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Now that Bernie Sanders has brought forward his progressive agenda, including real health care reform through a single-payer universal Medicare for all program, the knives are out from the Republicans as well as Hillary Clinton to distort and discredit this option with false information and voodoo numbers.
Rationing, the hated R-word, evokes widespread resentment and debate whenever mentioned in connection with health care in the U. S. There are many who hold that we don't ration care now, never want to, and that the free market will work its magic and be fair to all without rationing if we just keep the government out of health care.
There is a pervasive myth generated by conservatives that the private sector is more efficient and less bureaucratic than government. This is perpetuated by those seeking to continue the "free market" in U. S. health care, which simultaneously exploits public programs through privatization of Medicare and Medicaid.
While it is tempting to look at just one metric--the decline in numbers of the uninsured, this is a trap if used to deceive ourselves as to the success of the ACA. As the above examples indicate, we still have a long way to go before we can say that we have reformed U. S. health care in the public interest.
You might think that we learned the lesson of discredited managed care in the 1990s. The term "managed care" is confusing to many, but really amounts to managed reimbursement rather than managed care, whereby a set prospective annual payment is made by federal/state governments, as in the case of Medicaid managed care (MMC), to cover whatever services patients will receive over the coming year.


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Wholesale Case Top Speed Police Mini Cooper Electric RTR RC Car (6 Units at $8.50 Each)
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Sorry ,,getting to this revue a little late.
I liked this hotel a lot. It was very clean and modern. The suite was very nice..big in size. It would have probably have been bigger but we had a room with 2 beds instead of one king. We had no choice in rooms because we booked thru expedia/hotwire. We did ask at the front desk but unfortunately none were available. Anyway, room had a micro and a fridge and a desk etc.
The front desk person was extremely nice and welcoming. The lobby was lovely and 24 hour coffee. We enjoyed the pool and Jacuzzi. Overall we had a very pleasant stay although we were only there one night we would definitely recommend this hotel!!
- Official Description (provided by the hotel):
- When it comes to hotel accommodations in the Northwest Austin area, your Choice is clear. The recently renovated Comfort Suites NW Lakeline has everything you need, and nothing you don't. No matter what is bringing you to the Austin area, the location of our property will put you within a 15 mile radius of some of Central Texas's favorite attractions; just a few would include the Cedar Park Center, the state Capitol, Lake Travis, the Domain, Zilker Park, Austin Convention Center, Circuit of Americas track, and Dell Diamond. At this property, we offer all Suite accommodations with King or Two Queen beds. All of our rooms are equipped with a mini fridge and microwave. A wide range of other amenities are offered on site like, free hot Breakfast every morning, complimentary Wi-Fi, Business Center, Fitness Center, Guest Laundry, and an outdoor Pool and Spa. Come treat yourself to the Comfort Suites NW Lakeline, and experience the service and comfort you want, at a price you can afford. ... more less
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For questions about the biological aspects of alcohol, such as metabolism, mechanisms of effect, etc. Questions should be carefully written to focus on the biology. Chemistry questions are off-topic.
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What are the survival limitations of alcohol?
This question was inspired by watching one of the Pirates of the Caribbean movies; particularly where Jack Sparrow allegedly survives on a desert island by finding an unlimited supply of rum. I've ...
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Why does alcohol cause the hemolysis of RBC in a large proportion?
I had today an experiment that we put 95% alcohol to the blood which made it completely transparent so hemolysis must have occurred. I started to think about the reasons. I think that this is because ...


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Our new cash office, "Bayangol Duureg" cash office, started its operation from 28th March 2016 at Bayangol District's "Center of Capital Service"
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Steve Zuckerman | Resume | Photos
Enhance your page with a resume!
Steve Zuckerman
Mr. Zuckerman's most recent theatrical project is the premiere of Garry Marshall's EVERYBODY SAY CHEESE at the Falcon Theatre in Los Angeles. He directed Shem Bitterman's MAN.GOV and IN HARM' S WAY which received excellent reviews in Los Angeles at Circus Theatricals and were presented in NY in the Fall of 2008. He also just mounted Michael Zettler's WAITING FOR MERT at the Yale Cabaret Hollywood. Mr. Zuckerman has directed NUTS by Tom Topor on Broadway which received two Tony nominations and a Drama Desk Award. As Associate Director of New York's WPA
theater he directed many productions including the premieres of NUTS, Steve Metcalf's THE INCREDIBLY FAMOUS WILLY RIVERS, Shem Bitterman's TEN BELOW and Israel Horovitz's UNEXPECTED TENDERNESS and NORTH SHORE FISH. His collaboration with Mr. Horovitz also resulted in the Off Broadway premieres of TODAY I AM A FOUNTAIN PEN, A ROSEN BY ANY OTHER NAME and THE CHOPIN PLAYOFFS. He directed a filmed version of NORTH SHORE FISH for Showtime which starred Mercedes Ruehl, Peter Riegert and Tony Danza. His revivals of Tennessee
Williams' VIEUX CARRE and Ibsen's WHEN WE DEAD AWAKEN with Kim Hunter both received Drama Desk Awards. He has been Director of Play Development at The Circle in the Square Theatre and Artistic Director of the Impossible Ragtime Theatre in New York and the Sharon Playhouse in Connecticut. He has directed at the Manhattan Theater Club, Circle Rep and many regional theatres including Baltimore's Center Stage, The La Jolla Playhouse and the Yale Cabaret. Mr.
Zuckerman was trained at the University of Michigan and the Yale School of Drama.
IMDb is not responsible for the accuracy or completeness of the contents of this page, which have been supplied by a third party and have not been screened or verified.


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Are Lobsters Ambidextrous?: An Imponderables Book (Paperback)
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Ponder, if you will ...Has anyone ever seen a live Cornish game hen?Why do quarterbacks say "Hut"?Why do "sea" gulls congregate in parking lots of shopping centers?What does the "Q" in Q-Tips stand for?
Pop culture guru David Feldman demystifies these questions and much more in Are Lobsters Ambidextrous? Part of the Imponderables(r) series the unchallenged source of answers to civilization's everyday mysteries and charmingly illustrated by Kassie Schwan, this book provides you with information that encyclopedias, dictionaries, and almanacs just don't have. And think about it, where else are you going to find out what happens to the caffeine left over from making decaffeinated coffee?
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Feldman is the author of the Imponderables series, as well as Who Put the Butter in Butterfly? and How to Win at Just About Everything. He has a master's degree in popular culture from Bowling Green State University in Ohio and consults and lectures on the media. He lives in New York City.


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December 13th, 2012
Re: Isay and the APsaA — as a former psych student, I can testify to the innate conservatism of many psychoanalysts. For many of them, Freud is closer to being god than God is, and he has a set of anointed disciples whose theories set the parameters of the discipline for decades. Even though Freud himself never formulated a theory of homosexuality, the disciples did.
And don't forget that many of them were deriving substantial income from treating gay patients.
Ben in Oakland
"I was having fun and this dude ended up dying." If ever there was a psychopathic personality, helvey is its epitome– or it's nadir. He should never, EVER be released, or permitted among even barbaric human beings.
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In this original BTB Investigation, we unveil the tragic story of Kirk Murphy, a four-year-old boy who was treated for "cross-gender disturbance" in 1970 by a young grad student by the name of George Rekers. This story is a stark reminder that there are severe and damaging consequences when therapists try to ensure that boys will be boys.
When we first reported on three American anti-gay activists traveling to Kampala for a three-day conference, we had no idea that it would be the first report of a long string of events leading to a proposal to institute the death penalty for LGBT people. But that is exactly what happened. In this report, we review our collection of more than 500 posts to tell the story of one nation's embrace of hatred toward gay people. This report will be updated continuously as events continue to unfold. Check here for the latest updates.
In 2005, the Southern Poverty Law Center wrote that "[Paul] Cameron's 'science' echoes Nazi Germany." What the SPLC didn"t know was Cameron doesn't just "echo" Nazi Germany. He quoted extensively from one of the Final Solution's architects. This puts his fascination with quarantines, mandatory tattoos, and extermination being a "plausible idea" in a whole new and deeply disturbing light.
On February 10, I attended an all-day "Love Won Out" ex-gay conference in Phoenix, put on by Focus on the Family and Exodus International. In this series of reports, I talk about what I learned there: the people who go to these conferences, the things that they hear, and what this all means for them, their families and for the rest of us.
Prologue: Why I Went To "Love Won Out"
Part 1: What's Love Got To Do With It?
Part 2: Parents Struggle With "No Exceptions"
Part 3: A Whole New Dialect
Part 4: It Depends On How The Meaning of the Word "Change" Changes
Part 5: A Candid Explanation For "Change"
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And don't miss our companion report, How To Write An Anti-Gay Tract In Fifteen Easy Steps.
Anti-gay activists often charge that gay men and women pose a threat to children. In this report, we explore the supposed connection between homosexuality and child sexual abuse, the conclusions reached by the most knowledgeable professionals in the field, and how anti-gay activists continue to ignore their findings. This has tremendous consequences, not just for gay men and women, but more importantly for the safety of all our children.
Anti-gay activists often cite the "Dutch Study" to claim that gay unions last only about 1½ years and that the these men have an average of eight additional partners per year outside of their steady relationship. In this report, we will take you step by step into the study to see whether the claims are true.
Tony Perkins' Family Research Council submitted an Amicus Brief to the Maryland Court of Appeals as that court prepared to consider the issue of gay marriage. We examine just one small section of that brief to reveal the junk science and fraudulent claims of the Family "Research" Council.
The FBI's annual Hate Crime Statistics aren't as complete as they ought to be, and their report for 2004 was no exception. In fact, their most recent report has quite a few glaring holes. Holes big enough for Daniel Fetty to fall through.


text:
MIAMI -- Florida International's 3-year-old football program
was placed on three years probation by the NCAA on Monday after an
internal investigation revealed an assistant coach committed
offseason football workout violations.
The unidentified assistant coach -- fired by the school --
supervised and conducted offseason practice, illegal under NCAA
rules. The sanctions don't include any loss of scholarships or
reductions in postseason play or television appearances.
"It is my sense that the NCAA's decision was to give us one
year of probation for each of the seasons in which a violation
occurred," FIU athletic director Rick Mello said. "We are
relieved to have all of this behind us and that no student-athletes
will be negatively impacted."
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"It was very important to our institution as we went through
this procedure that we set a serious tone for the future of our
program," Mello said.


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This course presents law as an evolving social institution, with special emphasis on the legal regulation of business. It considers basic concepts of law and legal process, in the U.S. and other legal systems, and introduces the fundamentals of rigorous legal analysis. An in-depth examination of contract law is included.
Legal Studies & Business Ethics
This course introduces students to important legal and ethical challenges they may face in business. Its focus includes theories of ethics and their application to case studies in business, including current business ethics events. We begin with questions about individual value, purpose, and responsibility. We then look at concrete questions about the obligations of corporations, managers and employees. Do corporations have any obligations besides making money for their shareholders? If a multinational operates in a country where child labor is the norm, does that make it acceptable for the company to hire children? The material covered is intended to help prepare students to recognize and manage ethical issues as they arise. Class sessions will consist of collaborative case discussions, exercises, debates, and discussions of theoretical frameworks for interpreting ethics. An emphasis will be placed on class discussion.


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Mononuclear cytotrophoblasts of the human placenta proliferate rapidly, subsequently fuse, and differentiate to form multinucleated syncytiotrophoblast with induction of aromatase (hCYP19A1) and chorionic gonadotropin (hCGβ) expression. Using microarray analysis, we identified members of the miR-17~92 cluster and its paralogs, miR-106a~363 and miR-106b~25, that are significantly downregulated upon syncytiotrophoblast differentiation. Interestingly, miR-19b and miR-106a directly targeted hCYP19A1 expression, while miR-19b also targeted human GCM1 (hGCM1), a transcription factor critical for mouse labyrinthine trophoblast development. Overexpression of these microRNAs (miRNAs) impaired syncytiotrophoblast differentiation. hGCM1 knockdown decreased hCYP19A1 and hCGβ expression, substantiating its important role in human trophoblast differentiation. Expression of the c-Myc proto-oncogene was increased in proliferating cytotrophoblasts compared to that in differentiated syncytiotrophoblast. Moreover, c-Myc overexpression upregulated miR-17~92 and inhibited hCYP19A1 and hCGβ expression. Binding of endogenous c-Myc to genomic regions upstream of the miR-17~92 and miR-106a~363 clusters in cytotrophoblasts dramatically decreased upon syncytiotrophoblast differentiation. Intriguingly, we observed higher levels of miR-106a and -19b and lower aromatase and hGCM1 expression in placentas from preeclamptic women than in placentas from gestation-matched normotensive women. Our findings reveal that c-Myc-regulated members of the miR-17~92 and miR-106a~363 clusters inhibit trophoblast differentiation by repressing hGCM1 and hCYP19A1 and suggest that aberrant regulation of these miRNAs may contribute to the pathogenesis of preeclampsia.
The multinucleated syncytiotrophoblast of the human placenta is formed by fusion of underlying proliferating cytotrophoblasts. This multinucleated cell layer, which covers the chorionic villi, is bathed in maternal blood and performs several essential functions to ensure growth and survival of the developing embryo. These include transport of O2 and nutrients and synthesis and secretion of syncytiotrophoblast-specific protein and steroid hormones, including estrogen and progesterone. Synthesis of estrogens from C19 steroids is catalyzed by aromatase P450 (P450arom, product of the hCYP19A1 gene). The ability of the human placenta to synthesize estrogens is vastly increased after the ninth week of gestation (1), in association with cytotrophoblast invasion and enlargement of the uterine arterioles, increased blood flow, and O2 availability to the floating chorionic villi (2, 3). Trophoblast stem cells and cytotrophoblasts do not express hCYP19A1/aromatase; however, when cytotrophoblasts fuse to form multinucleated syncytiotrophoblast, aromatase is markedly induced (4, 5). The exceptionally high levels of placental aromatase likely function to metabolize large amounts of C19 steroids produced by the human fetal adrenals (e.g., dehydroepiandrosterone), thus preventing conversion of these steroids to active androgens, which can masculinize the fetus. Biologically active estrogens and their metabolites formed by placental aromatase may also enhance angiogenesis and uteroplacental blood flow and reduce systemic vascular resistance (6–10).
While different genetic programs have been found to be switched on and off during syncytiotrophoblast differentiation, the regulatory mechanisms that affect these events remain incompletely defined. We previously observed that when midgestation human trophoblasts were cultured in a hypoxic (2% O2) environment, syncytiotrophoblast differentiation and induction of hCYP19 gene expression were prevented (3). These inhibitory effects of hypoxia were associated with increased expression and binding of the basic helix-loop-helix (bHLH)-zipper transcription factors USF1 and -2 to two E boxes within placenta-specific hCYP19 exon I.1 and its 5′-flanking region (11). Conversely, increased O2 tension promoted enhanced USF1/2 polyubiquitination and degradation via the proteasome pathway (12). We therefore suggest that with the gestational increase in placental vascularization and increased O2 availability to trophoblast cells, the inhibitory USFs are degraded, which may allow stimulatory transcription factors to bind to these E boxes and/or to other response elements and promote placenta-specific expression. Placental estrogens are believed to play an autocrine role in trophoblast differentiation (13, 14). In recent studies, we have established crucial roles for estrogen receptor α (ERα) (15) and estrogen-related receptor γ (ERRγ) (16) in the O2-mediated induction of CYP19 expression during human trophoblast differentiation.
The transcription factor glial cells missing 1 (GCM1) has been shown to play a fundamental role in trophoblast differentiation and syncytiotrophoblast formation. GCM1 knockout mice die at midgestation due to the failure of a functional placental labyrinth, analogous to the human syncytiotrophoblast layer (17, 18). Human GCM1 (hGCM1) induces the expression of syncytin A, a gene involved in trophoblast fusion (19), and regulates expression of placental growth factor (20). While the hCYP19A1 promoter has been shown to have a GCM1 binding element that is required for reporter activity (21), a critical role for endogenous hGCM1 in the regulation of CYP19A1 expression in human syncytiotrophoblast has yet to be established.
MicroRNAs (miRNAs) are regulatory, small noncoding RNAs of 20 to 25 nucleotides (nt) in length, with a fundamental role in different aspects of cell biology, including cell cycle regulation, differentiation, apoptosis, and maintaining stemness (22). MicroRNAs function to repress gene expression by binding via their seed sequences to complementary sites in the 3′ untranslated regions (3′ UTR) of target mRNAs. This results in degradation of the mRNA target and/or inhibition of translation (23). Individual miRNAs can bind to and regulate networks of mRNAs with related biological functions and can act as rheostats or as on-off switches of gene expression, while multiple miRNAs can target a single mRNA. MicroRNAs have been found to play important roles in cell differentiation (24–26) and cancer (27, 28) and have been found to have particularly important roles in female reproduction (29–31).
Thus, to further define the cellular mechanisms that underlie human trophoblast differentiation and induction of CYP19A1 gene expression, in the present study, we have investigated the potential role of miRNAs. Interestingly, we have uncovered a novel role for the c-Myc-regulated microRNA-17~92 (miR-17~92) cluster and its paralog miR-106a~363 in directly targeting and inhibiting hCYP19A1 and hGCM1 gene expression in cytotrophoblasts and thereby repressing human trophoblast differentiation and aromatase expression. We suggest that dysregulation of this pathway may have an important consequence in the pathogenesis of preeclampsia, a pregnancy-induced hypertensive disorder in which the placenta plays a central role.
Term placental tissues from control normotensive women and preeclamptic women were obtained at the time of cesarean section. The placental tissues were immediately placed in RNAlater (Qiagen, Foster City, CA) solution and snap-frozen at −80°C.
Midtrimester human placental tissues were obtained from Advanced Bioscience Resources (Alameda, CA) in accordance with the Donors Anatomical Gift Act of the State of Texas. Protocols were approved by the Institutional Review Board of the University of Texas Southwestern Medical Center at Dallas. Cytotrophoblasts were isolated from midgestation human placenta and placed in primary culture, as described in detail previously (5, 32). Briefly, the placental tissues were washed with Hanks' balanced salt solution (pH 7.4) (Life Technologies, Grand Island, NY) and then finely minced and digested with 0.125% trypsin in Hanks' balanced salt solution at 37°C for 20 min. At the end of the digestion, the supernatant was collected, layered over 10 ml of serum, and then briefly centrifuged at 1,000 × g. The resulting pellet was suspended in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies), filtered, and layered over a Percoll gradient (70% to 5%). This procedure was repeated three times with the pellet from the original trypsin digestion. The gradients were centrifuged at 1,200 × g for 20 min at room temperature, and cells in the middle layer (density, 1.045 to 1.062 g/ml) were collected, washed, and counted. The cells were then resuspended in DMEM supplemented with 10% fetal bovine serum (FBS) and 1.2% antibiotic/antimycotic solution (Life Technologies) and plated at a density of 2 × 106 cells per 35-mm culture dish or 15 × 106 cells per 100-mm dish. The cells were cultured overnight; the medium was then changed to DMEM containing 2% FBS. For expression of miRNA mimics or miR-19b inhibitor, freshly isolated cytotrophoblasts were transfected with either 10 nM miRNA mimic (Qiagen), LNA hsa-miR-19 family inhibitor (Exiqon, Woburn, MA), LNA scrambled control (Exiqon), or AllStars Negative control (Qiagen) using the HiPerfect transfection reagent (Qiagen). Cells were harvested for RNA and protein analysis 24, 48, and 72 h posttransfection.
Total RNA, including small RNAs, from trophoblast cells cultured for 24, 48, or 72 h was extracted using the miRNeasy minikit (Qiagen) according to the manufacturer's instructions. Quantification of miRNAs was carried out by TaqMan real-time PCR (Applied Biosystems). Primer sets specific for the placenta-specific hCYP19A1 transcript, hCYP19I.1 (15), along with constitutively expressed RPLP0, were designed utilizing the Primer Express software program (PE Applied Biosystems, Boston, MA): hCYP19I.1 forward primer, 5′-ACG GAA GGT CCT GTG CTC G-3′; reverse primer, 5′-GTA TCG GGT TCA GCA TTT CCA-3′; RPLP0 forward primer, 5′-TGC ATC AGT ACC CCA TTC TAT CA-3′; reverse primer, 5′-AAG GTG TAA TCC GTC TCC ACA GA-3′.
The relative abundance of each transcript was determined by qRT-PCR using previously published methods (15). All primer sets produced amplicons of the expected size and sequence. The relative fold changes were calculated using the comparative cycle times (CT) method with RPLP0 as the internal reference.
Nuclear and cytoplasmic extracts were prepared from human trophoblast cells according to a protocol described previously (15). Protein concentrations were determined by using the Bradford assay (33) (Bio-Rad) and resolved by electrophoresis on 4 to 12% Bis-Tris gels (Invitrogen). Rabbit polyclonal c-Myc antibody (9402) was obtained from Cell Signaling (Beverly, MA). Antiaromatase (A7981) was obtained from Sigma (St. Louis, MO), anti-GCM1 from Aviva Systems Biology (San Diego, CA), anti-lamin A/C (catalog no. 05-714) from Upstate (Lake Placid, NY), and anti-β-actin (ab8227) from Abcam (Cambridge, MA). Horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse IgGs from GE Healthcare (Little Chalfont, Buckinghamshire, United Kingdom) were used as secondary antibodies. The membranes were incubated with enhanced Supersignal West Pico chemiluminescent substrate (Thermo Scientific, Rockford, IL) and exposed to X-ray film.
Twelve hundred base pairs of the 3′ UTR of human CYP19A1 and 500 bp of the 3′ UTR of the human GCM1 were amplified from placental genomic DNA and cloned downstream of luciferase in the p-MIR-REPORT miRNA reporter vector (Applied Biosystems). JEG3 cells maintained in RPMI medium supplemented with 10% fetal bovine serum were transfected with either the empty pMIR-REPORT luciferase plasmid (LUC) or with LUC containing the 3′ UTR of the CYP19A1 or hGCM1 gene (LUC-CYP19A1 or LUC-GCM1) with or without the mutated miRNA response element (MRE) (LUC-CYPmut or LUC-GCMmut). Nontargeting control (NTC) or miRNA mimics of miR-19b or miR-106a and a plasmid expressing beta-galactosidase (β-Gal) to correct for transfection efficiency were cotransfected. Forty-eight hours after transfection, cells were harvested and luciferase and β-Gal activities were measured. The mutation of the MRE site in placenta-specific hCYP19 3′ UTR was generated using the QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA). All constructs were confirmed by DNA sequencing.
Recombinant adenoviruses expressing human c-Myc were obtained from Vector Biolabs (Philadelphia, PA). A cytomegalovirus β-gal-containing (CMV-β-gal) adenovirus was kindly provided by Joseph Alcorn (University of Texas Medical School, Houston, TX). Recombinant adenoviruses were used to infect freshly isolated cytotrophoblasts at a multiplicity of infection (MOI) of 2.0. Lentiviral vectors containing short hairpin RNAs (shRNAs) targeting GCM-1 in pGIPZ vectors were obtained from a recombinant lentivirus shRNAmir library (Open Biosystems) (34). Lentiviruses expressing shRNAs for GCM1 were produced in human embryonic kidney (HEK)-293T cells by calcium phosphate transfection of the plasmids pMD2.G and psPAX2 and specific human GIPZ lentiviral shRNA for GCM1, according to a protocol described earlier (16). Lentiviral supernatants were collected after 24, 48, and 72 h of culture. The viral particles were concentrated by ultracentrifugation, and the titer of the viral stock (MOI or number of transducing units per cell) was determined according to the protocol recommended by Open Biosystems (Thermo Scientific). As a control, cells were infected with nonsilencing GIPZ lentivirus shRNA. Two different shRNA vectors were used for silencing of GCM-1. The following GCM1 shRNA clones were used: Open Biosystems catalog no. RHS4430-99166548 and RHS4430-98703843.
Human trophoblasts were cultured for 5 h or 72 h in DMEM containing 2% FBS. Utilizing the ChIP kit (Millipore, Temecula, CA), chromatin immunoprecipitation (ChIP) assays were performed as described previously (15). Precleared cross-linked chromatin was immunoprecipitated using either c-Myc antibody or nonimmune rabbit IgG control (sc-2027; Santa Cruz Biotechnology, Santa Cruz, CA). Quantitative RT-PCR using primers that flanked E boxes corresponding to putative Myc-responsive elements (identified by using the Genomatrix MatInspector program; Genomatrix Software GmbH, Munich, Germany) in the genomic regions of the miR-17~92 and the miR-106a~363 clusters was used to assess the fold enrichment of the immunoprecipitated protein-DNA complex for endogenous c-Myc. Primers used in this assay were the following: miR-17~92 E box 1f, AAAGGCAGGCTCGTCGTTG; miR-17~92 E box 1r, CGGGATAAAGAGTTGTTTCTCCAA; miR-17~92 E box 2f, CTCGACTCTTACTCTCACAAATGG; miR-17~92 E box 2r, GCTACTGGTGCAGTTAGGTCC; miR-106A~3631f, GAGAGGGGGAGTCCAAAATC; miR-106A~3631r, TGGTTTCAACCAAATCCTGA; miR-106A~3632f, GACTGGAGCGCGTGGTGTGA; miR-106A~3632r, CTCCCGTTGAAGTCCGCGAGC.
Quantitative PCR was performed using SYBR green PCR master mix (Applied Biosystems) on a Bio-Rad CFX384 real-time PCR detection system. Signals were normalized to input samples and expressed relative to that for control IgG. Samples from three independent ChIP experiments were analyzed.
Data are expressed as means ± standard deviations (SD). Differences between groups were analyzed by Student's t test. Statistical significance was set as P values of <0.05, and each experiment was performed at least three times (from three independent cell preparations) in triplicate.
To identify miRNAs that are differentially regulated during human trophoblast differentiation, RNA isolated from human cytotrophoblasts before (0 h) and after 24 and 48 h of culture was sent to LC Sciences (Houston, TX), which carried out miRNA microarray using quadruple replicates of the 0-h-, 24-h-, and 48-h-of-incubation time points. We detected 16 miRs that were significantly downregulated and 9 miRs that were significantly upregulated >2-fold at both 24 and 48 h, compared to results at 0 h (a heat map of all significantly regulated miRNAs is shown in Fig. S1 in the supplemental material). Shown in Fig. 1A is a heat map of several members of the miR-17~92 cluster and its paralogs miR-106a~363 and miR-106b~25 that were found to be significantly downregulated during human trophoblast differentiation. In the human genome, the miR-17~92 cluster encodes six miRNAs (miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92a-1), while its paralog, the miR-106a~363 cluster, also encodes six miRNAs (miR-106a, miR-18b, miR-20b, miR-19b-2, miR-92a-2, and miR-363). Using TargetScan v5.1 analysis, we identified several predicted and confirmed mRNA targets of these miRs (Fig. 1B). ERα and SRC-3 are known targets of the miR-17~92 and miR-106a~363 clusters (35, 36). MicroRNAs in these clusters that share similar seed sequences are grouped in Fig. 1C. miR-106a and miR-19b are predicted to target hCYP19A1 mRNA. In previous studies, we observed that hCYP19A1 expression is highly upregulated during syncytiotrophoblast differentiation (5) and the estrogens formed via the action of hCYP19A1/aromatase act in a positive feed-forward manner through ERα to further promote syncytiotrophoblast differentiation and the induction of hCYP19 expression (15). Furthermore, miR-19b, a member of the miR-17~92 cluster, is also predicted to target hGCM1, a transcription factor critical for trophoblast differentiation (18) and upregulation of hCYP19I.1 promoter activity (21). As hCYP19I.1 expression increased during syncytiotrophoblast differentiation (Fig. 1D), downregulation of several members of the miR-17~92 cluster and its paralog miR-106a~363 was confirmed in cultured trophoblasts using TaqMan-based RT-qPCR (Fig. 1E). Interestingly, JEG-3 cells, which express low levels of aromatase and are cytotrophoblast-like, manifested significantly higher expression of these miRNAs than syncytiotrophoblast (Fig. 1D and andE).E). Thus, we have clearly demonstrated that members of the miR-17~92 cluster and its paralog miR-106a~363 are highly expressed in cytotrophoblasts and their expression decreases as the cells differentiate to form syncytiotrophoblast.
Since miR-17, -20a, and -106b are predicted to target hCYP19A1 mRNA, contain identical seed sequences, and were significantly downregulated along with miR-19b during syncytiotrophoblast differentiation, we initially analyzed the effects of overexpression of miR-19b and -106a on their predicted target, hCYP19A1. Nontargeting control (NTC) or miRNA mimics of miR-19b or -106a were transfected into freshly isolated placental trophoblasts. After 72 h of culture, the cells were harvested for RNA; although TaqMan qRT-PCR analysis revealed a 100-fold increase in either miR-19b or miR-106a expression (data not shown), a significant decrease in hCYP19I.1 mRNA expression was observed in cells transfected with miR-19b but not in cells transfected with nontargeting control or miR-106a (Fig. 2A). Since miRNAs are also known to inhibit mRNA translation, we performed immunoblot analysis of aromatase and observed that overexpression of either miR-19b and -106a greatly decreased endogenous aromatase protein expression in syncytiotrophoblast (Fig. 2B, top panel). The immunoblot results of three independent experiments quantitated using the software program Image J demonstrated that these miRNAs significantly decreased aromatase protein expression (Fig. 2B, bottom panel). To inhibit endogenous miR-19b expression, LNA scrambled control (Con) or LNA miR-19 inhibitors were transfected into freshly isolated trophoblasts. After 72 h, the cells were analyzed for expression of miRNA by TaqMan qRT-PCR analysis and aromatase protein by immunoblot analysis. Inhibition of miR-19b specifically decreased miR-19b (and had no effect on miR-106a expression) and increased aromatase protein expression compared to results for cells transfected with a LNA scrambled control (Fig. 2C). Using TargetScan prediction software, we identified 2 binding sites for miR-17, -20a, and -106a and one for miR-19b in the 3′ UTR of hCYP19A1 mRNA, as shown in Fig. 2D. To demonstrate that miR-106a and miR-19b directly regulate hCYP19A1 expression, we transfected a reporter construct containing the hCYP19A1 3′ UTR subcloned downstream of the luciferase gene (LUC/CYP19) together with miRNA mimics into JEG-3 cells and observed a significant reduction in luciferase activity compared to results for cells transfected with nontargeting control miRNA (Fig. 2D). Notably, no reduction in luciferase activity was detected when the putative miRNA binding sequence in the hCYP19A1 3′ UTR luciferase reporter construct was mutated (LUC/CYP19mut) (Fig. 2D). These data suggest that hCYP19A1 expression is negatively regulated in human trophoblasts via direct interaction of both miR-106a and miR-19b miRNA response elements in the hCYP19A1 3′ UTR.
As mentioned, GCM1 is a transcription factor critical for trophoblast differentiation and fusion (18), as well as hCYP19I.1 expression (21). Accordingly, we observed that hGCM1 protein expression increased rapidly in cultured human trophoblasts, reaching peak levels at 24 h. Although GCM1 expression declined at later time points, it remained at markedly higher levels than in the cytotrophoblasts prior to culture (Fig. 3A). Since miR-19b is predicted to target hGCM1, mimics of miR-19b, -106a, or NTC were transfected into freshly isolated human trophoblasts. After 72 h of culture, the cells were harvested and analyzed for hGCM1 mRNA and protein. While qRT-PCR analysis revealed no change in hGCM1 mRNA expression, overexpression of miR-19b and -106a mimics caused a significant decrease in human chorionic gonadotropin β (hCGβ) mRNA, a marker of syncytiotrophoblast differentiation (Fig. 3B). However, immunoblot analysis revealed that overexpression of miR-19b but not miR-106a markedly decreased endogenous hGCM1 protein expression in syncytiotrophoblast (Fig. 3C, top panel). The inhibitory effect of miR-19b on hGCM1 protein expression was confirmed by Image J analysis of immunoblots from three independent experiments (Fig. 3C, bottom panel). In contrast, transfection of cultured human trophoblasts with the LNA miR-19b inhibitor increased GCM1 protein expression in cells compared to that in cells transfected with LNA scrambled control (Con) (Fig. 3D). Thus, miR-19b targets hGCM1 in human trophoblasts. While hCGβ is not a predicted target of these miRNAs, its expression was probably decreased by miR-19b and miR-106a transfection as a consequence of reduced expression of either hGCM1 or hCYP19.
To demonstrate that miR-19b directly targets hGCM1 mRNA, we transfected a hGCM1 3′ UTR luciferase reporter construct (LUC/GCM) together with miRNA mimics into JEG-3 cells and observed a significant reduction in luciferase activity in cells cotransfected with miR-19b, compared to that in cells cotransfected with nontargeting control (Fig. 3E). On the other hand, no reduction in luciferase activity was detected upon cotransfection of miR-19b when the putative miR binding sequence in the hGCM1 3′ UTR luciferase reporter construct was mutated (LUC/GCMmut) (Fig. 3E). These findings suggest that hGCM1 expression is translationally suppressed in human cytotrophoblasts by increased levels of miR-19b binding to a response element in its 3′ UTR.
Having established that miR-19b directly targets hGCM1 expression in human trophoblasts, we further investigated the functional role of endogenous hGCM1. While cotransfection studies in a choriocarcinoma cells line indicated that hCYP19I.1 promoter activity was upregulated by hGCM1 (21), a role for endogenous hGCM1 in the regulation of hCYP19A1 in primary human syncytiotrophoblast has yet to be established. Therefore, we utilized an RNA interference approach to knock down hGCM1. Two different lentivirus constructs carrying hGCM1-targeting shRNA (GCM shRNA) were used to infect freshly isolated human cytotrophoblasts at a multiplicity of infection (MOI) of 0.5 and 1.0. Parallel dishes of cells were infected with lentiviruses expressing a nontargeting shRNA, as a control. After 72 h of culture, a significant knockdown in hGCM1 mRNA and protein expression was observed. This was associated with a corresponding decrease in hCYP19I.1 and hCGβ mRNA expression, compared to results for cells infected with control lentivirus (Fig. 4A). A decrease in endogenous hGCM1 protein in cells infected with lentiviruses expressing hGCM1 shRNA was confirmed by immunoblot analysis (Fig. 4B). These findings clearly identify an essential role for hGCM1 in regulating hCYP19I.1 and hCGβ expression during human trophoblast differentiation and suggest that miR-19b-mediated repression of hGCM1 in cytotrophoblasts contributes to suppression of hCYP19I.1 and hCGβ expression.
c-Myc belongs to a family of helix-loop-helix/leucine zipper transcription factors and together with its obligatory binding partner, Max, regulates cell proliferation, transformation, growth, differentiation and apoptosis (37). Increased c-Myc expression is evident in the proliferative cytotrophoblasts of human placenta (38). Interestingly, studies have shown that c-Myc directly binds and activates expression of the miR-17~92 cluster in immortalized B lymphocytes (P493-6 cells) stably expressing a tetracycline-regulated c-Myc transgene (39). Thus, we explored a role for c-Myc in differentiation of human primary trophoblast cells in culture. During trophoblast differentiation, c-Myc mRNA (Fig. 5A) and protein expression (Fig. 5B) were markedly decreased. To determine whether c-Myc overexpression in cultured trophoblast cells altered expression of miR-17~92 and miR-106a~363 family members and whether this occurred at the transcriptional level, freshly isolated cytotrophoblasts were infected with recombinant adenoviruses expressing c-Myc or β-Gal, as control, and cultured for 24, 48, or 72 h. Effects of c-Myc overexpression on levels of mature miRNAs in the miR-17~92 and miR-106a~363 clusters was analyzed, as was expression of pri-miR-17~92. Expression of hCYP19I.1, hCGβ, and c-Myc mRNA levels was also analyzed by qRT-PCR. As can be seen in Fig. 5C, c-Myc overexpression significantly increased mature miR-17, -19, -20a, and -106a expression after 48 h and 72 h of culture. Notably, increased expression of pri-miR-17~92 was evident within 24 h of culture in cells overexpressing c-Myc (Fig. 5D), indicating that c-Myc transcriptionally activates expression of the miR-17~92 cluster. Importantly, this was associated with a marked inhibition of hCYP19I.1 and hCGβ expression (Fig. 5E), suggesting that increased c-Myc expression in cytotrophoblasts upregulates miR-17~92 and miR-106a~363 cluster expression, which in turn blocks the induction of hCYP19I.1 and hCGβ. Since hCYP19I.1 mRNA levels also were inhibited within 24 h of culture in cells overexpressing c-Myc, it remains possible that c-Myc also directly represses hCYP19A1 gene expression. However, when the cMyc-transduced cells were cotransfected with the miR-19b inhibitor, aromatase and GCM1 protein expression was greater than that observed with c-Myc alone (Fig. 5F). This suggests that c-Myc repression of GCM1 and aromatase expression in the cultured trophoblasts is mediated in part via induction of the miR-17-92 cluster expression. When human trophoblasts were transfected with c-Myc small interfering RNA (siRNA) to knock down endogenous c-Myc expression, significant decreases in expression of mature miR-17, -19b, -20a, and -106a, as well as pri-miR-17-92, were observed after 72 h of culture (see Fig. S3 in the supplemental material).
To determine whether c-Myc increases miR-17~92 and miR-106a~363 expression by directly binding to the genomic regulatory regions, we performed ChIP assays with human trophoblasts before and after differentiation in culture. Chromatin was cross-linked in cells before culture (0 h), after 5 h (a time point at which we previously observed early chromatin modifications associated with differentiation) (15), or after 72 h of culture, when the cells have differentiated to form syncytiotrophoblast. After immunoprecipitation with anti-c-Myc antibody or control IgG, PCR was carried out using primers that amplify two genomic regions containing E boxes within intron I of MIR17HG, the gene coding for the miR-17~92 cluster (Fig. 6A), that are conserved between human and mouse and that have been previously demonstrated to recruit c-Myc (39). We also identified putative c-Myc binding sites upstream of the miR-106a~363 genomic cluster (Fig. 6C) and designed primers to amplify two of these regions. Using ChIP, we observed increased binding of endogenous c-Myc (relative to the IgG control) to E box 1 within the miR-17~92 cluster genomic region (Fig. 6B) in freshly isolated cytotrophoblasts; relative binding activity markedly declined after 5 h of culture and was undetectable by 72 h. No specific c-Myc binding to E-box 2 was observed. Increased binding of endogenous c-Myc (relative to the IgG control) to the MIR106A1 region upstream of the miR-106a~363 cluster in freshly isolated cytotrophoblasts also was observed (Fig. 6D); again, c-Myc binding declined rapidly and was undetectable after 5 h of culture. Binding of endogenous c-Myc to the MIR106A2 genomic region was undetectable at any time point. These findings provide strong evidence that these miRNAs are directly upregulated by c-Myc in cytotrophoblasts; as c-Myc expression decreases upon trophoblast differentiation, expression of the miR-17~92 and miR-106a~363 clusters declines, releasing repression of their target genes.
Since overexpression of miR-19b and miR-106a in trophoblasts inhibited expression of their direct targets, hGCM1 and hCYP19, as well as hCGβ, a critical marker of trophoblast differentiation, we next examined the morphology of the human trophoblasts in which these miRNAs were overexpressed. Freshly isolated human cytotrophoblasts were transfected with nontargeting control (NTC) or with mimics of miR-19b or -106a. After 72 h of culture, the cells were stained with hematoxylin and eosin and viewed by light microscopy. When the cells were transfected with NTC miRNA, there was clear syncytium formation; however, when cells were transfected with either miR-106a or miR-19b, syncytium formation was markedly reduced (Fig. 7A). The Image J program was used to quantify the percentage of syncytiotrophoblasts compared to the total number of cells; statistical analysis indicated that miR-106a and miR-19b transfection significantly decreased syncytium formation (Fig. 7B). c-Myc overexpression decreased syncytium formation, while in the presence of miR19b inhibitors, this inhibition was prevented (see Fig. S4 in the supplemental material). This effect of miR-19b inhibitor may be due to increased expression of GCM1 in the cultured cells (Fig. 3D).
In consideration of the potential roles of the miR-17~92 and miR-106~363 clusters in human trophoblast differentiation and the impairment of trophoblast differentiation in preeclampsia, we next investigated expression of members of these miRNA clusters and their targets, hCYP19A1 and hGCM1, in placentas of preeclamptic and normotensive women near term. RNA and protein from term placentas of 8 preeclamptic and 8 gestation-matched normotensive women were analyzed for miR-106a and miR-19b expression using TaqMan-based qPCR; aromatase/CYP19A1 and hGCM1 protein expression were analyzed by immunoblotting. We observed significantly higher levels of miR-106a and -19b expression in placentas from preeclamptic women than in placentas from gestation-matched normotensive women (Fig. 8A). Correspondingly, aromatase and hGCM1 protein expression in protein extracts of preeclamptic placentas compared to that in normotensive placentas was significantly decreased (Fig. 8B and andC).C). However, expression of CYP19A1 and hGCM1 mRNA was not significantly altered (see Fig. S5 in the supplemental material). These findings suggest that aberrant expression of members of the miR-17~92 and miR-106a~363 clusters may contribute to the pathogenesis of preeclampsia.
In this study, we have analyzed the miRNA profile of cytotrophoblasts versus that of syncytiotrophoblast and demonstrated that members of the miR-17~92 cluster and its paralogs, the miR-106a~363 and miR-106b~25 clusters, are expressed at relatively high levels in rapidly proliferating cytotrophoblasts and that their expression declines in association with syncytiotrophoblast differentiation (Fig. 1). The miR-17~92 cluster, also known as oncomir 1, is among the most potent of oncogenic miRNAs (28, 40, 41). It is overexpressed in a variety of human cancers, including lung carcinomas, B-cell lymphomas, and retinoblastomas (42–44). Overexpression is frequently due to amplification of its locus (44). These miRNAs are known to contribute to tumorigenesis by targeting the cyclin-dependent kinase inhibitor CDKN1A (p21), a potent negative regulator of the G1-S checkpoint; thus, increased expression of these miRNAs enhances cell cycle progression (45). miR-19 also targets PTEN, a critical negative regulator of the highly oncogenic prosurvival P13K/Akt signaling pathway (46). Thus, it is likely that the relatively high levels of expression of all three miRNA clusters observed in human cytotrophoblasts promotes their proliferation and prevents syncytiotrophoblast differentiation. Notably, a subset of cytotrophoblasts in placental villi have been equated to tumor cells because they rapidly proliferate, invade maternal blood vessels, and form aggregates that attach the fetus to the maternal uterine wall (47). Thus, our findings further support the concept that cytotrophoblasts share properties with tumor cells in their similarly high expression levels of these miRNAs.
Despite these well-studied actions of the miR-17~92 cluster in tumorigenesis, a role for miRNAs in this cluster during normal development also has been established. Loss of function of the miR-17~92 cluster in mice resulted in smaller embryos and postnatal death associated with lung hypoplasia, cardiac defects, a block in B-cell development, and abnormal skeletal mineralization and patterning (48). While mice lacking the miR-106a~363 cluster do not manifest any phenotypic abnormalities, embryos that are doubly deficient in both the miR-17~92 and miR-106a~363 clusters die prior to embryonic day 15.5 (E15.5), suggesting that these miRNAs functionally cooperate (48). The miR-17~92 cluster is highly expressed in embryonic lung, and expression decreases toward term. Moreover, overexpression of these miRNAs in lung epithelium of transgenic mice resulted in severe developmental defects with enhanced cell proliferation and inhibition of epithelial cell differentiation (49). The miR-17~92 cluster also was found to be downregulated in myeloid progenitors undergoing macrophage differentiation (50), and studies of developing mouse embryos suggest that the miR-17~92 cluster inhibits stem cell differentiation (51).
In the present study, we have clearly established that members of the miR-17~92 and miR-106a~363 clusters are highly expressed in cytotrophoblasts and that expression declines upon terminal differentiation to syncytiotrophoblast. Members of these miR clusters were found to target hCYP19A1/aromatase (Fig. 2) and hGCM1 (Fig. 3) directly, and their overexpression in cytotrophoblasts prevented syncytiotrophoblast differentiation (Fig. 7). Our novel findings indicate that miR-106a and miR-19b can both directly target hCYP19A1, while miR-19b directly targets hGCM1. Since miR-17, -20a, -20b, -106a, -106b, and -93 all share the same seed sequence, whereas miR-19a and miR-19b share a different seed sequence, they all can be expected to target hCYP19A1 and may cooperatively promote hCYP19A1 gene repression. Moreover, we confirmed that miR-17 also exerts an effect similar to that of miR-106a to target aromatase protein expression and inhibit trophoblast differentiation (see Fig. S2 in the supplemental material).
GCM1 functions as an important transcription factor during formation of the mouse labyrinth; fusion of chorion trophoblast cells to form syncytiotrophoblast was blocked when GCM1 was ablated (17). Our results indicate that knockdown of hGCM1 in cultured human trophoblasts inhibits the induction of hCYP19I.1 and hCGβ (Fig. 4), known markers of trophoblast differentiation. Moreover, miRNA overexpression mimicked the effects of hGCM1 knockdown by inhibiting trophoblast differentiation (Fig. 3). Notably, miR-18a and miR-19a also target and repress expression of estrogen receptor α (ERα) and were found to inhibit neuroblast differentiation (52). In previous studies, we observed that ERα is expressed in human trophoblasts and was upregulated during differentiation in culture (15). Furthermore, estrogens acting via ERα upregulated hCYP19A1 expression and contributed to trophoblast differentiation in a positive feed-forward manner (15). Thus, the miR-17~92 and miR-106a~363 clusters may also inhibit trophoblast differentiation by inhibiting ERα expression in cytotrophoblasts. Another studied target of the miR-17/20/106 family is steroid receptor coactivator 3 (SRC-3), which serves as a coactivator for several nuclear receptors and other transcription factors, including ERα (35). Although we have yet to establish a functional role for SRC-3 in human trophoblasts, we observed that SRC-3 mRNA levels were upregulated in association with syncytiotrophoblast differentiation and the decline in miR-17~92 expression (see Fig. S6 in the supplemental material). Moreover, SRC-2 and SRC-3 have been reported to act cooperatively to promote mouse labyrinthine trophoblast development (53). Consequently, multiple miRNAs of the miR-17~92 and miR-106a~363 clusters may converge to inhibit trophoblast differentiation by targeting several key mRNA targets.
The c-myc proto-oncogene encodes a basic helix-loop-helix leucine zipper transcription factor that modulates diverse cellular processes, including proliferation, growth, apoptosis, and differentiation (37). In human placenta, c-Myc was observed to peak at 4 to 5 weeks of gestation and to be highly expressed in the proliferating cytotrophoblasts (38). In the present study, we detected relatively high levels of c-Myc mRNA and protein expression in freshly isolated cytotrophoblasts, which declined upon syncytiotrophoblast differentiation in culture (Fig. 5A and andB).B). Elevated expression of the miR-17~92 and miR-106a~363 clusters in cytotrophoblasts was associated with increased binding of endogenous c-Myc to a previously identified conserved region in the first intron of the miR-17~92 cluster (39) (Fig. 6B) and to a genomic region upstream of the miR-106a~363 cluster identified in the present study (Fig. 6C). Endogenous c-Myc binding to these genomic regions declined rapidly within 5 h of culture. While elevated c-myc mRNA expression was still evident at 24 h (Fig. 5A), a pronounced reduction in c-Myc protein levels was evident at this time point (Fig. 5B). Several posttranslational modifications, including phosphorylation, acetylation, and ubiquitylation of c-Myc, are known to control its transcriptional activity (54); one or more of these modifications may be responsible for the rapid decline in DNA binding activity to the miR-17~92 and miR-106a~363 clusters. Notably, targeted deletion of the c-myc gene in mice resulted in apparent placental defects by E10.5, with markedly decreased cell proliferation and inhibition of branching morphogenesis (55).
c-Myc has been shown to directly activate transcription of the miR-17~92 cluster (39) and thereby promote cell proliferation, survival, angiogenesis, and metabolic reprogramming in a number of tumor cell lines (56). In cultured human trophoblasts, we found that c-Myc overexpression upregulated miR-17~92 and miR-106a~363 transcription (Fig. 5C). Thus, our findings support the important role of c-Myc in the increased transcription of the miR-17~92 and miR-106a~363 clusters in proliferating cytotrophoblasts, thereby suppressing translation of CYP19A1 and hGCM1 and inhibiting trophoblast differentiation (Fig. 9).
Preeclampsia, characterized by the onset of hypertension and proteinuria after 20 weeks of gestation, affects 6 to 8% of all pregnancies in the United States and is a leading cause of maternal and neonatal morbidity and mortality (57–59). In preeclampsia, the placenta is relatively hypoxic and cytotrophoblast differentiation into invasive cells is inhibited. Thus, fewer arterioles are invaded, and O2 availability to the hormone-producing syncytiotrophoblast is reduced (60). Recently it was observed that miR-17, -20a, and -20b, encoded within the miR-17~92 cluster, were significantly increased in placentas of preeclamptic women and that these miRNAs directly target Eph receptor B4 (EPHB4) and ephrin B2 (61), which are critical for vascular patterning and trophoblast invasion during placentation (62). In another study, miR-18a and -19a were among 22 miRNAs significantly upregulated in preeclamptic placentas (63). Importantly, in the present study, we observed that miR-106a and miR-19b were expressed at higher levels in placentas of preeclamptic women than in placentas of gestation-matched normotensive women (Fig. 8A). Elevated expression of these miRNAs may reflect upregulated expression of the miR-17~92 and miR-106a~363 clusters and/or a relative increase in the proportion of undifferentiated cytotrophoblasts in preeclamptic placentas. Since our primary cell culture studies suggest that increased miR-17~92/106a~363 expression prevents syncytiotrophoblast differentiation, it may be difficult to clearly establish a cause-effect relationship.
While the molecular basis for placental dysregulation in preeclampsia remains unclear, our findings indicate that increased miR-106a and -19b expression in preeclamptic placentas was associated with decreased expression of aromatase (Fig. 8B). Importantly, placental aromatase deficiency has been found in women with preeclampsia (64), and a hCYP19A1 gene polymorphism associated with decreased aromatase levels has been reported to occur more commonly in preeclamptic patients (65). Notably, hGCM1, which we also found to be a target of miR-19b, was found to be markedly decreased in placentas of women with preeclampsia (66). We also observed decreased GCM1 protein levels in placentas from preeclamptic women (Fig. 8C). Moreover, reduced GCM1 expression in mouse placenta was associated with defective syncytiotrophoblast differentiation and pregnancy outcomes resembling preeclampsia in women (67). In light of our important findings and to further understand the significance of increased miRNA expression in preeclampsia, our future work will include analysis of clinical endpoints of preeclampsia in a transgenic mouse model of miR-17~92 overexpression.
In summary, the present findings provide evidence for a critical inhibitory role of a c-Myc/miR-17~92/-106a~363 signaling pathway in the regulation of syncytiotrophoblast differentiation and the expression of hGCM1 and hCYP19A1 in human placenta (Fig. 9). Early in the first trimester, when the placenta is poorly vascularized and relatively hypoxic, c-Myc expression is elevated, resulting in increased transcription of the miR-17~92 and miR-106a~363 clusters. We suggest that elevated expression of c-Myc and these miRNA clusters in cytotrophoblasts promotes "stemness," exemplified by increased expression levels of the stem cell factor, achaete-scute homologous protein 2 (ASCL2/Mash-2) (3). Increased expression of these miRNA clusters may promote increased cytotrophoblast proliferation via targeting of p21 (45) and suppression of hGCM1 and hCYP19A1 to block syncytiotrophoblast differentiation. After 9 to 10 weeks of gestation, increased cytotrophoblast invasion of the spiral arteries results in increased placental perfusion and enhanced oxygen tension. This is associated with suppression of ASCL2 and c-Myc expression and decreased expression of the miR-17~92/106a~363 clusters, which permits upregulation of hGCM1 to promote syncytiotrophoblast differentiation and increased hCYP19A1 expression. We suggest that aberrant regulation of this novel signaling pathway interferes with normal induction of trophoblast differentiation that may contribute to the pathogenesis of preeclampsia.
We gratefully acknowledge the expert assistance of Jo Francis Smith in isolation and culture of human placental cells.
This work was supported by NIH grant R01 DK031206.
Published ahead of print 25 February 2013
Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01228-12.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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This possibility was highlighted at the conclusions of the October 2013 European Council, which stated that "part of the European Structural and Investment Funds (2014-2020) should be used for ICT education". The following funds could be considered:
Have a look at other types of funds
The Erasmus+ programme aims to boost skills and employability, as well as modernise Education, Training and Youth work. €14.7 billion is dedicated for Erasmus+ programme 2014-2020. Erasmus+ will support transnational partnerships among Education, Training and Youth institutions and organisations in order to tackle the skills gaps. Organisations interested in Erasmus+ funding can contact the National agencies for Erasmus.
The Employment and Social Innovation (EaSI) programme is a financing instrument at EU level to promote a high level of quality and sustainable employment, guaranteeing adequate and decent social protection, combating social exclusion and poverty and improving working conditions. The total budget for the EaSI programme 2014-2020 is €919.47 million.
As of January 2014, programmes form the three axes of EaSI support the following areas:
To apply for funding, eligible organisations must respond to a call for tender or/and to a call for proposals.
To apply for funding, eligible organisations must respond to a call for tender or/and to a call for proposals.
Your First EURES Job is a job mobility scheme to help young Europeans find a job, traineeship or apprenticeship in other EU countries. The scheme is part of the Youth on the Move package and the Youth Opportunities Initiative. It could be incorporated into national Youth Guarantee schemes that include cooperation with other EU countries. The 2015 target for Your first EURES job is to help some 5 000 people find jobs in EU countries other than their own. The initiative involves only a limited number of employment services.
The European Commission has published a call for tenders, which aims at selecting a service provider for the organisation of a new major communication and awareness-raising campaign in Europe on 'E-skills for jobs'.
The duration of the campaign will be 2 years (2015–2016). The budget available in this call for tenders is EUR 2,000,000. The deadline for applying is 7 October 2014.
For more information click here.
Several projects are in place to support the Grand Coalition, in particular:
Support Services for the Grand Coalition for Digital Jobs. The European Commission has launched a call for tenders for a service contract with a total price that cannot exceed One million Euros today. The deadline is 3 May 2016. For more information please click here.
The Secretariat of the Grand Coalition for Digital Jobs was created in 2014, with a budget of €1 million from the Competitiveness and Innovation programme over 2 years (February 2014 - February 2016).


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text: Web-based Electronic Health Record (EHR) & Practice Management SolutionIn an increasingly complex healthcare environment, McKesson Practice Choice™ can help you keep things simple. This web-based electronic health record (EHR) and practice management system was designed with the independent practitioner in mind and features a graphical interface that makes it easy to navigate to the different work areas: clinical, scheduling and accounting. Because it's Internet-based, there's no need to worry about backing up databases or downloading software, which helps free you up to focus on patient care.Intuitive and efficient, McKesson Practice Choice does more than maintain records and protect cash flow; it has the power to improve the quality of your patient interactions. Offering you flexible access to the office, McKesson Practice Choice is an easy-to-use solution that streamlines workflow and enhances care quality.When Care is your Priority and Simplicity is your ChoiceSimplify your administrative overhead and learning curve with just a single solution integrating EHR, practice management, patient portals, e-prescribing and claims. Simply log on to our secure system to access and manage your practice's data. Updates and upgrades to the program will stream automatically to your computers, so manual reinstallations are never required. As a Web-based subscription service, there are no costly servers to maintain, no IT staffers to hire and no backups to schedule.You compose your progress notes carefully, which is why we've carefully constructed the Smart Note™ technology that powers McKesson Practice Choice. It allows you to document on a single screen while pulling and pushing data from anywhere in the patient's chart. Search codes using natural terminology and view cross-sectional chart summaries. Everything you enter automatically flows data into all pertinent fields throughout a chart. You can even create a template that suits your note-taking preferences. You'll spend less time charting and more time interacting with your patients. One choice for connecting providers, payers and patients.McKesson is familiar with the ways clinical and business operations overlap in the small-practice environment. That's why McKesson Practice Choice integrates your office's primary functions, connecting data and streamlining practice-wide processes in one easy-to-use system.With 20+ years developing practice management and EHR technologies, McKesson understands the juggling act of the independent medical office, and is committed to utilizing technology to help make your life easier, flexible and more efficient. That's why McKesson Practice Choice is more than a combined EHR and practice management product — it's a comprehensive full practice solution.McKesson Practice Choice Advanced: Combination of Technology & ServicesWith the McKesson Practice Choice Advanced™ solution, you gain a fully integrated EHR and powerful practice management system combined with a virtual back-office team to help you manage cash flow. This powerful combination of technology and services will help you improve the clinical, administrative and financial sides of your practice – at the same time, from a single source: McKesson Practice Choice Advanced. As a leader in revenue cycle management (RCM) services across the healthcare continuum, McKesson Business Performance Services will bring to your practice the people, processes and technology needed to help you increase revenues, control costs and reduce regulatory risks.


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Japan getting multi-coloured PSPs for Christmas
SCEJ just announced five new PSP bundles for Japan, featuring white, blue, black, red and Monster Hunter shades. See them on Kotaku.
The non-game bundles are ¥19,800 (US$219.50). The Monster Hunter Pack's ¥18,500 ($205). They're all out in December.
Sometimes we include links to online retail stores. If you click on one and make a purchase we may receive a small commission. For more information, go here.


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S. C. J. Plastics LimitedView mapKirti Nagar, New Delhi, Delhi(Verified Supplier)
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The SCJ group is a leader in the field of color and additive masterbatches, PVC Compounds, Co-Extruded Sheets and Telecom Cables, providing the plastic processing industry with quality products both for standard, as well as tailor made applications. With a three-decade strong foundation, SCJ is now poised on the threshold of creating a global niche with products Our commitment to society and a Green World, has led us to develop additives and masterbatches which are environment friendly. In India, the group operates from strategic locations in Delhi, Baddi (Himachal Pradesh), Daman and Goa. These diverse manufacturing facilities are equipped with the latest state of the art machinery and enable SCJ to respond on the spot to the demands of the market and make available 24x7 back-up support advantages to its' valued customers.
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Near Kirti Nagar
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Delhi, India
Near Kirti Nagar
New Delhi - 110015
Delhi, India
Send your enquiry to this supplier


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Rashad Richey's Review: Should the DNC Recognize the Black Lives Matter MovementJoin our poll. Should the Democratic National Convention recognize the Black Lives Matter Movement as it aims to unify around nominee Hillary Clinton.
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Rashad Richey's Review: Do You Think Republicans Truly Want to Unite America?Join our poll: Do you think Donald Trump and the Republican party truly aim to unite America?
Atlanta Weighs in on the Killing of 3 Baton Rouge Police OfficersAtlanta citizens react to the shooting deaths of three Baton Rouge Police officers.
Judge Hatchett to Represent Castile FamilyJudge Glenda Hatchett discusses why she chose to represent the family of Philando Castile and what she has in the works to bring those accountable to justice.
What is Our Next Move?Atlanta NAACP Chapter President Richard Rose, discusses the future protests and boycotts in response to last week's police shootings.
Robert Patillo Asks: What Happens To A Dream DestroyedAttorney Robert Patillo wants to know- What happens to a dream destroyed? Does it dissolve into the ether of time? Or continue forever like the scheme of a rhyme?
The AutoNsider: Driving While Black and Confronted by PoliceThe police killings of Alton Sterling and Philandro Castile have affected thousands of people around the country and has everyone thinking about what to do if pulled over by a police officer. Daryl Killian addressed the issue on AutoNsider
Al Sharpton: Let's Talk About DallasBlack Lives Matter, But Violence and Murder is NOT the Answer, Sharpton Discusses with a Prolific Panel and with WAOK Guests to Discuss an Alternative.
He Did Everything Right and was Still MurderedRashad Richey discusses the shooting death of Philando Castile, another black man murdered by police, a day after the shooting death of Alston Sterling.
APS Chairman Discusses Lead Found in School's WaterChairman Courtney English discusses the elevated levels of lead found in the water of numerous APS schools.
The Movement With Dr F.Keith Slaughter : Authors Corner With Jeevan BrownWhat do you get when you have a story of Rape, STD'S, Racism and more? you get "A Lesson Learned" by Jeevan Brown who was highlighted today on The Movement with Dr F.Keith Slaughter in the Authors Corner. check out the interview....


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Chris Picton
From Wikipedia, the free encyclopedia
Christopher James "Chris" Picton is an Australian politician representing the South Australian House of Assembly seat of Kaurna for the South Australian Branch of the Australian Labor Party since the 2014 state election.[1]
Prior to being elected to parliament, Picton was Chief-of-Staff for his predecessor John Hill and later a staffer for Commonwealth Health Minister and Attorney-General Nicola Roxon.[2]
- ^ 2014 SA election: Antony Green ABC
- ^ Bowe, William. "Electorate: Kaurna". Crikey. Private Media Pty Ltd. Retrieved 16 December 2014.
External links[edit]
|South Australian House of Assembly
|Preceded by
John Hill
|Member for Kaurna
|This article about an Australian Labor Party politician is a stub. You can help Wikipedia by expanding it.


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Gaming desktops with AMD Zen chips will be hard to come by this year
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As the U.S. heads toward a contentious national election in November, 15 states are are still clinging to outdated electronic voting machines that don't support paper printouts used to audit their internal vote counts.
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BYU rugby: Cougars beat Life University to advance to 7th straight championship game
Paul Meyers
MARIETTA, Ga. — BYU's top-ranked rugby team traveled to No. 2 Life University for a Division 1 semifinal and beat the Mid-South champions 26-20 to advance to their seventh straight national championship game Saturday at Rio Tinto Stadium.
In a very physical and tactical match BYU took an early lead and held on late to beat a strong Life team on its home field.
Trailing 26-5 the Running Eagles clawed their way back into the game behind two tries by All-American Cameron Dolan and flanker Andrew McNeil.
After an additional penalty goal by fullback Joe Cowley, Life was on the verge of coming back in the final quarter of the game.
But BYU, which played the majority of the game in its own half, would regain possession and hold on to the ball to run out the clock and win the game following a clearing kick from center Paul Lasike.
"We had to earn this one," BYU captain Ryan Roundy said. "Life is a very good team. And there's not a lot of teams, if any, who could come in here and beat them. I'm really proud of our guys for their guts and determination."
The Cougars started the game fast from an early try by prop Ray Forrester in the fourth minute. A tough-angled conversion by scrumhalf Shaun Davies had BYU leading 7-0 in Marietta.
Life would come back quickly with a chip kick that bounced back into the arms of Cowley, who then passed to a supporting scrum half Cornelius Dirksen. He scored Life's opening try in the 10th minute. BYU would still lead 7-5 after the Life try was unconverted.
But as Life controlled more field position and possession the BYU defense was committed and repelled the Life attack on several occasions.
Eventually the BYU defensive patience led to three tries from the Cougars that would prove to be the difference in the game.
The first came from fly half Dylan Lubbe, who intercepted a Life pass to sprint the remaining 60 meters for the try. And the remaining two were from center Paul Lasike manufacturing a duo of tries late in the first half.
"Those last two tries were big obviously," said BYU head coach David Smyth. "Not only did we hold on because of those tries, but both came on the back of some tough defense, which was the name of the game."
The Cougars now move on to the national championship game for the seventh straight time Saturday at Rio Tinto Stadium in Sandy.
BYU will face a familiar foe in Arkansas State, a team that is making its first ever national title game appearance.
BYU has faced Arkansas State twice before — the first time in 2009 as the Cougars marched on to their first national title and most recently last year when the Cougars came from behind to beat the Red Wolves 36-15.
For ticket details and information on the Collegiate D1-A National Championship go to www.riotintostadium.com.
BYU 1st XV vs. Life
- Mikey Su'a
- Ishmael Tilialo
- Ray Forrester (Joey Mount @ 30′)
- TJ Allred
- Kyle Sumsion
- Kumi Tua'one (Chad Harker @ 55′ front row sub)
- Hynie Leaaetoa
- Ryan Roundy ©
- Shaun Davies
- Dylan Lubbe
- Malosi Te'o
- Seki Kofe
- Paul Lasike (Chris Wernli @ 50′ blood sub)
- Will Taylor
- Tua Laei
BYU 26 Tries: Forrester 4′; Lubbe 13'; Lasike 37', 40' Conversions: Davies 3/4 (4', 13', 37') Penalties: Davies 0/1 (78')
LIFE 20 Tries: Dirksen 10'; Dolan 43'; McNeil 56' Conversions: Cowley 1/3 (56') Penalties: Cowley 1/3 (60')
The BYU Rugby program is one of the school's most winning sports teams with just 19 losses in over 20 years. BYU Rugby won the 2009 Collegiate National Championship and has promoted 28 players to All-American status. For more go to byurugby.com
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text:
"At PARLEZ WIRELESS we strive to be the best wireless location - period. We offer service that differentiates with standards that we all live by in order to treMore
$1 mil. - $5 mil.in Revenue
5 - 10EmployeesContact people at this company
4312 Innes RoadOrleansOntarioK4A 5E6Canadaview map
(613) 824-0388(613) 824-2184Fax
Enterprise_Iteration-NT.1545[01] SEIDXWK-31