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I've had anxiety for as long as I can physically remember. It's always been lingering around, not always giving me too much of a problem, but definitely rearing its head from time to time and showing up in annoying ways. With anxiety for a lot of people (myself included) depression can be a serious comorbid problem that pops up somewhere along the way. This can seriously make things more difficult than they were before, and honestly, can make life really difficult to live. Sadly, this is a really common problem that so many people struggle with, but next to no one wants to talk about.
Oh well, I'm going to talk about it, because it's important. I'll throw some caution to the wind and just be candid.
Anxiety and its related illnesses have left me laying in bed all day more days than I'd care to admit. I really had a hard time during my high school years, and really ended up feeling like I was someone on the outside looking in. Things looked up for me once I started university, but it was around then that I stumbled upon someone describing the idea of "No zero days" and my outlook on my problems changed drastically. I would love to think that this little technique has saved me a lot of problems and sadness along the way.
I wish I had a link to where I found this technique, but it's likely lost somewhere in the archives of reddit. If you know me personally, you know that I pretty much live for reddit, and for once, it came in clutch with an idea that is really useful.
In my eyes, the idea of a "no zero day" is something that comforts me when I can't really pull things together as much as I would like to on a normal day. All I have to do is one thing that is productive, which constitutes the day as a "no zero day". For me, usually it's making something to eat, cleaning up a little, or doing some homework. For others, it might be getting up and taking a shower or something else. It really is vastly different for everyone.
I love "no zero days" because it takes back the little sense of control that I can, in fact, make something positive happen in my life on the days that are more difficult than others. It keeps the day from being a total wash, while also not putting exorbitant pressure to be perfect on the days that are a little more down than others.
Like I said, it really has changed my life for the better, and helps me to get moving on days its too hard. Completing one task for me often leads to completing another, and gets me up and moving and on a much more positive path than I would have been on if I had stayed in bed or not left my apartment. It puts the power back in my hands, on days I feel like I have no control at all. Then, it's really up to me on how much I can complete, or if it's day than I should stay in and just try my best to take care of me. Both options are positive and fine by me.
So, how can you have a "no zero day"? It really is all up to you. Pick one task to complete, and then reassess and see if you feel up to another. No task is too small (or too large) and everything counts. Getting up and moving really can make a difference in your mood and overall health. Remember it's okay to have some days where you just stay put too!
Do you know someone who struggles with similar issues, or do you yourself? What are your favorite ways to combat anxiety or other similar conditions? Share in the comments below, I would love to know and try out some new techniques!
Happy Tuesday!
38 comments on "No Zero Days – My Mindset on Living With Anxiety"
I like your suggestion in one task at a time. I experience anxiety too. Not severely but definitely noticeable. To the point where I can get nothing done sometimes especially if I feel overwhelmed with too much to do. I try to distract myself as Best as i can with Something entirely different. This helps most times.
Thanks for stopping by Sandy!
I too struggle with anxiety. I worry about what WILL happen in the future and about things that are out of my control. Control seems to feed my anxiety. What I do to tame my anxiety is read lots of inspirational quotes to help change my mindset and to put things into perspective. I try to live in the present moment and trust that the universe has "got this." From my experience I learned that everything turns out the way it is supposed to be. So I plan the unexpected and expect to be surprised. 🙂
That's so awesome that you found something that works for you Christine!
I give you so much credit for your honest – living with anxiety can be so hard – I have a close coworker who has very similar struggles & she's read all sort of books on the topic – she especially loved "The Power"
I'll have to look into it!
I'm so happy your doing better!
Thanks Mary!!
I hope your next zero day provides all of the comfort that you need. You are brave for speaking up, and I wish you days of bravery ahead. <3 <3
Thank you Dionne <3
This sounds like such a good idea. I plan on implementing this into my off days. Thank you so much for posting this!
I hope it works for you Brenda!
yes yes yes! I hope for many more "no zero days" if you need it. and a relaxing day if you do. speaking about anxiety really helps people that are dealing with it that don't know it yet. while i have never dealt with this issue, i know a lot of my friends in college have.
It affects more people than you would think!
One of the things I love most about blogging is that you get to know there are more people out in this world that can relate to you than you think. Everyone struggles with something and it's wonderful that you're sharing your story with others. I wish you the best of luck with all you do! 🙂
Thanks Cassie!
Such a great technique for managing anxiety, I actually use a similar technique and make sure to leave myself time to relax to not over work myself. I also find that staying active has made a huge difference with managing any anxiety or stress I may be experiencing. Great post!
Staying active is so pivotal.
I like this idea of no zero days! I've been struggling with anxiety to! I always take a really short nap when I feel anxious and it really helps:) Or when I can't take a nap I try to think of beautiful things.
I hope you feel better! Once you find something that works, you'll feel much much better.
I think I've had anxiety longer than I even knew how to identify it. In fact, I'm still trying to figure out how it works. I don't have it like most people, like how you describe but it's definitely there. I really love how you're not afraid to talk about it. I wish more people would seriously talk about it. When I talk about it I get those looks – you know the ones. It's like I'm crazy for feeling this way and only people who have it understand. I'm so happy that you've found something that works for you and gives you a lifeline. Because even if it's small it helps.
Hi Christina;
Talking about things like anxiety is so important because it still holds some silly stigma when it shouldnt. I hope you find something that helps; it takes time but its worth it!
I have suffered from anxiety myself and just want to tell you that there will be good days and bad days. Don't give up and you'll be just fine
I love the idea of this! For me, I've found that if I force myself to go to the gym (even if that's ALL I do), it helps tremendously! So I guess I've been having "no zero days" and not even realizing it!
I like the idea of a no zero day — doing at least one productive thing that makes you feel better (no matter how big or how small).
I had not known this before, but I realized few years back that what I had been living with all my life was anxiety. I have learned to work on it and this past year has been better. A lot of meditation, saying "no" to anything that brings stress and living in the moment has helped. I love your post!
Thank you for sharing this. I've struggle with anxiety many times throughout my life and it's a very scary time. What gets me through tough times is listening to some music to calm me down.
I have a friend that suffers from anxiety and she gets relief by meditating.
I love the no zero day idea. When I get anxiety attack I usually put on some music, go to a peaceful and quite room and calm myself down. But I will definitely give it a go!
I suffer from anxiety. I've been doing meditation quite a bit lately which seems to help. I'm glad the no-zero day concept has really been helpful to you.
I spend 15 minute each day just focusing on my breathing, I find this helps me a lot.
I like that "no zero days." Making a continuous list of things to do helps with that! Great suggestion!
Oh goodness, this resonates with me so much. I've suffered from anxiety for years and finally have it under control but I totally understand what you mean. Head up and don't give up! http://www.aniatravels.com
Thanks for sharing, a lot of people suffer silence.
love love love this more than I can say. depression and anxiety can be debilitating – it's taken more days than I'd like from me too. finding just one thing to accomplish can be all the difference!
I have not had anxiety before but would imagine it to be a daily struggle and I admire you for courage and strength to be so outspoken about this and share your thoughts and journey.
From time to time, my head can mess up my plans this "no zero days" is so cool technique. When I have days like these I love to make myself do something that I love and enjoy or going somewhere.
Living with anxiety is a battle within itself, but the best way to overcome is to BREATHE and do things one step at a time.


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There are realities we cannot avoid. One of them is the importance of self-esteem.
Regardless of what we do or do not admit, we cannot be indifferent to our self-evaluation. However, we can run from this knowledge if it makes us uncomfortable. We can shrug it off, evade it, declare that we are only interested in "practical" matters, and escape into baseball or the evening news or the financial pages or a shopping spree or a sexual adventure or a drink.
Yet self-esteem is a fundamental human need. Its impact requires neither our understanding nor or consent. It works its way within us with or without our knowledge. We are free to seek to grasp the dynamics of self-esteem or to remain unconscious of them, but in the latter case we remain a mystery to ourselves and endure the consequences.
~ Nathaniel Branden, The Six Pillars of Self-Esteem

subset: commoncrawl


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Cancers and tissue stem cells (SCs) share similar molecular pathways for their self‐renewal and differentiation. The race is on to identify unique pathways to specifically target the cancer, while sparing normal SCs. Here, we uncover the transcription factor Runx1/AML1, a known haematopoietic and leukaemia factor, albeit dispensable for normal adult SC homeostasis, as being important for some mouse and human epithelial cancers. We implicate Runx1 as a SC‐intrinsic gene in mouse hair follicle and oral epithelia by genetic lineage tracing in adulthood. Runx1‐expressing SCs, but not other cells that ectopically upregulate Runx1 by injury and inflammation, are at the skin tumour origin. Runx1 loss impairs tumour initiation and maintenance and the growth of oral, skin, and ovarian epithelial human cancer cells. Runx1 stimulates Stat3 signalling via direct transcriptional repression of SOCS3 and SOCS4 and this is essential for cancer cell growth. Thus, Runx1 is a broader epithelial SC and cancer factor than previously recognized, and qualifies as an attractive potential target for both prevention and therapy of several epithelial cancers.
There is a Have you seen? (November 2012) associated with this Article.
Increasing evidence, including genetic lineage tracing, seems to support the theory that some cancers, such as skin basal and squamous cell carcinoma (SCC) (Youssef et al, 2010; Lapouge et al, 2011; White et al, 2011), intestinal cancer, adenocarcinoma, and leukaemia originate in tissue stem cells (SCs; Visvader, 2011). Alternatively, some cancers might arise through de‐differentiation of cells that hijack the normal SC self‐renewal pathways (Visvader, 2011). In either case, for cancer therapies to work we must identify factors that are essential for the survival and proliferation of cancer cells, yet dispensable for normal SCs. There could be selective pressure among cancer cells to preserve the genomic locus of factors essential for their own growth and survival, and thus these factors might not surface in conventional human genetic mutation screens. In fact, such factors could be difficult to identify without in depth knowledge about the genetic control of normal and cancer SCs, which are difficult to access directly in humans.
We recently identified Runx1 as a factor involved, but dispensable for mouse skin epithelial hair follicle SCs (HFSCs) proliferation in vivo (Osorio et al, 2008). In contrast, Runx1 was strictly required for mouse skin papilloma and SCC formation and for cultured keratinocyte proliferation (Hoi et al, 2010). This makes Runx1 an attractive potential target for cancer that would not affect normal tissue homeostasis. However, the significance of our mouse finding to human cancer biology remained unclear. Human skin SCC is the second most common cancer after skin basal cell carcinoma (BCC), with ∼0.8 million new cases per year in the United States alone, of which ∼8% are metastatic and cannot be treated with current methods (Alam and Ratner, 2001). Head and neck (oral) SCC are extremely invasive and currently uncurable, and have several regulatory pathways in common with skin SCC (Molinolo et al, 2009).
Runx1 is recognized as a human leukaemia and haematopoietic stem cell factor (Mangan and Speck, 2011), but its role in human SCC and potentially other epithelial cancers is poorly understood. Recently, Runx1 SNPs were associated with human colon and rectal cancer (Slattery et al, 2011) as well as with prostate cancer (Yeh et al, 2009; Huang et al, 2011), and Runx1 was found expressed in endometrial and ovarian cancer (Planagumà et al, 2006, 2011). Moreover, triple negative (−Her2, −ER, −PR) human breast tumour growth in culture depends partially on Runx1 expression (Wang et al, 2011).
This evidence began to suggest that the leukaemia factor Runx1 might be more broadly implicated in cancer than previously recognized. Here, we present a breadth of evidence that Runx1 is overexpressed in a significant fraction of human epithelial cancers, and is essential for the growth and survival of three of them: skin SCC, oral SCC, and in part for ovarian cancer. Our lineage tracing data in adult mice show for the first time by lineage tracing that Runx1 is expressed in adult HFSCs and in oral epithelium SCs. The oral epithelium SCs were previously poorly characterized. Runx1 appears essential for solid tumour initiation and maintenance by upstream stimulation of Stat3 signalling, a central cancer pathway implicated in several epithelial cancers and in leukaemia (Chan et al, 2004a, 2004b; Yu and Jove, 2004; Aziz et al, 2007; Chan et al, 2008; Kim et al, 2009; Li et al, 2011). Together with the notion that Runx1 is not critical for normal adult tissue function, our work places this gene as an important candidate for epithelial cancer targeting.
Runx1 is overexpressed in various epithelial tumours
Previously, we found Runx1 expressed in the same pattern in normal mouse and human skin (Hoi et al, 2010). Runx1 has a dynamic expression in the hair follicle during its homeostatic cycle (hair cycle), which consists of the morphologically distinct stages of growth and proliferation (anagen), regression (catagen), and rest (telogen) (Supplementary Figure S1; Tumbar, 2012). During quiescence, Runx1 marks a subset of the hair follicle bulge and hair germ cells where the SCs reside (Osorio et al, 2008). Moreover, Runx1 protein is detected in the bulge during anagen when HFSCs self‐renew (Osorio et al, 2008; Zhang et al, 2009). The embryonic precursors of HFSCs express Runx1 (Osorio et al, 2011). Lineage tracing to formally prove Runx1 expression in adult HFSCs has not yet been done, but loss of Runx1 at different stages of the hair cycle delays HFSC activation from quiescence (Osorio et al, 2008), and slows down bulge cell proliferation during anagen (Hoi et al, 2010). Finally, we found Runx1 expressed at high levels in mouse skin papilloma and SCC, and Runx1 deficiency impaired mouse skin tumorigenesis (Hoi et al, 2010).
To examine the expression pattern of Runx1 in human epithelial cancers, we first surveyed the Oncomine public database, which contains microarray data comparing primary tumours and normal tissue. We found Runx1 to be overexpressed in several types of cancers in 47 studies and underexpressed in only 5 out of 138 studies analysed. The data are summarized in Figure 1A and Supplementary Table S1. Notably, comparing tumour to normal tissue Runx1 was among the top 1% of all overexpressed genes in head and neck, and skin SCC, and was highly expressed in several other epithelial tumours such as oesophageal SCC, cervical carcinoma, and colon adenocarcinoma. Next, we verified Runx1 expression by western blot and immunofluorescence staining of a panel of human cancer cell lines from breast (MCF‐7; MDA‐MB‐231), prostate (PC‐3; DU‐145), colon (HCT116; CCSC, Sikandar et al, 2010), ovary (OVCAR‐3; SKOV3), head and neck (oral) SCC (SCC66, SCC74, SCC125; White et al, 2007), and skin SCC (SCC13; Rheinwald and Beckett, 1980, 1981), as well as a mouse skin SCC cell line (Pam212; Yuspa et al, 1980). All cancer cell lines tested expressed more Runx1 relative to normal mouse keratinocytes, while we detected close to no Runx1 expression in mouse fibroblast, which served as negative control (Figure 1B–D; Supplementary Figure S2A and B). Attesting to the specificity of our antibody, we detected no Runx1 signal by immunostaining of knockout (KO) mouse keratinocytes (not shown). The different cancer cell lines showed variable levels and some contained only a subfraction of Runx1‐positive cells. Notably, all head and neck and skin SCC cell lines showed a consistently high level of Runx1 expression.
Finally, we checked for Runx1 expression in human primary tumours of skin cylindroma, skin SCCs, and BCCs, as well as head and neck (oral) SCCs by immunofluorescence staining. We found Runx1 expressed in a subset of tumour cells including the proliferative edge, and these cells co‐localized in part with Ki67 (a proliferation marker), K15 (an HFSC marker), or K8 (a malignancy marker; Casanova et al, 2004). The results are detailed in Table I, Figure 1E, and Supplementary Figure S2C.
The widespread expression of Runx1 in human epithelial cancers uncovered here, together with the strict requirement of Runx1 for the formation of mouse skin SCC (Hoi et al, 2010) warranted further examination of a potential role of Runx1 in human solid tumours and prompted us to delve deeper in the essentials of its mechanism. Specifically, we asked whether Runx1 expression is (a) required at the tumour initiation, promotion, or maintenance stage; (b) found in normal SCs and in cells of tumour origin; (c) ectopically raised by injury and inflammation in cells of tumour origin; (d) essential for human epithelial cancer cell growth and survival; (e) coupled with known signalling pathways of epithelial cancers.
Runx1 is required for tumour initiation but is dispensable for promotion
Previously, we showed that conditional deletion of Runx1 in mouse epidermis using a constitutively active K14‐Cre allele drastically reduced papilloma and skin SCC formation (Hoi et al, 2010). If Runx1 is to be used as a cancer target, then it is important to determine the stage at which is acting in tumorigenesis (initiation, promotion, and maintenance). Thus, we deleted Runx1 by Tamoxifen (TM) injection in BL6 mice that carry the K14‐CreERT2; Runx1fl/fl alleles at three different stages of tumorigenesis induced by a 2‐step carcinogenesis protocol (Figures 2A and 5A). The KO targets the Runt domain in exon 4 rendering a null allele (Growney et al, 2005). Four days after TM injection, Runx1 KO was efficiently induced in over 90% of all HFs (Figure 2B). We applied 9,10‐dimethyl‐1,2‐benzanthracene (DMBA) to induce point mutations in the DNA at first telogen (PD21) and 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) for the following 20 weeks to promote the mutation by robust proliferation of skin epithelial cells (Abel et al, 2009). As expected, after 20 weeks of TPA treatment, there were fewer tumours in the Runx1 KO mice TM treated at PD17 relative to both control groups (TM no CreER and oil only) (Figure 2C, top and middle; Figure 2D and G). We refer to this treatment as the initiation scheme (Figure 2A). Tumours that did form in the KO mice expressed Runx1 at high levels, likely from the rare cells that escaped Cre activity, developed later on and were 'young' in their development, as indicated by K10 expression (Figure 2C, middle; Supplementary Figure S2D; Santos et al, 1997). These data from inducible KO mice with a BL6 background were in line with our previous results obtained from mice carrying the K14‐Cre allele, which were a mix of CD1 and BL6 genetic backgrounds, activated in embryogenesis (Hoi et al, 2010).
To differentiate between a role in tumour initiation and promotion, we injected TM 2.5–3 weeks after DMBA application when HF bulge cells divided at least once having passed through a full anagen (promotion scheme; Figure 2A and B). Surprisingly, Runx1 KO mice and control mice developed comparable numbers of tumours at a similar rate (Figure 2C bottom; Figure 2E and G), indicating that Runx1 is dispensable for tumour promotion or growth at least in this genetic background. However, 1 month after the final TPA treatment tumour counts dropped by 53% in the Runx1 KO mice as compared to 33% in WT mice (Figure 2E). Importantly, the only surviving tumours were those that still expressed Runx1 due to inefficient Cre activity (Figure 2C, bottom).
To check the robustness of our phenotypes in a uniform genetic background highly prone to skin cancer (Abel et al, 2009), we backcrossed our mice for five generations with Fvb background mice. As before, we found impaired tumorigenesis when Runx1 was deleted prior to tumour initiation (Figure 2F). This was true whether the Runx1 KO was induced 2 days before or 2 days after DMBA application either in telogen (PD18‐20) or in anagen (PD21‐23). When Runx1 was deleted 2.5 weeks after tumour initiation, 53% of tumours completely lacked Runx1 expression at week 7.5 of TPA treatment (Figure 2H and I). This was unlike our data from the initiation scheme, when no Runx1‐negative tumours could be detected. This demonstrated that past the initiation stage tumours do not strictly require Runx1 for their growth under TPA treatment and was consistent with our data from the promotion scheme in the BL6 background. Suggestively, these Runx1‐negative tumours were smaller in size (Figure 2I, bottom; Supplementary Figure S2E) and their number decreased steadily from 7.5 to 15 weeks of TPA treatment (Figure 2H), indicating that tumours are short lived in the absence of Runx1. Lower levels of Ki67 in these KO tumours support this loss, while apoptosis assayed by Caspase 3 staining does not seem to play a significant role (Figure 2I; Supplementary Figure S2F). Co‐incidentally, the cancer SCs previously defined as a population of CD34‐expressing papilloma cells (Malanchi et al, 2008), were greatly diminished in tumours that lacked Runx1 expression (Figure 2I and J; Supplementary Figure S2G). In general, cancer SCs are thought to maintain tumours and cause their re‐occurrence for extended periods of time; CD34+ cancer SCs have been shown to initiate SCC formation upon serial transplantation for at least three generations (Malanchi et al, 2008). Our finding suggests that Runx1 expression is important for the maintenance of the CD34+ tumour SCs. Moreover, Runx1 is only essential for tumour growth in the absence of a proliferative agent such as TPA, which can at least temporarily overcome Runx1 loss.
Overall, our experiments clearly distinguish an irreplaceable function of Runx1 in tumour initiation from a somewhat more context‐dependent function in tumour promotion.
Runx1‐expressing cells are HFSCs
We previously reported that Runx1 loss impaired adult HF bulge‐cell proliferation (Osorio et al, 2008; Hoi et al, 2010), but it remained unclear whether this effect is SC intrinsic or environmental, and if Runx1 is actually expressed in the adult long‐term HFSCs, although we did find it expressed in the embryonic precursors of HFSC (Osorio et al, 2011). To address this question here, we used lineage tracing involving an inducible CreER recombinase previously knocked into the Runx1 genomic locus (Samokhvalov et al, 2007) and ROSA26R (Soriano, 1999) reporter mice. We injected TM at PD17 in these mice and sacrificed them at first telogen (PD21), first anagen (PD35), and third anagen (∼1‐year old). In skin sections stained with X‐Gal from the first telogen we observed labelling in ∼50% of HFs marking ∼5 cells per bulge (Figure 3A–C). Later on at the first and third anagen, we found prominent trails of X‐Gal+ cells in the ORS (marked by K14) and the differentiated portion of the HF co‐localizing with AE13 and AE15, as well as small clones of cells in the bulge (Figure 3B, top; Figure 3C–E). These X‐Gal+ cells were completely absent in mice injected with oil only (Figure 3B, bottom). These data demonstrate for the first time by lineage tracing that original Runx1‐expressing adult bulge cells are long‐term self‐renewing and differentiating HFSCs.
Runx1‐expressing HFSCs are at the origin of skin tumours
Since Runx1 is expressed in adult HFSCs, which can generate skin SCC, and Runx1 is important for tumour initiation it follows that it might be expressed in cells of tumour origin. However, epithelial skin tumours can originate in both HF and epidermis (Youssef et al, 2010; Lapouge et al, 2011; White et al, 2011). Our data so far suggest that tumour cells might hijack the self‐renewal/proliferation pathways of normal HFSCs to promote their growth. Since Runx1 expression is confined to the HF in normal skin, it was intriguing to find that all skin tumours in our initiation scheme expressed high levels of Runx1 (Hoi et al, 2010). It is possible that some, but not all skin epithelial tumours generated in our protocol originated in HFSCs that normally expressed Runx1 and the remaining cells of tumour origin acquired ectopic Runx1 expression near the point of tumour initiation due to inflammation or injury induced by the treatment. We aimed to understand when Runx1 expression is aberrantly acquired in tumours and to what extent Runx1+ cells contribute to the cells of tumour origin. This would provide the context in which tumours are initiated and promoted.
The strategy was to induce genetic lineage tracing via TM injections in the Runx1‐CreER; ROSA26R mice at different stages of tumorigenesis. We injected TM at PD17/18 in Runx1‐CreER; ROSA26R mice and initiated the tumours with DMBA at PD21 (Figure 4A, Bulge scheme). This resulted in an average of 4.45 X‐Gal+ cells/bulge in 80% of the HFs and 20% infundibular (Figure 4B and C, Bulge scheme) but no interfollicular epidermis labelling. After 20 weeks of TPA treatment, whole‐mount X‐Gal staining of the mouse back skin revealed 3/89 positive tumours in the TM‐injected animals, as opposed to 0/71 in the oil controls (Supplementary Table S2). The positive tumours were completely blue suggesting monoclonal expansion, and expressed Runx1 (Figure 4D, Bulge scheme; Supplementary Figure S3A). Of important note only ∼2.3% of all bulge cells were X‐Gal labelled upon TM induction due to low efficiency of the Runx1‐CreER driven recombination. Using previously engineered Runx1‐LacZ knock‐in (KI) mice that report endogenous Runx1 promoter activity and expression (North et al, 1999) we estimate a conservative 50% of all bulge cells as normally being able to express Runx1 (Supplementary Figure S3B). These should have been labelled by the Runx1‐CreER induction and we can thus infer that a significant fraction (∼70% if not more) of the skin tumours in this procedure may arise from Runx1+ bulge cells. The incidental labelling in 20% of infundibuli make these cells as potential alternative candidates for the origin of at least some of the tumours obtained in our treatment.
Tissue injury and inflammation has long been recognized as an oncogenic factor. Intriguingly, we found that injury or short‐term TPA treatment induced ectopic Runx1 expression in the hair infundibulum and epidermis just adjacent to the HF (Figure 4E and F; Supplementary Figure S13C). Micro‐injury may be responsible for the 20% infundibular labelling present in our previous experiment. We asked whether the infundibular cells rendered to express Runx1 upon TPA induction or injury are SCs and may contribute to tissue homeostasis, injury repair, or tumour formation. Injection of TM in Runx1‐CreER; ROSA26R mice at the onset of the second telogen (PD38‐45) following ∼2.5 weeks of TPA treatment (Figure 4A, Infundibular scheme) resulted in the selective labelling of the majority of infundibular cells in each HF (Figure 4B, C, Infundibular scheme) but no labelling in the bulge or the interfollicular epidermis (n∼1000). Two weeks after TM injection, we sacrificed mice that had been injured by a penetrating cut and examined skin sections by X‐gal staining. We determined that this population contributed neither to HF homeostasis (away from cut) nor to healing of the epidermal wound (Figure 4G and H). Therefore, we conclude that cells ectopically expressing Runx1 in the infundibulum do not acquire tissue SC properties.
We next asked if aberrant expression of Runx1 in these more differentiated HF cells had an oncogenic effect in the experimental conditions of our 2‐stage skin carcinogenesis protocol. DMBA treatment 4 days after or 2.5–3 weeks before labelling by TM injection, followed by 20 weeks of TPA to generate tumours did not lead to any fully or partially X‐Gal+ papilloma (Figure 4A and D, Infundibular schemes). These data together with the fact that we had achieved a much greater labelling efficiency in the infundibulum versus the bulge labelling schemes (Figure 4B and C), suggest that the ectopic Runx1 expression in populations of non‐SCs do not render these cells capable of forming tumours. Additionally, the results indicate that Runx1 is essential for tumour formation only in HFSCs but not sufficient outside this niche, even if those more differentiated short‐lived cells had been previously initiated with a mutagenic drug.
Absence of Runx1 leads to tumour regression
The data thus far demonstrated that Runx1 is in the cells of tumour origin, that these cells must be HFSC to generate tumours, and that Runx1 is essential in the initiation stage of tumour formation. Moreover, the loss of Runx1‐negative tumours in the absence of a proliferative agent (Figure 2H) suggested a possible role for Runx1 in tumour maintenance.
To address this question, we attempted to remove Runx1 from tumours after they had formed and monitored their number and size over time. To remove Runx1, we used topical applications and monthly injections of 4‐OHT in acetone directly into tumours (Figure 5A). One month after the first injection Runx1 was lost in 50% of 29 tumours analysed. We next measured the volume of 72 treated tumours bi‐weekly for 26 weeks. We found that ∼75% of WT tumours (acetone only) increased 940% in size while 80% of 4‐OHT injected tumours decreased to 8% of their original size, suggesting that Runx1 loss induced roughly 120‐fold shrinkage of the tumour size (Figure 5B–D). This is likely an underestimation, as all remaining tumours in Runx1 KO mice at 26 weeks showed Runx1 expression due to incomplete KO (not shown). In conclusion, the removal of Runx1 from developed epithelial skin tumours drastically reduces the size of these tumours.
Runx1 is important for oral tumour formation in LSL‐KrasG12D mice and is expressed by oral epithelial SCs
Next, we aimed to expand our understanding of Runx1 role in SCs and tumours beyond that of skin SCC and HFSCs. Runx1 appeared highly expressed in oral epithelium human SCC lines and primary tumours (Figure 1). DMBA, which induces the initiating mutation in the two‐stage carcinogenesis protocols utilized here, has been shown to target the Ras genes with high frequency (Abel et al, 2009) and a considerable fraction of human oral and skin SCCs show mutation in one of the Ras genes (Pierceall et al, 1991; Spencer et al, 1995). Expression of an activated form of K‐Ras (KRasG12D) initiates squamous tumour formation in the oral and anal epithelium as well as in the skin (Vitale‐Cross et al, 2004; Gilad et al, 2010; Lapouge et al, 2011). To connect our findings with genetic pathways of human SCC, confirming that tumours with activated Ras depend on Runx1 for their formation, and to analyse the role of Runx1 in the oral epithelium tumour formation, we utilized LSL‐KRasG12D mice that can be activated by TM induced Cre activity (Jackson et al, 2001). We generated K14‐CreERT2;LSL‐KrasG12D;Runx1 KO, wild‐type, and heterozygous mice and injected TM at PD21 to induce either the Runx1 KO, or K‐Ras activation, or both. The KrasG12D mice rapidly developed sublingual and hard palate oral papillomas (Figure 6A–C) at low TM doses as previously reported (Gilad et al, 2010). Few K14‐CreERT2‐negative mice developed tumours as well, indicative of low frequency, leaky, non‐Cre‐dependent K‐Ras activation. Oral tumour formation severely impaired the mice's ability to take in food, ultimately leading to premature death of these mice, prior to skin tumour formation. Although we observed oral or anal tumours in all KrasG12D mice, tumour formation and subsequent death were significantly delayed in Runx1 KO mice. Moreover a dosage‐dependent effect was apparent in mice with a single intact copy of Runx1 (Figure 6C). These data extend our results on the importance of Runx1 from skin to oral and anal epithelial tumorigenesis. Moreover, they underscore the function of Runx1 in a genetic context (KrasG12D) highly common in human skin and head and neck (oral) and skin SCC, complementing our 2‐step skin carcinogenesis data.
Given Runx1 function in oral tumorigenesis, we examined its expression in the oral epithelium using the Runx1‐LacZ KI. At PD38 Runx1 expression is in the basal layer of the palate epithelium (Figure 6D). This area was previously shown to contain label‐retaining cells, which were putative SCs (Bickenbach and Mackenzie, 1984).
Next, we traced the lineage of oral Runx1+ cells using mice containing the inducible Runx1‐CreER and the tdTomato reporter (Madisen et al, 2010). Labelling was induced mostly in the basal layer of the hard palate 4 days after TM but not oil injection (Figure 6E). At 18.5 weeks after TM injection we detected rare clones consisting of basal layer (un‐differentiated) and supra‐basal (differentiated) cells (Figure 6E). This is the first study showing that indeed a subpopulation of oral basal layer cells differentiate and self‐renew for extended periods of time. Oral epithelium SCs express Runx1, making this the third adult tissue SC type with lineage tracing evidence for Runx1 expression, after HFSCs (this work) and HSCs (Samokhvalov et al, 2007).
Runx1 expression in colon and intestine
Because we observed Runx1 expression in several human cancer cell lines of epithelial origin other than skin and oral, we decided to analyse Runx1 endogenous expression in a third tissue: the colon and intestine. We used the Runx1‐LacZ KI mice at PD38 and found X‐gal signal, and thus endogenous Runx1, in villus cells but more importantly, at the base of every crypt (Figure 6F) where intestinal SCs reside (Barker et al, 2008). We confirmed this by staining using a Runx1 antibody in wild‐type intestinal tissue (Figure 6G and H) and in Lgr5CreER‐IRES‐GFP mice. Indeed, some Runx1 expressing crypt cells co‐localize with GFP, suggesting they may be SCs (Figure 6I). Notably, staining of Runx1 and K8, an epithelial marker, in wild‐type mice revealed an overall pattern of Runx1 expression that is similar to HFs: Runx1 is lightly expressed in a few SCs at the base of the crypt (analogous with the hair bulge) and strongly expressed in the activated cells or transit amplifying cells in the upper crypt (analogous with the hair germ). Extremely low labelling efficiency of Runx1‐CreER in the intestine and colon with tdTomato, YFP or ROSA26R reporter prevented lineage tracing of these Runx1+ cells. It is possible that by studying the isoform structure of Runx1 in the intestine and different CreER KI strategy may overcome this limitation. These data suggest that Runx1 may be expressed in the intestine in a subfraction of the LGR5+ SCs.
Human cancer cells require Runx1 for growth
Thus far we presented evidence that Runx1 is a SC factor for at least two epithelial tissues, and is important for tumour initiation/formation in these tissues. Runx1 is also expressed in several kinds of human epithelial cancer cells (Figure 1E) and tumours, as shown by Oncomine data (Figure 1A). To test the significance of Runx1 in human epithelial cancers, we knocked down its expression in several cancer cell lines and monitored their cell growth and survival.
We used Luciferase shRNA (Luc) as a control and two Runx1 shRNAs. Runx1‐1 and Luc expression, but not Runx1‐2, can be assessed by detection of GFP (Materials and methods). Both Runx1 shRNAs (but not Luc) knocked down Runx1 levels (Runx1KD), albeit to a different degree, as determined by immuno‐fluorescence staining with a Runx1‐specific antibody (Figure 7A and B). Skin and head and neck (oral) SCC cell lines with Runx1KD failed to grow, confirming an essential role for Runx1 in tumour maintenance as seen in mouse papillomas (Figure 7C and D; Figure 5). Ovarian cell lines show a reduction in growth, whereas colon, breast, and prostate cancer cell lines were not affected (Figure 7D). Notably, cells react at different degrees to the two Runx1 shRNAs, likely due to variable knockdown efficiencies of the two target sequences. The effects of Runx1KD can be rescued by the overexpression of Runx1 (Figure 7C; Wang et al, 2011).
Since all cancer cell lines expressed Runx1 and their tissues of origin also displayed suggestive Runx1 expression we briefly looked into a possible mechanism for resistance to loss of Runx1. In a recent study in breast cancer Runx1 levels were inversely correlated with FOXO1 expression, compensating for Runx1 loss (Wang et al, 2011). However, we could not find a correlation between Runx1KD resistance and FOXO1 in any of the cell lines analysed here (Supplementary Figure S4A). Overall, these results show that loss of Runx1 is detrimental to growth of three human epithelial cancer cell types: head and neck (oral) SCC, skin SCC, and in part to ovarian cancer cells.
Runx1 acts upstream of Stat3 in cancer cell growth and survival
To understand the mechanism by which Runx1 affects cancer cell growth in mice and humans, we considered a possible interaction with Stat3, which appears to play a similar role as Runx1 in regulating the hair cycle, and in tumour formation (Sano et al, 1999, 2000; Chan et al, 2004a, 2004b; Osorio et al, 2008; Kim et al, 2009). The activated form of Stat3—pStat3 T705—is upregulated in skin upon TPA treatment in WT mice (Chan et al, 2004a). This upregulation appeared more variable and overall reduced in the Runx1 KO skin (Hoi et al, 2010), and is lost in tumours that form after loss of Runx1 during the tumour promotion stage (Figure 7E). Given the essential role of Stat3 in proliferation and survival of human and mouse SCC (Bito et al, 2011; Rho et al, 2011), this may explain why the Runx1‐deficient tumours obtained here in the promotion scheme were short lived and could not be maintained in the absence of TPA (Figure 2H). Given the complex role of the microenvironment in regulating Stat3 activation, the manner in which Runx1 interacts with the Stat3 signalling pathway, and the functional relevance of this interaction to Runx1 mechanism of action are unclear. For example, Stat3 downregulation in the Runx1 KO skin treated with TPA could be an indirect effect to reduced inflammation potentially driven by a paracrine function downstream of Runx1, rather than a direct interaction of Runx1 with the Stat3 pathway itself.
To analyse whether Runx1 directly regulates Stat3 activation, we utilized cells removed from the complex skin and tumour environment and created inducible KO K14‐CreER;Runx1fl/fl keratinocytes (iKO). After 4 days of 4‐OHT treatment, Runx1 mRNA and protein level were significantly reduced as shown by western blot and by QRT‐PCR (Figure 7F and G). Moreover, western blots for Stat3, pStat3 T705, and pStat3 S727 revealed a selective and striking de‐phosphorylation of T705 upon Runx1 loss (Figure 7F). In this form, Stat3 is inactive because it cannot dimerize and translocate to the nucleus (Ihle, 1996).
To analyse if the disruption of the Jak/Stat pathway is responsible for the growth defect of normal and cancer cell lines with Runx1 deficiency, we attempted to restore Stat3 activity in Runx1KD cells. To this end, keratinocytes and Pam212 cells were either co‐transfected with Runx1‐1 shRNA and cStat3, the constitutively active form of Stat3 (Bromberg et al, 1999) or treated with Oncostatin M (OSM), a small signalling peptide that activates Stat3 signalling (Ishihara and Hirano, 2002). In both cases, the Runx1‐deficient cell lines were then able to form large colonies (Figure 7D), and showed increased levels of T705 pStat3, but not Runx1, relative to untreated cells (Figure 7H).
OSM activates Jak and Tyk kinases through its receptors that in return activate the Stat3 and the MAP kinase pathway (Erk1/2) (Ishihara and Hirano, 2002). To see if the effect of OSM can be attributed to Stat3 or whether Erk phosphorylation plays a role in the rescue of growth defects in our cells, we examined P‐Erk1/2 level by western blotting. We fail to see an increased phosphorylation of Erk1/2 in keratinocytes (Figure 7H), which suggests that it is the Stat3 activation that rescued the Runx1‐deficient growth defect, further supporting the similar effect obtained with the cStat3 (Figure 7C).
Finally, we employed the human cancer cell lines that previously showed dependence on Runx1 levels, and found that OSM rescues their growth to different degrees (Figure 7D), with the most striking effects on skin and head and neck SCC lines and a milder effect on ovarian cancers. Hence, in normal keratinocytes as well as in three epithelial cancer cell types Runx1 loss appears to impair growth by directly preventing Stat3 activation.
Runx1 stimulates the Jak/Stat pathway by repression of SOCS3 and SOCS4
Next, we attempted to understand the mechanism by which Runx1 may stimulate Stat3 signalling. Since our western blots suggested stable Stat3 protein levels upon Runx1 loss, it seemed likely that some members of the Jak/Stat pathway involved in T705 phosphorylation might be Runx1 transcriptional targets. To find such putative targets, we surveyed the status of the Jak/Stat pathway using SA‐Biosciences RT2 Profiler PCR array and compared mRNA levels in Runx1‐CreER;Runx1fl/fl and Runx1fl/fl keratinocytes after 4 days of 4‐OHT treatment. A volcano plot showing the fold changes between WT and KO versus the associate P‐value for each tested gene revealed three genes significantly upregulated in the iKO cells: Crk, SOCS3, and SOCS4 (Figure 7I; raw data in Supplementary Table S3). Crk is a proto‐oncogene thought to stimulate proliferation (Tanaka, 2009), and as such its upregulation cannot explain proliferation defects but is likely an attempt to compensate for Runx1 loss. On the other hand, SOCS3 and SOCS4 suppress Stat3 T705 phosphorylation via interference with Jak2 and EGFR kinase activity, respectively (Larsen and Ropke, 2002; Segatto et al, 2011).
To test if Runx1 directly binds to the SOCS3 and SOCS4 promoters to represses their transcription, we employed two independent K14‐CreERT2;Runx1fl/fl keratinocyte lines and performed chromatin immunoprecipitation (ChIP) using Runx1‐specific antibodies in KO and WT (Runx1fl/fl) cells. We followed with QRT‐PCR using primers encompassing SOCS3 and SOCS4 genomic regions containing Runx1 binding sites (Figure 7J). Our data showed enrichment from one primer pair in each of the SOCS4 and SOCS3 gene loci (P=0.04 and 0.07, respectively). We used a region of the Gapdh promoter free of conserved Runx1‐binding sites as a negative control (Figure 7K). Binding of Runx1 on the SOCS3 gene promoter, although here showed a P‐value slightly lower than the conventional 0.05, was also reported by ChIP Seq data on a blood cell line (Wilson et al, 2010). Thus, we suggest that Runx1 represses SOCS3 and SOCS4 expression, likely via direct binding to their promoters, and ultimately upregulates Stat3 activity by enhancing phosphorylation at T705.
If Runx1 loss induced defects were mediated through SOCS3 and/or SOCS4, as indicated by the QRT‐PCR and ChIP experiments described above, then simultaneous knockdown of SOCS3 or 4 and Runx1 should rescue the cell growth defect. This could be measured by the presence of GFP+ cells, which indicate Runx1 deficiency (Materials and methods). As expected, cells transfected with control Luc shRNA but not those transfected with Runx1‐1 shRNA and the empty plasmid backbone (pLKO1) did not show a population of GFP+ keratinocytes or Pam212 cells after puromycin selection. In contrast, addition of either SOCS shRNA instead of the empty plasmid backbone rescued the GFP+ (Runx1‐deficient) population, in which cells with high SOCS were able to grow without Runx1 (Figure 7L; Supplementary Figure S4B). Knockdown of SOCS3 or 4 alone did not affect cell growth as expected from low or absent SOC3/4 expression in WT cells and as shown in Supplementary Figure S4C and through equal amounts of GFP− cells in Luc+backbone or Runx1+SOCS shRNA‐transfected cells. These data together suggested that Runx1 is a direct transcriptional repressor of SOCS4 and potentially SOCS3, which results in activation of Stat3 and thus allows growth of normal and cancer cells (Figure 8). In conclusion, Runx1 repression is accompanied by activation of SOCS genes, inhibition of Stat3 activity, and growth impairment of some epithelial cancer cells.
Here we uncover Runx1, as a broader epithelial tissue SC and tumour factor than previously recognized. We not only confirmed its expression in adult mouse HFSCs by direct lineage tracing, but also we implicate Runx1 in previously uncharacterized oral epithelium SCs. We showed that mouse oral epithelium tumours, like their skin counterparts (Hoi et al, 2010, and this work), also appear dependent on Runx1 for their growth. We find that increased proliferation due to ectopic injury‐induced expression of Runx1 in skin non‐SCs does not render those cells tumorigenic, while Runx1‐expressing SCs are at the tumour origin. We implicate Runx1 as a key molecule upstream of a central epithelial cancer pathway, Stat3 (Li et al, 2011), which Runx1 activates by direct transcriptional repression of SOC3/4. We documented for the first time the expression of Runx1 in primary skin and oral human epithelium SCC and suggest that several more human epithelial cancers may express Runx1. We demonstrate that three different human cancer cell types (skin SCC, oral SCC, and ovarian) depend upon Runx1 for their normal growth. We find Runx1 important for both skin tumour initiation and maintenance, while we previously showed that Runx1 was not essential for adult tissue maintenance (Osorio et al, 2008). This differential requirement for Runx1 places it as a promising target for skin, and potential other epithelial cancers.
Runx1 has been intensively studied for the past 4 decades as a cancer gene frequently mutated in acute myeloid leukaemia, which when studied in mice turned out to work as a master regulator of haematopoiesis (Mangan and Speck, 2011). Taking the opposite direction—from mouse to human—we first demonstrated that Runx1 is important for the timely emergence and maturation of HFSCs in embryogenesis (Osorio et al, 2011) and the activation and proliferation of adult HFSCs (Osorio et al, 2008). Moreover, Runx1 appears essential in mice for skin squamous epithelial tumour formation (Hoi et al, 2010). Importantly, with this work we now take Runx1 back to humans where we analysed the role of Runx1 in several epithelial cancers.
Runx1 surfaced among the top 10% highly expressed genes in 1/3 of all 138 applicable microarrays studies of human cancers found by us in the Oncomine database. These included the skin SCC and BCC, oral SCC, breast, oesophageal, lung, colon, and pancreatic cancer among others. We detected Runx1 expression by immunofluorescence staining of several kinds of skin human primary tumours as well as in oral epithelium SCCs. Moreover, we found Runx1 overexpressed in a whole panel of established human cancer cell lines. Importantly, Runx1 appears to be required for growth of skin and head and neck (oral) SCC, and to some extent ovary cancer cells, suggesting Runx1 as a novel putative target for treating these cancers. In contrast, although Runx1 was expressed at high levels it was dispensable for growth of breast, prostate, and colon cancers. In light of a recent study of breast cancer, these cells may have learnt to live without Runx1 by upregulating compensatory pathways (Wang et al, 2011). However, it appears that FOXO1, shown to compensate for Runx1 loss in MCF10A‐5E breast cancer cells, is not upregulated in the cells we analysed here. This information will be important for future treatment of some cancer types in which a combination drug therapy against Runx1 and compensatory pathways may be required.
An attractive clue positioning Runx1 as a promising therapeutic target is our demonstration that loss of Runx1 shrinks tumours in mice and impairs human and mouse tumour cell growth in vitro. This potential adds an important angle to the role of Runx1 in tumour initiation, which could be exploited for tumour treatment and prevention, respectively. Runx1 loss is not essential for normal skin and oral epithelium homeostasis, and deletion of Runx1 in adult mice via a ubiquitous β‐Actin‐CreER allele or Mx1‐Cre had mild effects on the mouse physiology (Appleford and Woollard, 2009; Hoi et al, 2010; and our unpublished data). The tolerance of the tissue to Runx1 loss in HFSCs is probably due to the unique protective niche environment, which is disrupted in cell culture and in tumours. Disrupting Runx1 binding to the DNA via specific drugs is already considered as a potentially beneficial approach to treating leukaemia (Mangan and Speck, 2011). A more targeted approach for local treatment of accessible skin or head and neck tumours would be even less invasive.
How is Runx1 working in regulating tumour formation and maintenance? One possibility is that Runx1 is expressed in the cell of tumour origin and remains expressed in some of the progeny cells subsequently generated. At least in the skin, HFSCs are known to be at the origin of some SCC (Youssef et al, 2010; Lapouge et al, 2011; White et al, 2011). Previously, we reported Runx1 expressed in a fraction of cells in bulge and hair germ cells, the regions of the HF where SCs are known to reside. However, it is unclear whether all cells in these regions are SCs. Even though loss of Runx1 impairs proliferation of HFSCs it was possible that Runx1 modulates the SC environment from neighbouring cells. For example, Runx1 is able to regulate production of several secreted Wnt molecules during embryogenesis (Osorio et al, 2011). Our lineage tracing work directly demonstrates here for the first time that adult Runx1‐expressing cells are long‐lived, self‐renewing and differentiating HFSCs, which are at the tumour origin. Moreover, Runx1 also appears to be a marker for oral epithelium SCs. Our data suggest some overlap of Runx1 and LGR5 expression, and LGR5 is a well‐recognized intestinal SC marker. Further substantiation by lineage tracing is needed for the intestine since the existing Runx1‐CreER is extremely inefficient in this organ.
Using skin as a model system we demonstrate that Runx1‐expressing HFSC is at the origin of tumours, and these tumours were likely monoclonal. It is recognized that epidermal cells may form skin SCC (Lapouge et al, 2011), and even though Runx1 is not expressed in the epidermis in normal conditions it appears that in our experimental conditions all mouse tumours expressed Runx1. We therefore hypothesized that a population of epidermal cells rendered by inflammation or injury caused by the carcinogenic treatment to ectopically express Runx1 might also generate tumours. However, we found this population located in the infundibulum of the HF to be short‐lived progenitors that did not contribute to long‐term homeostasis and injury repair. Moreover, this population was unable to generate tumours in our experimental conditions, even in the context of an initiating mutation induced by drug treatment. This underscores the crucial importance of the intrinsic epigenetic context or cellular make‐up to initiation of carcinogenesis and adds to the theory that SCs, and not differentiated cells, are the most likely initiators of cancer (Magee et al, 2012).
Runx1 is known to regulate cell survival, proliferation, differentiation, and cell‐cycle progression (Blyth et al, 2005; Mikhail et al, 2006; Friedman, 2009). Moreover, Runx1 directly binds and represses the promoter of cyclin‐dependent kinase inhibitor p21 in skin epithelial cells (Lee J and Tumbar T, submitted; Hoi et al, 2010). Thus, it seemed surprising that Runx1 was essential for tumour initiation but dispensable for tumour promotion, in which over 50% of the tumours in Runx1 KO mice actually lacked Runx1. However, these tumours were lost after we ceased the TPA treatment, demonstrating that during the proliferative/maintenance stage other strongly proliferative agents can compensate for Runx1 loss. These short‐lived Runx1‐deficient tumours displayed downregulation of activated Stat3 and a striking absence of CD34+ cells, which were previously deemed cancer SCs, important for tumour growth and maintenance (Malanchi et al, 2008). These data together may suggest that Runx1 is expressed in the HFSCs that generate the tumours, and is continuously required in these tumours for the maintenance of their long‐lived tumour SC population and for continuous tumour growth. Moreover, Runx1 role in this process is likely mediated via stimulation of Stat3 signalling.
Phosphorylation of Stat3 at T705 and S727 is necessary for full activity of Stat3 (Yokogami et al, 2000). However, phosphorylation at S727 alone prevents activation at T705 and thus locks Stat3 in its inactive state in the cytoplasm (Gartsbein et al, 2006). Upon Runx1 loss we observed a loss of T705 phosphorylation while S727 maintained its levels, indicating a double‐safe mechanism to keep Stat3 inactive. Active Stat3 is associated with psoriasis in humans and mice (Sano et al, 2005) and its increased activity has been repeatedly associated with skin SCC formation (Chan et al, 2004a, 2004b, 2008; Aziz et al, 2007; Chan et al, 2008). Like Runx1, Stat3 is also considered essential for tumour initiation and maintenance by directly targeting the HFSCs in mouse models (Kim et al, 2009). Activated Stat3 is found in the vast majority of human oral cancers as well as in breast, lung, ovarian, pancreatic and prostate cancers and myelomas, leukaemias, and lymphomas (Yu and Jove, 2004). High levels of activated Stat3 have also been reported in oral SCC cell lines, and proliferation of these SCC cells could be further increased with OSM (Douglas et al, 2004), a potent activator of the Stat3 pathway. This connects well to our finding that addition of OSM rescues the proliferation defect of skin and oral SCC and ovarian cancer cells upon Runx1 loss, through activation of Stat3.
Here, we expose a novel regulatory pathway for Stat3 activity. We show that Runx1 regulates Stat3 activation by transcriptional repression of Stat3 inhibitors SOCS3 and SOCS4. Conversely, loss of Runx1 results in upregulation of SOCS3 and SOCS4 and Stat3 inactivation, which is detrimental to cancer cell growth. Importantly, we show that this mechanism is conserved in humans where it affects skin SCC, and head and neck (oral) SCC, and ovarian cancer. The connection between Runx1 and Stat3 is a putative axis in regulation of cell proliferation and tumorigenesis, suggesting a potential benefit for investigating Runx1 status in Stat3‐dependent cancers. If Runx1 is upstream of Stat3 and potentially other major cancer pathway, such as Wnt signalling as we previously showed (Osorio et al, 2011), then it may be more beneficial to target the master regulator, Runx1, rather than its individual target signalling pathways.
Materials and methods
Human samples were obtained de‐identified and stained as described in the supplement. K14‐CreERT2, Runx1fl/fl, Runx1‐CreER, LSL‐KrasG12D, Lgr5CreER‐IRES‐GFP, ROSA26R, and tdTomato mice were crossed to obtain the various experimental mice. CreER activation by Tamoxifen (Sigma) and 2‐step carcinogenesis DMBA/TPA protocol were previously described (Hoi et al, 2010). The Cornell University IACUC approved all the mouse work.
Rescue assays
All knockdown and rescue assays were duplicated or triplicated. Mouse keratinocytes or Pam212 was co‐transfected with 1 μg DNA of each shRNA (Runx1‐1 or Luc in pLKO1‐pig) and cStat3 (Addgene plasmid 8722; Bromberg et al, 1999). At 90% confluency, we select for shRNA containing with puromycin. Oncostatin M (OSM) (Cell Signaling, human: #5367; mouse: #5371) was used at 10 ng/ml media at the time of puromycin selection. pLKO1‐puro only or mouse‐specific SOCS3 or 4 shRNA was co‐transfected with pLKO1‐pig Runx1‐1 and Luc shRNA. All transfected cells will be puromycin resistant; however, only if growth of Runx1‐1 (or Luc) KD cells is permissible, GFP+ cells are observed.
RNA from iKO cells treated with ethanol or 4‐OHT (Sigma) for 4 days was extracted using Trizol and the RNeasy Mini kit (Qiagen) and converted to cDNA with the iScript kit (Bio‐Rad). The mouse Jak/Stat RT2 Profiler PCR array (PAMM‐039) was purchased from Qiagen, used as instructed, and the data were analysed using proprietary bioinformatics tools provided by Qiagen. Details for Runx1 QRT‐PCR were described previously (Hoi et al, 2010).
In all, 0.5–1 × 107 iKO or WT keratinocytes treated with 4‐OHT for 4 days were used as starting material. SOCS3 and SOCS4 genomic regions were pulled down with 5 μg Runx1 antibody (Abcam ab23980). Control ChIP reactions were set up with 4 μg rabbit‐IgG (Imgenex 20304) and 3 μg H3 (Abcam ab1791).
Data was processed and analysed in Excel or R. The survival package in R was used for Kaplan–Meier analysis.
Conflict of Interest
The authors declare that they have no conflict of interest.
Supplementary Information
Supplementary Information [emboj2012270-sup-0001.pdf]
Supplementary Tables S1‐S2 [emboj2012270-sup-0002.xls]
We thank the Victorian Cancer Biobank, Drs Jonathan Vogel, Agnes Kobielack and Ralf Paus for human tumour samples; Drs Igor Samokhvalov, Elaine Fuchs, Nancy Speck, Pierre Chambon, and Alexander Nikitin for transgenic mice. We thank Dr Stuart Yuspa for the mouse skin SCC cell line Pam212; Dr James Rheinwald for the human skin SCC cell line SCC13; Dr Susanne Gollin for the human oral SCC cell lines SCC66, 125, and 74; Dr Xiling Shen for colon CCSC and HCT116 and Dr Alexander Nikitin for all other human cancer cell lines. The Stem Cell and Transgenics Core at Cornell aided in the virus production. Funding for this work was from NYSTEM Grant C024354 and NIH Grant R01AR053201 to Tudorita Tumbar.
Author contributions: DJM performed and evaluated hair follicle stem cell homeostasis lineage tracing; TSL executed all skin tumor lineage tracing experiments and significantly contributed to monitoring the KrasG12D mice. CS designed the project, performed, analysed, and interpreted all other experiments and wrote the manuscript. TT designed the project, analysed, and interpreted data and wrote the manuscript.
- Copyright © 2012 European Molecular Biology Organization


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System Requirements For now, this utility only works on Windows Vista, Windows 7, Windows Server 2008, Windows 8, and Windows 10. The architecture of Windows Error Reporting has been revamped with a focus on reliability and user experience. This allows distributing solutions as well as collecting extra information from customers (such as reproducing the steps they took before the crash) and providing them with support links. WER uploads additional CAB files that can contain data about the same event types as an earlier uploaded report. http://devstude.net/windows-error/windows-server-2008-system-queued-windows-error-reporting.php
Not to be confused with the Dr. The service is provisioned to receive and process well over 100 million error reports per day, which is sufficient to survive correlated global events such as Internet worms.[6] Buckets[edit] In the Maximum size of the archive, in files MaxQueueCount REG_DWORD Range of possible values: 1–500. This setting is not supported in the HKEY_CURRENT_USER registry hive. Bonuses
Because of that, there are some third party alternatives which allow users to also submit crash reports to the developers of the crashing software. However, it is often only the developer who will really understands the contents of the .wer file. The default is 10. And weaknesses in the expanding heuristics, which result in mapping more than one bug into the same bucket.
If the bypass is disabled or not configured as a policy setting, WER throttles data by default. Windows Error Reporting runs as a Windows service and can optionally be entirely disabled. In order to start using it, simply run the executable file - AppCrashView.exe The main window of AppCrashView contains 2 pane. Windows Error Reporting Disable Windows 10 Over half of all Microsoft Office XP errors were fixed with Office XP SP2.[20] Success is based in part on the 80/20 rule.
When it's turned on, the odd and even rows are displayed in different color, to make it easier to read a single line. Windows Error Reporting Location Problem Reports and Solutions (new in Vista) A new feature in Windows Vista is Problem Reports and Solutions in the Control Panel under the System and Maintenance category (if you don't Yes No Additional feedback? 1500 characters remaining Submit Skip this Thank you! https://msdn.microsoft.com/en-us/library/windows/desktop/bb513613(v=vs.85).aspx Kinshuman is the original designer of Windows Error Reporting in Vista which is the same design and implementation that is present in current Windows versions. [3] Contents 1 History 1.1 Windows
Microsoft. 11 March 2014. Windows Error Reporting Tool In earlier OSs prior to WindowsVista, the process usually terminated silently without generating an error report in these conditions. A new Control Panel applet, "Problem Reports and Solutions" was also introduced, keeping a record of system and application errors and issues, as well as presenting probable solutions to problems. It's possible that in future versions, I'll also add support for Windows XP/2000/2003 by using Dr.
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Version 1.15 Added /ReportsFolder command-line option, which allows you to specify the exact reports folder you want to load, for example: AppCrashView.exe /ReportsFolder "c:\temp\wer" Version 1.12 Fixed bug: The crash items this contact form It's very easily done for wifi networks (properties > set as metered connection) and there is a simple hack for Ethernet here:…[Read more] 0 Adam Bertram wrote a new post, Set See also[edit] Winqual Dr. Translate all string entries to the desired language. Windows Error Reporting Windows 10
If you don't specify this option, the list is sorted according to the last sort that you made from the user interface. If this parameter is not specified, the profiles folder of the current operating system is used. /ReportsFolder
Cancel TwitterFacebookGoogle+PowerShellCloud ComputingWindows 10Windows Server 2016Active DirectoryGroup PolicyVirtualizationSecurityMore Toggle navigation Site-Wide Activity Blog Wiki Forum About AboutAuthorsMembersContactSponsorsWrite for 4sysops Login Register RSS Free Windows Error Reporting (WER) viewing tool - AppCrashViewHome Windows Error Reporting Service Windows 10 Client-side software detects an error condition, generates an error report, labels the bucket, and reports the error to the WER service. This feature enables the collection of the actions performed by a user while encountering a crash so that testers and developers can reproduce the situation for analysis and debugging.[5] System design[edit]
Version 1.00 - First release. About WER Windows Error Reporting (WER) is a flexible event-based feedback infrastructure designed to gather information about the hardware and software problems that Windows can detect, report the information to Microsoft, Note that this behavior changed with Windows Server 2008 and Windows Vista with SP1. Windows Error Reporting Log Also there are some difficulties mentioned above to get access to WER data for independent software vendors, especially for small one and open source teams.
This service is available for all products, even those that do not qualify for the Microsoft Certified Products list—although we strongly recommend that you submit your products to the Windows Hardware So, if you have identified a recurring application error / problem report with an Enterprise Vault process, then now is a good time to temporarily enable advanced WER settings as explained Downloads and tools Windows 10 dev tools Visual Studio Windows SDK Windows Store badges Essentials API reference (Windows apps) API reference (desktop apps) Code samples How-to guides (Windows apps) Learning resources Check This Out An article in the New York Times confirmed that error reporting data had been instrumental in fixing problems seen in the beta releases of WindowsVista and Microsoft Office 2007.[21] Privacy concerns
For example, if two different bugs crash inside strlen function because they call it with corrupted string there will be only one bucket for both. Ideally, each bucket contains crash reports that are caused by the same bug. This documentation is archived and is not being maintained. Required fields are marked * Notify me of followup comments via e-mailName *Email *Website Recently Active Members Subscribe to NewsletterEnter your email address:You can unsubscribe anytime!Site Wide Activities [RSS] Viewing 1
Once that occurs the crash details get logged in the Application Log as an Error event. AppCrashView also allows you to easily save the crashes list to text/csv/html/xml file. Note that per-process settings will override any global settings that exist For more information, see Collecting User-Mode Dumps. However, the freeware utility has a few advantages.
For instance, in 2002, Steve Ballmer noted that error reports enabled the Windows team to fix 29% of all WindowsXP errors with Windows XP SP1. One of those hats is typically the "new project" hat. AboutLatest PostsMichael PietroforteMichael Pietroforte is the founder and editor of 4sysops. Microsoft.
Added 'Auto Size Columns+Headers' option. I prefer using Problem Reports and Solutions versus Application Log to get crash details. Examples: AppCrashView.exe /shtml "f:\temp\crashlist.html" /sort 2 /sort ~1 AppCrashView.exe /shtml "f:\temp\crashlist.html" /sort "Process File" /nosort When you specify this command-line option, the list will be saved without any sorting.


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Staff Profile: Dr. Alan Marvell
Senior Lecturer in Events Management
Senior Lecturer in Events Management
Leisure Industries Management
As a Senior Lecturer with experience in events and tourism I enjoy researching and teaching about an industry that is constantly evolving, providing new challenges and opportunities.
I teach Events Management with a focus on business events, responsible events and marketing. I am a Chartered Geographer and have a research interest in how places are constructed through marketing and events. I also work with local businesses to promote the Cotswolds as a visitor destination.
Chartered Geographer (CGeog) Royal Geographical Society
University of Gloucestershire Teaching Fellow, 2017
University of Gloucestershire Learning for Sustainable Futures Award 2015-16, Responsible Events
Senior Fellow of The Higher Education Academy (SFHEA)
Fellow of the Royal Geographical Society (FRGS)
Fellow of the Tourism Society (FTS)
Geographical Association
Association for Events Management Education
Research methods
Risk management
Industrial placement
Planning business events
Contemporary issues in events management
Strategic communications
Dissertation and investigative study
Destination marketing
Undergraduate, Postgraduate
Place promotion, branding and identity through (sub)urban development
Pedagogy, scholarship, student-led learning and engagement with place through fieldwork
Place promotion through events and tourism
Employability and partnerships with industry
More publications from Dr. Alan Marvell can be found in the Research Repository.
Vice-Chair of the Post-16 and Higher Education Committee, Geographical Association
Membership of the Editorial Advisory Board of the Journal of Hospitality, Leisure, Sport & Tourism Education and the journal Geography.
Director and Trustee of the Geographical Association
Event Management at Coventry University


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The gargantuan 440-foot yacht of Yuri Scheffler, a billionaire Russian vodka tycoon (Stolichnaya or "Stoli"), is currently floating a hail-mary throw from where the Miami Heat play in Downtown Miami.
"Serene" cost Yuri $330 million and can be chartered for a weekly fee of $5 million. The beast of the sea is docked on a piece of land directly across from the north side of American Airlines Arena. If you're attending Friday's night game, you should check it out.
Some of the ridiculous features/photos are below (via Yacht Charter Fleet):
- Sundeck with wet bar, pizza oven and Teppenyaki grill
- Indoor climbing wall
- Dedicated children's playroom
- Two helicopter landing pads
- Fully-equipped health spa and beach club
- Multiple swimming pools
- Underwater viewing room
- Full conference room


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I found out on Tuesday that it looks like we won't be getting back into Flintstone this year, after all. I am heartbroken, again, but I keep telling myself there is a reason. I've never been one to believe that "everything happens for a reason" but maybe there is one this time. I'm not so sure. I tried so hard not to get my hopes up for fear of this, but just couldn't help myself. I kind of thought it seemed so simple and easy because God had set it up. Obviously, I was wrong. How are you supposed to know when to just wait and trust God to handle things or when to be pro-active?? Sometimes, waiting seems like laziness. When do you know??? I am so confused. :(
The new speech therapist we were supposed to get called on Tuesday, too. She can only see Colt on the days that I work, because she is in a different county on my off days. So. That's disappointing. We're going to try to see the Speech Pathologist at the clinic, but there is no guarantee that she will have an opening, since we didn't jump on the chance in the beginning. Our case manager was supposed to call yesterday, but she's out of town, so she may be behind. I am getting very discouraged here. Ryan and I both keep dreaming (a few times a week) that Colt says words, but it seems we are going backward again. We did get a new sign, "potty", and he used it last night after he pooped, so that was exciting for us. We obviously need some excitement in our lives if we get excited over that. How do you get a child who can't talk to tell you he has to potty? The sign, I guess, but I don't know how to talk to him about how to tell me BEFORE he goes potty. Ryan and I have been trying to teach Colt the sign for "I love you" but it involves too many fingers for now. We do it anyway. We're still stuck at the "progress plateau." This is the point where I always get discouraged and want to quit. However, I can't quit on Colt, so we'll push on and hope for some words!
Most of my readers probably live around here, but if you don't, we've been having some seriously rainy weather around here lately. I mean, solid rain for as long as I can remember. It is really getting old and I love a good rainy day or two. I don't know if my mood is due to the dreariness or not, but for the past week or two, I have been in a terrible funk. I don't want to get up and once I am up, I feel like I just fumble through the day. Yesterday, I had this great idea to get out of the house and go to the mall to let Colt play on the indoor play area. He played about 20 minutes, then tugged my arm to leave. He's never done that. Maybe he is in the funk too. :( While at the play area, I found myself looking around at all the other moms, in their little groups, skinny and dressed all cute and there I was, without a shower in yesterday's jeans. I felt so out of place and lost. All the other moms seemed to "get it." I don't "get it." Bah. Maybe it's just the weather getting me down or maybe I really have no idea what I am doing. I'm seriously leaning toward the latter.
Colt has also been waking up religiously at 3:30 in the morning, for no apparent reason. When he wakes up, I lie there for a good hour or more after he goes back to sleep, tossing and turning with all those terrible thoughts that come to you in the middle of the night. You know, the bad ones that make you feel even worse about yourself. I hate those. Why do they always haunt you in the middle of the night? Sigh. I think if Colt didn't wake up, I would sleep okay. I hope he gets out of his funk soon too, maybe it will help me with mine. I am exhausted and down in the dumps. Say a little happy prayer for us, if you get a chance. It would be greatly appreciated.
The only sunshine I have seen in far too long.
The highlight of my week...Dad's cows got out. I got all 4 of them back in the fence. By myself.
Take that. Moo.


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Neutral color and natural texture come together in this reversible throw rug. Woven using 100% natural wool, its an eco-friendly essential that's designed to last. We recommend a rug pad to extend the life of your rug and keep it securely in place.


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Steven Gerrard will not be taking the MK Dons job after ending talks with the League One club.
The Buckinghamshire-based outfit are on the hunt for a replacement for Karl Robinson, but as they sit in the relegation zone, they're struggling to tempt some of the high-profile names they would like.
Of Ryan Giggs, Frank Lampard, and Gerrard, the latter was the only one that ever looked likely, as he did speak to chairman Pete Winkleman.
Neither party has officially confirmed why Gerrard didn't want the role, apart from the obvious. Not only are the Dons in the third tier of English football – just about – they are also a truly unfashionable club, and the former Liverpool skipper will be aware of their unpopularity.
The Dons have never really shaken off the vitriol attached to their controversial move from Wimbledon, and for the first time in their history, they're now sitting below the reformed club – AFC Wimbledon – in the Football League.
It's also thought Gerrard would like to try his hand at a Premier League club, as he's understandably very ambitious having won the Champions League, among other domestic trophies, during his time at Anfield.
Yet, none of this seems to be the reason why he's snubbed Winkleman. According to the Mirror, it's because Stevie G would like more time before rushing into a decision after leaving LA Galaxy.
In fact, he's yet to even say whether he'll hang up his boots. At present, Liverpool seems the most likely destination for him, as Jurgen Klopp will give him until after Christmas before discussing a possible coaching role with him.
The England side is another option, but again, that will all have to wait until Gareth Southgate is officially appointed as permanent boss so he can have more of a say in choosing his staff.
Check out Football Advisor for profitable betting predictions


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Have you read, "Mayhem of the Miserables!" available @ http://www.smashwords.com/books/view/52075
Published: Sunday, Jul 3, 2011, 15:09 IST / Place: Mumbai | Agency: PTI.
Neeraj Grover.
Around 50 supporters of slain TV executive Neeraj Grover, today held a protest rally from Dheeraj Solitaire building, where he was stabbed to death, in suburban Malad to Tulip Star Hotel in Juhu, demanding justice.
The protest rally was led by filmmaker Ashok Pandit for the gruesome murder of Grover.
"Earlier in the day, police picked up 15 supporters and took them to Bangur Police station, as they said that they did not have permission to hold the rally. Later, I called up Congress MP Sanjay Nirupam and he intervened in the matter. After talking to a senior police officer, we were granted permission to hold the protest rally," Pandit said.
Full Story at,


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I recently moderated the Mergers and Acquisitions panel at the annual meeting of startup founders from Europe and the U.S. backed by Bertelsmann's Digital Media venture fund, which our law firm represents. While the discussion covered numerous topics, I was struck by how many questions our panel received when we ticked through the litany of the most common mistakes we've seen founders regret during M&A deals.
Interestingly, few of these mistakes had been made during the deal. Rather most would be filed under "ounce of prevention/pound of cure" because they'd been made long before the deal. Our panel consisted of a founding CEO and a CFO from two different companies that the fund had backed to a successful sale of each of those two startups, as well as an M&A investment banker (formerly a VC). There were definitely times when either the CFO, the CEO/Founder and/or the banker nodded and uttered something to the effect of "we did that" in response to mentioning these issues, however, I am not attributing these problems to any specific company, founder or fund.
Scraps from the cap table


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As interest continues to build about the entire process of book publishing, I have been working to bring everything to authors that they need to succeed. As a result, there are now five classes addressing different aspects of publishing. They are offered by the Community Learning Department of COCC and will be held during the Fall session.
For those needing to edit graphics for their book, attend:
Photoshop Elements Beginning: September 30 and October 7 and
Photoshop Elements Intermediate: October 28 and November 4.
There will be a class on self-publishing titled Publish and Sell Your Book on:
October 11 and October 25.
Interested in having a publishing company bring your book to Market? Try The Key to Publishing. It is about locating an agent and publishing house as well as query letters and submission packages. It will be held on November 6 and November 13.
If you want to stay current in marketing activities, check out Blogs and Podcasts on November 9 and November 16.
To enroll, contact COCC.
Monday, May 08, 2006
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The UCLA National Writing Project sent me to Temple City today for a workshop. The main focus was narrative writing. In addition, I showed the teachers various strategies to get students to write.
I can't believe how engaged and involved the teachers were throughout the session. They were respectful, understanding and willing to listen to new ideas.
It was honor to present to such an inspiring group.
Thank you!
Tuesday, July 28, 2015
Presenting in Temple City
Posted by Sean Killeen at 3:39 PM
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Jewelry Junkie Women's Black Faceted Rosary Necklace
Jewelry Junkie
- Jewelry Junkie Women's Black Faceted Rosary Necklace
- Item # 2000231243
$38.99 Original Price -$6.49 Sale Savings
$32.50 Final Price
17% Total Savings
Additional Information
Additional Product Data
- Aptos Styles Search: 2000231243
Features & Description
- Faceted rosary necklace
- KC gold-plated pendant
- Approx. 24" with a 6" drop
- Adjustable chain attached
Add the perfect finishing touch to your ensemble when you pull on this necklace from Jewelry Junkie. This necklace features a gold-plated pendant that hangs at the end. The rosary design of this necklace will add a unique flair to any outfit in your closet. Draw the eye of everyone in the room when you walk in wearing this necklace from the Jewelry Junkie.


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a few other surprising
ways to repurpose:
{ wood boxes as stairs }
{ old piping as bed frame }
{ truck side mirror as tabletop mirror }
{ truck springs as stools }
{ shipping pallet as a potting bench }
couldn't love these ideas more.
unfortunately, the only thing i can't figure out how to repurpose is my time.
{ although, there's a little voice whispering in my head
saying that i could be building these things
instead of blogging about them. sssshhh. }


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I really enjoy these anal beads. I also love the colour of them! Makes for a super cute/fun toy.
Easy to insert, comfortable to play with. Definitely a good impulse buy.
This was the perfect little G spot finder! Yummy!
Product Description
Ideal for many levels of anal experience, but absolutely perfect for beginners, the X-10 Beads range gradually from a very small (1/4 inch) starter bead to a more filling final sphere, letting cautious players ease very gently into the insertion process. Seamless and supple, the entire strand is pliable and extremely easy to handle, a large ringed handle provides a sturdy grip at all times.
Of a body safe, phthalate free PVC, the Beads are easy to clean and maintain- simple soapy water or a good toy cleansing formulation will see this backdoor piece thoroughly sanitized and ready to play. Always use lots of a good water based lubricant with the Beads for maximum comfort and pleasure.
- Length: 12.25"
- Insertable Length: 10.5"
- Girth: 3.25" at largest point
- Width: 0.9" at largest point
- Materials: PVC
- Special Features: Flexible, Non-Phallic, Phthalate Free
- Color: Purple
Product Code: SE-1233-14-2
UPC Code: 716770017925
Customer Reviews
These anal beads are great. I'm giving it 3 stars because it doesn't give you that kick when you're pulling the beads out. Check out the butt plugs if that's what you're looking for.
Nice feeling, and great price. Quite narrow, so it's easy to use.
Great toy, allows to experiment with varied sizes and depth. New to anal play? This is a good place to start.
I was new to anal play and thought this was a good place to start. Ok quality for the price. I find the length is too long to get to the larger beads. Overall fun and I am looking for something different in the future.
I added this onto my order last minute and didn't really think too much of it , I'm glad I did though cause my gf loves it :)
These feel great both going in and coming out. However, because of the length and the stiffness of the tip, you have to be careful as to the position. Not to say the perfect position cannot be found because when you do, they feel great!
These looked less intimidating on the screen than when I unpacked them. as a beginner, i liked that they started small...but wow, was it ever long! but, even as a beginner, the length was not uncomfortable...so, don't be scared, just try them!
A great choice for beginners, but i find that the beads arent much of a challenge. I recommend buying this in conjunction with a larger set.
I'm 28 and bought this as my first anal toy. I have to say, it was WELL worth the $10 I paid for it! My boyfriend and I love using this during our sex sessions! It makes it exciting just thinking about it!
The first night we used it I didn't realize by bf had put 8 of the beads into my ass so quickly.....maybe I was more relaxed then I realized! Its a great starter, but I'm now looking for something bigger and better. I've "outgrown" these beads fast ;)
These beads are good for the price. A staple to any sex toy collection. Downside is they feel a bit hard and there are plastic ridges that can poke you.
This was my first anal toy. Before this I had not experienced anything back there but fingers. I like this much better. I would definitely suggest this for anal beginners!
Wonderful toy. Great for beginners
I enjoyed these beads, but I enjoyed the price even more. I have to admit that although I am experienced in the area of anal play & sex, I never tried beads before because I just wasn't convinced they would feel amazing. This is now by-far, one of my favorite purchases. And a plus my spouse loves them too & found them to be very sexy.
Loved these anal beads! I'm quite experienced with anal play. These are amazing when your just about to orgasm, you remove them. I like to remove them at a medium pace. It is just so enjoyable. I think I'm going to look for something a little larger next time.
i recently started exploring the whole anal play and i was curious on how it felt i was not disappointed! they felt smooth the balls aren't big so it was a pleasurable experience i recommend this to someone who wants to try them out for the first time
These might be good to use for someone who is new to anal, but the cheap construction (seams) coupled with the inflexible material really takes away from the experience. I wouldn't recommend inserting these completely, as you'll most likely feel the tiny end poking your deep innards.
The price is hard to beat, but with toys (anal especially) it's worth it to pay a bit more.
I'm not sure why these are rated so highly, the ones I received are rather large, long, stiff and rough, I'm actually concerned they may damage something in there. Go with silicone or jelly instead.
This was the perfect little G spot finder! Yummy!
Easy to insert, comfortable to play with. Definitely a good impulse buy.
I prefer softer beads, but if you like the tougher material, you'll like these!
Very small, even if I am a beginner. Don't buy if you are already into anal.
This was the first anal toy I ever purchased. It opened up a whole new world for me and my s.o. the way it graduates in size makes it less intimidating and a good way to get use to having something in there. This is also my s.o's fave toy for me to use on him.
It's great for beginners since the first beads are tiny.
Well made
I really enjoy these anal beads. I also love the colour of them! Makes for a super cute/fun toy.
First anal toy and well received. Gradual sizes and pliable material made for fun time. Now however interested in graduating to different/bigger adventures. Perfect for first time use.
Fun! Wanted something a bit longer than my smaller and similar toy. Fun to use, easy to clean.
pinkcherry has awesome toys ever
Wife loved them. Starts nice and small. Quality toy. Great length. Good for both of us.
She like it very much
I have many toys for anal play and these still remain to be one of my favourites


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For so many years, Cagayan de Oro has been known as the City of Golden Friendship.
And with a booming tourism industry and a robust local economy, the city's progress is marked with several infrastructure projects basically changing the city's skyline as shown above – a view from Macajalar Bay. Named as the Most Competitive City in the Philippines and lately, one of Emerging Cities of the Future by no less than the United Nations, Cagayan de Oro will always remain to be a City by the Bay.
photo by Vincent Tom Udasco


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Join me in reading through the Bible in One Year! We will follow a reading plan together and go beyond just reading the Bible, to studying it. We will post about the passage and learn from each other as we grow in our walk with the Lord. Join me in the pursuit of discernment!
Friday, February 8, 2013
8 February 2013 ~Roxie
4 comments:
- LaughingLady said...
I guess the reason the concept of "waiting on God" is mentioned regularly all throughout the Bible must be because it's so terribly contrary to human nature! True patience and contentment are hard to come by.
Beautiful post, Roxie!
- February 8, 2013 at 6:42 AM
- Miriam said...
I love words like abide too. So much nicer than continue in, live in, or stay with. Or wait. Thanks for the reminder of Martha and Mary. I tend to be Martha, and when I have time to be Mary, I look for entertainment instead, thinking to myself I need a break from all the Martha.
- February 8, 2013 at 8:15 AM
- TammyIsBlessed said...
So true Roxie, beautifully put.
MacArthur stressed the point that because we have the hope of eternal life and His returning, that should inspire us to live godly lives, to pursue holiness.
Our remaining steadfast in our faith and Christ keeping us steadfast is again like the two pedals of a bike. His power inspires our belief and action.
- February 8, 2013 at 4:39 PM
- Pamela said...
I'm more of a "Martha" myself and I loathe waiting. I do, however, love what I learn while I'm waiting. That song in the move Fireproof "while I'm waiting" has such powerful lyrics that remind me that I am not in control and that I just need to wait on HIm.
- February 10, 2013 at 1:45 PM


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Long before Robert Pelton, the convicted Soviet spy, was receiving clandestine calls on a wall phone at the Pizza Castle in Falls Church, spies had been making their way across the Washington metropolitan area, il,7p dropping packages at trees in Poolesville, giving CIA officers the slip at a French restaurant, making contact with the KGB at Zayre's.
It's part of "tradecraft" -- the art of making the right connection at the right time.
"In general, the principles of selecting drop sites haven't changed in decades," says Roy Godson, GeorgetownXL professor and director of the Washington office of the National Strategy Information Center. "Just the specific sites change."
Godson, an intelligence expert, has interviewed numerous current and former intelligence officers. "Almost all substantial intelligence organizations use one or another form of drop sites," Godson says, referring to the "live drop," where a spy meets someone, perhaps his case officer, and the "dead drop," where a spy leaves something to be picked up. "I just don't want to get into any specific sites."
But there are basic rules. "The KGB and the GRU Soviet military intelligence send people to school to learn how to do this," says Godson. "You don't just decide, 'Oh, let's go to that place.' "
For example:
Pick a place easy to get into, easy to get out of and hard to be followed into.
"Favorite drop sites in the Washington area are shopping centers," Godson says. "It would range from shopping centers to the woods. Those fit the pattern of difficult to survey, lots of entrances and exits, lots of people passing through."
Pick a place where you belong. In other words, if you're not a punk rocker, don't make a drop at the 9:30 club.
"It's a matter of the individuals you're dealing with," says Samuel Halpern, a retired CIA officer. "Are you going to find them in the library or museum, the department store or supermarket? Where is it more likely for someone to see them?
"If the Soviet case worker is meeting an American, where is the FBI likely to drop its surveillance? That's why it's so much more convenient to use drop sites -- preferably dead. Then if something goes wrong, only one party gets caught . . . The idea is not to be conspicuous. You don't want to stand out like a sore thumb."
Don't leave a trail of bread crumbs.
"It's frequent to use the public telephones and use them from one phone to another," says John K. Greaney, executive director of the Association of Former Intelligence Officers. "The reason is that it's very difficult to monitor all phones. If you get a warrant, it's usually limited to a specific phone for a specific person."
Stay away from heavily secured areas.
"With greater surveillance around public buildings, you're not going to go to an airport or a railroad station or even a museum these days," says Halpern. It's not that guards are actively looking for you. It's just that they might remember you.
"Try going into even a lovely-looking museum like the Air and Space Museum these days," says Halpern. "The entrances are narrowed down to one entrance and one exit . . . You don't want to be narrowed down to one entrance where a security guard is looking at people and packages and briefcases and could later remember your face when he's looking through photographs and identify you."
Don't be seen in the same place twice.
"Any popular restaurant could be a live drop site, but you wouldn't want to use the same one again and again," Godson says. "You're going to bring surveillance down on you. People are going to see you keep going to the same shopping center again and again."
Soviet Embassy officials may travel unrestricted only within a 25-mile radius of Washington. So that's one consideration if an American is spying for them.
No place is so pregnant with possibilities for drop sites as wooded areas: "It's very difficult to follow someone going into the woods," says Godson, who has even seen such spy tools as hollowed-out rocks and empty soft-drink cans with false bottoms: "They make them in the KGB labs -- for secreting messages, packages or money."
Confessed spy John Walker was typical.
On the evening of May 19, 1985, he drove to rural Poolesville in Montgomery County, stopping and getting out of his van many times to shake a potential tail. But FBI agents, trained in spotting dead drops, combed Walker's path until they found a tree on Partnership Road that they deduced was a reference point.
Thirty feet away, at a telephone pole with a "no hunting" sign on it, was a crumpled grocery bag -- inside was "trash" on top of secret Navy documents wrapped in plastic.
"In every way, it looked like a Soviet drop site," FBI counterintelligence agent Bruce Brahe II testified during the espionage trial of Walker's brother Arthur. John Walker has said that from 1968 to May 1985 he made about 30 drops at various places in suburban Washington.
Though John Walker had most of his face-to-face meetings with Soviet contacts in Austria, he once met a Soviet contact in a Zayre department store in Northern Virginia. Walker carried a copy of Time magazine to identify himself.
More spies who got caught before they came in from the cold:
Former CIA officer David Barnett, who pleaded guilty in 1980 to selling secrets to the Soviets, was given a prearranged dead drop site by his Soviet contact -- near Lock 11 on the C&0 Canal -- and the numbers of two telephones outside a gas station in Annandale.
Barnett, who lived in Bethesda, used neither. Instead he called his contact on the last Saturday of each month from a public phone in the Bethesda Medical Building.
David Truong, one-time antiwar activist who was convicted of spying for Communist Vietnam along with former USIA officer Ronald L. Humphrey, was said in his indictment to have turned over stolen U.S. documents at such meeting places as the Landmark Shopping Center in Alexandria.
Waldo H. Dubberstein, a former Pentagon intelligence analyst indicted in 1983 on charges of selling U.S. military secrets to Libya through ex-CIA officer Edwin P. Wilson, allegedly received his payments at meetings in the Washington Hilton and the Key Bridge Marriott. (Dubberstein was found dead, an apparent suicide victim, in his apartment building the day after he was indicted.)
To be sure, nothing is certain in the world of tradecraft.
Soviet defector Vitaly Yurchenko was eating dinner with his CIA security officer in the Georgetown restaurant Au Pied de Cochon in 1985 when Yurchenko said, "If I'm not back in 15 minutes, don't blame yourself." Yurchenko walked out, making his way to the Soviet compound near Calvert Street and Wisconsin Avenue -- where he redefected. Afterward the restaurant put a little brass plaque over the booth where he sat. It reads, "Yurchenko's Last Supper, Saturday, November 2, 1985."
And some spies did not take elaborate precautions:
Edwin G. Moore II, a former CIA officer convicted in 1977 of attempting to sell documents to the Soviets, left a package of documents, a demand for money and an offer of more documents outside a Soviet residence here. A Soviet employe found the package and, thinking it was a bomb, called the police.