration, Sima Qian started to compile Shiji, which became known as the Records of the Grand Historian.
Sima's Shiji is respected as a model of biographical literature with high literary value and still stands as a textbook for the study of classical Chinese. His works were influential to Chinese writing for two thousand years.
Sima Guang and Comprehensive Mirror in Aid of Governance
The Zizhi Tongjian, or Comprehensive Mirror in Aid of Governance, was published in 1084 in the form of a chronicle.
In 1065 AD, Emperor Yingzong of Song Dynasty ordered the great historian Sima Guang (1019–1086 AD) to lead other scholars in the compilation of a universal history of China. The task took them 19 years to complete.
The Zizhi Tongjian records Chinese history from 403 BC to 959 AD, covering 16 dynasties and spanning across almost 1,400 years, and contains 294 volumes and about 3 million Chinese characters.
The Simas were originally from Xia County in Shǎn Prefecture, and claimed descent from Sima Fu in the 3rd century. A famous anecdote relates how the young Sima Guang once saved a playmate who had fallen into an enormous vat full of water. As other children scattered in panic, Sima Guang calmly picked up a rock and smashed a hole in the base of the pot. Water leaked out, and his friend was saved from drowning.
At age 6, Sima Guang once heard a lecture on the 4th-century BC history book Zuo Zhuan. Fascinated, he was able to retell the stories to his family when he returned home. He became an avid reader, "to the point of not recognizing hunger, thirst, coldness or heat".
Sima Guang obtained early success as a scholar and officer. When he was barely twenty, he passed the Imperial examination with the highest rank of jìnshì -metropolitan graduate, and spent the next several years in official positions.
Translation by staff editor


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Drew Barrymore was right on trend with her pretty floral shift dress on The Tonight Show last week. Her actual dress is from J.Crew, but frugalistas might prefer our more affordable take on her look.
drew barrymore's spring blooms
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Rotisserie Chicken and Mashed Cauliflower
1 rotisserie chicken (Publix is Gluten Free)
1-2 bags of cauliflower rice fresh or frozen
1 unsalted gf chicken stock (Kitchen Basics)
Green veggie of your choice
Prepare the cauliflower according to bag directions (I use chicken stock in place of water). Drain and pour into a deep bowl.
Add a tablespoon or 2 of mayo or sour cream, or butter and your favorite seasoning (garlic or Wildtree Rancher Steak Rub are my favorites. Even horseradish) plus salt and pepper to taste.
Mix it all together with a hand mixer. Add a little chicken stock at a time to achieve the texture you prefer. Dinner will be ready to serve in 10 minutes. You can also pour the chicken stock into a sauce pan before you start any of your prep. Heat it over medium heat to allow it to reduce while everything else is cooking. Season it with garlic salt and pepper. Drizzle over your mashed cauliflower and chicken for another delicious layer of flavor.


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Located near the Melrose shopping neighborhood, the new 3.1 Phillip Lim store and showroom announces the designer's west coast debut with the transformation of 5000 square foot auto body shop into a flowing interior.
By Para-project. read on for rest of project description and project fact sheet
The experience is a curious indulgence taking clients from the sunny LA sky into the intimate ambiance of Phillip Lim.
An Undulating Wall: Amoeba, Niches and Nooks
By introducing a thick curving wall within the existing building envelope one large main space unfolds into four smaller niches. Mirrors along the walls enhance the spatiality of the store and create visual continuity between adjacent spaces.
The curving geometry and thickness of the wall allows for the conventional methods of lighting and display to be rethought. With most of the spotlights within the thickness of the wall, the ceiling is free of the clutter of typical track lighting. A single, continuous light-diffusing membrane floats above the space adding to the intimate atmosphere while giving the space a sense of lightness.
Liners: Atmospheres + Ambiances
Like the construction of a garment, the curving wall has an inner 'lining' and outer 'lining.' The pyramidal texture on the walls is actually soft to the touch creating a sensation of being both hard and soft. The smaller niche spaces are each lined with different materials; wallpaper, Spanish cork, leather herringbone, and bamboo creating a variety of tactile vignettes for the clothing to be displayed in.
A Blank Facade
As an alternative to the typical window display, the exterior façade has no openings except the entrance. Instead of windows, the façade is surfaced in a stark yet supple pattern of concrete tiles shaped like pillows. The emphasis of the blank façade acts like a 'palette cleanser' as one transitions from the hustle and bustle of the street-scape to the intimate interior.
Project description © PARA-Project, LLC.
In Collaboration with: Office/Giancarlo Valle
Location: 631 N. Robertson Blvd, West Hollywood, CA
Program: Fashion Boutique Flagship Store, Office, Showroom
Area: 5000 sf. interior, 2000 sf. exterior
Completion: 2007-2008; 8 months
Design Team: Dominic Leong, Jonathan Lott, Brian Price, Giancarlo Valle
Staff: Andrew Seiger, intern
Executive Architect: Leong Architects, Inc.
Consultants: MKM Associates, Structural Engineer
Client: 3.1 Phillip Lim
Contractor: Hinerfeld-Ward Inc.
All Images © Iwan Baan


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I have joined with Free Is For Me to bring you this great giveaway. Free Is For Me recently had the pleasure of reviewing this awesome toy! Her kids went crazy when she told them that she was receiving a Happy Napper from Coppin's Gifts to review! They literally waited by the door to see which Happy Napper would become their new pal. Happy Nappers come in 8 different pals including monkey (hut), penguin (igloo), dragon (castle), ladybug (bungalow), unicorn (palace), bear (log cabin), cow (barn) and the one my daughters received the dog (house) and if you are the winner you can choose one of the above for yourself! To read the full review on how awesome these toys really are head over here and leave a comment and get a bonus entry!
Happy Nappers Giveaway
If your kids have been begging for a Happy Napper or if you just want to get an early start for Christmas this is your chance. Thanks to Coppin's Gifts they are offering our readers a chance to win a Happy Napper of their choice ($24.95 value!)
Giveaway Information: Giveaway is open ONLY open to residents of the 48 contiguous US states and must be 18 years or older. Giveaway will begin at 12:01 am on 8/10/2012 and expire on 12:01 am on 9/01/2012. One winner will be chosen to win a Happy Napper ($24.95 value) of their choice from Coppin's Gifts. The winner will have 24 hours after notification to contact Free is for Me or we will choose another winner. The winning prize will be shipped directly from Coppin's Gifts and Free is For Me or
Special thanks: Special thanks go out to all our special co-hosts who without them this would not be nearly as fun for all of us! Take some time to look at their pages as they are also offering other great giveaways! Saving Common Cents, Saving Makes Cents, Kara's Deals and Steals, The Momma and Boys, Freebies with Attitude, Sweet N Sour Deals, The Frugal Navy Wife, Lynchburg Coupon Mama, Missi Rose's Coupon Spot, Mommy Coupon Swappers, Trinkies, Two Chix Beauty Fix, Saving Money Can Be Fun, Frugal-Shopping, Our Wolves Den, Young Mom's Deals, Avon by Cristin Miller, Frugal Family Fun, Annalisa Price It works! distributor, & Julie Gonzalez, Avon Representative.
Disclosure: Free Is For Me received a free product from Coppin's Gifts to review and write an honest review of their product. They were not compensated for this review and all opinions and thoughts are their own. Saving Money Can Be Fun is not responsible for shipping this prize, the winning Happy Napper will be shipped directly from Coppin Gifts,
a Rafflecopter giveaway


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Vickie Henderson has published a most remarkable sketchbook of her sketches and journal pages of the red-shouldered hawk through its nesting season.
I first encountered Vickie Henderson on Cathy Johnson's Sketching in Nature blog, and have followed Vickie's blog and sketchbook ever since.
I hesitated buying the sketchbook, but then I decided it was a must have for personal reasons: I love hawks, all species, and I wanted to see Vickie's work on paper and not on the computer screen. It was a wise decision. The book is beautifully done--both the sketches and the printing itself.
Check Vickie's work and you'll be impressed not only with her skill but the number of bird species she spies in her native Tennessee.
Sketchbook: Red-shouldered Hawk Territory
Posted by Zoe, ontheroad at 4:41 PM
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Mike From the MK Job Show & Elizabeth From EWR Alliance
The MKFM Lunch Break
- Podcast Home
- Download Episode
- Subscribe to this podcast
Wednesday, February 12th, 2020 - 7 minutes
Mike from the MK Job Show and Elizabeth from EWR Alliance talk to Lia about the region's most attended recruitment event coming to Middleton Hall on 27th and 28th March.
Sponsored Stories You May Like
Local News
Four new coronavirus cases in Milton Keynes today
Four new coronavirus cases have been identified in Milton Keynes today.
Milton Keynes Council Leader says increase of positive coronavirus cases is "concerning"
In his weekly COVID-19 video update, Peter Marland warns residents, "If people continue to not follow the guidance and cases continue to rise it is highly likely that restrictions will be reimposed here."
"GO AND GET IT CHECKED!" Greg Rutherford urges men to check their testicles
Olympic athlete, Strictly Come Dancing Star and local family man Greg is encouraging men to check their testicles for lumps, after he reveals he ignored his.
Appeal for witnesses following assault in Milton Keynes
Thames Valley Police is appealing for witnesses following an assault in Milton Keynes. At 1.07am on Tuesday (11/8), two people were involved in an altercation on Bradwell Road, New Bradwell.
Here are all the events restarting at Parks Trust parks in MK - including pond dipping and bug hunting!
The Parks Trust in Milton Keynes are now able to start re-introducing some small-scale events back into their parks.
Oakgrove School students celebrate 'fantastic' A-Level Results
Natural Sciences at Cambridge, Law at Oxford and History at Durham are just some of the exciting paths of further study students will be undertaking, following excellent A level results achieved at Oakgrove
Watchdog threatens MK Council with contempt of court over Blakelands freedom of information request
Residents have won another battle in their fight to get to "the truth" over how a giant warehouse was permitted near their homes.
Developers prepare to submit plan for first 800 homes in Milton Keynes Eastern Expansion Area
Developers have asked planners at Milton Keynes Council for their opinion on the potential environmental impacts of building up to 800 new homes on agricultural land south of Newport Pagnell.
Appeal for witnesses following assault in Milton Keynes
Thames Valley Police is appealing for witnesses following an assault in Milton Keynes. It happened between midnight and 6.30am on Sunday (9/8).
Green light given for new community hub in Great Linford
Planning permission has been given for an impressive new community building in Great Linford Parish, with work due to start at the end of September.
Destination Milton Keynes appoints new Membership Manager
Destination Milton Keynes (DMK), the official tourism and information service for the city, has announced the appointment of Kelly Wesley as the new Membership Services manager.
Watling Academy prepares to welcome first pupils
Watling Academy will open its doors to Year 7 pupils in September from a purpose-built facility at Fairfields Primary School.
You May Have Missed
'Scum' spray-painted onto Milton Keynes couple's store window as well as other graffiti
A Milton Keynes couple have had the word 'Scum' spray painted onto their store window, as well as other graffiti on their store sign.
Milton Keynes local coronavirus case numbers do NOT include Drive-Through test centre results
A national newspaper has revealed that government coronavirus case numbers on a local authority level do not include any tests from local drive-through test centres and some home testing kits.
The areas in Milton Keynes where the most deaths involving coronavirus have sadly happened
The Office for National Statistics have revealed new localised data on the number of deaths which have taken place involving coronavirus.
Deadly blue-green algae appears in another Milton Keynes park as warning sent to dog owners
Deadly blue-green algae has been identified in another Milton Keynes park as dog owners are warned to keep their animals out of the lake.
Cafe Rouge to close in Milton Keynes as owners of chain enter administration
Cafe Rouge in Milton Keynes will be closing permanently as the owners of the chain enter administration.
Coronavirus test centre employee's outcry over being charged to park whilst at work in Milton Keynes
Employees at the drive-in coronavirus test centre in Central Milton Keynes have to pay to park each day, it has been revealed.
Cat is stabbed twice in Milton Keynes
Last week, a family discovered that their pet cat had been stabbed in each of her back legs.
EXCLUSIVE: McDonalds to restart breakfast service in Milton Keynes from July 8th
MKFM understands that McDonalds will be re-starting their breakfast service in all Milton Keynes stores from 8th July.
- How To Listen To MKFM
- MKFM and GDPR Data Regulation
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© 2020 MKFM Limited - Radio Made In Milton Keynes. Powered by Aiir.


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August 8 & 9, 2015
SkyDive for Kids
Children's Advocacy Center of Benton County
Join Children's Advocacy Center of Benton County for a day of excitement, anticipation, and hope as 30 to 40 jumpers pledge to raise $1,000 and skydive at SkyRanch in Siloam Springs to raise funds and awareness for abused children in Northwest Arkansas. Lunch will take place from 11 am to 1 pm and the entertainment will be continuous as each of the jumpers make the climb to 10,000 feet on Saturday and Sunday. With the help of jumpers and the NWA community, Children's Advocacy Center of Benton County hopes that the literal leaps of faith will indeed forever alter the journey of not just one but hundreds of children in Benton County.
For more information, visit www.cacbentonco.com.