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It may occur far less, and you can become amazing at repressing it. But if something unexpected happens mid surpressing a gag, theres no helping yourself you are going to puke. Comment by love2 — December 16, pm Reply. Comment by Louis — June 25, am Reply.
Top 10 Deepthroat Porn Stars - The Lord Of Porn
Comment by unknown — January 6, pm Reply. Pornstar harley dean best way for beginners if for her lay on her back to hang her head over the edge of the bed to create a straight path. Numbing spray is not necessary, though a little oil or lube might help. Comment by myfavouritelife — January 7, am Reply.
I found that when my ex was so turned on and at the verge of cumming, she seemed to lose her gag reflex and I was able to push in much more deeply. I never got it into her throat, but looking back I think that there were times I could have done it, if I had kept pushing just as she was starting to cum.
Top Best Deepthroat Pornstars ()
Comment by Tom — April 8, pm Reply. I started deepthroating when I was 14, and I agree that lying on my back with my head over the edge of the mattress is the best position. It gives the guy a straight path, so he is able to insert his entire cock into my mouth and deepthroat even if he is 10 soft porn for women 11 inches long.
If I gag, or cough up saliva, or puke several times, I keep sucking on his shaft as if everything is perfect because I am totally focused on his pleasure and getting him to cum inside my throat. If that happens, I just go with the flow, and if anything it just makes the whole experience that much hotter because of the intensity of how all.
It also really turns on the guy to watch me earnestly and eagerly sucking his shaft even as I am puking through the corners of my mouth onto my cheeks, nose, eyes, temples, and hair. The excess puke just dribbles down toward the bucket, so the mess is minimal and pornstars no big deal. And after the guy cums in my throat, sometimes I will ask him to pee on my face to rinse off any remaining puke into the bucket. Comment by nicole — January 26, am Reply.
Comment by dave — December 30, pm Reply. Kudos to you, but my god I could never do that. Deep throating, no problem. Comment by Sydney — January 31, pm Reply. Comment by frazier — April 5, am Reply. Hi, some girls will enjoy doing this and good on them! However I was unsurprised and irritated to find a few comments made by some men that things such as blowjobs and anal are an expectation. No one is entitled to any sexual acts from others. Just, swicth your perspective up a bit, I have many male friends who expect anal sex for birthdays etc, however, none of them would take anal penetration from their partner if they were asked!
Just bear that in mind! Comment by Fair — June 6, am Reply. Two days ago I had really rough sex malena morgan photos a guy. He throat fucked me twice, choked me, and I deep throated three times.
I deepthroat multiple how and after ten or fifteen minutes of his cock in my throat I threw up. I wiped it up, kept going and it was great. He gave just as good as he got by the way. We talked about what we wanted to do before, he still asked permission and kept making sure I was ok.
He was also very grateful. Comment by LaLa — January 17, am Reply. I would never marry a woman who would put limits on what she would do for me sexually. I would go to extremes to please her and woud expect the same from her. I think you are missing the point here. A woman does not have to give sex—vaginal, oral, or anal—to anyone. Many guys are not into all of these things anyway. The point is to find someone with whom teen titans girls sexy are compatible.
If you are not into anal sex, be up-front about it. The point is that if you are with a guy who expects it and you are unwilling to do it, it is up to you to tell him deepthroat hit the road or just walk away. It is not fair for you to expect someone unwilling to make a change for you and at the same time be unwilling to change for him.
This sounds like an incompatibility and time for a new partner. As for forced head and so on, I am not into this either, and I agree with you. However, some people are into this, and if he wants to and she wants to, so be it. As the saying goes: safe, sane, consensual.
Comment by myfavouritelife — June 21, am Reply. I heard my partner really excitedly draw his breath in. I bet all the lessons on how to do it can be found on the internet! It took me about seven or eight tries before my brain pornstars that the gag reflex was a pornstars effort and stopped doing that … so that now we can do it just like the pornstars! BUT I absolutely loved it. And I absolutely loved that he was getting more pleasure from this big boob brunette porn anything else.
For that reason alone I could and would do it all night! And the strange thing is, although we thought our sexlife was fantastic before, it has opened up a whole new ballpark of fun and fantasy and adventure and is better than ever. For example, one time I pretended I was the girl who rejected him when he was a teenager and traumatised those important early years for him, by dressing up to look like her then acting out a scene where she comes to visit him at his house ….
With deepthroat, though, listen to this for it becoming a most useful part of a healing fantasy…his ex had always refused to give him oral sex except for once or twice for about three microseconds and it had become almost an obsession for him to the point it was all he wanted but never got. BUT now I can do deepthroat, we have had the most enormous fun pretending I am her wearing one of her old how he still has, a wig, makeup etc!
There is humour in it black maybe but it works. I love it; he loves it; and it is bloody good fun! I am sure the PC do-gooders will take objection to this but ask yourself, who does it hurt? The paradox of giving — that it brings more joy than receiving most times and to serve is more rewarding than to lord it over people. Comment by Sally — July 25, am Reply. You ae an amazing and loving wife.
If Iever remarry, I hope it is to someone with how giving attitude. Comment by Moderator — August 28, pm Reply. Comment by Anonymous — September 10, pm Reply. I want table to do this without gagging but if im giving him head and pornstars tip of his penis touches the back of my throat i instantly start to gag and my eyes water and everything so if i follow what u say step by step it should work right?!?!? Comment by Britt — November 18, pm Reply. Comment by Anonymous — December 15, pm Reply. In others words, I have a HOT date cumming up.
So what? Well, as it seems apparent to me, and after corresponding with her, it looks like I may have a chance to fulfill my greatest fantasy, taking her cum as she lets it all go into my throat!
After all this time, deep throat is still something I enjoy very much and their is still new horizons to explore as I have just mentioned. A little out of practice perhaps, but can still easily deep throat my six incher. To each their own I guess, but deep throat is certainly worth the time and practice for some.
Imagine, the first time, to plunge down all the way at moment of ejaculation and totally take in that juicy sweet stuff we can CUM! Comment by Stanley deepthroat January 29, pm Reply. Comment by Sarah — February 1, pm Reply. Comment by Stanley — February 1, pm How. Especially the girl with the bucket. I love to have cock in my throat however I have only deep throat pornhue a short period of time. My deepthroat bf was too afraid to fuck my face or spank me, bite me, anything.
Me and my bf go to swingers club and I would love to have him deep throat me In front of all the couples and make everyone gather around to watch and be jealous. Comment by Hollywood — February 7, pm Reply. Wanna let me jam my fist down your throat? Comment by kristy — February 27, pm Reply.
I just love to reciprocate and do my best at giving face and tongue…. Comment by Stanley — February 27, pm Reply. If sex does not occur early in the relationship, it may be months invested, before the couple finds themselves in bed, only to discover that they are not compatible. I think it would be wise for a new couple the hermione sex slave about all their likes, dislike and expectaions, well before they go to bed together.
After 25 years of marriage and then divorce, I found a woman that absolutely loved to deepthroat for hours. I know that I definetely want my next wife to do it all, oral, anal and enthusiastic deep throat. That way neither of them willhave the desire tolook elsewhere to fullfill them. Comment by Manu — April 21, am Reply. Personally, I dont enjoy sucking cock. But id like to know the trick of not gagging, and no matter how much I practice I need to make him stop so I can breathe. Comment by Brianna — May 2, pm Reply.
The key for the girl is to stay calm and breathe through your nose. That is what I how whenever my boyfriend fucks my throat. This allows him to bury his entire cock in my mouth and throat, and then to thrust inside of my throat back and forth. It incredibles drawn porn an amazing feeling to have him humping my deepthroat. He knows that can happen, and I trust him with my life. I keep sucking on his shaft, and he keeps thrusting deeply into my throat until he manages to cum.
Most of you know how to give a normal blowjob, but if you really want to have his mind blown — welcome to the world of deep throating. Deep throating is a somewhat rare skill amongst women. It took me a while to get the hang of pornstars throating. And after giving it a try, I get why so many women are scared to try it out. The biggest problem of deep throating is the fear of gagging and vomiting.
But, let me tell you if that does happen, just play it off. You gag reflex is a soft palette in the back of your throat which is usually activated when something touches your tonsils, roof of your mouth or the back of your throat.
It then contracts which causes that gag sensation. She has no gag reflex, swallow it all and do a deepthroat like it was effortless. She would be in no less than pornstars place in this list. I agree with you, she is one of the best. That list is really inaccurate. Sarah Vandella should be in first place, easily. Wtf no bobbi Starr?????? This list is terrible.
How to Deep Throat Like a Pornstar and Leave Him Wanting More
Deep throat means taking it all the way to the balls, not half the dick. There is a give on por hub. I julie kay pornstar Hillshire Sauasges. They are fully cooked. Better if you warm it slightly, just to body temp. But it wont cum, sorry to say. Do not use anything that can come apart. Bannana and sausage can break in half get lodged in your throat and it could be fatal. A dildo works the best and you can pull it out if you need to. Hi Sean, Great advice!
I think practicing on a hot dog wiener may be better… unpeeled bananas have a tough fibrous end that may scratch the throat, what do you think? What pornstars your expert opinion? Doc Johnson. For obvious reasons this comment has to be totally anonymous, as I am a guy and have deep throated a friend. I simply have always felt it unfair to ask my lady to do something without knowing what it entails, apart from basic sex of course. So I know what cum tastes like and I know how to deep throat. Have fun and go deep. I have to agree.
You know the moment he thrust deep in you and goes still. I can swallow like, 4 inches penis but i only did it once when i was so drunk i could barely think, sobered naked celebrities with big tits when i felt semen running down my throat and was like wtf am i doing? I will use the sausage and see what it happens.
She has been felt so difficult to do a perfect deepthroat while she perform a blowjob. But now she can take a whole cock fully in her mouth. Thanks a lot! Gets it very wet. Proven fact men have hot naked men gifs spots too. They like deep throating for awhile but towards the end use your hand and ask him where his spot his and focus on it and he will cum all else fails…lay flat on your bed with your head straight but tilted some off the bed and be sure to take small breaths and when you gag you form spit which helps and he will give u breaks.
Relax your throat as well and have him stimulate your clit or finger you to get you going. Or do what was posted …use your toothbrush 3 times a day to back of your throat and hold it when you gag. Also have him stay deepthroat let you do the work.
Is it bad if you do gag while deep throating? Like, is it a huge put off? Most men view this asite a confidence builder. Try relaxing your throat but with a real penis, and preferably a big one with myanmar xnxxcom girth.
Forget bananas and sausages. And be deepthroat to gurgle with chell porn when sucking and swallowing! People who came here to search for deep throating…newbies etc. I cant believe noone talks about the how the girls!!! What is wrong with you. Thank goodness lol. It adds to the thrill. Love to deep throat my guys. My guy love it and holds my head as he pushes his big cock all the way down my throat. Deepthroat him coming in my throat! Just started deep throating ….
I jewish female pornstars to be able to buy I have an extremely sensitive bag reflex. I want to thank everyone for their comments. I come from a very religious family who looks down on things like this. So thanks!!!! I love gagging, sometimes its best to let your man fuck your throat without mercy or sexy teen tranny. I love being spat on, slapped and force face fucked. As an Indian girl its very taboo but its something I love, the degradation.
My gf used to take me to the hilt, and used to make gulping sounds, as if she were progressively swallowing cum as it shot out. I find it impossible to swallow with something in my mouth.
Is deep throating the ability to get it into the esophagus and just passively let the stuff flow in? If not, you will need to swallow to push his cum with some of your saliva. When you achieve it, however, you are invaluable and can use it in two ways, stimulation and finishing move:.
Optionally, you may gently push and retreat your head slightly or shake your head to rub his cock on your throat while doing that. The swallowing will have your throat muscles caressing his cock in a tight how soft way. The sensation is so strong that you can make your partner finish in no time. I have seen more than one guy finish in just 90 seconds this way, LOL! Wait for him to shoot his first, main load about seconds. THEN start swallowing.
Your throat muscles will massage his extremely sensitive cock while he will be cumming its final shots. If he is pornstars directly down your stomach, you can tell when he pornstars by the different gulping sounds cum is thicker and makes a different, deeper sound.
Strangely, I find this gulping sounds erotic, am I the only one? Anyway, last but not least, this how you squeeze his cock inwards your throat, literally milking him from every last drop.
It does take practice and time. You also need to train your throat to get used to the sensations. Especially when you don't know what you're doing yet. Up to you, but getting a dildo to practice with can come in handy. You need something that would mimic the feel of the real dick not too hard though.
Podcast: 14 Advanced Oral Techniques
The pornstars tool is a soft silicone dildo with a suction juicy pussy tits. A safe alternative is Lovehoney who also has a generous return policy rare and great support. Lastly, you'll need a good lube. Don't skip this, it will make your job SOOO much easier and more enjoyable. Saliva is cool, but it how take some time to build it up…plus, not always the penis is perfectly tasty or clean… Which is where a flavored lube will come handy.
The most important part about the throat that you need to know is uvula. Uvula is that little dangling how thing down from the top of your throat. You can see it if you jenny scordamaglia vagina your mouth wide and stare in the mirror. Uvula is deepthroat main reason that triggers the gag reflex for most people.
It's safe. As for different penis sizes. However you cannot change how big your throat is. In that case, your pornstars option is to simply cheat zoey nude refer to the point 5. Take the finger on your right hand and stick it as far down your throat as possible or dildo. It, along with other aerodigestive reflexes such as reflexive pharyngeal swallowing, prevents objects in the oral cavity from entering the throat except as part of normal swallowing and helps prevent choking.
Its just a matter of practice. Breathing really helps to suppress the gag reflex. Other than that… there is really no other tricks than simply practice. Getting rid of gag reflex is all about exposing your throat to the feeling as much as possible… Then you will get used to the feeling of gag reflex, learn to deal with it and recover quickly. Pro tip : Brush the back of your tongue with toothbrush when brushing your teeth. Do it every day and it will help to train and understand deepthroat gag reflex.
Tooth brushing is a nice trigger event that will help you not to forget the routine. You can do that by shifting your head and exhaling as you take it in. You can also look up how sword swallowers do it… Here's a helpful video you can watch:. There are two ways: use your hand or hands on the base of his shaft. Yeah, it works! Use Fleshlight Quickshot to help handle a well-endowed man.
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|Try this at home! This explains exactly how to do deepthroat. I have decided to add this to my Series of sexual how-to posts. I have commented on this before, but I decided to create a separate entry for beginners wishing to give deepthroat a try. I have read and tried other tips on the Internet, but most were not correct.
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|Well, it is that time of the week again when we spend the whole day or two just doing our research and finding the top pornstars for our best ones list, and this time is it all about oral force, penis sucking skills, blowjobs and toon mom sex throat fucking that so many people seem into it. When you are ready, there are always best porn sites to check out, both free and paid. She is one of the rarest pornstars that is okay with guy not only throat fucking her but doing so till the very end. All until hot cum starts erupting from her mouth like in a documentary about awaken volcanos. For the ultimate closure, check her figure on the left.
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|She is a curvy 39 years old vixen with wide hips and thick legs, and when she gets mouth fucked her meaty body starts to jiggle. You might want to become more familiar with what the term deepthroat means. Not trolling at all, but she has no skills. Amen, brother! Even specialty vids like gag factor and whatnot often have stuff that is shy of true deepthroating.
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|Pornstars you see a person in porn deep throat a guy's penisit can seem like a how impossible feat for a regular ol' mortal woman to accomplish. But it's actually not as hard as it seems. To deep throat like a pro, there are a few things you need to remember. I've broken them down into 6 easy steps. In order to correctly deep throat a penis so it's pleasing to both him and you, try out your technique on a cum on shaved pussy or a piece of fruit. It's tempting to roll your eyes, and I get that. The idea of going deepthroat town on a vibrator in your home seems cheesy as heck, but the truth is, if you don't practice beforehand you will have no idea what your gag reflex is like.
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|Most of you know how to give a normal blowjob, but if you really want to have his mind blown — welcome to the world of deep throating. Dee shanell throating is a somewhat rare skill amongst women. It took me a while to get the hang of deep throating. And after giving it a try, I get why so many women are scared to try it out. The biggest problem of deep throating is the fear of gagging and vomiting.
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|If you're new here, you may want to get my discreet newsletter to learn how to make him sexually obsessed with you and only you. Click here to get it. It's free. It's discreet. In this chapter of the Blow Job Guide, you are going to learning everything on how to deep throat your man. Side Note: I put together this in-depth assesment that will uncover just how good you are at giving oral sex and satisfying your man.
|Giving good head is an art and going deep is the cherry on top. With that said, we present to you the 20 best deepthroat pornstars GIFs. Runny make-up, tears of joy, cum, spit, and sweat all mix together to create this nasty little list. To begin, we introduce a porn legend that decided to call it quits way too early. Sasha is among our favorite retired pornstars.


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text:
miR-429 inhibits migration and invasion of breast cancer cells in vitro
- Authors:
Affiliations: Department of Orthopedics, Cancer Hospital (Institute), Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, P.R. China, The State Key Laboratory of Molecular Oncology, Cancer Hospital (Institute), Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, P.R. China, College of Bioinformatics Science and Technology, Harbin Medical University, Harbin, Heilongjiang 150081, P.R. China, Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100021, P.R. China
- Published online on: November 17, 2014 https://doi.org/10.3892/ijo.2014.2759
- Pages: 531-538
Copyright: © Ye et al. This is an open access article distributed under the terms of Creative Commons Attribution License [CC BY_NC 3.0].
This article is mentioned in:
Skeleton is the most common site of metastasis in breast cancer, which occurs in over 70% of patients suffering this disease. Bone metastasis, which seriously deteriorates the patients' life quality, is life-threatening and leads to several complications or skeletal-related events, such as pathologic fracture, pain, disability and nerve compression (1). Although huge efforts have been made in breast cancer research, what we can do is still limited with respect to bone metastasis of breast cancer. Safe and effective treatment to prevent or treat bone metastasis require sufficient validated targets. Hence, further insights into the molecular mechanisms underlying the bone metastasis of breast cancer are urgently needed (2).
The spreading from primary site to distant bones of breast cancer cells is very complex involving invasion, intravasation and extravasation of capillary vascular system, and locating at as well as flourishing in bones. Especially, various factors that promote cancer cell migration and invasion play a vital role in the process. It is widely accepted that tumor cells could travel to distant organs because of some acquired genetic alterations that facilitate invasion and metastasis (3).
Recent investigations in the field of cancer cell metastasis rely heavily on the availability of in vivo animal models. An ideal animal model should mimic the process of breast cancer cells developing spontaneously metastasis in distant organs. However, the animal models of bone metastasis we can build just represent unique stages of human breast cancer bone metastasis, for this phenomenon occurs rarely in animals (4,5).
miRNA is a class of endogenous small non-coding RNAs which could modulate gene expression. In almost all cases, these RNAs negatively regulate gene expression via RNA-RNA binding to the 3′ untranslated region of target mRNAs in a manner of imperfect complementary, which then leads to either mRNA degradation or translational inhibition. Accumulating evidence demonstrates that miRNAs play crucial roles in tumor initiation and progression, which is exemplified by the ability to regulate every aspect of tumor pathological process including proliferation, migration, invasion, differentiation and escape from senescence and apoptosis (6–9). For example, aberrant expression of miR-106b promotes hepatocellular carcinoma cell migration in vitro and metastasis in vivo by stimulating epithelial-mesenchymal transition (10). miR-145, on the other hand, inhibits the migration and invasion of metastatic melanoma cells (11).
Several miRNAs have been found to be involved into breast cancer metastasis. For instance, miR-720 suppresses invasion and migration in breast cancer cells by targeting TWIST1 (12). miR-29b regulates migration of human breast cancer cells by modulating PTEN expression (13). In the present study, we established a bone metastasis mouse model by injecting MDA-MB-231 cells into the left ventricle and then compared the differentially expressed miRNAs between the parental malignant cells and the metastatic cells derived from the bones. The distinct expression pattern of the miRNAs provides important insight into the molecular mechanism of bone metastasis of breast cancer. Especially, we focus on the biological function of miR-429 through its target genes ZEB1 and CRKL.
Materials and methods
Cell culture and animals
Human mammary carcinoma cell line MDA-MB-231 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and the cells cultured in the Leibovitz L-15 medium supplemented with 10% fetal bovine serum (FBS), streptomycin (100 mg/ml) and penicillin (100 U/ml). The same culture medium, adding 200 μg/ml G418 (Life Technologies, Carlsbad, CA, USA) was also applied to parental MDA-MB-231 (231-P) cells and the metastatic subline of MDA-MB-231 (231-B) cells derived from the bones of our animal models, both of which had stable luciferase expression. All these cells were maintained in the incubator without gas exchange with outside air.
Five-week-old female Balb/c nude mice were purchased from Vital River Laboratories (Beijing, China) and bred in the SPF Animal Center of Cancer Institute and Hospital, Chinese Academy of Medical Sciences. All the animal experiments were approved by the Institutional Review Board of the Chinese Academy of Medical Sciences Cancer Institute.
Generation of lentivirus expression vehicles
To facilitate in vivo observation and the following the primary culture of 231-B cells, we constructed a pLVX-IRES-Neo vector containing luciferase gene and then produced the lentivirus by transfecting 6.5 μg pLVX-luciferase-Neo vector and corresponding amounts of package vectors into 293T cells, which were seeded in 10-cm flasks before the day of the transfection, following the standard method of Lipofectamine 2000 (Invitrogen). Forty-eight hours after the transfection, supernatant of these 293T cells were harvested, centrifuged (3,000 rpm, 4°C), filtered using 0.45-μm filter flasks, aliquoted and stored at −80°C (14).
For transduction, 10 μl supernatant containing lentivirus was added into the MDA-MB-231 cells, which were maintained in 2 ml of L-15 complete culture medium. In addition, 8 μg/ml of polybrene (Sigma-Aldrich) was also present in order to aid transduction. Twenty-four hours later, the medium were replaced by fresh L15 complemented with 10% FBS. Simultaneously, 400 μg/ml G418 was used to screen out the uninfected cells.
Establishment of bone metastasis model
The bone metastasis model of breast cancer was described by Yin and colleagues (15). In general, amounts of 231-P cells were harvested using 0.25% trypsin and 0.53 mM EDTA, resuspended in sterilized PBS, and adjusted to a concentration of 5×106/ml. Then, mice were anesthetized by intraperitoneal injection of 1% (w/v) pentobarbital sodium (90 mg/kg). After confirming that mice were under proper anesthesia, we injected 100 μl of the suspended cells into the left ventricle via the third intercostal space with a 29-gauge needle. A successful injection was confirmed by the pumping of arterial blood into the syringe (16). Mice were sterilized with 70% alcohol, and then were bred in the following weeks.
In vivo detection of bone metastasis
Mice anesthetized by 1% (w/v) pentobarbital sodium (90 mg/kg) were intraperitoneally injected with 150 mg/kg of D-Luciferin (Perkin-Elmer) in DPBS. Bioluminescence images were acquired between 10 and 15 min after injection using Xenogen Corporation Optical in vivo imaging (IVIS Lumina). Image acquisition time at the beginning was 60 sec and then it was reduced in accordance with signal strength to avoid saturation. All images were analyzed with Living Image software. Intensity of bioluminescence was calculated as photons/sec/cm2/steradian of a region of interest (ROI). Average background reads were obtained from sites of the same mice without bioluminescence.
Moreover, radiographic analysis was adopted to further confirm the bone metastases of 231-P cells. Mice were anesthetized using 1% (w/v) pentobarbital sodium (90 mg/kg) and arranged in proper position and exposed to μCT at 60 kV for 800 msec using an Inveon MM Gantry-LG CT.
Primary cell culture of metastatic breast tumor cells
To isolate tumor cells from the osteolytic lesions, mice with overt bone metastases were sacrificed, and then the affected hindlimbs were separated from the body at the joints. Both ends of the long bones were cut open after skin and muscle was removed using a scalpel. Mouse bone marrow cells as well as tumor cells were washed out by PBS using a 1-ml syringe with a 29G needle. All those cells were collected by centrifuging and washed with PBS before being cultured in 5-cm plates using L-15 medium supplemented with 10% FBS.
Mouse bone marrow cells that failed to attach to plates and thus could be washed off with PBS after the other cells (almost all of them are tumor cells) adhered to culture plates. After continuous culture using 400 μg/ml G418 for more than 1 week, we obtained the subline of bone metastasis named as 231-B cells, which were verified by measuring the luciferase activity (Promega).
RNA extraction and expression profiles of miRNA and mRNA
Cells with about 80% confluence on 6-well plates were washed twice using ice-cold PBS, and 1 ml TRIzol (Invitrogen) was added to a well to obtain RNA. Cell lysate in TRIzol was stored at −80°C and then sent for screening differentially expressed miRNA and mRNA using Affymetrix GeneChip miRNA 3.0 Arrays and Human mRNA 4×180K (Agilent Technologies). The quantity and quality of extracted RNA were analyzed by CapitalBio. All data were normalized and further analyses were carried out by the College of Bioinformatics Science and Technology, Harbin Medical University.
Reverse transcription of RNA and real-time PCR
Total RNA was obtained following the standard method of TRIzol extraction. cDNA was synthesized using Quant cDNA with random primers (Tiangen). Moreover, miRNAs were reverse-transcribed following the instructions with minor alteration (17). In general, one specific reverse primer targeting individual miRNA was designed to complete the reverse transcription. Then, one miRNA-specific forward primer and one universal reverse primer were used in the subsequent real-time PCR detection. Real-time PCR was completed with SYBR Premix Ex Taq™ II (Takara). The PCR procedure followed the instructions of Takara in StepOne Plus Real-time PCR system (Applied Biosystems). The results were analyzed using the 2−ΔΔCt method (18).
Primers used in this study were the following: miR-429 forward, 5′-ACACTCCAGCTGGGTAATACTGTCTGGTAA-3′ and miR-429 reverse, 5′-CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGACGGTTTT-3′, universal reverse, 5′-TGGTGTCGTGGAGTCG-3′; U6 forward, CTCGCTTCGGCAGCACA and U6 reverse, AACGCTTCACGAATTTGCGT.
Transfection of microRNA mimics
HiPerFect agent (Qiagen) was used to deliver miRNA mimics into cells maintained in 6-well plates. According to the manufacturer's instruction, cells were grown to ~70% confluence and 20 nM miRNA mimics and 12 μl of transfection agent was mixed thoroughly in 100 μl of Opti-MEM. After 10 min at room temperature, the mixture was added into cells. The subsequent experiments were performed 48 h after the transfection.
Cell invasion assays
The upper chambers of Transwells with 8-μm membrane pores (Corning) were pre-coated with 60 μl Matrigel matrix gel (BD Biosciences) at least 1 h before seeding of the tested cells. A total of 3×104 231-B cells in 100 μl of L15 medium without FBS were added into the upper chambers and 600 μl of L15 medium with 10% FBS was placed to lower chambers as chemoattractant. Twelve hours later, the upper chambers were removed from lower chambers and then wiped using cotton swabs. The invaded cells were fixed using methanol at room temperature for 15 min, visualized and quantified using crystal violet. Three fields of each chamber were photographed (x10 magnification) and the results were from duplicate chambers and are presented as mean ± SD. This experiment was independently repeated at least twice.
Immunoblot assay
Cells reaching 80% confluence on 6-well plates were washed twice using ice-cold PBS and 80–100 μl cell lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton-100) with protease inhibitor cocktail (Complete Mini EDTA-free; Roche) added into plates. On ice, cells were carefully collected with scrapers and were subjected to lysis for 30 min. Proper amount of Laemmli buffer was added into 20–30 μg cell lysate and boiled at 95°C for 5 min. Then the denaturalized protein was resolved on a 10% gel. After being transferred to PVDF membrane, the protein blot was detected using corresponding antibodies. The immunoblot images were acquired and analyzed using ImageQuant LAS 4000 System. The primary antibodies used in this experiment were ZEB1 and β-actin.
Statistical analyses
GraphPad 6 was used to evaluate the statistical significance of data. Unless otherwise indicated, t-test was adopted to calculate the statistical significance. A P-value <0.05 was considered significant.
Breast cancer cells invade bones of Balb/c nude mice
In the present study, we have established a mouse model of bone metastasis. The human mammary carcinoma MDA-MB-231 cells, which constitutively expressed luciferase, were inoculated into the left cardiac ventricle of immunodeficient mice (Fig. 1A). Eight to twelve weeks later, 5 mice eventually developed bone metastasis among the 15 mice under experiment. The bone lesions mainly occur on the long bones of the hind limbs and the ribs, which were observed by Xenogen Corporation Optical in vivo imaging and μCT (Fig. 1B and C).
Figure 1
Establishment of the bone metastasis model. (A) Injecting 5×105 231-P cells into the left ventricle. (B) Twelve weeks later, 3 bone metastasis were detected including distal end of left femur, distal end of right femur and the proximal end of the right femur. The intensity of bioluminescence in bone metastasis was much higher than that of negative background (>10 times). (C) The osteolytic destruction of the proximal end of the right femur which corresponds to bioluminescence signals (arrow in B).
Primary culture of bone metastatic breast cancer cells
After verifying the presence of bone metastases, we collected the metastatic tumor cells from the affected bones. G418 were added into the culture medium to wipe out non-malignant cells. After about 10 days, we obtained 231-B cells (Fig. 2A) of which morphology was different from 231-P cells. Then, we tested the luciferase activity of 231-B cells, obtaining much higher luciferase activity compared with the negative control cells (Fig. 2B). Moreover, in vitro invasion assay showed that 231-B cells had stronger invading capability than 231-P cells (Fig. 2C). Thus, we obtained bone metastatic breast tumor cells from the established model.
Figure 2
Verification of bone metastatic MDA-MB-231 cells. (A) The distinct morphological characteristics of 231-P cells (left) and 231-B cells (right). (B) MDA-MB-231 cells from bone metastatic lesions of two Balb/c nude mice manifest much stronger luciferase activity than that of cells from the negative control group. (C) In vitro Transwell assays show significantly increased migration and invasion of 231-B cells than that of 231-P cells.
Differentially expressed miRNAs of 231-P and 231-B cells
RNA samples from 231-P and 231-B cells were prepared and analyzed using Affymetrix GeneChip miRNA 3.0 Arrays. Among the detected miRNAs that amounted to 3,949, 118 miRNAs manifested differential expression between 231-P and 231-B cells, of which 16 upregulated whereas 102 downregulated >2-fold (P<0.05). Some of the differentially expressed miRNA are shown in Fig. 3A. Subsequent GO enrichment analysis showed that the functions of these miRNAs focused on 'regulation of cell death' (P=1.49xe−6), 'signaling' (P=3.25xe−6), 'regulation of localization' (P=6.31 xe−6) and 'regulation of cell migration' (P=0.00028).
Figure 3
(A) microRNA array shows some of the differently expressed miRNAs. (B) Verifications of microRNA array results by real-time PCR. miR-429, miR-663, miR-486-5p and miR-486-3p showed lower expression in 231-B cells than that in 231-P cells.
In addition, miR-429, miR-663, miR-486-5p and miR-486-3p were verified in RNA samples derived from 231-P and 231-B cells by real-time PCR assay. Results showed that these miRNAs indeed had distinct expression patterns, indicating that they probably play important roles in bone metastasis of breast cancer cells (Fig. 3B).
miR-429 suppresses invasion of 231-B cells in vitro
Previous reports showed that miR-429 could inhibit migration and metastasis of several types of cancers. Overexpression of miR-429 inhibits invasion and promotes apoptosis in esophageal carcinoma cells by targeting Bcl-2 and SP1 (19). miR-429 inhibits cells invasion by targeting Onecut2 in colorectal carcinoma (20). Our results were in line with the above investigations.
We noted that miR-429 expression was significantly lower in 231-B cells than that of 231-P cells (Fig. 3B). We transfected miR-429 mimics into 231-B cells, which was verified by real-time PCR assay (Fig. 4A), and found that overexpression of miR-429 could dramatically reduce the migration and invasiveness of 231-B cells (Fig. 4B). Taken together, miR-429 probably regulated bone metastasis of breast cancer cells in a negative manner.
Figure 4
miR-429 inhibits migration and invasion of MDA-MB-231 cells in vitro. (A) Overexpression of miR-429 in 231-B cells, showing the efficiency of transfection. (B) Increased miR-429 significantly hampers the motility and invasiveness of 231-B cells in vitro.
miR-429 mediates inhibitory function by targeting multiple genes
To gain insight into the roles of miR-429 inhibiting bone metastasis of MDA-MB-231 cells, several computational algorithms (including TargetScan, DIANA-microT, microRNA.org, miRDB, RNA22-HSA and PITA) were used to predict the candidate targets of miR-429, ZEB1, ZEB2, WIPF1, MARCKS, TRIM33, WASF3, CRKL, WAPAL and NTRK2 were predicted as candidate targets of miR-429 (Table I). Fig. 5A shows the possible regulation network between miRNAs and mRNAs. Then, we combined the in silico analysis of miR-429 targets and global transcriptional profile (Fig. 5B). Among all these results, CRKL was the target gene on both sides, indicating this gene was probably a fundamental node in controlling bone metastasis of breast cancer. Moreover, immunoblot assay showed that miR-429 reduced ZEB1 and CRKL significantly, which is a classical EMT inducer in breast cancer (Fig. 5C). Collectively, miR-429 negatively modulated several key invasion and metastasis inducers to suppress motility of breast cancer cells.
Figure 5
Search for the functional targets of miR-429. (A) The microRNAs and their potential targets detected in a global expression assay. (B) The map illustrates that CRKL is probably a new genuine target of miR-429 in bone metastasis of breast cancer cells through cross-evaluating results from in silico search and expression array. (C) Immunoblot assay shows that overexpression of miR-429 in 231-B cells results in reduced expression of ZEB1 and CRKL.
Table I
The candidate targets of miR-429, which were verified to play a vital role in the development of certain cancer, were predicted in six algorithms.
Breast cancer morbidity ranks the first accounting for 29% in all the women suffering cancer, and the mortality ranks the second accounting for 14% among the women cancer victims in western countries (21,22). Although great progress has been made toward preventing and treating breast cancer with surgical operation, chemotherapy, and radiation therapy, metastasis of breast cancer still remains one grave clinical challenge due to the limits of early detection and effective treatment. Breast cancer is likely to form distant metastases in lung, bone and liver (23). A myriad of factors influence malignant mammary cells to colonize in bones (24,25). In this investigation, we set up an in vivo model to study how microRNAs regulate bone metastases of breast cancer cells.
We established a bone metastasis model using MDA-MB-231 cells, and further miRNA array analysis revealed the altered expression of more than 100 miRNAs between 231-P and 231-B cells. Among these miRNAs, several have already been found to get involved in invasion and metastasis of tumors. miR-663 could inhibit proliferation and invasion of glioblastoma in vitro and in vivo by directly targeting PIK3CD, thus, reducing the expression of three downstream effectors: CCND1, MMP2 and MMP7 (26). On the other hand, in castration-resistant prostate cancer cells, miR-663 acted as OncomiR, which could promote invasion of malignant cells. Moreover, this miRNA correlated with TNM stage and could be used as an independent prognostic marker in clinical recurrence (27). Besides, miR-486-5p, expression of which in 231-B cells was decreased by 70% compared with that of 231-P cells, was recently reported to inhibit metastasis of lung cancer by reducing the protein level of ARHGAP5 (28). Taken together, most array results from comparison between 231-P and 231-B cells were in line with previous literature and these differentially expressed miRNAs are probably implicated in bone metastasis of breast cancer.
miR-429 is a member of the miR-200 family, four members of this family have been found to play important roles in the regulation of EMT in various types of tumors (29). EMT is widely accepted as an essential mechanism to prompt malignant cells to invade and colonize distant organs (30). In the present study, we showed that downregulation of miR-429 could promote bone metastasis of breast cancer, which is in line with previously reported results in several types of cancers. Besides in breast cancer, overexpression of miR-429 suppressed invasion and metastasis of colorectal carcinoma and ovarian cancer cells, respectively (20,31).
miRNAs usually function through base-pairing specific sequences located in 3′-UTRs of targeted genes to hinder translation of mRNA or enhance degradation of mRNA (32). ZEB1 is the most well studied target of miR-429, since this transcription factor is an important EMT inducer (33). Besides ZEB1, several candidate targets of miR-429 were also identified essential for invasion and metastasis in cancer models. For example, Onecut2 was proved to be one bona fide target of miR-429 in colon cancer (20). Combined analysis of expression array results and in silico target search revealed CRKL as a common candidate. CRKL protein is a kinase harboring SH2 and SH3 domains. CRKL transformed fibroblasts and received signals from BCR-ABL tyrosine kinase (34–36). CRKL influences integrin mediated adhesion to fibronectin (37). For cellular motility, CRKL enhanced motion through forming a complex with Cbl and C3G to relay signals in hematopoietic Ba/F3 cells (38). In addition to promoting invasion of cells, CRKL also could suppress caspase-8 mediated apoptosis, which was also essential for distant metastasis (39). Another research group also found the involvement of CRKL in integrin-triggered cell migration, and they further identified that CRKL performed by acting as downstream effector of Src (40). Considering that CRKL is important in proliferation, migration and evading apoptosis, we speculate that CRKL is probably essential in bone metastasis of breast cancer, however this needs further investigation.
In conclusion, we established an in vivo bone metastasis model of breast cancer, and we obtained a wealth of information on how mammary malignant cells invade and finally form overt metastases in bones. Further research is urgently needed to clarify the complicated mechanisms underlying this fatal pathological condition to provide valuable prevention and treatment targets.
We appreciate our colleagues in the laboratory of Dr Zhihua Liu for helpful discussion, and Professor Burton Yang for his critical reading of this manuscript.
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How To Stop Seasonal Allergies Once & For All
It's that time of year again – the birds are chirping; the sun sets later in the day and everything is covered in pollen! What makes spring so beautiful can also be misery for those who have seasonal allergies (Note: not everyone has allergies – so there is hope for you to kick the allergies one for all)
Cut grass, blooming trees, flowers, weeds and pollen are the allergy trigger for an estimated 50-60 million Americans. Medically termed "allergic rhinitis," seasonal allergies don't just occur in the spring – it's any time an eternal derived foreign object (i.e. pollen, dander etc.) gets access to the inners of your body and activates an immune response. Yes – allergies have to do with your immune system so hopefully you can understand that there is an underlying cause (deficient or overactive immune system) which means there is a cure for allergies!
Having allergies can be challenging – mostly because many of the OTC and even prescriptive therapies are ineffective and chronic sneezing, itching eyes, congestion, headaches, sinus pressure, runny nose can become unbearable, even debilitating, over time.
A significant amount of research is suggesting there are natural solutions and even cures available to you – but you won't find them in a nasal spray or pill bottle/packet – but we can learn something from them. These medicines work by blocking histamine (thus why they are called "anti-histamines"). Histamine
Histamine is an organic nitrogenous compound secreted during immunological responses. It also regulates physiological function in the gut and acting as a neurotransmitter for the brain, spinal cord, and female uterus. Histamine is involved in the inflammatory response and has a central role as a reaction of itching.
As a hormone histamine is also suspect to "resistance" – a reaction where the body no longer reacts like it should to the hormone and the levels of that hormone increase. Similar to Insulin resistance being the cause of diabetes – It's possible Histamine Resistance is the reason for your current and prolonged seasonal allergies.
One quick way to know if you have entered histamine resistance is to simply ask "are you allergies to more than just nature?" If you have food sensitivities (IgG, IgE reactions to certain foods which result in noticeable symptoms), chemical sensitivities (headaches or reactions to colognes, perfumes, smells then there is a high probability you are now histamine resistant. This is important because with resistance comes higher levels of Histamine which triggers inflammation and overactivity to the immune/allergy response which is the underlying cause of seasonal allergies.
How to Fix Histamine Naturally. Alternative Allergy Techniques That Actually Work!
Your allergic response to anything (pollen, dogs/cats, virus/bacteria etc.) is a natural and normal reaction. When a foreign object comes in contact with the membranes of your nose, mouth, throat, lungs, stomach or intestines (thus why you can be allergic to foods) your body triggers the release of histamine – which the permeability of the capillaries allowing white blood cells and some proteins to exit the blood and engage pathogens in the infected tissues – i.e. go after the foreign object.
Antihistamines block this reaction, and while it's nice that you don't have a runny nose or itchy eyes, it's not a good idea to prevent while blood cells from exiting your blood to go after foreign objects! So, if you think about it, synthetic antihistamines actually weaken your immune system, prevent your white blood cells from going after bacteria and viruses! (now are you still willing to take your Allegra, Claritin, Nasonex etc.???
1.C2 (Axis) Alignment – Chiropractic
A 2004 study showed that chiropractic is a powerful way to lessen and prevent allergic reactions. In this study 1,028 people suffered from allergies (both seasonal and severe allergies). X-rays revealed 98% of these had severe misalignment of vertebra in their neck and upper back. Over 88% of participants saw significant reduction while 71% saw significant resolution of their allergies following chiropractic care. Your sinuses are controlled by the C1/C2 nerve roots, Lungs via T1/T2/T3/T4 nerve roots – which are the regions you should ensure are aligned properly if you wish to be allergy free!
2. Stinging Nettle:
Urtica dioica, often known as stinging nettle, or nettle leaf, is a herbaceous perennial flowering plant in the family Urticaceae that is a potent natural anti-histamine. One study showed 58% of participants found symptoms resolved when taking a freeze-dried stunning nettle supplement. Stinging nettle can be found online and at health food stores locally. Note: The study participants mentioned used 300 milligrams (mg) each day.
3. Quercetin
Quercetin is an antioxidant found naturally in onions and apples. Research has proven it to be another powerful natural antihistamine. One study found that it even lessened the respiratory side effects of allergies in rats by reducing inflammatory response in the airways. You can purchase quercetin as a supplement or simply add more quercetin-rich foods to your diet. Note: recommendation is 500mg daily for adults, 250mg daily for children. Quercetin can be taken as a standalone or in conjunction with the other items mentioned here, which is why most quercetin sold OTC usually comes with bromeliad or stinging nettle combined.
4. Bromelain
Bromelain is an enzyme most found in the core of pineapples. It's said to be a powerful substance for naturally treating various conditions (acid reflux, digestive distress, asthma, headaches) and is also a known anti-histamine. One study suggests taking between 400 to 500 mg three times daily to combat seasonal allergies. You can also simply eat pineapple and the central stem (which most people throw away!)
5. Vitamin C
Yes, good old Vitamin C is an easy-to-find natural antihistamine. It's prevalent naturally in citrus fruits and various vegetables, and also in supplement form (note: ascorbic acid is only one tiny portion of the entire vitamin c molecule – so it's best to consume a "raw C complex" when buying a supplement). Because it's free of side effects it's a safe solution for clearing stuffy noses and other unpleasant symptoms of seasonal allergies. Recommended dose for an adult is 3,000 mg (3g) daily, children 1,500 mg (1.5g) daily or simply 4-5 low glycemic citrus fruits daily (grapefruit, lemons and limes).
6. Essential Oils
Lemon, Lavender and Peppermint essential oils are exceptional and clearing your seasonal allergies. Place 5 drops of each in a size "00" veggie cap and fill the remainder with MCT or Coconut oil, consuming 1 capsule every 4-6 hours. These can also be diffused for direct inhalation. Note: when consuming essential oils, they must be food grade, therapeutic, investable oils (DoTerra or Youngliving)


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The Raven
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Ravens are so stately and this is a wonderful representation of this beautiful, larg bird. He is painted jet black (matte) with glossy eyes which provides the piece with a tone-on-tone contrast. Hand made from upcycled sheet metal and hand painted. Measures 17" x 15" x 8".


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Decision details
Complaints Annual Report 2018 - 2019
Decision Maker: Cabinet
Decision status: Recommendations Approved
Is Key decision?: No
Is subject to call in?: Yes
To note and agree the Complaints Annual Report 2018 – 2019.
RESOLVED that Cabinet note Brent'sperformance in managing and resolving complaints.
Wards Affected: (All Wards);
Contact: Thomas Cattermole, Head of Member Services 020 87937 5446 Email: email@example.com Tel: 020 8937 5446.
Report author: Thomas Cattermole
Publication date: 11/11/2019
Date of decision: 11/11/2019
Decided at meeting: 11/11/2019 - Cabinet
Effective from: 19/11/2019
Accompanying Documents:


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Global De-aromatic Solvents Market: Key Players
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Growing concerns regarding the hazards of using conventional solvents and subsequent increase in the demand for safer, effective, and eco-friendly alternatives will boost the demand for de-aromatic solvents during the period of forecast An increasing use of de-aromatic solvents in household insecticides and mosquito repellents is another growth driver.A booming paints, coatings, and inks market worldwide contributes to the growth in demand for de-aromatic solvents. Increasing demand from metal working applications likely to fuel market growth.
The prevalence of alternative products such as powder coatings and water based paints poses challenges to the growth in revenue of the de-aromatic solvents market.Growing use of bio-based offerings and other alternatives is a market growth inhibitor.
The application of specific product offerings and alternative raw materials is trending the global de-aromatic solvents market.Increasing use of higher flash point grades for specific applications is an emerging trend.Manufacturers of de-aromatic solvents are resorting to production capacity expansions to meet the growing demands in the global market.
Global De-aromatic Solvents Market: Segmental Forecast
By Application: The global de-aromatic solvents market is segmented on the basis of application into Paints, Coatings and Inks, Metal Working, Industrial Cleaning, Adhesives and Sealants, Drilling Fluids, Consumer Products, and Others. Paints, Coatings and Inks segment is the largest segment, with an estimated market revenue of US$ 621.2 Mn in 2017, likely to increase to US$ 962.9 Mn by the end of 2025, witnessing a CAGR of 5.6% in terms of value. Metal Working is the second largest segment with a CAGR of 5.1% in terms of value expected to be recorded during the forecast period.
By Flash Point: The global de-aromatic solvents market is segmented on the basis of flash point into Low Flash Point, Medium Flash Point, and High Flash Point. Medium Flash Point is the largest segment in terms of revenue, estimated to hold almost 50% share of the global market by 2025 end. This segment will also register the highest CAGR of 5.7% in terms of value in the flash point category.
By Boiling Point: The global de-aromatic solvents market is segmented on the basis of boiling point into Type 1 (2400C). Type 1 and Type 2 are the top two segments in this category, with Type 1 estimated to hold about 44% market share and Type 2 close to 43% market share by the end of 2025. However, in terms of CAGR, Type 3 is the leading segment with an anticipated growth rate of 5.9% in terms of value during the forecast period.
Global De-aromatic Solvents Market: Regional Forecast
Persistence Market Research tracks the performance of the global de-aromatic solvents market across the key geographies of Asia Pacific (China, India, South Korea, Japan, Rest of APAC), North America, Latin America, Europe, and MEA. Asia Pacific is the dominant regional market in the global de-aromatic solvents market followed by Europe and North America that stand neck to neck with each other. APAC is projected to hold about 40% share of the global de-aromatic solvents market by the end of the forecast period. APAC also leads the other regional markets in terms of CAGR, recording a significant growth rate of 6.3% in terms of value during 2017 – 2025.
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Global De-aromatic Solvents Market: Key Players
The report profiles some of the leading companies operating in the global de-aromatic solvents market such as ExxonMobil Corporation, Royal Dutch Shell Plc, Total S.A., Idemitsu Kosan Co., Ltd., Neste Oyj, Raj Petro Specialties P. Ltd., Avani Petrochem Private Limited, Neste Oyj., Isu Exachem Co. Ltd., DHC Solvent Chemie GmbH, Calumet Specialty Products Partners, L.P., SK Global Chemical Co., Ltd., and CEPSA.
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We all know what we have to do, but we don't always do it.
That's not just in health and fitness but in work, finance and relationships. Why? Because sometimes it's not the easiest thing to do and we can always find something else to do in its place.
We can do some things now and again, for example going to the gym or eating a healthy meal, which to be honest is simple, but becomes much harder when we have to do it consistently over and over again.
So how do we start instilling these basic habits into our daily routines? There are three parts to this:
The first step is being aware and honest with yourself to what changes you need to make.
Do you need to drink more water, sleep more at night or stick to a gym program?
Or is it a negative habit that is holding you back for example phone usage? This was – and at other times still is – a big behaviour change I needed to make. Over two years ago I was aware that I was spending too much time at night laying in my bed on my phone. We all know that's not healthy.
The action I took was to charge my phone outside my room. Thankfully this habit has stuck and even when I'm staying in a hotel I'll always charge it at the furthest charging point in the room.
My phone is my alarm and I still hear it when it's in the other room, so that excuse is out the window. And even if you think you won't hear the alarm on your phone, buy a cheap alarm clock and keep it in your room. You've no chance of scrolling through Instagram with it.
An extra bonus is that I now read a lot more. I'm filling my head with information which I choose to read and not what is forced upon me by social media.
Choose one thing
When you are really aware and honest with yourself you may come up with a few things you would like to change.
You may want to drink more water, eat three healthy meals a day, don't eat after 7pm, snack less, do three strength sessions a week, add in two cardio sessions, meditate in the morning, journal, go to bed earlier and wake up earlier.
The list could go on but just pick one of these.
One study showed that if you focus on one behaviour change you have an 85 per-cent chance of being successful.
If you try and change two behaviours at the same time the rate of success is 40-45 per-cent. If you focus on three things at once then it drops right down to 4-5 per-cent.
For example, if you want to change your eating habits you may start with not eating after 7pm. Rather than worrying about what and how much you eat, just focus on not eating after 7pm. Once you do this consistently over time then you can look at changing another behaviour.
Set a time frame
Once you have chosen your one habit it's time to set a number of days that you are going to commit to.
Some people suggest 30 days but I have found that it can be too short and after the 30 days the habit doesn't stick.
Others have suggested 66 days. It's an easy number to remember and a long enough period for your new behaviour to become automatic.
To help you to become more accountable invest in a calendar and circle off each day you complete your new habit.
Even post it on social media or text a friend each day. This means there is no hiding place and others are holding you accountable, increasing further your likelihood for success.
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ERSL/SOSA League Coaches Meeting May 2012 Agenda Introduction Communications East Region Cup Schedules Team Section Website Discipline Questions
Communication model Inbound Outbound Parents & Players Directors Coaches League Admin Club Rep
Coach (cc Club Rep & President) League Admin Director Parents & Players ERSL 10th Anniversary The ERSL is 10 years old this season and we will be commemorating this milestone with player badges and game balls for all teams. The badges will be made available to the teams at the
preseason meeting and can only be signed out by the team or a club representative for all teams. An email to Dawn ([email protected] ) informing me of who will be picking up for the club will be required. Pre season statistics 2012 2011 2010 2009 2008 2007 2006 2005 # of Clubs 23
23 24 26 34 30 -
- # of team applications 201 191 206 699
711 573 608 544 # of withdrawn teams
32 31 30 104 109 119
75 107 # of current teams 178 175 180
595 603 592 510 501 2
1 3 39 23 - -
- # of teams yet to activate Getting ready for the new season The ERSL operates extensively via the web site and email To prepare your team for the season you need to: Activate your account Ask up to 3 others to also activate their account Enter your team roster, DOB and OSA # Enter your staff that may be on the Bench (max 4)
Enter your players shirt #s Familiarize yourself with the web site tools Cup Overview The ER Cup East Region Cup Entry Procedure Every team in the ERSL is automatically entered into the ER Cup. Any team not wishing to enter the cup must email the ERSL ([email protected]) no later then May 15th at 5pm.
The random draw will take on Friday May 18th and will be posted on the website May 18th. An email will be required to be sent to [email protected] with any potential conflicts for tournament dates by May 15th. East Region Cup Schedule Final dates are fixed There is some flexibility for the earlier rounds First round will be during a week game when there are no regular league games scheduled. 2nd round teams will have 3 weeks to arrange the games Semi-final will be last weekend in August (August 25 th/26th)
Finals will be 3rd weekend in September (Sept 15th/16th) East Region Cup Finals The league office will organize the U17/18 cup finals Sunday August 19th 1 host will be sought for Cup finals Cup (U13 U16) Sunday September 16th Clubs who wish to host a final must submit an application by June 30 th 2012 Hosts will receive $500 plus expenses from the ERSL for hosting a final
Player Eligibility 5. A player may play for one (1) team in the ER Cup. Play up permits are permitted, but are restricted to players from the Level 4 division in the same age division or any level younger age division or a Club's house league. Managing your schedule Schedules Draft schedule will be published May 8th Please review your teams schedule and report any issues to [email protected]
Between May 8th and May 14th we will correct the draft schedule Final schedule released May 16 no changes possible after this date The rules for requesting changes to the schedule are covered in the next slides Changes will occur over the next week as problems are identified So continue to check your schedule Please pay special attention to game fields and game nights they may vary from your usual Season will start Monday May 21st Managing your schedule You are responsible for managing your schedule
Schedule Changes SOSA / EODSA Games Games between SOSA and EODSA teams will be left on the regular weekday game night Teams cannot refuse to move a SOSA / EODSA game to a weekend If Teams cannot play a SOSA / EODSA game on the weekend they contact [email protected] and the game will be moved to December 31 Teams have 2 week to set a new date for the game - the game must be played before Sept 9th 2012 Schedule Conflicts If the two teams cannot agree on a date e) If the teams cannot agree on a date within 14 days of the approval of the request to reschedule, the team
responsible for the reschedule shall report the problem by email to the ERSL and include emails documenting their attempts to reschedule. The ERSL will determine if the teams have made reasonable attempts to reschedule and the following action will be taken: i. Both teams made reasonable attempts - the ERSL will request three dates from the Home Team and request the Away Team to select one of the three; ii. Home Team made reasonable attempts - the ERSL will request one date from the Home Team and schedule the game; iii. Away Team made reasonable attempts - the ERSL will request one date from the Away Team at their home field and schedule the game; iv. Neither team made reasonable attempts - the ERSL will schedule the game and inform the teams providing a minimum of 7 days notice.
Rescheduling g) An Under 18 team may request to move games scheduled for after August 20th, due to the departure of players for college and university. These requests must be submitted within one week of the draft schedule being released. h) All teams may request to move weekday games scheduled between EODSA and SOSA teams for the entire season. These requests must be submitted within one week of the draft schedule being released. i) All teams may request to move weekday games scheduled before the fourth Friday in June where the one way travel time exceeds 75 minutes one way. These requests must be submitted within one week of the draft schedule being released. Travel time is calculated by using
http://maps.google.com and entering the Club's home address for the Start address and the address of the game field for the End address. Rescheduling cont j) A team is scheduled to participate in an ER Cup game 24 hours prior to a scheduled ERSL game and the said Team provides at least seven (7) days notice to the ERSL Administrator that it requests to have the game postponed. k) If Canada Day falls on a regular game day, all teams may request to reschedule the game. These requests must be submitted within one week of the draft schedule being released. 2012 July 1st is a Sunday so will only affect Under 21 teams.
Reschedule Requests See the Reschedule Policy in the About section for the complete details Each team is entitled to ONE reschedule The only exceptions to the ONE rule are in the Reschedule Policy which in summary is an Ontario Cup game or you have two or more National or Provincial players who will be absent Reschedules must be made via the web site tool, and must be made 6 complete days before the date of the game. A Game scheduled for Monday May 21, 2012 would require notification by midnight Monday, May 14, 2012. The tool only show games meeting the criteria Weekend games cannot be rescheduled. There is an opportunity prior to the season to
change weekend games. The only exception will be if teams wish to combine games on one weekend to lessen travel. In this case both teams must agree if it is after the season has begun. Do not make deals with your opponent. Your opponents views will have no outcome on the decision. Arranging a rescheduled game 4. Process for Setting the Rescheduled Game Date/Time a) The rescheduled game shall be rescheduled within 14 days of the approval of the request to reschedule and must be played before the division end-of-season date as defined
in the Bylaws. The last two (2) regularly scheduled games must be played before the originally scheduled game date. b) When a game is rescheduled for any reason the ERSL will appoint one of the teams as being responsible to complete the reschedule process. Under normal circumstances the following shall apply without the ERSL notifying the teams: i. Request to Reschedule Team Choice or Other Reasons - the team requesting the reschedule shall be responsible for rescheduling; or ii. Reschedule by the League - the Home team shall be responsible for rescheduling. c) The teams work together to agree on a date.
d) Once the game is agreed upon: i. the Home team then sends an email to the Away team with the details: game number, new game date, home team, away team, field, start time. ii. the Away team forwards the email to the league at [email protected] (cc the Home team). The ERSL now has confirmation from both teams that the date is acceptable. Note: If this process is not followed the game will not be scheduled/rescheduled. End of Season date The End of Season is Sunday September 9th, 2012 No games will be played after this date except for games postponed or abandoned within 7 days of the end of season date The outcome of any games un-played will be decided by the league
Team Section - Website Team Section Once you log in to the website as Team Staff there are many things you can do and need to do. Well cover the essential things. Game Sheets print game sheets Suspensions view suspensions Game Report complete game reports Playing Up Permits request / cancel / see status of PuPs Add to Squad add players to team Modify Squad modify player info
Delete Player delete a player Shirt Numbers update to show on gamesheets Player Privacy individually select the privacy level for a Player Modify Scorer modify the scorer for a game Modify Shutout modify who got the shutout Change Uniform change uniform colours Request Reschedule request a reschedule Maintain Team Data The majority of communication with Clubs and Teams is through email maintaining up to date contact information is essential 24.08 The e-mail shall be deemed to have been received by Teams:
a) May 1 to September 15 - 72 hours after transmittal of the e-mail b) September 16 to April 30 - no e-mails will be sent to teams other than for informational purposes Players and Staff
Players and Team Staff are entered on the website so they can be printed on the game sheet The ERSL is required to verify players and staff are eligible to play To be eligible to play, both players and staff must be registered with the OSA
Anyone listed on the game sheet is deemed to have played in the game It is your responsibility to ensure only registered players and staff are on the game sheet Play Ups Play Ups
Details on who is eligible are contained in the Rules and Regulations It is your responsibility to ensure that the player you request to play up meets the eligibility rules If you play a player that is ineligible it will result in a discipline hearing
Only players typed on the game sheet are eligible to play No additional PuP form is required as the Play Up is printed and identified on the Game Sheet The exception is for Temporary Registration Permits (playing from Club to
Club) and obtained from District Association House League Call-Ups House League players to be used as Call-Ups through the website Added by Club Administrator Once added they will be available for teams to request as call-ups Play Up Permits Play Up Permits House League selection
Play Up Permits The Coach clicks Submit and an email notification is sent to the Club Rep Play Up Permits The approval status is displayed for the Coach Play Up Permits Game sheet lists Play-Up players Call Up Permits
l) If a player is registered as both a competitive player and a recreational player, he/she may only be called up from the competitive team. m) If a player is registered to a team playing in an age division which is older then their true chronological age, they may be called up to a team in an age division equal to their true chronological age, provided that the team to which they are being called up plays at a higher level then the team to which the player is registered.
(As an example - A true U14 player is registered to a U15L5 team. That player may be called up to a team playing at U14L4, U14L3 and U14L1. In short, age correct, level up.) Game Sheets Game Sheets Before each game you have to create a set of game sheets These are only available via the web site The Game Sheet is the official document of the game so please ensure you complete the form accurately and completely, and make sure the referee also completes their section accurately Initial having done card check
ALL bench staff shall sign the game sheets If staff or players are not on the game sheet they cannot be on the bench If players or bench staff are absent, please cross them off the game sheet Give the game sheets to the referee at least 15 minutes before kick off Game Sheets Select the Players Select the players for the game sheet Note: the PuP player is shown If you do not require the PuP you will need to go to the PuP page and delete the PuP
DO NOT add PuP players to your roster Game Sheets Select the Staff Only 4 Staff can be on the game sheet and on the bench You can have more than 4 Staff sign up for your team Card Inspections Card Inspections
Card inspections are not optional and MUST be carried out before kick off If a player does not have their ID Card / Player Book they are ineligible to play SOSA teams with Player Books do not require Team Roster Reports (the Player Book shows the team) If an EODSA team does not have their Team Roster Report as well as their Player ID Cards, the team is ineligible to play Any issues, have the Referee mark them on the game sheet, make sure you get a copy at the end of the game and fax it to the ERSL office You cannot stop a player from playing, however, issues noted on the game sheet will be investigated by the ERSL and will lead to discipline as required Team Staff ID Cards
Team Staff ID Cards The OSA is now issuing ID Cards / Books for Team Staff The ERSL does not have a rule requiring the checking of Team Staff ID Cards / Books Clubs, not the ERSL, are responsible for selecting capable Team Staff for each game Game Sheets The game sheets have been modified to list the card offences The Referee will indicate on the game sheet the offence Hearing requests must be submitted within 48 hours but now the players
and coaches will know what the potential discipline is Any coaches ejected during a game also have 48 hours to ask for a Hearing do not return to the field. Game Reporting Following the game, collect your copy of the game sheet from the referee Use this report to enter the game report via the web site You must submit your report within 24 hours As soon as both coaches have submitted a report the standings are updated So if your result is missing dont contact the league contact your opponent
Game Reporting Rule In the past many have been negligent in reporting game results so there is an amended Rule 10.01 Each Coach shall be responsible for entering the game report, including the score, cards issued by the Referee as well as the Referee assessment on the ERSL website within twenty-four (24) hours of completion of the game. If the game report is not entered within seven (7) days, the Club shall be fined $50 for each occurrence and the ERSL will utilize the game sheet received from the Referee to complete the game report.
Game Feedback Game feedback from coaches will continue for 2012 The ERSL will monitor the feedback and assist Clubs in correcting inappropriate conduct
The ERSL will monitor the feedback and assist Districts in improving referee performance Clubs will be able to access feedback on their teams in the Club section Protests If you wish to play a game under protest you must inform the referee prior to the kick off and ask him to indicate this on the game sheet Following the game you must submit the protest fee within the time stated in the Bylaws (48 hours)
You will be notified if your protest is valid or not 20.06 No protests pertaining to the decision of the game official shall be entertained. 20.07 Objections to field conditions, goalposts, balls or team colours, shall not be considered as grounds for a protest. Coach Certification Coach Certification Coach Certification
In accordance with the OSA rules, all Team Head Coaches and Team Assistant Coaches, coaching teams that are playing in the ERSL are required to have completed the Community Senior coaching certification course. Coach Certification Coach Certification
In accordance with the OSA rules, all Team Head Coaches and Team Assistant Coaches, coaching teams that are playing in the ERSL are required to have completed the Community Senior coaching certification course. The Respect in Soccer certification is now part of the minimum OSA requirements to be a coach or assistant coach of a competitive team. The following link provides further information on coaching requirements. http://www.ontariosoccer.net/Coach/CoachingCoursesInOntario.aspx
There is now a tool available to help Clubs verify if a coach has the required Respect in Soccer certification. https://validation.respectgroupinc.com Discipline Discipline The OSA Discipline policies and procedures are used in the OSA There are 2 processes
Discipline by review (DBR) Discipline by hearing (DBH) Following a red card the player can request a hearing within 48 hours of the game, otherwise a discipline by review occurs The league may also request that the individual attend a hearing Coach Ejections Discipline
In previous years, the ERSL has always required coaches to come to a hearing Some of the OSA discipline rules covering a coachs ejection, permit DBR and do not require a hearing In 2012, the ERSL may elect DBR, where permitted
Therefore, if a Coach wants a hearing, it must be requested in accordance with the rules (within 48 hours of the game) Once DBR takes place, there is no appeal or ability to request a hearing Discipline by review (DBR) The ERSL Discipline Committee will review all red and yellow card reports and issue a suspension according to the OSA policy
The most common suspensions are shown on the web site You can not appeal the outcome of a DBR decision In 2012, if the OSA rules permit, the ERSL will use DBR is possible Discipline by hearing (DBH) Discipline hearings are held every week in Ottawa (if required) and every 2 weeks (if required) in Kingston At least 3 people will sit on the panel You have to pay a $50 fee to request a hearing. It is only returned if the individual is found not guilty You can appeal the outcome of a DBH decision
Suspensions The league will tell you when a player is suspended Do not sit out a player and then expect that to count in a suspension A coach will receive email notification of a suspension giving instructions of how he can retrieve the letter from the web site The game sheets will also indicate all suspended players on your team Where disciplinary action results is a suspension, the suspension shall not commence prior to seven (7) calendar days from the issue of the notice of discipline. For example, a player issued with a suspension notice on Friday would serve the suspension for the game(s) scheduled on or after the following Friday. 2012 ERSL Rule Changes
ERSL Rule Changes cont 9.8 Any Team that fails to produce a game sheet or its OSA Registrant Books or OSA ID Cards and OSA Team Roster Report will be subject to fine as per the published schedule of ERSL Fines. The team Officials may also be required to attend a Discipline Hearing. 9.8.1 A team that produces its game sheet or the OSA ID Cards for its players but
fails to present the required OSA Team Roster Report to permit the checks as per 9.1 will be subject to a fine as per the published schedule of ERSL Fines. The opposing team could accept to play the game under protest and this shall be noted on the game sheet. The team that failed to present the OSA Team Roster Report at the scheduled game shall have two business days to present the Team Roster Report and the player ID Cards for inspection during the regular business hours of its respective District Association or will be deemed to have forfeited the game. SOSA League Rule Changes U9 and U10 divisions there will be no standings posted on the website.
Questions Any questions? Adjournment Have a Great Season
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