text:
- HubPages»
- Health»
- Mental Health
Catch Yourself Before You Fall Into An Addiction
How many people do you know that have fallen into an addiction? They don't get addicted because they are out for a death wish I can tell that much. I had fallen into an alcohol addiction myself, but over many years of drinking alcohol I had fallen and caught myself and picked myself up.
Catch yourself before you fall into an addiction. These addictions whether be drugs, alcohol, hoarding, sex, eating and many more things that people get addicted to are controllable if you have the strength to walk away and not let those demons inside you take over your life.
I was one of those people that just didn't have the strength to say no and walk away from the demons that controlled my life. I admit, I was weak and didn't really care what happened to me, but as I didn't know, I wasn't only ruining my life I was ruining the ones that loved and cared for me, my family and wife.
The way to catch yourself before you fall into an addiction is to don't start doing drugs or drinking alcohol to start with. Yes, I know, you may think I am talking crazy and how can we predict if we will get addicted to any type of drug when first starting our experimenting with drinking or using.
Don't get me wrong I am not anti-drinking because I loved to drink and loved how it made me feel, but the point is I didn't know when to stop. I didn't know when enough was enough. I didn't know how to catch myself before I became fully addicted to alcohol. I'm sure any alcoholic, such as myself, the first thing we think of when we open our blood shot eyes in the morning is what time is it and when should I start drinking alcohol again?
Now being clean and sober and looking back on those dim days of my life I can not believe I let those demons control my life like they did and how they control millions of other people all over the world the same way.
It is pitiful and sad that this liquid can destroy a humans life and everything that belongs to us humans, including spouses, families and friends.
We must be stronger than our addiction. Many of us will learn the hard way and in some case it will unfortunately be death by addiction. A overdose, drinking so much that you get alcohol poisoning and losing our good health, our looks, our families and friends all for the love of alcohol.
Think about it as I did. Is it really worth all of that because we are just pain weak and can't stand up to our demons and shut them down in their tracks. Learn the word "NO." Learn to live without your crutch.
Remember God gave all of us only one life, so we should use this beautiful gift of life to the fullest and not destroy it with drugs and alcohol. If you are addicted to anything, think about giving your only life a second chance.
Wise words, Marky.
Thank you for sharing! I am always happy to hear these success stories...but if you don't mind me asking, what do you think started your addiction? Why couldn't you easily stop? Did you just not see a better life for yourself?
Hi Mark, thanks for sharing this hub and your strength with us. Life is so precious.
Thanks for the motivations. I've been trying to stop smoking for a while now, but learning to say no is the hardest part. You're hub gives me much hope. God Bless
Being from an alcoholic family I relate very well, it is very destructive to oneself, and also everyone around you. Fortunately it is a death wish, and even to others around you when you don't value their lives by driving them in a car impaired, or many other scenarios with guns, drugs, etc. Thanks for writing this hub! Posted on my facebook! :)
I believe one of the factors that contribute to people becoming addicted to a substance or behavior has to do with the common belief that as an individual, we are stronger than those "others" who become addicted.


text:
14-Nov-2013 Palo Alto, CA- Marilyn Ehrhardt, CEO and President of Sabianet, was invited to speak at the Hispanic IT Executive Council's Q4 IT Leadership Summit. She was joined by other industry leaders in Healthcare Information Technology and Telecommunications: Romulo Juarez, Managing Director, Technology Growth Platform Lead for Connected Health, Accenture; Juliana Su, Chief Operations Officer, Openet; Aloha McBride, Federal Health Sector Lead, Ernst & Young LLP. Ehrhardt spoke with insight and passion on the empowerment of consumers through usage of technology in the Healthcare Industry citing two real-life examples that brought home the message that Information technology combined with Telecommunications technology reduces the cost of healthcare but even more critical--it saves lives! Given the widespread concerns on healthcare costs, access to healthcare professionals and Insurance, Marilyn Ehrhardt emphasized that we are only scratching the surface on how technology can provide solutions with impact to these concerns and clearly stated that empowering individuals to participate in their healthcare via Technology is the key.
One of the cases Ehrhardt spoke of demonstrated a clear pathway to cost reduction through empowerment of caregivers of chronic care patients utilizing health information technology and a smartphone. The patient, having chronic hypertension combined with depression had a prescription medication multi-drug interaction that led to a heart attack and congestive heart failure. After successful treatment of these events, the caretaker took simple steps to establish a Personal Health Record for the patient that could be accessed via Smartphone. This single up-to-date view of the patient's current health conditions aided all providers to remain on the same page, having a clearer, fuller picture of the patient. The smartphone, which is always with the patient, is a unique delivery platform for access to this critical information and for keeping it up to date. This chronic patient, after having costly initial encounters with the healthcare system has managed to avoid having any other costly medical events due to the simple use of technology. Still, there is much to be improved, stressed Ehrhardt, and the major challenge for IT professionals in the Healthcare Industry is to create a consumer experience that is simple and easy to use by ALL stakeholders in the system. Additionally, with today's vast collection of structured and unstructured data, IT Professionals must begin to create integrated platforms that can ingest and integrate volumes of data from Big Data with traditional structured data to produce contextual snapshots of what is needed by a patient, in his current condition, in his current location, with pertinent history that can be easily delivered to healthcare professionals.
The other case Ehrhardt described showed how healthcare can be administered to save lives using technology in a mobile setting. She describes an event that occurred on a transatlantic flight where a person on the plane was experiencing chest pains. A physician, seated nearby, had a smartphone that had a prototype device/application that was capable of producing a real-time electrocardiogram (EKG or ECG, similar to the now available product from AliveCor). The physician was able to diagnose the patient with acute STEMI, a critical condition of the heart. With this information, the pilot conducted an emergency landing and the patient's life was saved. With technology, we are empowering the healthcare community, patients and providers with the ability to diagnose and treat health issues outside of traditional venues like the physician's office or the emergency room. Healthcare is becoming manageable everywhere.
Healthcare and personal health awareness is becoming manageable on a personal 24 hour clock with new devices being introduced frequently. Devices available today, such as those provided by Fitbit and devices or sensors available in the future that are embedded in clothing will monitor and collect your body's vital statistics. These small bits of data will be accumulated to Big Data stores in the cloud for trends and analysis. This is not only valuable from a personal perspective, but this data can be de-identified, aggregated across a population and studied for health trends, locational outbreaks and other even now unidentified requirements for the improvement of healthcare, medicines, treatments and outcomes.
Sabianet is a strategic transformation and information technology firm servicing the Healthcare and Telecommunications industries. We help hospitals, medical centers, clinics, and physicians transform their information systems to improve information capture, include mobility, collaboration, and security, resulting in improved quality, cost and access. More at www.sabianet.com. Sabianet does not endorse or recommend any products mentioned in this article and the citing of same is for illustrative purposes only.
Sabianet offers a range of exciting job oportunities. Take a look at our current openings and apply today.

subset: commoncrawl


text:
Download Fancy Walking Smiley apk 2.0 free for Android smartphone
Posted by Aerotech Solutions in Social | June 28, 2014 | 1 Comments
Apk file size: 1.5 MB
This is a beautiful app which enables you to see fancy smileys everywhere, no matter on which application or screen you are!
A 3d animated fancy smiley walks on your screen globally. It will surely bright up your eyes and minds every day!
You can also select any custom picture as walking smiley.
Download this application and see magic and impress your friends!
Whats new
Some update
Aerotech Solutions part of our Social and have average installs from 5000 to 10000. Last Update June 28, 2014. Google play rating is 74.1538. Current verison is 2.0. Actual size 1.5 MB.
Similar applications
1 Comments
Kandace Park
Love this. Thank you very much


text:
Aedon Charles Green
- 2 years old
- Date of birth: Jan 13, 2012
- Date of passing: Apr 2, 2014
|Let the memory of Aedon be with us forever
This memorial website was created in memory of our loved one, Aedon Green, 2, born on January 13, 2012 and passed away on April 2, 2014. We will remember him forever.
Memorial Tributes
"I miss you bubba. I love you so much."
"I love you son and there isn't a moment in any day that I don't miss your presence and your beautiful light."
Leave a Tribute:
Invite your family and friends
to visit this memorial:
to visit this memorial:
Subscribe to receive e-mail notifications when others contribute to this memorial.
708 views
Have a suggestion for us?


text:
A NEW website has been launched giving mums-to-be information about maternity services on offer in Eastbourne. The website, www.esht.nhs.uk/maternity, includes virtual tours of the DGH's maternity unit and similar departments in East Sussex to help mums-to-be decide where they would like to give birth.
There is also a wide range of information on subjects like what happens prior to labour, when to go to hospital and when to call a midwife if giving birth at home. The website also contains sections on pregnancy scanning, schedules of antenatal appointments and a downloadable antenatal class booking form.
Debra Young, head of midwifery at the trust, said, "Many families have access to the internet so this is a chance to provide information and ensure women are able to make an informed choice about maternity care."


text:
SUZHOU, China--(EON: Enhanced Online News)--Suzhou-based MabSpace Biosciences announced that the preclinical data for its lead program MSB2311, a 2nd generation humanized PD-L1 blocking antibody with a unique pH-dependent antigen binding property, will be presented at the upcoming 2017 Inaugural American Society of Clinical Oncology (ASCO)-Society for Immunotherapy of Cancer (SITC) Annual Meeting on February 23rd-25th, 2017 in Orlando, Florida, US. The data provide a body of evidence that MSB2311 has significantly higher activities in inducing complete responses in humanized syngeneic tumor models than existing PD-L1 antibodies on the market at both low and high doses. MSB2311 has the potential of being a best-in-class PD-L1 antibody in achieving better efficacy and safety profile with lower doses required.
"Developing well-differentiated, lifesaving and highly affordable therapeutics is our mission. Using our proprietary immune tolerance breaking technology we generated a novel antibody with pH-dependent PD-L1 binding that can recycle within the tumor"
MabSpace is scheduled to present a poster on Thursday, February 23rd with the title of "Characterization of a Humanized PD-L1 Blocking Antibody with pH-Dependent Antigen Binding in a Humanized Syngeneic Tumor Model" by Qian et al., Abstract 177907, Poster #81, Board C10, 11:30-1:00 PM & 5:30PM-6:30 PM.
"Developing well-differentiated, lifesaving and highly affordable therapeutics is our mission. Using our proprietary immune tolerance breaking technology we generated a novel antibody with pH-dependent PD-L1 binding that can recycle within the tumor," commented Xueming Qian, Ph.D., Founder, Chairman and CEO, MabSpace Biosciences. "This differentiated property of MSB2311 presents an opportunity to provide cancer patients with an agent of better efficacy and safety profile than existing PD-L1 antibodies yet highly affordable. We are honored to be selected for presentation and look forward to sharing our data at this inaugural ASCO-SITC meeting."
MabSpace plans to start FIH study for MSB2311 later this year. MSB2311 is the first therapeutic antibody in its pipeline which consists of a panel of antibodies targeting various components of the tumor microenvironment, including both antagonistic and agonist antibodies. MabSpace plans to move two antibody programs to IND filing each year. These agents will allow MabSpace to develop its proprietary combinations for the treatment of cancer. MabSpace will focus on the development in China and find partners for ex-China development of these agents.
MabSpace Biosciences is a biotechnology company focused on the discovery and development of antibody therapeutics for oncology and fibrotic disorders leveraging its immune tolerance breaking technology (IMBT)-based antibody discovery platform and translational sciences capability. This technology enables the generation of antibodies with diverse target binding epitopes and well differentiated properties. MabSpace is headquartered in BioBay, Suzhou Industrial Park, China and has an R&D facility with fully-integrated in vitro and in vivo pharmacology profiling capabilities. Visit http://www.mabspacebio.com for more information.


text:
Sunday's post was about a miracle I experienced.
What has been bugging me lately is how I freely write about miracles but I don't write posts about God. I needed help. So I put a survey up for a week and asked that people email me with what their conception of God is. I appreciate all who participated.
I realized in the process why God is a tricky subject for me. I have my convictions. But before I experienced God, I was deaf to the people who promoted their beliefs. It seemed the more they pushed their agendas, the deeper I sank in agnosticism.
I don't want to construct an argument on the subject. Arguing does not have a place in my spiritual practice. It was my favorite Pantheist Walt Whitman who wrote this in the introduction to Leaves of Grass:
Argue not concerning God."
I do want to show people that belief in and of itself can become a powerful tool in your life.
Here are the results of the survey. The 5 options summarized the conceptions of God I have heard most frequently in recovery. The sample size was 22 responses.
I wanted my first post about God not to be just what I thought about God. So here are 8 people across 4 countries who have found faith. I placed my thoughts at the bottom. I hope you find it useful in your journey.
The survey was limited, as Colin was quick to point out!
Colin Chatburn
the short answer does not explain all. i think taoist (the way) some think you can't define. basically a mix.
Abbie Wirick
(Abbie blogs: click here / Abbie's FB page: click here)
I believe in God, as in Jesus Christ, the Holy One.
It took me many years before I was able to enter a church building for any reason other than a meeting.
The people in AA told me that I only had to change one thing: everything. I took that to mean I needed to reexamine my concept of God, too.
I had been taught about the angry and terrifying God that hated me for my humanness.
I eventually came to believe that my continued interest in having a relationship with Him was a result of His drawing me to Himself. I researched for myself, rather than listening to other people telling me Who and What God was.
I'm still looking and praying. I had an experience when I was a teenager that has never left me, even after my determination to be as "evil" as I could be (because I was clearly not capable of being "good") for many years.
I was involved in an event that lasted for years where the Spirit of God moved and changed people's lives forever.
I cannot deny what I know to be true.
At the same time, I don't talk about my convictions/experience much, because I know I would never have stayed in the rooms if they talked about Jesus, cos I'd been severely abused in His name.
I cannot think of AA and Jesus without being reminded of my Dad.
Once he went into treatment, he traded the spirit of RELIGION for the Spirit of Christ.
He became the man he'd never thought he COULD be, after working the steps. Dad was a step-Nazi, but more importantly, he found his ministry. In keeping with the traditions, he did not talk about Jesus in the meetings, but before and after… 🙂
His Sponsees were introduced to that One who has all power…
Dad was such a fun-loving, charismatic guy, that he had many men asking him to Sponsor them.
A Higher Power Poem
by the man who wrote the fictional step series: Destiny Awaits
join the author's Obsession Challenge here
Omnipresent & undetectable
The underlying current that gently pulls us, the force which attracts and repels.
Like vapour trails in the sky, propelling yet dissipating, it makes us go here and work there.
It works without credit, unbeknown, selflessly steering, never wrong.
Juanita Johnson
From reading the Bible to singing hymns, I just believe God is. He is the Great I Am. He is the Creator of all things. He is my heavenly Father. He is love. He is every good and perfect gift. I tear up to think that He loves all of us so much that He provided a Savior that we might be made whole again. Find and read the lyrics to "Who Am I?" I have always believed the Bible is truth and that is reassuring in every way.
Read HD's story here via the Recovery Revolution
I believe in God. And I call this being, force, power, or whatever it/he/she is, "God." Something out there is much bigger than I am and created all the wonder of this world. FAITH is, by definition, a mystery. I don't need the answers, but I believe it.
Girl in Therapy (A Recovering Codependent)
Her website is here
My hope and sometime mantra is that "The Universe Will Provide".
I think of my Higher Power in the larger sense of the Universe. I'm agnostic, so my view of "God" doesn't lie in religion.
Mark Goodson
A personal relationship with God is the single difference between how I lived my life before, and how I live my life now. I can also write with confidence that not having that relationship was miserable, and having that relationship is joy. So I know that God is good and God wants me to be happy.
I call my God Jesus Christ. I go to a Baptist worship every Sunday when we are in town. Although, I don't consider myself Baptist. I'm a believer who needs some good orderly direction every day. Church helps me with that.
I stopped doubting that my higher power is Jesus Christ when I had a vivid dream. I was in a cave and looking for Christ's body to prove it never resurrected. No luck. Then I felt a warm sensation and realized there was a light behind me, the way you do when someone uses flash photography behind you. When I turned around I was awake, bolt-upright in bed.
I know that no one could convince me of God. I had to be physically persuaded by a moving spirit. So I do not claim to have the answer. As HD wrote, God is by definition a mystery. And mysteries are no longer mysteries once they are solved.
When I hear someone believes in Krishna or Buddha or Yahweh or Muhammad or Fellowship or Nature, I believe them. To me, people don't need to say "Jesus Christ" to act for or believe in the name of God.


text:
Stem cells have a potential of gene therapy for regenerative medicine. Among various stem cells, spermatogonial stem cells have a unique characteristic in which neighboring cells can be connected by intercellular bridges. However, the roles of intercellular bridges for stem cell self-renewal, differentiation, and proliferation remain to be elucidated. Here, we show not only the characteristics of testis-expressed gene 14 (TEX14) null spermatogonial stem cells lacking intercellular bridges but also a trial application of genetic correction of a mutation in spermatogonial stem cells as a model for future gene therapy. In TEX14 null testes, some genes important for undifferentiated spermatogonia as well as some differentiation-related genes were activated. TEX14 null spermatogonial stem cells, surprisingly, could form chain-like structures even though they do not form stable intercellular bridges. TEX14 null spermatogonial stem cells in culture possessed both characteristics of undifferentiated and differentiated spermatogonia. Long-term culture of TEX14 null spermatogonial stem cells could not be established likely secondary to up-regulation of CDK4 inhibitors and down-regulation of cyclin E. These results suggest that intercellular bridges are essential for both maintenance of spermatogonial stem cells and their proliferation. Lastly, a mutation in Tex14+/− spermatogonial stem cells was successfully replaced by homologous recombination in vitro. Our study provides a therapeutic potential of spermatogonial stem cells for reproductive medicine if they can be cultured long-term.
Citation: Iwamori N, Iwamori T, Matzuk MM (2012) Characterization of Spermatogonial Stem Cells Lacking Intercellular Bridges and Genetic Replacement of a Mutation in Spermatogonial Stem Cells. PLoS ONE 7(6): e38914. https://doi.org/10.1371/journal.pone.0038914
Editor: Shree Ram Singh, National Cancer Institute, United States of America
Received: February 9, 2012; Accepted: May 15, 2012; Published: June 13, 2012
Copyright: © 2012 Iwamori et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: These studies were supported in part by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01HD057880). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Stem cells are defined as cells that possess both self-renewal activity and differentiation potential. Because of these characteristics, stem cells are expected to be therapeutic targets for regenerative medicine and can be used in the future for the correction of genetic defects [1], [2]. Once the mutation is repaired, either the stem cells or differentiated cells could be transferred to the individual to reconstitute or correct the damaged cells or organs. At present, there have been several reported trials utilizing stem cells as a means of regenerative therapy[3]–[5]. One problem for the utilization of stem cells for regenerative medicine is the difficulty of culture and expansion of stem cells in vitro. Indeed, stem cells used in the regenerative therapies described above are embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), for which efficient culture systems have been established[3]–[5]. Among the various stem cells, spermatogonial stem cells (SSCs) are the only stem cells derived from an adult animal and for which both long-term culture systems and transplantation techniques have been developed [6]–[9].
SSCs arise from gonocytes during postnatal development of the testis, possess self-renewal potential, and allow spermatogenesis to continue throughout adult life [10], [11]. SSCs can be isolated from both postnatal and adult testes and be maintained long-term in vitro in an undifferentiated state in the presence of glial cell line derived neurotrophic factor (GDNF) [7]–[9]. SSCs can reconstitute spermatogenesis when they are transplanted into germ cell-depleted testes [6]. A number of genes, including cell surface markers, transcription factors, and other proteins, have been identified as markers of undifferentiated spermatogonia, and some of these proteins are essential for self-renewal of SSC, although the molecular mechanisms regulating SSCs still remain elusive [12]–[27].
During spermatogenesis, a unique structure called the intercellular bridge is observed [28], [29]. As germ cells begin the process of differentiation, they are connected with neighboring sister germ cells through the intercellular bridge. We previously identified testis-expressed gene 14 (TEX14) as an essential component in the intercellular bridges during spermatogenesis [30]–[32]. When we produced a knockout of TEX14, spermatogenesis was arrested and only spermatogonia remained even in 1-year-old testes [30]. Moreover, most PLZF-positive spermatogonia existed as single cells in TEX14 null testes, suggesting that TEX14 null SSCs were at least maintained even in aged testes. However, whether SSCs without intercellular bridge can self-renew, differentiate, and proliferate remains unclear.
Herein, we analyzed the characteristics of testes without intercellular bridges by examining the expression of SSC marker genes, established long-term cultures of SSC with a mutation in Tex14, and showed that we could correct a genetic defect in Tex14+/− spermatogonia by homologous recombination to reveal the therapeutic potential of SSCs for regenerative medicine.
Materials and Methods
Animals and Cell culture
TEX14 heterozygous mice [30], which were C57BL/6J: 129S5/SvEvBrd hybrid background, were crossed to actin-GFP transgenic mice. Tex14+/− and GFP double-mutant mice were backcrossed to DBA strain for at least two generations before they were intercrossed. All mouse experiments were performed in accordance with protocols approved by the Institutional Care and Use Committee of Baylor College of Medicine.
Relative expression patterns of the indicated genes in Tex14+/− (blue) and Tex14−/− (red) testis at 8 weeks of age (A) and in CD9-positive Tex14+/− (blue) and Tex14−/− (red) spermatogonia (B) were quantitatively analyzed. All of the expression levels were normalized to Gapdh expression. * indicates that there is significant difference (P<0.05).
Establishment of spermatogonial stem cell lines was performed as previously described [7]. Briefly, testes and tails were collected from GFP-positive pups at postnatal 7 days and processed for establishment of spermatogonial stem cell lines and genotyping, respectively. After removal of the tunica, isolated testes were treated with a two-step enzymatic digestion with collagenase and trypsin [33]. Testicular cells were suspended in SSC media supplemented with 10 ng/ml rat glial cell derived neurotrophic factor (GDNF; R&D systems), 20 ng/ml mouse epidermal growth factor (EGF; eBiosciences), 10 ng/ml human basic fibroblast growth factor (bFGF; eBiosciences), and 103 U/ml mouse leukemia inhibitory factor (LIF; Millipore) and seeded onto gelatinized dish. After seven days of culture, SSCs were collected by gentle pipetting and seeded onto inactivated mouse embryonic fibroblast (MEF) feeder layers. SSCs were cultured for at least one month before use in additional experiments.
Magnetic Activated Cell Sorting
Testicular cells prepared as described above were incubated with rat anti-CD9 antibodies (BD Biosciences) on ice for 30 min, followed with anti rat IgG microbeads (Miltenyi Biotec) on ice for 20 min. CD9-positive cells were separated by MACS cell separation system (Miltenyi Biotec) according to the manufacturer's instructions.
A. Detection of intercellular bridge proteins in Tex14+/− and Tex14−/− testis. Immunofluorescence images of CEP55 (red) and TEX14 (green) and nucleus (DAPI) in 8-week old Tex14+/− (+/−) and Tex14−/− (KO) testis are shown. Insets indicate higher magnification images. B. Detection of intercellular bridges in short term culture of Tex14+/− and Tex14−/− spermatogonia. GFP (green) positive spermatognia were cultured and stained with TEX14 antibody (red) at second passage. Arrows indicate TEX14 positive intercellular bridges. C. Cytoskelton of short-term culture of Tex14+/− and Tex14−/− spermatogonia. Immunofluorescence images of TUBA (alpha-tubulin: green) and ZBTB16 (red) in Tex14+/− (+/−) and Tex14−/− (KO) spermatogonia are shown. D. Morphology of established spermatogonial stem cells in culture. Typical morphologies of cultured wildtype and Tex14+/− SSCs at 3 months of culture are shown.
Relative expressions of SSC markers (A) and cell cycle regulators (B) in Tex14+/− (blue) and Tex14−/− (red) SSCs were quantitatively analyzed. All of the expression levels were normalized to Gapdh expression. * indicates that there is significant difference (P<0.05).
Quantitative RT-PCR
Total RNA from testes and SSCs were extracted using the RNA mini and micro kit (QIAGEN), respectively, according to the manufacturer's instructions and reverse transcribed using Superscript III reverse transcriptase (Invitrogen) and an oligo-dT primer (Invitrogen). PCR amplifications were performed using SYBR Green PCR Master Mix (ABI) and analyzed by the ABI 7500 sequence detection system. The primers used in the experiments were shown in Table 1.
For visualization of intercellular bridges, cells were fixed with 2% paraformaldehyde for 10 min and permeabilized with 0.5% NP-40 for 15 min. After incubation with goat anti-TEX14 antibodies [30] at 4°C overnight, intercellular bridges were visualized by Alexa 594 conjugated anti-goat IgG (Invitrogen). For immunofluorescence of SSCs in culture, fixed and permeabilized cells were incubated with primary antibodies at 4°C overnight, followed by Alexa 488 and Alexa 564 conjugated secondary antibodies (Invitrogen). Following primary antibodies were used; rabbit anti-ZBTB16 (PLZF; Santa Cruz Biotechnology), mouse anti-ZBTB16 (PLZF; Calbiochem), mouse anti-TUBA (alpha-tubulin; Santa Cruz Biotechnology), rabbit anti-ARF (Abcam), mouse anti-INK4A (p16; Santa Cruz Biotechnology), mouse anti-CDKN1A (p21; Santa Cruz Biotech.), and mouse anti-CCNE (cyclin E; Santa Cruz Biotech.) antibodies. For immunofluorescence of tissue sections, paraformaldehyde fixed sections were retrieved by microwave, and then incubated with goat anti-TEX14 [30] and guinea pig anti-CEP55 [32] antibodies overnight at 4°C, followed by Alexa 488 and Alexa 594 conjugated secondary antibodies (Invitrogen) for 1 hour at room temperature. Fluorescent sections were mounted with VECTASHIELD containing DAPI (VECTOR Laboratories).
Generation of a Genetic Rescue Construct and Homologous Recombination in SSCs
A rescue construct was generated as follows: 8.3 kb of the 129S7/SvEvBrd-Hprtb-m2 genomic region containing exons 9 and 10 of the mouse Tex14 gene [34] was inserted into pBluescript SK containing diphtheria toxin A for negative selection (pDTA.3 kindly provided by Dr. Pumin Zhang, Baylor College of Medicine) and a loxP-flanked Pgk1-Neo cassette was inserted 2 kb downstream of exon 10 of Tex14. The linearized rescue construct was electroporated into Tex14+/− SSCs. SSC clones were selected in SSC media supplemented with G418 and 6-thioguanine. Targeted clones were screened by genomic PCR using primers shown in Table 1.
Protein expression of cell cycle regulators in Tex14+/− and Tex14−/− SSCs were analyzed by immunoblot (A) and immunostaining (B). Immunofluorescence images (B) of indicated cell cycle regulators (green) and ZBTB16 (red) in Tex14+/− (+/−) and Tex14−/− (KO) SSCs are shown.
A. Strategy of gene replacement of the Tex14 mutation. The Pgk-Hprt, which was inserted into exon 10 of the Tex14 gene, was replaced with exon 10 of Tex14 and a loxP flanked Pgk-Neo cassette. Locations of primers used for genotyping and predicted size of the PCR products are shown. B and C. Genotyping of the Tex14 mutation. Mutation of the Tex14 gene was confirmed by genomic PCR using primer pairs F1 and R1, F2 and R2 (B), and F1 and F2 and R2 (C). D. Genotyping of the replaced allele of Tex14 in Tex14+/− SSCs. Genomic PCR using indicated primer pairs and conditions are shown. Lane 1: Tex14+/− SSCs, Lane 2: unreplaced SSC clone, Lanes 3–5: replaced SSC clone. E. Long range genomic PCR to confirm homologous recombination. Genomic PCR using indicated primer pairs are shown.
Molecular Analysis of SSC Markers in Tex14 Null Testes
Expression patterns of genes, known to be transcriptionally active in spermatogonial stem cells, were analyzed in TEX14 null testes, because characteristics of Tex14−/− spermatogonial stem cells, such as self-renewal activity, differentiation potential, and proliferative activity, remained elusive. For the expression of cell surface markers of spermatogonial stem cells, Cd9, Gfra1, and Ret were significantly increased in Tex14−/− testes, although Epcam was decreased (Fig. 1A). Up-regulation of Gfra1 and Ret suggests enrichment of undifferentiated spermatogonia in Tex14−/− testes, since Gfra1 and Ret are receptors for GDNF, which is an essential growth factor for SSCs. For the expression of intercellular markers of spermatogonial stem cells, Zbtb16 (also called Plzf), Stra8, and Taf4b were increased in Tex14−/− testes, whereas Bcl6b, Id4, and Nanos2 were decreased in Tex14−/− testes (Fig. 1A). Enhanced expression of the three former genes suggests that undifferentiated spermatogonia might be enriched in Tex14−/− testes. Suppressed expression of the latter three genes, however, suggests that the ratio of undifferentiated spermatogonia to differentiated spermatogonia remaining in Tex14−/− testes might not be very high, or that spermatogonia remaining Tex14−/− testes might have both differentiated and undifferentiated characteristics even if they were existed as single spermatogonia. Expression of Ddx4 (also known as Mvh) was extensively lower in Tex14+/− testes, probably because Ddx4 is expressed not only in spermatogonia but also in later stage germ cells, and there were only spermatogonia enriched in Tex14−/− testes (Fig. 1A).
Next, spermatogonia were enriched from whole testis using CD9 as a marker of undifferentiated spermatogonia. When expression of cell surface markers was analyzed, Epcam was significantly lower in Tex14−/− spermatogonia, and Gfra1 and Ret were significantly higher (Fig. 1B). However, differences of expression of those genes between Tex14+/− and Tex14−/− spermatogonia were decreased. For the expression of intracellular markers, expression of Zbtb16, Bcl6b, and Nanos2 were not significantly changed between Tex14+/− and Tex14−/− spermatogonia (Fig. 1B). Although Id4 expression was significantly lower in Tex14−/− spermatogonia, the difference between Tex14+/− and Tex14−/− spermatogonia was decreased (Fig. 1B). When differentiation related markers were analyzed, Neurog3 expression was higher in Tex14−/− spermatogonia, but Kit expression in Tex14−/− spermatogonia was as high as Tex14+/− spermatogonia (Fig. 1B). These results for the expression profiles in enriched spermatogonia suggest that spermatogonia lacking intercellular bridge might not be very different from normal spermatogonia and that CD9-positive Tex14−/− spermatogonia might have both undifferentiated and differentiated characteristics.
Morphological Analysis of Tex14 Heterozygous and Homozygous Mutant SSCs in Culture
The disappearance of intercellular bridges in Tex14−/− testes was previously confirmed by immunostaining of TEX14 and CEP55, both of which are essential components for intercellular bridges to be stabilized [32]. There is no clear localization of both TEX14 and CEP55 in Tex14−/− testes, indicating that no stable intercellular bridges form in Tex14−/− testes (Fig. 2A).
Before establishment of long-term cultures of spermatogonial stem cells, characteristics of spermatogonia without intercellular bridges were analyzed in short-term cultures of Tex14−/− spermatogonia. Surprisingly, Tex14−/− spermatogonia could form chain-like structure, even though no intercellular bridges formed (Fig. 2B). Tex14−/− spermatogonia could also form cluster-type colonies, although the size of these clusters was small and the number of cells in these clusters was less than ten (data not shown). When intercellular bridges were visualized in isolated Tex14+/− and Tex14−/− spermatogonia using TEX14 staining, clear TEX14-positive intercellular bridges were detected in both chains and clusters of Tex14+/− colonies but not in Tex14−/− colonies (Fig. 2B). To confirm lack of intercellular bridges in Tex14−/− spermatogonia, alpha-tubulin (TUBA) in cultured spermatogonia was determined. TUBA expression in Tex14+/− spermatogonia was observed to be stretched and showed that cells were connected with neighboring cells, whereas TUBA expression in Tex14−/− spermatogonia was round shaped and showed that each cell was not connected with neighboring cells (Fig. 2C). These results indicate that Tex14−/− spermatogonia definitely lack intercellular bridges although they could form small clusters and chain-like structures without interconnections to neighboring cells.
Molecular Analysis of Tex14 Heterozygous and Homozygous Mutant SSCs in Culture
Gene expression of several genes, which are essential for spermatogonial stem cells, was examined in short-term cultures of spermatogonia. A number of stem cell related genes (Epcam, Ret, Zbtb16, Bcl6b, Taf4b, Neurog3, and Stra8) were significantly upregulated in Tex14−/− spermatogonia, while differentiation related genes (Kit and Ddx4) were simultaneously upregulated (Fig. 3A). The expression of Id4 and Nanos2, whose expression are restricted to single cells and clusters with less than four aligned spermatogonia, respectively, were not significantly changed in Tex14−/− spermatogonia, even though single spermatogonia were enriched in Tex14−/− intercellular bridge less spermatogonia (Fig. 3A). Upregulation of Neurog3 and Stra8 was also intriguing, because these genes are known to be expressed at a higher level in differentiating spermatogonia rather than undifferentiated spermatogonia [17], [25], [35]. These results suggest that Tex14−/− spermatogonia are already committed to differentiation, even though they possessed characteristics of undifferentiated spermatogonia.
We also analyzed the expression of cell cycle related genes in Tex14−/− spermatogonia. While expression of CDK4 inhibitors, p16 Ink4a and Cdkn1a, are increased, p19 Arf and Cdkn1b were decreased in Tex14−/− spermatogonia (Fig. 3B). When expression levels of cyclins were analyzed, cyclin D1 and cyclin D2 were significantly upregulated, and cyclin D3 and cyclin E were not detected in Tex14−/− spermatogonia (Fig. 3B). Down-regulation of p19 ARF, cyclin D3, and cyclin E and up-regulation of p16 INK4A and CDKN1A were confirmed at protein level (Fig. 4). Notably, CDKN1A in Tex14+/− spermatogonia was undetectable, whereas CDKN1A in Tex14−/− spermatogonia was high, suggesting that lack of intercellular bridges inhibit cell proliferation by induction of CDKN1A. Down-regulation of cyclin E could be induced by inhibition of CDK4 activity. These results suggest that cell cycle progression of Tex14−/− spermatogonia was inhibited by two different CDK4 inhibitors even though CDK4 activity in Tex14−/− spermatogonia could be activated by expression of its related cyclins.
Establishment of Tex14 Heterozygous and Homozygous Mutant SSCs in Culture
We next attempted to establish long-term cultures of Tex14−/− and Tex14+/− spermatogonial stem cells. We successfully established long-term cultures of spermatogonial stem cell from wild-type and GFP-positive Tex14+/− mice (Fig. 2D). Tex14+/− spermatogonial stem cells showed similar colony morphology as wild-type cells (Fig. 2D). However, Tex14−/− spermatogonia could not be maintained for long-term, although they could form colonies and grow for short-term as shown above.
Replacement of the Genetic Defect in Tex14 Heterozygous Mutant SSCs in Culture
To replace the genetic defect in Tex14+/− spermatogonial stem cells in which exon 10 was replaced with a Pgk1-Hprt cassette in one allele of genome, a replacement construct to "knockin" exon 10 into the deleted genome was generated (Fig. 5A). In this replacement construct, the Pgk1-Neo cassette was flanked with loxP sequences so that it could be removed after insertion into the genome. After transfection of this replacement construct into Tex14+/− spermatogonial stem cells, the stem cells were expanded and genotyped using genomic PCR. Seventeen colonies could be expanded and one colony was confirmed to have correct replacement of the deleted exon 10 of Tex14 in the genome (Fig. 5). Although these genetically replaced spermatogonial stem cells were transplanted into germ cell-depleted testes, unfortunately no pups were obtained.
In the present study, characteristics of spermatogonia without intercellular bridges were analyzed. By gene expression profiling, Tex14−/− spermatogonia possessed not only characteristics of undifferentiated spermatogonia but also characteristics of differentiated spermatogonia. To conclude if spermatogonial stem cells without intercellular bridges could self-renew or not, further experiments using transplantation technique could be required. However, it is likely impossible to evaluate self-renewal activity of Tex14−/− SSCs by detection of reconstituted colonies after transplantation because our results suggest that proliferative activity of Tex14−/− SSCs is low and Tex14−/− SSCs may only form small colonies. At least in vivo, Tex14−/− SSC are able to self renew, since there are a number of spermatogonia even in one year-old Tex14−/− testes [30].
In our study, one surprising finding is the formation of chain-like colonies by Tex14−/− SSCs, although our previous reports showed that most spermatogonia in Tex14−/− testes lack chain formation and exist as a single state but not in paired and aligned forms as observed in wild-type adult testes [30]. In addition, we confirmed Tex14−/− spermatogonia were not connected with neighboring cells, even if they form chain-like structure. This suggests that there may be adhesion proteins expressed on the surface of the dividing SSCs that allow them to adhere to one another in a chain-like structure. Alternatively, the chain-like structure may be beneficial for survival and/or cell division of the SSCs because of paracrine growth factors secreted from neighboring SSCs. There may also be some differences in the characteristics between spermatogonial stem cells in vivo and in vitro. All postnatal male germ cells are surrounded by Sertoli cells in vivo, whereas cultured SSCs are free to move and to divide and their proliferation could be accelerated in vitro. As we previously showed, TEX14 functions as a stabilizer of intercellular bridge to avoid cell abscission by interaction with and inhibition of CEP55 function [32]. Midbody formation occurs during cellular division even in Tex14−/− spermatogonia. As shown in Figure 2A, there was no localization of CEP55 to midbodies in 8-day old testes where spermatogonia actively proliferate. These results suggest that proteins required for cell abscission might not be expressed at very high levels in the Tex14−/− midbody, and this midbody might act as a transient intercellular bridge. Alternatively, intercellular bridge might regulate the movement of spermatogonia, because previous reports showed that chains of spermatogonia extensively move around in seminiferous tubules [36], [37]. Tex14−/− spermatogonia might not be able to move actively after cell division because of a lack of intercellular bridges.
Tex14−/− spermatogonia display both activation and repression of proliferative activity according to our expression profiling. Tex14−/− spermatogonia can proliferate even though expression of CDK inhibitors were activated and cyclin E expression was repressed, because some chains of Tex14−/− spermatogonia were detected in the culture in vitro. However, there must be a reduced rate of proliferation since the Tex14−/− colonies were smaller in short-term culture, and Tex14−/− SSCs could not be expanded in long-term culture. Otherwise, Tex14−/− SSCs could proliferate in their first few divisions and might subsequently enter cellular senescence. Since down-regulation of cyclin E expression could be affected by up-regulation of CDK inhibitors, especially CDKN1A, up-regulation of CDKN1A may be critical for inhibition of cellular proliferation of Tex14−/− spermatogonia. As shown in Figure 2C, cytoskelton of Tex14−/− spermatogonia were dramatically changed by lack of intercellular bridges. Checkpoint mechanisms monitoring cellular cytoskelton might induce up-regulation of CDKN1A. Formation of intercellular bridges, at least, could be important for continuous proliferation of spermatogonia.
An attempted replacement of a genetic defect in spermatogonial stem cells was also presented in this study. This is the first report showing a genetic correction in vitro using spermatogonial stem cells, although there are reports of homologous recombination in spermatogonial stem cells [38], [39]. Unexpectedly, Tex14−/− spermatogonial stem cells could not be expanded, although there are many spermatogonia in Tex14−/− testes. Therefore, we used Tex14+/− spermatogonial stem cells as a model of genetic correction. If spermatogonial stem cells could be easily expanded from infertile animals or patients, this methodology could be applicable. Of course, there are some ethical issues that need to be considered if this technique is used in the therapy of infertile patients since this method could produce genetically engineered offspring. Therefore, at this time, this technique is appropriate for therapy of infertile animals except for human. If rare animals are infertile or subfertile because of genetic defects, this methodology would be very powerful to solve this problem.
Author Contributions
Conceived and designed the experiments: NI MMM. Performed the experiments: NI TI. Analyzed the data: NI MMM. Wrote the paper: NI MMM.
- 1. Hochedlinger K, Rideout WM, Kyba M, Daley GQ, Blelloch R, et al. (2004) Nuclear transplantation, embryonic stem cells and the potential for cell therapy. Hematol J 5: S114–117.K. HochedlingerWM RideoutM. KybaGQ DaleyR. Blelloch2004Nuclear transplantation, embryonic stem cells and the potential for cell therapy.Hematol J5S114117
- 2. Lerou PH, Daley GQ (2005) Therapeutic potential of embryonic stem cells. Blood Rev 19: 321–331.PH LerouGQ Daley2005Therapeutic potential of embryonic stem cells.Blood Rev19321331
- 3. Rideout WM III, Hochedlinger K, Kyba M, Daley GQ, Jaenisch R (2002) Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy. Cell 109: 17–27.WM Rideout IIIK. HochedlingerM. KybaGQ DaleyR. Jaenisch2002Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy.Cell1091727
- 4. Hanna J, Wernig M, Markoulaki S, Sun CW, Meissner A, et al. (2007) Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 318: 1920–1923.J. HannaM. WernigS. MarkoulakiCW SunA. Meissner2007Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin.Science31819201923
- 5. Howden SE, Gore A, Li Z, Fung HL, Nisler BS, et al. (2011) Genetic correction and analysis of induced pluripotent stem cells from a patient with gyrate atrophy. Proc Natl Acad Sci U S A 108: 6537–6542.SE HowdenA. GoreZ. LiHL FungBS Nisler2011Genetic correction and analysis of induced pluripotent stem cells from a patient with gyrate atrophy.Proc Natl Acad Sci U S A10865376542
- 6. Brinster RL, Avarbock MR (1994) Germline transmission of donor haplotype following spermatogonial transplantation. Proc Natl Acad Sci U S A 91: 11303–11307.RL BrinsterMR Avarbock1994Germline transmission of donor haplotype following spermatogonial transplantation.Proc Natl Acad Sci U S A911130311307
- 7. Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, et al. (2003) Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod 69: 612–616.M. Kanatsu-ShinoharaN. OgonukiK. InoueH. MikiA. Ogura2003Long-term proliferation in culture and germline transmission of mouse male germline stem cells.Biol Reprod69612616
- 8. Seandel M, James D, Shmelkov SV, Falciatori I, Kim J, et al. (2007) Generation of functional multipotent adult stem cells from GPR125+ germline progenitors. Nature 449: 346–350.M. SeandelD. JamesSV ShmelkovI. FalciatoriJ. Kim2007Generation of functional multipotent adult stem cells from GPR125+ germline progenitors.Nature449346350
- 9. Ko K, Tapia N, Wu G, Kim JB, Bravo MJ, et al. (2009) Induction of pluripotency in adult unipotent germline stem cells. Cell Stem Cell 5: 87–96.K. KoN. TapiaG. WuJB KimMJ Bravo2009Induction of pluripotency in adult unipotent germline stem cells.Cell Stem Cell58796
- 10. Oatley JM, Brinster RL (2008) Regulation of spermatogonial stem cell self-renewal in mammals. Annu Rev Cell Dev Biol 24: 263–286.JM OatleyRL Brinster2008Regulation of spermatogonial stem cell self-renewal in mammals.Annu Rev Cell Dev Biol24263286
- 11. Caires K, Broady J, McLean D (2010) Maintaining the male germline: regulation of spermatogonial stem cells. J Endocrinol 205: 133–145.K. CairesJ. BroadyD. McLean2010Maintaining the male germline: regulation of spermatogonial stem cells.J Endocrinol205133145
- 12. Kanatsu-Shinohara M, Toyokuni S, Shinohara T (2004) CD9 is a surface marker on mouse and rat male germline stem cells. Biol Reprod 70: 70–75.M. Kanatsu-ShinoharaS. ToyokuniT. Shinohara2004CD9 is a surface marker on mouse and rat male germline stem cells.Biol Reprod707075
- 13. Tokuda M, Kadokawa Y, Kurahashi H, Marunouchi T (2007) CDH1 is a specific marker for undifferentiated spermatogonia in mouse testes. Biol Reprod 76: 130–141.M. TokudaY. KadokawaH. KurahashiT. Marunouchi2007CDH1 is a specific marker for undifferentiated spermatogonia in mouse testes.Biol Reprod76130141
- 14. Ryu BY, Orwig KE, Kubota H, Avarbock MR, Brinster RL (2004) Phenotypic and functional characteristics of spermatogonial stem cells in rats. Dev Biol 274: 158–170.BY RyuKE OrwigH. KubotaMR AvarbockRL Brinster2004Phenotypic and functional characteristics of spermatogonial stem cells in rats.Dev Biol274158170
- 15. Kanatsu-Shinohara M, Takashima S, Ishii K, Shinohara T (2011) Dynamic changes in EPCAM expression during spermatogonial stem cell differentiation in the mouse testis. PLoS One 6: e23663.M. Kanatsu-ShinoharaS. TakashimaK. IshiiT. Shinohara2011Dynamic changes in EPCAM expression during spermatogonial stem cell differentiation in the mouse testis.PLoS One6e23663
- 16. Naughton CK, Jain S, Strickland AM, Gupta A, Milbrandt J (2006) Glial cell-line derived neurotrophic factor-mediated RET signaling regulates spermatogonial stem cell fate. Biol Reprod 74: 314–321.CK NaughtonS. JainAM StricklandA. GuptaJ. Milbrandt2006Glial cell-line derived neurotrophic factor-mediated RET signaling regulates spermatogonial stem cell fate.Biol Reprod74314321
- 17. Oatley JM, Avarbock MR, Telaranta AI, Fearon DT, Brinster RL (2006) Identifying genes important for spermatogonial stem cell self-renewal and survival. Proc Natl Acad Sci U S A 103: 9524–9529.JM OatleyMR AvarbockAI TelarantaDT FearonRL Brinster2006Identifying genes important for spermatogonial stem cell self-renewal and survival.Proc Natl Acad Sci U S A10395249529
- 18. Buaas FW, Kirsh AL, Sharma M, McLean DJ, Morris JL, et al. (2004) Plzf is required in adult male germ cells for stem cell self-renewal. Nat Genet 36: 647–652.FW BuaasAL KirshM. SharmaDJ McLeanJL Morris2004Plzf is required in adult male germ cells for stem cell self-renewal.Nat Genet36647652
- 19. Costoya JA, Hobbs RM, Barna M, Cattoretti G, Manova K, et al. (2004) Essential role of Plzf in maintenance of spermatogonial stem cells. Nat Genet 36: 653–659.JA CostoyaRM HobbsM. BarnaG. CattorettiK. Manova2004Essential role of Plzf in maintenance of spermatogonial stem cells.Nat Genet36653659
- 20. Oatley MJ, Kaucher AV, Racicot KE, Oatley JM (2011) Inhibitor of DNA binding 4 is expressed selectively by single spermatogonia in the male germline and regulates the self-renewal of spermatogonial stem cells in mice. Biol Reprod 85: 347–356.MJ OatleyAV KaucherKE RacicotJM Oatley2011Inhibitor of DNA binding 4 is expressed selectively by single spermatogonia in the male germline and regulates the self-renewal of spermatogonial stem cells in mice.Biol Reprod85347356
- 21. Sada A, Suzuki A, Suzuki H, Saga Y (2009) The RNA-binding protein NANOS2 is required to maintain murine spermatogonial stem cells. Science 325: 1394–1398.A. SadaA. SuzukiH. SuzukiY. Saga2009The RNA-binding protein NANOS2 is required to maintain murine spermatogonial stem cells.Science32513941398
- 22. Falender AE, Freiman RN, Geles KG, Lo KC, Hwang K, et al. (2005) Maintenance of spermatogenesis requires TAF4b, a gonad-specific subunit of TFIID. Genes Dev 19: 794–803.AE FalenderRN FreimanKG GelesKC LoK. Hwang2005Maintenance of spermatogenesis requires TAF4b, a gonad-specific subunit of TFIID.Genes Dev19794803
- 23. Yoshida S, Takakura A, Ohbo K, Abe K, Wakabayashi J, et al. (2004) Neurogenin3 delineates the earliest stages of spermatogenesis in the mouse testis. Dev Biol 269: 447–458.S. YoshidaA. TakakuraK. OhboK. AbeJ. Wakabayashi2004Neurogenin3 delineates the earliest stages of spermatogenesis in the mouse testis.Dev Biol269447458
- 24. Sadate-Ngatchou PI, Payne CJ, Dearth AT, Braun RE (2008) Cre recombinase activity specific to postnatal, premeiotic male germ cells in transgenic mice. Genesis 46: 738–742.PI Sadate-NgatchouCJ PayneAT DearthRE Braun2008Cre recombinase activity specific to postnatal, premeiotic male germ cells in transgenic mice.Genesis46738742
- 25. Zhou Q, Li Y, Nie R, Friel P, Mitchell D, et al. (2008) Expression of stimulated by retinoic acid gene 8 (Stra8) and maturation of murine gonocytes and spermatogonia induced by retinoic acid in vitro. Biol Reprod 78: 537–545.Q. ZhouY. LiR. NieP. FrielD. Mitchell2008Expression of stimulated by retinoic acid gene 8 (Stra8) and maturation of murine gonocytes and spermatogonia induced by retinoic acid in vitro.Biol Reprod78537545
- 26. Shinohara T, Avarbock MR, Brinster RL (1999) beta1- and alpha6-integrin are surface markers on mouse spermatogonial stem cells. Proc Natl Acad Sci U S A 96: 5504–5509.T. ShinoharaMR AvarbockRL Brinster1999beta1- and alpha6-integrin are surface markers on mouse spermatogonial stem cells.Proc Natl Acad Sci U S A9655045509
- 27. Toyooka Y, Tsunekawa N, Takahashi Y, Matsui Y, Satoh M, et al. (2000) Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development. Mech Dev 93: 139–149.Y. ToyookaN. TsunekawaY. TakahashiY. MatsuiM. Satoh2000Expression and intracellular localization of mouse Vasa-homologue protein during germ cell development.Mech Dev93139149
- 28. Huckins C, Oakberg EF (1978) Morphological and quantitative analysis of spermatogonia in mouse testes using whole mounted seminiferous tubules, I. The normal testes. Anat Rec 192: 519–528.C. HuckinsEF Oakberg1978Morphological and quantitative analysis of spermatogonia in mouse testes using whole mounted seminiferous tubules, I. The normal testes.Anat Rec192519528
- 29. Greenbaum MP, Iwamori T, Buchold GM, Matzuk MM (2011) Germ cell intercellular bridges. Cold Spring Harb Perspect Biol 3: a005850.MP GreenbaumT. IwamoriGM BucholdMM Matzuk2011Germ cell intercellular bridges.Cold Spring Harb Perspect Biol3a005850
- 30. Greenbaum MP, Yan W, Wu MH, Lin YN, Agno JE, et al. (2006) TEX14 is essential for intercellular bridges and fertility in male mice. Proc Natl Acad Sci U S A 103: 4982–4987.MP GreenbaumW. YanMH WuYN LinJE Agno2006TEX14 is essential for intercellular bridges and fertility in male mice.Proc Natl Acad Sci U S A10349824987
- 31. Greenbaum MP, Ma L, Matzuk MM (2007) Conversion of midbodies into germ cell intercellular bridges. Dev Biol 305: 389–396.MP GreenbaumL. MaMM Matzuk2007Conversion of midbodies into germ cell intercellular bridges.Dev Biol305389396
- 32. Iwamori T, Iwamori N, Ma L, Edson MA, Greenbaum MP, et al. (2010) TEX14 interacts with CEP55 to block cell abscission. Mol Cell Biol 30: 2280–2292.T. IwamoriN. IwamoriL. MaMA EdsonMP Greenbaum2010TEX14 interacts with CEP55 to block cell abscission.Mol Cell Biol3022802292
- 33. Ogawa T, Arechaga JM, Avarbock MR, Brinster RL (1997) Transplantation of testis germinal cells into mouse seminiferous tubules. Int J Dev Biol 41: 111–122.T. OgawaJM ArechagaMR AvarbockRL Brinster1997Transplantation of testis germinal cells into mouse seminiferous tubules.Int J Dev Biol41111122
- 34. Wu MH, Rajkovic A, Burns KH, Yan W, Lin YN, et al. (2003) Sequence and expression of testis-expressed gene 14 (Tex14): a gene encoding a protein kinase preferentially expressed during spermatogenesis. Gene Expr Patterns 3: 231–236.MH WuA. RajkovicKH BurnsW. YanYN Lin2003Sequence and expression of testis-expressed gene 14 (Tex14): a gene encoding a protein kinase preferentially expressed during spermatogenesis.Gene Expr Patterns3231236
- 35.
Zhou Q, Griswold MD (2008) Regulation of spermatogonia. Q. ZhouMD Griswold2008Regulation of spermatogonia.StemBook 2010/07/09 ed.Cambridge (MA): Harvard Stem Cell Institute. Cambridge (MA): Harvard Stem Cell Institute.
- 36. Yoshida S, Sukeno M, Nabeshima Y (2007) A vasculature-associated niche for undifferentiated spermatogonia in the mouse testis. Science 317: 1722–1726.S. YoshidaM. SukenoY. Nabeshima2007A vasculature-associated niche for undifferentiated spermatogonia in the mouse testis.Science31717221726
- 37. Nakagawa T, Sharma M, Nabeshima Y, Braun RE, Yoshida S (2010) Functional hierarchy and reversibility within the murine spermatogenic stem cell compartment. Science 328: 62–67.T. NakagawaM. SharmaY. NabeshimaRE BraunS. Yoshida2010Functional hierarchy and reversibility within the murine spermatogenic stem cell compartment.Science3286267
- 38. Kanatsu-Shinohara M, Ikawa M, Takehashi M, Ogonuki N, Miki H, et al. (2006) Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells. Proc Natl Acad Sci U S A 103: 8018–8023.M. Kanatsu-ShinoharaM. IkawaM. TakehashiN. OgonukiH. Miki2006Production of knockout mice by random or targeted mutagenesis in spermatogonial stem cells.Proc Natl Acad Sci U S A10380188023
- 39. Kanatsu-Shinohara M, Kato-Itoh M, Ikawa M, Takehashi M, Sanbo M, et al. (2011) Homologous recombination in rat germline stem cells. Biol Reprod 85: 208–217.M. Kanatsu-ShinoharaM. Kato-ItohM. IkawaM. TakehashiM. Sanbo2011Homologous recombination in rat germline stem cells.Biol Reprod85208217


text:
All Down the Valley
first edition
1952 · New York
by BILLINGS, Henry
New York: Viking Press, 1952. Very Good/Very Good. First edition. Very good in very good dustwrapper. Rubbed and bumped along spine ends and corners. Dustwrapper torn along top edges, yellowed along spine. Please Note: This book has been transferred to Between the Covers from another database and might not be described to our usual standards. Please inquire for more detailed condition information. (Inventory #: 260674)


text:
Cheap Removals Croydon – bestremovalists.com.au
Croydon is a suburb of Melbourne that is quite appealing to anyone looking to relocate. For one, there are plenty of business opportunities here as evidenced by the many traders of food, clothing, beauty services, movies, etc.
In terms of leisure, there are a few that come to mind: the Amdale Centre and Civic Square, Croydon Park, Croydon Gymnasium Facility, Croydon Leisure and Aquatic Centre, Croydon Memorial Pool and the Wyreena Community Arts.
Croydon Movers can help you relocate to Croydon with ease and assurance. As professionals in this service, they have the expertise in transporting the customer's belongings safely from one place to another. They have the license as well as the insurance coverage demanded by law to assure their reliability and trustworthiness.
Avoid the fuss when you decide to move, get in touch with the Croydon Moving Company. Ask about packing and unpacking services as well to make relocating totally stress-free.


text:
Hey Joel Frazier here, thanks for checking out my web page.
I am 44 years old and passionate about reviewing a variety of products online.
I've reviewed 20 or so products in the 'Health & Fitness' niche, I have also suffered from Type 2 Diabetes problems myself and have found that some products available on the internet 'do' work.
I pride myself in providing visitors and readers with completely unbiased and honest reviews. I also believe in adding value to all my reader's lives.
Lastly, I started this blog to help others with their health and fitness goals and enjoy what this can bring to life. Hope you enjoyed my web page.


text:
I reckon the Washington Redskins are going to need a new fight song to go along with their new name, now This pisses me off.
I'm an 80's kid, and Joe Theismann (originally pronounced, Thees-man, before changing it to, Thighs-man, just so it would rhyme with, Heisman…as in, the Heisman Trophey…which he didn't win) was my idol. I had his jersey and everything. And, oh my God when Lawrence Taylor broke his leg…. my tiny little 4 year old heart broke into tiny little pieces. My hero was done, forever. I still wore his jersey, though.
I clearly remember hearing "Hail To The Redskins" playing during the football games. Hell, I remember hearing it on the radio. I sang along like a real badass, too. I still sing along to it, and sometimes I'll even do a little solo act when the spirit moves me.
So, what happens now? I guess with this new name change, I'll never hear it again. Glad that's been ripped away. *sarcasm*


text:
The Ward Line steamer OSCEOLA was battling a severe winter storm on Lake Superior on the night of Nov. 21, 1898. The ship, laden with salt, was bound from Sault Ste. Marie to Duluth when the gale struck from the northwest. Capt. J.C. McLeod turned the steamer into the teeth of the fury instead of taking a direct westerly track across the big lake.
McLeod kept the ship on that course throughout the night, not realizing how much headway the OSCEOLA was making against the storm. At 5:30 a.m. the steamer crunched to a sudden stop. She was aground on the south side of Mott Island. An inspection revealed no apparent structural damage. With the wind now howling directly out of the north, the captain reasoned that it might be possible to lighten the boat enough to work it back out into deep water.
The 16 members of the crew worked for hours, hoisting hundreds of barrels of salt from the hold and jettisoning them overboard. Before they had the job finished, the wind shifted and the seas began hammering the ship from starboard. McLeod decided it was too dangerous to stay aboard the stranded vessel.
The rocky island was only a few hundred feet away, but to the crew of the OSCEOLA it seemed like a hundred miles. The seas were so violent that every man felt in great danger as they struggled to reach the shore on a single lifeboat. A rope line was fastened to the little boat, with the other end tied to the side of' the steamer. When the first boat load of sailors reached shore. another rope was attached to something on the island.
The men then used the ropes to pull the life boat back and forth between the ship and shore until everybody was ashore. Their feet were on solid rock, but there was little comfort on the barren and uninhabited island. The sailors found the best shelter they could, gathered firewood and miraculously got a fire started.
Two days later the storm was over and the OSCEOLA was still in one piece. The crew returned to the ship and finished the job of throwing the cargo overboard. On the night of Nov. 23, after about 2000 barrels of salt were jettisoned. the ship floated free. (Author James Donahue shipwreck articles ran once a week in paper.)
Port Huron Daily Tribune
The Maritime History of the Great Lakes site is managed by Walter Lewis
Since 2005 this site has been the prototype for the digital collection management tools built for the Our Digital World (formerly the OurOntario project of Knowledge Ontario.)
As a prototype it is subject to occasional, unexplained interruptions in service ... because we keep trying new things.
My favourites lets you save items you like, tag them and group them into collections for your own personal use. Viewing "My favourites" will open in a new tab.
Login here or start a My favourites account.


text:
SCF Advanced Search
Pages: [1]
Topic: A Trojan purporting to be a video of the iPhone is at the center of a new pharmi (Read 1538 times)
0 Members and 1 Guest are viewing this topic.
Pages: [1]
With Quick-Reply you can write a post when viewing a topic without loading a new page. You can still use bulletin board code and smileys as you would in a normal post.


text:
[artist id="1998098"]T-Pain[/artist] is diligently at work on his next album, but it seems like the Tampa, Florida, native is already thinking about the marketing plan for the project.
"Revolver is coming," he told MTV News on Saturday, backstage at Chris Brown's "Virginia Stand Up!" benefit concert. "We're just trying to see. ... It's hard to sell records unless you're Susan Boyle, I guess. Nobody is selling records.
"I should compete on 'American Idol' the way they're selling records when they come off of there," T-Pain laughed. "I need to be a part of that competition. I don't know if they let you use Auto-Tune, but if they do, I'm a shoo-in. I'm two shoes in! I'm a pair of shoes in."
The computer-aided crooner hasn't released a project since he was embroiled in a pseudo-feud with Jay-Z after the rapper released his single "D.O.A. (Death of Auto-Tune)."
Pain initially announced his follow-up to 2008's Thr33 Ringz would be Revolver, although he flirted with the title UBER. But that project, which he said was going to be released last year, appears to have been shelved.
Recently, T-Pain announced that plans for a joint album with Lil Wayne are now on hold following the rapper's incarceration. He told MTV News in March that it didn't make sense to rush the project while Wayne was preparing to begin his prison sentence.
"The T-Wayne album, we're actually gonna wait until he gets out," Pain said at the time. "He has so much going on. He has Rebirth out right now. I don't wanna interrupt that. We just gonna wait until he gets out to really get in on that. We recorded a bunch of songs. But, right now, it's time for him and his personal sh--. We definitely got an album's worth of material, but it's only an album's worth. It's nothing to pick from. We just got an album. We can put out whatever we feel like and say, 'Hey, this is T-Wayne's album,' or we can actually try to make it good. Like we're actually trying to make an album and say, 'This is the good T-Wayne album.' Rather than just record 12 songs and say, 'Here you go, mutha----as! I know you want it. You gonna buy it anyway, just 'cause you think it's good.' "
Are you excited to hear T-Pain's next album? Let us know in the comments!


text:
Attendance at Bristol Mini Maker Faire 2015 is free, but to gauge numbers and make sure we have enough materials for everyone to take part in the various interactive activities we are encouraging people to book via Eventbrite.
If the event is much busier than we expected, we may prioritise those who have registered their attendance on Eventbrite, although registration is not a guarantee of entry at busy times.
For more information see the details on our Eventbrite listing here.


text:
- " Knock foes off their ft by blasting them backwards with a dose of quantum energy. "
- ―Instruction Manual Weapon Description [ItN]
The Alpha Temporal Repulsor[1] was a temporal shotgun used by Ratchet.[2][3] It used quantum energy to add power to its ammo.[3] It upgraded into the Quantum Repulsor at V3.[3]
In Game Summary
- " A shot-gun like weapon that knocks back enemies. It uses quantum energy to add more stopping power to it's deadly ammunition. "
- ―Holocard Description [R&C (PS4)]
- Ratchet & Clank: Into the Nexus (first appearance)
Notes and references
- ↑ Into the Nexus instruction manual
- ↑ "Traditional Ratchet and Clank Is Back With Into the Nexus" by Colin Moriarty
- ↑ 3.0 3.1 3.2 Ratchet & Clank Into the Nexus Demo Live Stream with Q&A from Brian Allgeier


text:
Mobile Massage Businesses Can Grow Revenue With A Mobile App!
As a Massage Therapist, your clients are busy and on the go and meeting their needs of course should be your main concern. Since people are using their Mobile devices to connect these days, it makes sense to use a mobile app to engage with your clients and make it easier for them to reach your business whenever they need your service.
3 Reasons Your Mobile Massage Business Needs An App to Grow Revenue
According to The American Massage Therapy Association's 2012 Research Report, the number of on-site mobile massages increased from 39 percent in 2010 to 50 percent in 2012.
This statistic illustrates the growing opportunities available for massage professionals looking to start a mobile massage business. These professionals typically do not operate out of an office or rented facilities. This requires them to adopt technologies that enable them to run their businesses profitably.
Mobile apps are one of these technologies.
Defined as applications developed for and downloadable to small handheld devices such as smart phones and tablets, mobile apps have become commonplace among small business owners.
According to the 2013 AT&T Small Business Technology Poll, almost one-third of small businesses surveyed use mobile apps. They cited time savings, increased productivity and cost-reduction as reasons why they use them.
Here are three reasons why apps help mobile massage therapists grow their business:
1. Accessibility.
Mobile apps provide instant access to their calendars, schedules, financial information and client data. This allows you to see your next appointment from your phone or tablet.
2. User-friendliness.
While accessing an online program via a Web site is possible through a smart phone, it's not as quick as through a mobile app. Mobile apps provide instant access to information without having to navigate a Web page. QuickBooks is a popular app among massage therapists in managing their accounting.
3. Client communication.
Apps that store client information—such as scheduling and e-marketing apps like Constant Contact—let you quickly compose and send out an e-mail blast right from your smartphone or tablet. They're especially beneficial when you need to communicate information to clients quickly—such as to publicize a one-day promotion—but are unable to access your laptop.
You can view the original article here.
Mobile Massage Business
Now is the time to build awareness for your Massage Therapy business. A mobile app gives any Massage Therapist the potential to develop a higher presence as well as strengthen their unique brand.


text:
WHAT a strange attitude the British display towards some of their national treasures. How could a building that represents a pivotal moment in architectural history end up rotting away through neglect at Hampstead's back door?
The Isokon apartment building is a Grade I landmark designed by Wells Coates, an idealistic engineer who believed in making houses that were entirely rational and convenient. He was interested in ship design and believed that homes could learn from the built-in cabin interiors with their fold-away functionality.
His building was the first purpose-built block of its type in the country, and one of the first to use concrete in such a revolutionary way. Agatha Christie described its effect as entering a "giant liner, which ought to have had a couple of funnels, and then you went upstairs through the door to your flat and there were the trees tapping at the windows".
It was 1934. The London Underground was spreading and cars were becoming the favourite middle-class toy.
The world was just waking up to the new ideas about the house as "a machine for living" that were emerging from the Bauhaus School of Design in Germany.
The Isokon flats offered collective living to the intelligentsia and a new aesthetic stripped of Edwardian and Victorian decoration.
More than half a century before serviced apartments properly arrived in London, Wells Coates had pioneered the idea. His block - at rents of £96 a year - contained 32 flats, most of them single-person, each linked by telephone to a central kitchen.
A dumb waiter took food up to each level, where there was a trolley with hot plates that fitted into a small pantry.
The penthouse was designed for the businessman Jack Pritchard, who lived there for years and welcomed many refugees from Nazi Germany, among them Marcel Breuer and Walter Gropius. Breuer designed the Isobar Club on the ground floor for residents.
Since its present owner, Camden Council, put the building up for sale at the end of last year there has been intense interest. Chris Flannery, a local architect who got wind of the opportunity two years ago, formed the Isokon Trust and put together funding and architectural expertise to save it.
"The bulk of the building would be flats but we would have two show flats with exhibitions, seminars and lectures about the building," he says. "We would like to re-open the Isobar, which was the Groucho Club of its day."
Camden Council, however, has to be seen to get good value for the flats, while avoiding the criticism that it is simply selling them off to the highest bidder. The selling agent, FPD Savills, has not set a guide price, though a figure of about £1m has been mentioned.
Savills' Noel de Keyzer is handling the sale. "It is a two-stage process," he says. "The first stage elicited 25 expressions of interest".
A meeting with the council whittled the list down to 10, who have now submitted detailed proposals and offers. "We want a scheme soundly financed and guaranteed to succeed," says Mr de Keyzer.
He admits that he was surprised by the 600 inquiries he received when he floated the building on the market. "We have had building trusts, housing co-operatives, national housebuilders and specialist overseas investors interested in letting."


text:
An effective email marketing strategy doesn't need to be complicated. In fact, the world we currently live and do ...
If you followed my blog post about trying to write 2,000-word blog posts, I would like to report that I am today fed up and won't do such foolish thing again. Srsly!
Well, it's been 2 months and all I can conclude is this: Your blog reputation wins, no matter how long or how useful it is. You may disagree, but this is what I have found out.
Here's what I've tried:
Test Site A: I write long blog posts (a couple 1,500+ words and one 2,000+ words) – with quality resources shared on each – on my 5-month old business blog with Domain Authority (DA) 22 and PageRank (PR) 2
Test Site B: I write long article (2,500+ words) on a brand new site with virtually no DA and PR.
Test Site C: As usual, I occasionally write less than 800 words blog posts on my 5-year old authority business blog with DA 49 and PR 4
After 2 months, I see that long blog posts have no correlation whatsoever with high search engine ranking. Of course this is non-scientific finding – so perhaps it's not accurate, but still, you can judge after the following report – let's move on, shall we?
Executive summary: Reputation wins!
Based on the test sites above, I find the following:
– Test Site A: My 600 – 800-word blog post ranks on the first page. My 2,000-word blog post (with proper research and plenty of useful resources, with good shares on social media) is indexed but it's nowhere to be found on Googles' first 2 SERPs for the target keywords!
– Test Site B's content is nowhere to be found on SERPs. It IS indexed, though…
– Test Site C: The best ranked page, which delivers considerable organic traffic – is a 600-word article. The 1,500-word one went viral, driving social media traffic even though it doesn't rank that well on Google.
Overall, Test Site C's articles get more shares, more buzz and higher search engine ranking better than Test Site A and B – regardless of articles' length.
I even hire a content specialist to write a 2,000-word article for me. It's a great read, and it cost me $50+. ROI: Zip, zero, zilch, nada.
Well, Neil Patel was spot-on! Although long copies are generally sitting high on Search Engine Result Pages (SERPs,) the ranking factor can't be decided by long copies in themselves.
As Google (and other SEs) uses many signals to rank your pages, word length is just a factor. Of course, it's proven that short blurbs will always get you slammed by Google Panda. But that doesn't mean a 2,000-word copy will rank you high.
Firstly, you need to write authoritative copy with minimal rambles. Secondly, your page must get online evangelists and influencers attention. This is difficult if you are practically a 'nobody' (like me!) If you are Neil Patel you will get attention. If you are Ivan Widjaya (me!), you will get attention too, but with much, much less significance.
So, again, Neil Patel is right: You need to have a strong social network to get your content buzzed. Minimal social presence, minimal article exposure, minimal conversion, low ranking. Period.
You also need to have a strong base of readers, who will link to you, share your blog post and so on. No readers, no traffic; no readers, no links (except you build links yourself.) Yes, it's that obvious…
Conclusion: Again, reputation wins!
So, it's no point for me to continue with my experiment. Writing 2,000-word content is tiring. Research for it is even more tiring. It's probably better for you to focus on growing your online reputation and create content that matters, rather than focusing on the length of your content. That's too shallow, IMO!
Content is king, indeed. If yours are authoritative, you will gain reputation (that's why you should have your name on prominent place – and link your name to your Google+ profile with rel="author". It's a good practice for building reputation online – and (hopefully) useful for your SEO.
But of course, content alone can't win you high search engine ranking and good social media popularity. You need to continue to build your reputation.
After you gain considerable reputation, THEN you can deliver more authoritative articles and have (way) much more people who will link to yours. Why? Because of your reputation and the quality of your content itself.
Again, you need both build your reputation AND your content quality. And be sure that you understand that quite often, your 2,000-word copies won't do better than your 600-word ones. But when you are THERE, your authoritative 2,000-word content will get you content high on SERP. The research has proven it.
So, what to do to get ranked well on search engines? Write content people will love. Period.
Ivan Widjaya
Founder/Editor AsepOnde.com
photo credit: strangejourney via photopin cc
Get updates and members-only tips, as well as freebies and special offers - sign up for our newsletter:
I build and manage blogs. I blog for brands. I have helped clients in getting found online.Learn moreContact me


text:
Control is one of humankind's greatest illusions. Let's face it—even with all the information available and expansive educational preparation—unexpected events often
Judith E. Glaser is the best-selling author of seven business books, including Conversational Intelligence. She is a pioneering and innovative change agent, consultant,
Here's why you might just want to take a breath before clicking send. Have you ever sent an email and immediately wanted to
Nine tips for keeping it real while engaging in meaningful communication. 1. Be present and welcoming Be 100% present. Set aside the usual
Pausing to let go of the stories we tell ourselves about our partner's actions and really listening to their feelings and needs can
The hardest part of communicating well is knowing when to speak, when to be quiet and when to wait and see—but mindfulness can
An interview with MIT professor Sherry Turkle about her book Reclaiming Conversation. What happens when we become too dependent on our mobile phones? According
In the face of internal and external pressure, these three tips can help you stand up for your values and have ethical courage.
Wondering what to do when you encounter mean people? Hint: Don't just turn the other cheek. "Why you gotta be so meeaann?" Taylor
Text messaging can breed disastrous misunderstandings between people. Here's how to stop that from happening. "How do you read emotions in text messages?"
Sign up for email updates
800 Bethany Drive
Scotts Valley,CA 95066
Username or Email Address
Remember Me
Don't have a 1440 account? Create an Account
Create a 1440 account to make checkout easier, manage your reservations, and more
First Name
Last Name
Confirm Email
Confirm Password
By creating an account you agree to 1440's standard
Terms of Service and Privacy Policy
Already have a 1440 account? Login
Your Name (required)
Your Email (required)
Sign up with your email and/or mailing address.
We look forward to keeping you posted--and inspired!
Add your account creation form script here.


text:
As I understood in C++11
int x;
decltype(x) y = x;
int y = x;
int x;
decltype((x)) y = x;
int& y = x;
decltype(auto) f1()
int x = 0;
return x; // decltype(x) is int, so f1 returns int
decltype(auto) f2()
int x = 0;
return (x); // decltype((x)) is int&, so f2 returns int&
decltype((x + x))
They wanted a way to get the type of declaration of an identifier.
They also wanted a way to get the type of an expression, including information about if it is a temporary or not.
decltype(x) gives the declared type of the identifier
x. If ypu pass
decltype something that is not an identifier, it determines the type, then appends
& or
&& depending on if it is an lvalue or rvalue.
Conceptually you can think of it as the difference between the type of a variable and the type of an expression. But that is not quite how the standard describes it.
They could have used two different keywords to mean these two things. They did not.


text:
Comment Re:Stupid claim (Score 1) 851
Also, most smart phones can be dropped or even accidentally put in water with the same survival rate as your dumb phone.
And unless you are constantly drunk, what does it matter? Or some super uncoordinated clumsy person -- gee, I hope they don't let you drive a car.
I've never dropped my phones. I had an HTC back before the iPhone, then an original iPhone and now a iPhone 4. I use minimal cases and never ever drop them. Not once have I broke a phone. All of them are still 100% functional.


text:
eSports on the rise
Brock Radford, Staff Writer
April 20, 2017
Filed under April, Opinion, Showcase
It's no question that sports at HB are a large part of students' lives, whether students are part of the team or just spectators. However, a new demographic is surfacing around the world: eSports, competitive video games played in front of large crowds of dedicated fans. The eSports...
Donate Life: you can't take them with you
Nicole Poitras, Co-Editor In Chief
April 19, 2017
Filed under April, Opinion, Showcase
Not long after birth, I was diagnosed with a life-threatening liver disease called biliary atresia. My liver was destroying itself, and the only way I could survive was if I had a transplant. Thankfully, I did not have to wait very long for an organ; the doctors quickly found that my father was a match....
SAT'S and how to prepare
Mitch Dreyer, Staff Writer
April 17, 2017
Filed under April, Features, Showcase
SAT's just hit students across the country, taking place on April 5. The national standardized test consists of a math, reading, and writing portion. Some juniors who took the test in the fall found it hard, while others thought it was a breeze. After receiving their results, students get to see in...
Becoming Blue
Connor Harris, Staff Writer
April 17, 2017
Filed under April, Opinion, Showcase
When I was a kid, I and many others dreamt of becoming police officers, and now that we are faced with the challenge of deciding our future professions, our dreams may become a reality. Many people often think about the kinds of experiences they'd have, the different jobs you might do. But not many people...
Eagle Spotlight: Sam St. Pierre
Colton Mailloux, Staff Writer
April 16, 2017
Filed under April, Features, Showcase
It seems this year that many Hollis Brookline have sprouted wings. Sam St. Pierre '17 is going to be honored for his accomplishments in Boy Scouts by receiving the rank of Eagle Scout. He, like many others, just needs to have his Eagle Ceremony to crown the achievement. "My project was to create an easy care lan...
An epic build: the library box fort
Rachel Marquis, Video Editor
April 15, 2017
Filed under April, Features, Showcase
During first period on Friday, March 31, an epic project for the HBHS library was completed. With an influx of new computers for lab 217 came an abundance of cardboard boxes and packing materials. These newfound assets were deemed appropriate for one thing only: a fort! Librarian Christine Heaton and a group of st...
How should I pick my college?
Kaitlin Johnson, Staff Writer
April 14, 2017
Filed under April, Features, Showcase
Throughout senior year, many students decide what colleges they want to attend. Applying to college is a long and challenging process, and takes time to fill out all of the registration. By the end of their application process, on May 1, they have to make a decision on what their first college choice...
Helping Hands
Anthony Tamposi, Staff Writer
April 13, 2017
Filed under April, Features, Showcase
Two Hollis Brookline graduates have now come back to the school and are working as paraprofessionals. When HB became their alma mater, they probably didn't expect to come back and have their professions here. Ian Tomer '14 and Morgan Elliott '11, who you may see around the school during the da...
Freezing teachers' plans
Parker Coady, Op/Ed Editor, Semester 2
April 12, 2017
While snow days can be a relaxing breath of fresh air for stressed students, the surprise days off are a double-edged sword for those with busy schedules. Snow days often muddle plans made by school teachers -- especially for those responsible for teaching AP classes. The "April Fool's Blizzard"...
Future publishers
Hannah Balfour, Staff Writer
April 8, 2017
Filed under April, Features, Showcase
At Hollis Brookline, many students are beginning to publish their creative works online. Some students at HB such as Alex Crupi '18, Allie Campbell '17, and Nicole Poitras '17, quickly got onto the web to create and upload articles and videos to the internet. How many people do you know that pu...


text:
Newly completed Science rooms in constant use.
The newly completed Science rooms are making a big impact on learning, allowing students to work intensively on a range of projects and investigations. The new Physics laboratories will soon be complete along with an exciting new data logging room for in depth experimental work. The Yr 12 students above are working on making detailed drawings of plant and animal tissue for an upcoming assessment. There are 30 microscopes set up in the Science Project Room at present, making rapid skill development possible here, something that is not available in many other schools.


text:
I'm not going to lie. It was the purple flecks. That's what got me to buy these beans, known as Dragon Tongue or Dragon Langerie. The beans' beautiful dappled exterior makes them hard to ignore as they sit among monochromatic green and yellow beans on the market stand.
As I paid for a bag at the Pinecrest Farmers Market I thought of how pretty they would look on the plate when I served them at home. Except at the time I didn't know cooking the beans would strip them of any shades of purple. That's not to say they didn't look nice on the plate in their yellow and greenish shades. They tasted great and that's what counts, right?
I could've eaten all of them raw. Crunchy and slightly spicy, I tried a few before throwing the rest into boiling water from where I could clearly hear them snapping. Pop. Pop. Pop. Cooked, they're just as good. They retain some of their crunch but lose a bit of the spice and become sweeter. Since I didn't really know what to do with them, I tossed them with a simple vinegar, olive oil and chives vinaigrette, which worked well. Now I want to go back for more and try to prepare them in other ways.
For now, here's a not-too-precise explanation of how I prepared my Dragon Tongue beans.
Dragon Tongue beans
Snap off the ends of the beans and wash. Bring a large pot of salted water to a boil. Add the beans and cook for 3-4 minutes. While the beans cook, fill a large bowl halfway with ice and cold water. When the beans are done, remove the pot from the heat, take out the beans with a slotted spoon and place them in the ice bath for about 3 minutes. Remove the beans from the ice water and lay out to dry on a paper towel or do like me and let them air dry in a colander.
For the vinaigrette, I mixed about 1 tablespoon of white wine vinegar with salt, pepper, chopped chives and a dash of sugar. Then I slowly added about 2 1/2 tablespoons of olive oil and whisked everything together. I did this very much to taste. Toss with the beans and serve.


text:
Udyr Humor Guide by mordhuehue
Not Updated For Current Season
This guide has not yet been updated for the current season. Please keep this in mind while reading. You can see the most recently updated guides on the browse guides page.
Thank You!
Your votes and comments encourage our guide authors to continue
creating helpful guides for the League of Legends community.
The True Udyr Build (Patch 999
Udyr Build
Jungle Role
Ability Sequence
Not Updated For Current Season
The masteries shown here are not yet updated for the current season, the guide author needs to set up the new masteries. As such, they will be different than the masteries you see in-game.
Threats to Udyr with this build
|this person is a fucking migget faggot whore nigger jew monkey sheephorse make sure to use the combo below
I hope you learned my tips and trix on my best jungle udyr
i am a very good challanger smurf elo guide and i show you how to play enjoy !
remember always farm and never gank!
Creeping / Jungling
on Udyr you wanna make sure to get a ancient coin so you can get movespeed and also im diamond player very good yes
remember to always farm and **** you know cause thats what trick28 does and you should too.
I am master tier
good bye
i pick clayovoronce becuase it looks nice and gets u viision anywhere you like yes and stuff
go check my mastery pls and rune cause they can really help you in the late game. i run ghost becuase i like the color blue and it's really cool how you can ghost and e it's amazing man
Skill Sequence
make sure to use all ur moves ok? are you still reading i hope you are ty i give u an eggsample of a udyr combo ok? look down below.
e - e - e - e - e - e - e - e - e - e - e - e - e - e - e - e - e - e - e - e - e - e - e - e - e - e - e - e - e - e that is the combo!
Team Work
league of legends does not require teamwork ok? yes yes yes
just make sure to not gank and you can win the game
you will not reported by team ok?


text:
Product Features
*Available for US Shipment Only.
If you like to be tricky and keep others off balance the Fox Sedated T-shirt is for you. You'll look good and won't believe how relaxed and calm you are. Then you can dive into action backed up by the mixture of cotton and polyester that let's you push the envelope.
Key Features of Fox Sedated T-Shirt:
- 50% Cotton / 50% Polyester Combed Ringspun Jersey
- 30/1 155g
- Screen print on front
All Gear by: Fox


text:
Mike The Knight And The Invisible Monster
Format: Paperback
Free Click & Collect
Check availability in your local store
Free Delivery Available
- Free Standard over £20
- Standard delivery £2.99
- Next working day £4.99
Product Information
Details:
 Theres a scary noise coming from below the bridge in the village so Mike vows to protect the people of Glendragon from whatever it is. But Mike enjoys the villagers praise so much, he puts off doing anything about the monster! Will Mike remember his mission and save the day?

 Ideal for:
 A brilliant picture book for children aged between 2 to 5 years.

 T
Stress-Free Returns
Subject to exceptions, we are happy to exchange or refund your purchase within 28 days of delivery. You can return items by post or to one of our stores.Go to full returns information
Together Rewards
5 points for every pound you spend
Rewards Points PLUS Great Prices - YES! We're proud to give even more back to you and reward all Together Rewards Card members with 5 points for every £1 spent, along with exclusive offers, bonus points promotions, member exclusive gifts and more! Interested? Order a card with your purchase and once you've registered and linked your Card, you'll receive 50 FREE points (worth 50p) and you'll receive Reward Vouchers every 3 months to spend on anything you like in-store and online! Click here to find out how to get your Together Card.