Podcasts about utrs

This month on Episode 72 of Discover CircRes, host Cindy St. Hilaire highlights four articles featured in the April 25th and May 9th issues of Circulation Research. This Episode also includes a discussion with Dr Sarah Costantino and Dr Francesco Paneni from University Hospital Zurich about their study, Chromatin Rewiring by SETD2 Drives Lipotoxic Injury in Cardiometabolic HFpEF     Article highlights: Laudette, et al. PCSK9 and Mitochondrial Cholesterol in Heart Yang, et al. Srsf3 Limits AS by Lengthening 3′ UTRs of mtARSs Li, et al.  CircCDYL Contributes to Cardiac Hypertrophy Zhakeer, et al. Treg Cells Regulate Pulmonary Venous Remodeling

New to self-employment and wondering when you should register? In this episode Dan discusses when you need to register and how it works. He chats about employment status, business names, UTRs…. all this and more on today's HeelanHub! www.heelanassociates.co.uk/podcast - the show for UK small business owners. info@heelanassociates.co.uk 02392 240040

In this informative session hosted by the National Association of Sessional GPs (NASGP), Victoria (Tori) Ferguson, a tax manager at Honey Barrett, shares essential advice for GP locums on managing their finances, tax obligations, and pensions. Tori, a chartered tax advisor, offers a comprehensive guide tailored to GPs who are new to locuming or those seeking a refresher on best financial practices.For further advice on these topics, NASGP members can access more resources and guidance on the NASGP website: https://nasgp.org.ukDuring the talk, Tori covers 10 key points to help locum GPs successfully manage their self-employment status. She explains the importance of setting up a personal tax account, notifying HMRC of self-employment, and staying on top of record-keeping and software tools like LocumDeck. Other essential topics include navigating the NHS pension scheme, understanding expenses for tax deductions, and planning ahead for tax payments, especially with looming changes such as Making Tax Digital.The session also delves into the nuances of claiming allowable business expenses, the significance of maintaining separate bank accounts, and the critical need for GPs to view themselves as both a doctor and a business. Tori emphasises the importance of investing in financial education to stay up-to-date with ever-changing tax laws, whether through self-learning or hiring a specialised accountant.The Q&A session further explores real-world concerns, such as handling fraudulent use of unique taxpayer references (UTRs) and dealing with the complexities of IR35 and employment status, highlighting Tori's expertise in resolving practical issues GPs may face.Intro 00:00Topics covered: 1. Setting up a personal tax account. 02:41 2. Informing HMRC about self-employment. 03:38 3. Importance of bookkeeping software. 05:23 4. Record-keeping essentials for locums. 06:25 5. Navigating expenses and the trading allowance. 08:30 6. Pension contributions and time limits. 12:00 7. Managing a separate business account. 15:15 8. Saving for taxes and avoiding pitfalls. 16:53 9. Viewing yourself as a business owner. 20:21 10. Investing in financial education. 21:52Q&A 25:41Highlights: • HMRC UTR fraud cases and how to handle them. 27:46 • Employment status and IR35 for locum GPs. 38:07For further advice on these topics, NASGP members can access more resources and guidance on the NASGP website: https://nasgp.org.uk#GPs #LocumTax #Pensions #NASGP #HoneyBarrett #SelfEmployment #TaxAdvice #GPFinance #MedicalAccountants #MakingTaxDigital #IR35 #LocumDeck

Founder of Tennis Neutral, Ric Curnow, joins CR contributors Richard Maj and Archit Suresh to discuss the inner workings of the Tennis Neutral App and why he came up with the idea to provide an accurate estimate of where the UTR neutral point lies in a match between players with different UTRs. You can check out the Tennis Neutral app on the Apple App Store or the Google Play Store, or on his website www.tennisneutral.com Don't forget to give a 5 star review on your favorite podcast app! In addition, add your twitter/instagram handle to the review for a chance to win some FREE CR gear!! This episode brought to you by: Tourna We are excited to partner with Tourna Tennis once again to share an exciting, longer lasting version of Tourna Grip, which Tourna has spent the last six years researching and developing their latest grip innovation: Tourna Tuff including: -same gold standard sweat absorption as Tourna Grip -same dry feel -same trademarked blue color -still gets tackier when you sweat PLUS new features: -If you didn't like the original Tourna Grip for its durability, try Tourna Tuff. -Stop blowing on your hands between every point and use a grip that absorbs your sweat! -Stop wiping your hand on your shirt, shorts, and towel constantly and get a grip that actually absorbs sweat! Plain and simple = Cracked Fans use Tourna Tuff. Tennis Point Discounted Tennis Apparel, Tennis Racquets, Tennis Shoes & Equipment from Nike, adidas, Babolat, Wilson & More! Visit their store today and use the code "CR15" at checkout to save 15% off Sale items. Some Exclusions (MAP Exceptions) apply and code will not work on those items. This code will add 1 FREE CAN of WILSON Balls to the cart at checkout. Tennis Channel Podcast Network Visit https://www.tennis.com/pro-game/podcasts/ to stay current on the latest tennis news and trends and enjoy in-depth analysis and dynamic debates.   Find Cracked Racquets Website: https://www.crackedracquets.com Instagram: https://instagram.com/crackedracquets Twitter: https://twitter.com/crackedracquets Facebook: https://Facebook.com/crackedracquets YouTube: https://www.youtube.com/c/crackedracquets Email Newsletter: https://crackedracquets.substack.com/ Learn more about your ad choices. Visit podcastchoices.com/adchoices Learn more about your ad choices. Visit megaphone.fm/adchoices

Insider Financial discusses the ongoing discussion around naked shorts and how the penny stock market is a pump and dump. To sign up for real-time alerts along with our FREE reports and eBook, go to: https://signup.insiderfinancial.com/ To sign up for FREE stocks and trade from 4am to 8pm on WeBull, go to: https://a.webull.com/i/insiderfinancial This video covers COSM, CORZQ, GVSI, MUN, GOVX, HLBZ, AMC, GNS, ENSC, UTRS, and AREB. Trading Pump and Dump Penny Stocks Disclosure: Insider Financial has not been compensated for this video. Insider Financial is not an investment advisor; this video does not provide investment advice. Always do your own research, make your own investment decisions, or consult with your nearest financial advisor. This video is not a solicitation or recommendation to buy, sell, or hold securities. This video is our opinion, is meant for informational and educational purposes only, and does not provide investment advice. Past performance is not indicative of future performance. For more information, please read our full disclaimer:https://insiderfinancial.com/disclaimer/ COSM stock, CORZQ stock, GVSI stock, MULN stock, GOVX stock, HLBZ stock, AMC stock, GNS stock, ENSC stock, UTRS stock, AREB stock, small caps, trading, otc stocks, otc stocks list , penny stocks , penny stocks list, NASDAQ penny stocks, NYSE stocks, NYSE penny stocks #pennystocks #trading #pumpanddump

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.23.517728v1?rss=1 Authors: Luisier, R., Andreassi, C., Riccio, A. Abstract: Background Neurons are morphologically complex cells that rely on the compartmentalization of protein expression to develop and maintain their cytoarchitecture. Targeting of RNA transcripts to axons is one of the mechanisms that allows rapid local translation of proteins in response to extracellular signals. 3' untranslated regions (UTRs) of mRNA are non-coding sequences that play a critical role in determining transcript localisation and translation by interacting with specific RNA binding proteins (RBPs). However, how 3'UTRs contribute to mRNA metabolism and the nature of RBP complexes responsible for these functions remain elusive. Results We performed 3' end sequencing of RNA isolated from axons and cell bodies of sympathetic neurons exposed to either Nerve Growth factor (NGF) or Neurotrophin 3 (NT3). NGF and NT3 are growth factors essential for sympathetic neuron development that act through distinct signalling mechanisms. Whereas NT3 is thought to act only locally, NGF signals back from axons to the cell bodies. We discovered that both NGF and NT3 affect transcription and alternative polyadenylation and induce the localisation of specific 3'UTR isoforms to axons. The finding that many transcripts with short 3'UTR were detected only in axons suggest that these may undergo local post-transcriptional remodelling. The integration of our data with CLIP-sequencing data revealed that long 3'UTR isoforms associate with RBP complexes in the nucleus, and once in axons, regulate cytoplasmic 3'UTR isoform cleavage into shorter isoform. Conclusions Our findings shed new light on the complex interplay between nuclear polyadenylation, mRNA localisation and local 3'UTR remodelling in developing neurons. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.17.512459v1?rss=1 Authors: Shi, B. Abstract: Epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) positively affect the initial control of non-small cell lung cancer (NSCLC). The rapidly acquired TKIs resistance accounts for a major hurdle in successful treatment. However, the mechanisms controlling EGFR-TKIs resistance remain largely unknown. RNA structures have widespread and crucial roles in various biological processes; but, their role in regulating cancer drug resistance remains unclear. Here, the PARIS method is used to establish the higher-order RNA structure maps of EGFR-TKI resistant- and sensitive-cells of NSCLC. According to our results, RNA structural regions are enriched in UTRs and correlate with translation efficiency. Moreover, YRDC facilitates resistance to EGFR-TKIs in NSCLC cells, and RNA structure formation in YRDC 3'UTR suppress ELAVL1 binding leading to EGFR-TKIs sensitivity by impairing YRDC translation. A potential cancer therapy strategy is provided by using antisense oligonucleotide (ASO) to perturb the interaction between RNA and protein. Our study reveals an unprecedented mechanism in which the RNA structure switch modulates EGFR-TKIs resistance by controlling YRDC mRNA translation in an ELAVL1-dependent manner. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

The Whole View, Episode 440: COVID-19 Vaccines Part 1 - mRNA Vaccine Technology Welcome back to episode 440 of the Whole View. (0:27)    Stacy explains that today's topic is one she and Sarah have received the most questions on possibly ever. Stacy also lets the audience know that this show will be a 2-parter, possibly a 3-parter depending on how deep in they get. This show has been long in the making because she and Sarah had to wait for the research publication. Then Sarah has done her own research on top of it to prepare for this show. Sarah shares that she's been following this topic for about a year now: ever since the novel coronavirus was sequenced. It's important they lay out the science for listeners, look at the technology and history of vaccines, answer the frequently asked questions, and bust the myths surrounding this topic. (2:08) She and Stacy decided to divide the show into multiple parts to take their time and do the subject justice. Stacy takes a minute to address how polarizing the word "vaccine" can be. And she and Sarah are aware of this. She wants to assure listeners they understand vaccines are a personal decision for everyone, just like every other health and medical choices are. Stacy and Sarah are here to provide the information you need to be an informed consumer.   Note On Vaccines In this episode, they will discuss the mRNA vaccine technology in the history of vaccines. (2:40) Next week's episode, Sarah and Stacy will go over the safety and efficacy data for the first two vaccines, Emergency Use Authorization, the Pfizer/BioNTech vaccine, and the Moderna vaccine. Sarah and Stacy will discuss their thoughts on vaccinations going forward. But Stacy reminds listeners that it's never aimed at telling others what to do.  She also reminds listeners that she and Sarah are not medical professionals. If you have questions regarding the vaccine for yourself or your family, discuss them with your doctor. There is a lot of information that is both true and not true floating around on the web.  Stacy is very excited to talk about the science and breakdown behind these vaccines and gives a little background on herself for context. Both Stacy and her mother have anaphylactic reactions to things like gluten due to multiple autoimmune disorders. Stacy has brought up to Sarah whether or not she thinks getting the vaccine is a good idea for someone with health issues like Stacy's mom. Stacy also wonders how having the coronavirus, but not having the antibodies, will affect her if given the vaccine.   Listener Questions Sarah reiterates just how many questions they've received from listeners around this subject. (5:10) She takes a moment to share a few she thinks accurately sum up what they want to cover in this episode. Mae wrote: I am sure you don't want to cover this topic, but you are a source I highly trust as I am sure a lot of your other followers do. Would you consider doing a show about the Covid vaccines out there? It's so hard to know what to believe these days.....Not looking to be told what to do, but merely to be presented the information as you do so well in breaking down the real science that is not filtered through such a biased lens. Meghan added: Can you please do an episode explaining the science behind vaccines, and explaining how they really work, including the new Covid one. You always do an excellent job of explaining things well in a relatively easy to understand way without shortcutting good science. Stacy assures listeners that they will do their very best to break everything down. However, you might still have questions or have heard something different that might conflict with prior information. Stacy encourages you to reach out via the contact forms on the website for any follow-up. If you're part of the Patreon family, use direct access to talk with Sarah and Stacy there. She also encourages listeners not to attack the topic on social media or to put too much emphasis on things you hear without any sources cited.   A Brief History of Vaccine Technology Sarah starts off by going way back into the history of vaccines. (8:27) The very first form of inoculation was called variolation. The first variolation for smallpox dates to the 1600s in China and Ottoman Empire and practiced first in Britain and colonial Massachusetts in 1721. They took the pus from someone suffering a natural smallpox infection. And then they'd would then rub it onto punctured or cut skin of someone who had never been exposed. If the procedure didn't kill you, you'd have immunity to the illness. However, Sarah noted it was pretty successful in terms of early inoculation. Sarah explains briefly how cell memory aids in fighting episodes of re-exposures. This is what gives us immunity or less a severe immune response when exposed. Development Of A Smallpox Vaccine Dr. Edward Jenner is considered the founder of vaccinology in the West. He noticed many milkmaids were immune to smallpox. He realized they were getting infected with cowpox (a related variola virus that is relatively harmless to humans), and the infection built an immunity to smallpox. In 1796, he inoculated his gardener's 8-year-old son by variolating cowpox pus from a milkmaid's hand. Jenner then demonstrated this immunity to smallpox by exposing the boy to smallpox 6 weeks later, and he didn't get sick. That's a lot of confidence! And also, not cool. Jenner then repeated this experiment multiple times over a couple of years with different people and published his methodology in 1798. He named his process vaccination because the cowpox virus is called vaccinia. Doctors started administering this as a smallpox vaccine all over the world in 1798. This is the first instance of understanding that exposing the body to a weaker version of a virus could stimulate enough of an immune response to tricker cellular memory. Over the 18th and 19th centuries, systematic implementation of mass smallpox immunization culminated in its global eradication in 1979. It took just about 200 years from the start of this vaccine to the eradication of smallpox. Other Vaccine Development Louis Pasteur's experiments spearheaded the development of live attenuated cholera vaccine in 1897. And then an inactivated anthrax vaccine in 1904.  Plague vaccine was also invented in the late 19th Century.  Between 1890 and 1950, bacterial vaccine development proliferated, including the Bacillis-Calmette-Guerin (BCG) vaccination, which is still in use today.  In 1923, Alexander Glenny perfected a method to inactivate tetanus toxin with formaldehyde. The same method was used to develop a vaccine against diphtheria in 1926. Pertussis vaccine development took considerably longer, and a whole-cell vaccine was first licensed for use in the US in 1948. mRNA vaccine technology Sarah tells the audience that many of the childhood vaccines given to children today were developed 70 – 100 years ago. There have been advancements in the vial today that are different from what was in the vial back then. However, the vaccine technology is pretty much the same now, and it was that then. Sarah underlines that mRNA vaccine technology was one of the biggest advancements since Jenner and Pasteur's experiments.   Modern Vaccines When looking at vaccines today, they all have the same basic ingredients (18:20) They all work by stimulating an immune response against what's called an antigen. An antigen is a bad thing that makes us sick. The body develops immunological memory by the adaptive immune system in response to the antigen.  It's the same way our immune system develops memory when we've been naturally infected.  But because vaccines use weaker viruses, it goes without the danger of natural infection. Vaccinations are very costly and big investments to undertake. So we really only develop vaccines against illnesses that are very, very bad and have a huge impact on society. Up until now, mRNA vaccine technology hasn't changed much since the 50s. Traditional vaccines contain three components: antigen, adjuvant, and additives to preserve/stabilize. AntigenThis is the thing we're developing immunity against. Antigens come in various types: live, attenuated virus; inactivated virus; inactivated toxins for bacterial diseases where toxins generated by the bacteria cause the illness; or parts of a virus-like split, subunit, or conjugate.   Adjuvants Stacy asks about adjuvants and what they do to cause the stimulation. (20:00) Sarah explains that adding a little bit of dead virus to our arm tissue isn't usually enough to trigger an immune response. An adjuvant is a compound (most commonly aluminum) that stimulates the immune system. And helps to develop a more robust immune response and stronger immunity against the antigen.  Adjuvants are why people often feel sick after a vaccine. It's not the virus causing the side effects, but rather the ramped-up immune system caused by the adjuvant. It's also why many people with autoimmune diseases experience a temporary flare after vaccination.  If you already have an immune system in overdrive due to an autoimmune system, it makes sense why autoimmune suffers would have more adverse reactions. Sarah feels it's important to note there is no science showing vaccines cause autoimmune diseases. However, because they're meant to cause an immune response, vaccines can make autoimmune diseases more noticeable. Sarah recommends this article as a source of more information about adjuvants. Additives Additives are preservatives, stabilizers, and residuals included in the vaccine. Sarah explains this is where there can sometimes be egg protein as a residual. So there are certain vaccines out there that people with egg allergies can't have. Sarah notes there is still one vaccine out there that uses Thimerosal as a preservative. But it has been mostly phased out since the 1980s. This is because Thimerosal contains traces of mercury. Stacy circles back to heavy metals and how often they talk about those as being bad.  She feels it's important to note that going through normal daily life, we encounter things like heavy metals in food and water. This is why we have livers: so we can flush them out of our systems naturally. It's why she and Sarah talk so much about taking care of our liver. So when we hear things like, "there's aluminum in this vaccine," it might come off as a red flag. We don't want to put that in our bodies. Stacy explains why these vaccines work to achieve the response it needs because you're right: your body does not want that aluminum in there. So it gets agitated and works a little bit harder to flush it out. And that's how the vaccine is able to create the body's immune response. Stacy shares one way she helps her body is to take extra care of her liver the weeks before getting a vaccine. That way, she could optimize her body's ability to flush out the substances it doesn't want in there. Sarah agrees that a great practice is to practice self-care, such as getting enough sleep and eating right before and after getting a vaccination.   Always a Cost-Benefit Analysis  Sarah explains that Stacy brought up a great point: there is always a cost-benefit to mRNA vaccine technology and other types of vaccines. (28:45) Sarah believes we are at a point now where most of us are disconnected from the actual impacts of viruses like polio and whooping cough. She shares that her grandfather survived polio when he was 14-years-old. He was wheelchair-bound for 2 years and walked with a cane or walker for the rest of his life. He also developed post-polio syndrome in old age, which caused heart failure. For Sarah, she is at the tail-end of people's age with a personal connection with some of these illnesses that we heard about. Gen X and younger generally don't understand a lot of the consequences that come with a lot of these diseases. Over a century ago, the infant mortality rate was over 20%. And the childhood mortality rate before age five was an additional disconcerting 20%. That's what vaccination has been able to do for us and society: give us more than a near 50% chance of reaching our 5th birthday. We only invest in vaccines for diseases with high mortality and/or morbidity. Sarah explains that mortality equals death.  Morbidity, on the other hand, anything bad that happens that's not death. It includes severe illness, complications, and lifelong health problems. For example, morbidity from mumps is basically zero. But 1 in 300 get encephalitis (or brain inflammation) while 1 in 10 men get orchitis (testicle inflammation) Measles mortality is 1 in 500, blindness is 1 in 500, encephalitis is 1 in 1000, and pneumonia is 1 in 20. So vaccinations aren't just reserved for high-mortality diseases, but also ones that have a high rate of complications that can impact the quality of life long-term. Safety Of Vaccine Technology Safety standards are much higher for vaccines than most medications because we give vaccines to healthy people.   Some of this was learned the hard way. For example, in April 1955, more than 200,000 children in five Western and mid-Western USA states received a polio vaccine in which was basically a bad batch.  The process of inactivating the live virus proved to be defective, so rather in inoculating the children from polio, it ended up giving them polio instead.  Within days there were reports of paralysis, and within a month, the first mass vaccination program against polio had to be abandoned. This became a huge issue in the medical community. And it ended up enacting a lot of change in terms of what was acceptable safety standards.  Sarah explains that now vaccine technology is at the safest point it's ever been. But there is such a thing as vaccine-induced injury. Vaccine-Induced Injury  Stacy thinks the realities of the few cases of negative outcomes of vaccines need to be explored. (34:35) Especially since they risk being taken out of context or misunderstood.  She wonders what Sarah knows about the frequency of these negative outcomes. And what the science sense about the risk of injury.  Sarah explains this is extremely well-tracked and well-studied. The phenomenon of vaccine-related injury is incredibly rare. But she explains we do need to acknowledge it exists. She attributes social media for taking these few and far between cases and inflaming them in public. This, in turn, has destabilized a lot of the trust the public has in vaccines, which can be very harmful. She explains that an adverse reaction is usually something like soreness near the injection site or a bruise, maybe a headache, or anything that doesn't feel normal. A serious adverse reaction is something that requires medical care and could potentially result in death. Because of this risk, Sarah believes it's very important to be aware of serious adverse reactions to ensure you're making decisions that are medically in your best interest. Sarah takes a few moments to summarize some of the more serious adverse reactions from commonly administered vaccines and the odds of experiencing one. Stacy feels it's super important to address the elephant in the room. And there is no science showing any sort of link between vaccines and autism.  Adverse reactions can occur from vaccination, but a huge amount of scientific information has really conclusively shown autism is not one of them.  For more on Vaccine-Induced Injury, Sarah recommends checking here for additional information. Vaccine And Autoimmune Diseases Stacy explains that in autoimmune diseases, we often see them "activate" due to an immune system flare up- for example, during pregnancy or nursing. This isn't to say that pregnancy or nursing caused the autoimmune disease. But rather, it triggered it to activate, and that's why we start noticing the symptoms around that time. She explains that this holds true with vaccines as well. If someone starts to notice autoimmune systems after receiving a vaccine, that vaccine itself didn't "cause" the immune disease. Rather, it agitated the immune system. And that agitation triggered the symptoms of an autoimmune disease that was already lying latent inside the body. Sarah adds there's no evidence saying people with autoimmune diseases should avoid vaccines. If anything, they may need more booster vaccines to reach adequate immunity due to the immune system already not functioning optimally. The Importance of Herd Immunity Sarah also reminds listeners that vaccines aren't actually about individual protection at all. (46:10) They protect you individually, sure, but the reason vaccines are so amazing (and why smallpox was able to be eradicated) is because of the creation of herd immunity. Herd immunity means enough of a community is immune to an illness (cannot get it and cannot pass it) that if there is an individual infection, the illness has nowhere to go. It's stuck. Herd immunity limits the path for the virus to spread and can be much more easily contained. Herd immunity also protects members in our community who might have some sort of medical issue that prevents them from being vaccinated themselves.  Sarah cites children with cancer are unable to get vaccinated due to their health issues. So being surrounded by people who cannot spread a life-threatening illness is very beneficial to their health and wellness. Smallpox, which had an incredibly high mortality rate and permanent scarring, no longer exists anywhere in the world because of vaccines! So while we might want the covid vaccine for individual protection, that's not the primary goal. The primary goal of vaccination is community protection.   How mRNA Vaccines Work mRNA vaccines are the biggest advance in vaccine technology since Louis Pasteur and Edward Jenner. (50:35) It can revolutionize not just immunizations but also cancer therapy and other drug development. Brief History of mRNA Vaccine mRNA stands for messenger RiboNucleic Acid. Our cells make as an intermediary between the DNA in our cell's nucleus and a protein.  It also functions as a set of instructions to make protein, which is the intermediate step between DNA and the protein it encodes The steps are: DNA - transcription -> RNA - translation -> protein Translation may occur at ribosomes free-floating in the cytoplasm. Or directed to the endoplasmic reticulum by the signal recognition particle. mRNA was first discovered in 1961 by Sydney Brenner at Cambridge and James Watson at Harvard. The concept of mRNA-based drugs occurred in 1989 when Malone demonstrated that mRNA could be successfully transfected and expressed in various eukaryotic cells under a cationic (positively charged) lipid package.  In 1990, in vitro-transcribed mRNA was sufficiently expressed in mouse skeletal muscle cells through direct injection. This became the first successful proof of the feasibility of mRNA vaccines. After the first mRNA-based drug company was established in 1997, many groups began to research and develop mRNA-based drugs.  So far, over twenty mRNA-based candidate drugs have entered the clinical trial stage. A big advance in 2005 when Katalin Karilo and Drew Weissman at the University of Pennsylvania showed how to modify mRNA to get into human cells without triggering an immune response. Major advances in lipid nanoparticle technology for the mRNA envelope over the last 4-5 years.   Last 4-5 years, improvements in mRNA vaccines increase protein translation, modulate innate, adaptive immunogenicity, and improve delivery. This mRNA vaccine technology has been perfected in just the last few years. This is why the Covid-19 vaccine was able to be developed so quickly. The technology we needed to create this vaccine was already primed and ready to go. How Do mRNA Vaccines Work? Sarah explains that the coolest part of mRNA vaccine is that they do not use adjuvants! (58:01) This is because adding the RNA to the cell nucleus is enough to trigger it to replicate. It doesn't need anything additional to trigger the immune response.  Two major types of RNA are currently studied as vaccines:  non-replicating mRNA which is what's in both the Pfizer/BioNTech covid-19 vaccine and the Moderna covid-19 vaccine virally derived, self-amplifying RNA.  Conventional mRNA-based vaccines encode the antigen of interest and contain 5′ and 3′ untranslated regions (UTRs).  Self-amplifying RNAs encode the antigen and the viral replication machinery that enables intracellular RNA amplification and abundant protein expression. The lipid envelope facilitates entrance into the cell via endocytosis and exit from endosome into cytoplasm This molecule provides the template in the cytoplasm of a cell for translation by the ribosome.  And tRNA into the encoded protein, making multiple copies of the protein from each mRNA template. The protein can then be presented to the immune system through MHC or, like both Pfizer/BioNTech and Moderna vaccine, the protein is transmembrane, so it presents itself!  Sarah explains that there were some human trials using mRNA vaccines to treat cancer patients.  So yes, as Stacy brings us, the technology is still pretty new. But this isn't the first time we're using mRNA technology. It's the first opportunity we've had to utilize the discoveries large-scale. Ingredients Of mRNA Vaccines Sarah explains that what makes this new vaccine technology so cool is how few ingredients it requires to make. (1:05:20) mRNA (rather than a live attenuated virus, dead virus, or split virus) Lipid nanoparticle envelope (rather than viral particles floating around a solution or viral vector-like adenovirus)LNPs often consist of four components:  an ionizable cationic lipid, which promotes self-assembly into virus-sized (~100 nm) particles and allows endosomal release of mRNA to the cytoplasm;  lipid-linked polyethylene glycol (PEG), which increases the half-life of formulations; cholesterol, a stabilizing agent;  and naturally occurring phospholipids, which support lipid bilayer structure.  It requires no adjuvant, which is SO COOL! Adding an adjuvant to the lipid envelope has been studied, but it doesn't seem to be necessary. This is because foreign mRNA and viral proteins are really good at eliciting an immune response. mRNA has self-adjuvant properties which activate strong and long-lasting adaptive immune responses through tumor necrosis factor-α(TNF-α), interferon-α(IFN-α), and other cytokines secretion by immune cells The foreign viral proteins are presented via MHC-I Lipid nanoparticles may have a little adjuvant activity in some circumstances. But basically, all of the immune stimulation is targeted against the foreign viral protein and mRNA! For example, here are all the ingredients for the Moderna Vaccine: The vaccine contains a synthetic messenger ribonucleic acid (mRNA) encoding the pre-fusion stabilized spike glycoprotein (S) of SARS-CoV-2 virus.  lipids (SM-102, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 [PEG2000-DMG], cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphocholine [DSPC]),  pH Buffering agents: tromethamine, tromethamine hydrochloride, (both drugs for metabolic acidosis) acetic acid, sodium acetate, (both naturally found in our blood) Cryo-stabilizer: sucrose Sarah jokes about how much she's nerding out about it.  Advantages Over the past decade, major technological innovation and research investment have enabled mRNA to become a promising therapeutic tool in vaccine development and protein replacement therapy.  The use of mRNA has several beneficial features over subunit, killed, live attenuated virus, and DNA-based vaccines.  Safety As mRNA is a non-infectious, non-integrating platform, there is no potential risk of infection or insertional mutagenesis.  Additionally, mRNA is degraded by normal cellular processes. And it's in vivo half-life can be regulated through the use of various modifications and delivery methods 9,10,11,12.  The inherent immunogenicity of the mRNA can be down-modulated to further increase the safety profile9,12,13.  2: Efficacy Various modifications make mRNA more stable and highly translatable9,12,13.  Efficient in vivo delivery can be achieved by formulating mRNA into carrier molecules, allowing rapid uptake and expression in the cytoplasm (reviewed in Refs 10,11).  mRNA is the minimal genetic vector; therefore, anti-vector immunity is avoided, and mRNA vaccines can be administered repeatedly.  mRNA vaccines expressing antigen of infectious pathogen induce both strong and potent T cell and humoral immune responses Even better for viruses requiring cellular immunity like coronaviruses. (Click here for more!) Production  mRNA vaccines have the potential for rapid, inexpensive, and scalable manufacturing, mainly owing to the high yields of in vitro transcription reactions. They are really fast to make. Moderna took 2 days to create the RNA sequence to produce the spike protein after sequencing the virus genome in January. Then shipped its first vial of vaccine to NIH for trials 41 days after that. This will also mean the vaccine can be modified for new strains (so far, not necessary), and we can get a vaccine even faster in the event of another pandemic! Myths About the mRNA Vaccines One of the biggest myths many people believe is that the vaccines were rushed. So we don't know if they're safe. (1:07:30) The unprecedented investment (funding) allowed for tests normally done serially to be done in parallel. And it allowed for manufacture (normally 6 months to a year) to be done during clinical trials rather than after. These vaccines build upon vaccine research from SARS and MERS and the knowledge base about coronaviruses from that research. So we've been researching it longer than people have known about the novel coronavirus. For example, it was already known that the spike protein bound with ACE2. And that's how SARS-CoV-2 infects cells. It also builds upon a tremendous amount of mRNA vaccine research and clinical trials of mRNA vaccines for cancer. mRNA vaccine technology allows for a fast process. It's also very inexpensive to make. Yay science! Vaccines have some of the most stringent safety standards in all of pharmaceutical development! They are given to healthy people, not sick, so tolerance for serious reactions is lower. Also, these vaccines were tested thoroughly and have exceeded the standards. No corners were cut! Yes, there are still some things we don't know (like whether or not you can get an asymptomatic case after you've been vaccinated and then spread the virus or how long immunity will last), but we do know that the safety profile is excellent. It's approved for 16 and over because they did tests on adults before children. In fact, the 12-15 age groups are being tested now. Final Thoughts One of the biggest reasons these vaccines were able to be produced so fast is because of the timing. (1:10:42) Scientists have been working on vaccine technology for centuries. And major advancements in the last 30 years have made it possible to produce both efficient and safe vaccines. This is why basic science funding is so, so important.  Sarah goes into why this basic funding is so important. Most funding is going to direct human relevance.  The science that these vaccines are based on comes down to a basic discovery and expanding human knowledge.  And only after the fact, we understood how it could be applied to improving human life.  So increasing funding for basic science discovery is very important to Sarah. Stacy also circles back to how mind-blowing that this basic science discovery could also further our advancement toward a cure for cancer.  She reminds listeners that there are two vaccines approved for disruption in the US. Next week, Sarah and Stacy with dive into the science and myths on those to bring you all the info you need to make your own decision. If you're curious how Sarah and Stacy really feel about this topic, pop on over to Patreon for more science talk and bonus content.   See you next week!  

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.06.371484v1?rss=1 Authors: Basu, I., Gorai, B., Chandran, T., Maiti, P. K., Hussain, T. Abstract: During translational initiation in eukaryotes, the small ribosomal subunit forms a 48S preinitiation complex (PIC) with initiation factors. The 48S PIC binds to the 5' end of mRNA and inspects long untranslated region (UTR) for the presence of the start codon (AUG). Accurate and high speed of scanning 5' UTR and subsequent selection of the correct start codon are crucial for protein synthesis. However, the conformational state of 48S PIC required for inspecting every codon is not clearly understood. Whether the scanning or open conformation of 48S PIC can accurately select the cognate start codon over near/non-cognate codons, or this discrimination is carried out only in the scanning-arrested or closed conformation of 48S PIC. Here, using atomistic molecular dynamics (MD) simulations and free energy calculations, we show that the scanning conformation of 48S PIC can reject all but 4 of the 63 non-AUG codons. Among nine near-cognate codons with a single mismatch, only codons with a first position mismatch (GUG, CUG and UUG) or a pyrimidine mismatch at the second position (ACG) are not discriminated by scanning state of 48S PIC. In contrast, any mismatch in the third position is rejected. Simulations runs in absence of one or more eukaryotic initiation factors (eIF1, eIF1+eIF1A, eIF2[a] or eIF2{beta}) from the system show critical role of eIF1 and eIF2[a] in start codon selection. The structural analysis indicates that tRNAi dynamics at the widened P site of 48S open state drives codon selection. Further, a stable codon: anticodon interaction prepares the PIC to transit to the closed state. Overall, we provide insights into the selection of start codon during scanning and how the open conformation of 48S PIC can scan long 5' UTRs with accuracy and high speed without the requirement of sampling the closed state for every codon. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.22.351072v1?rss=1 Authors: Coelho, V. L., Brito, T. F. d., Brito, I. A. d. A., Cardoso, M. A., Berni, M. A., Araujo, H. M. M., Sammeth, M., Pane, A. Abstract: Rhodnius prolixus is a Triatominae insect species and a primary vector of Chagas disease. The genome of R. prolixus has been recently sequenced and partially assembled, but few transcriptome analyses have been performed to date. In this study, we describe the stage-specific transcriptomes obtained from previtellogenic stages of oogenesis and from mature eggs. By analyzing ~228 million paired-end RNA-Seq reads, we significantly improved the current genome annotations for 9,206 genes. We provide extended 5' and 3' UTRs, complete Open Reading Frames, and alternative transcript variants. Strikingly, using a combination of genome-guided and de novo transcriptome assembly we found more than two thousand novel genes, thus increasing the number of genes in R. prolixus from 15,738 to 17,864. We used the improved transcriptome to investigate stage-specific gene expression profiles during R. prolixus oogenesis. Our data reveal that 11,127 genes are expressed in the early previtellogenic stage of oogenesis and their transcripts are deposited in the developing egg including key factors regulating germline development, genome integrity, and the maternal-zygotic transition. In addition, GO term analyses show that transcripts encoding components of the steroid hormone receptor pathway, cytoskeleton, and intracellular signaling are abundant in the mature eggs, where they likely control early embryonic development upon fertilization. Our results significantly improve the R. prolixus genome and transcriptome and provide novel insight into oogenesis and early embryogenesis in this medically relevant insect. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.12.247627v1?rss=1 Authors: Goepferich, M., George, N. O., Domingo Muelas, A., Bizyn, A., Pascual, R., Fijalkowska, D., Kalamakis, G., Müller, U., Krijgsveld, J., Mendez, R., Farinas, I., Huber, W., Anders, S., Martin-Villalba, A. Abstract: Autism spectrum disorder (ASD) is a neurodevelopmental disease affecting social behavior. Many of the high-confident ASD risk genes relate to mRNA translation. Specifically, many of these genes are involved in regulation of gene expression for subcellular compartmentalization of proteins. Cis-regulatory motifs that often localize to 3'- and 5'-untranslated regions (UTRs) offer an additional path for posttranscriptional control of gene expression. Alternative cleavage and polyadenylation (APA) affect 3'UTR length thereby influencing the presence or absence of regulatory elements. However, APA has not yet been addressed in the context of neurodevelopmental disorders. Here we used single cell 3'end sequencing to examine changes in 3'UTRs along the differentiation from neural stem cells (NSCs) to neuroblasts within the adult brain. We identified many APA events in genes involved in neurodevelopment, many of them being high confidence ASD risk genes. Further, analysis of 3'UTR lengths in single cells from ASD and healthy individuals detected longer 3'UTRs in ASD patients. Motif analysis of modulated 3'UTRs in the mouse adult neurogenic lineage and ASD-patients revealed enrichment of the cytoplasmic and polyadenylation element (CPE). This motif is bound by CPE binding protein 4 (CPEB4). In human and mouse data sets we observed co-regulation of CPEB4 and the CPEB-binding synaptic adhesion molecule amyloid beta precursor-like protein 1 (APLP1). We show that mice deficient in APLP1 show aberrant regulation of APA, decreased number of neural stem cells, and autistic-like traits. Our findings indicate that APA is used for control of gene expression along neuronal differentiation and is altered in ASD patients. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.28.224998v1?rss=1 Authors: Froehlich, J., Uyar, B., Herzog, M., Theil, K., Glazar, P., Akalin, A., Rajewsky, N. Abstract: Understanding how regulatory sequences control gene expression is fundamental to explain how phenotypes arise in health and disease. Traditional reporter assays inform about function of individual regulatory elements, typically in isolation. However, regulatory elements must ultimately be understood by perturbing them within their genomic environment and developmental- or tissue-specific contexts. This is technically challenging; therefore, few regulatory elements have been characterized in vivo. Here, we used inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3' UTRs of 16 genes in C. elegans. To quantify the consequences of mutations on expression, we developed a targeted RNA sequencing strategy across hundreds of mutant animals. We were also able to systematically and quantitatively assign fitness cost to mutations. Finally, we identified and characterized sequence elements that strongly regulate phenotypic traits. Our approach enables highly parallelized, functional analysis of regulatory sequences in vivo. Copy rights belong to original authors. Visit the link for more info

(1) How did Scientologists react to the obvious and serious anti-Scientology, unethical behavior that David Miscavige’s sister was involved in via drugs/alcohol when it was reported on back in 2013? I’d imagine it made the local paper due to the connection to the elephant in the area. I’d suppose UTRs, regular public and maybe some brave […] The post Critical Q&A #180 appeared first on Chris Shelton - Critical Thinker at Large.

Die miR-30-Familie gehört zu den am stärksten exprimierten microRNAs im Herzen. Expressionsanalysen zeigten, dass alle Mitglieder der miR-30-Familie bei humaner Herzinsuffizienz und bei Mäusen im Rahmen einer post-Infarkt- Herzinsuffizienz herunterreguliert werden. Überraschenderweise fanden wir auch bei physiologischer Hypertrophie nach Lauftraining eine Herunterregulation von miR-30. Dies könnte ein Indiz für eine kardioprotektive Funktion der Herunterregulation der miR-30-Familie im Rahmen von pathologischem Stress sein könnte. Im Einklang mit dieser Hypothese entwickelten transgene Mäuse mit Kardiomyozyten-spezifischer Überexpression einzelner miR-30-Familienmitglieder eine dilatative Kardiomyopathie und starben deutlich verfrüht. Anhand von Gen-Array-Analysen mit Herzen von transgenen miR-30-Mäusen sowie bioinformatischer Zielgensuche mit TargetScan konnten wir RGS2, DDAH1, EDNRA und ADRA2A als mögliche Ziel-Gene identifizieren, über die miR-30 potentielle kardioprotektive Effekte ausüben könnte. Durch weitere Analysen mit Luciferase-Assays konnten wir zeigen, dass die miR-30-Familie diese Gene tatsächlich direkt über ihre 3’-UTRs regulieren kann. Zusammenfassend lässt sich folgern, dass die Herunterregulation von miR-30 im Rahmen von kardialem remodeling einen kardioprotektiven Effekt haben könnte.

Background: miRNAs are small, non-coding RNA molecules that mainly act as negative regulators of target gene messages. Due to their regulatory functions, they have lately been implicated in several diseases, including malignancies. Roughly half of known miRNA genes are located within previously annotated protein-coding regions ("intragenic miRNAs"). Although a role of intragenic miRNAs as negative feedback regulators has been speculated, to the best of our knowledge there have been no conclusive large-scale studies investigating the relationship between intragenic miRNAs and host genes and their pathways. Results: miRNA-containing host genes were three times longer, contained more introns and had longer 5' introns compared to a randomly sampled gene cohort. These results are consistent with the observation that more than 60% of intronic miRNAs are found within the first five 5' introns. Host gene 3'-untranslated regions (3'-UTRs) were 40% longer and contained significantly more adenylate/uridylate-rich elements (AREs) compared to a randomly sampled gene cohort. Coincidentally, recent literature suggests that several components of the miRNA biogenesis pathway are required for the rapid decay of mRNAs containing AREs. A high-confidence set of predicted mRNA targets of intragenic miRNAs also shared many of these features with the host genes. Approximately 20% of intragenic miRNAs were predicted to target their host mRNA transcript. Further, KEGG pathway analysis demonstrated that 22 of the 74 pathways in which host genes were associated showed significant overrepresentation of proteins encoded by the mRNA targets of associated intragenic miRNAs. Conclusions: Our findings suggest that both host genes and intragenic miRNA targets may potentially be subject to multiple layers of regulation. Tight regulatory control of these genes is likely critical for cellular homeostasis and absence of disease. To this end, we examined the potential for negative feedback loops between intragenic miRNAs, host genes, and miRNA target genes. We describe, how higher-order miRNA feedback on hosts' interactomes may at least in part explain correlation patterns observed between expression of host genes and intragenic miRNA targets in healthy and tumor tissue.

The effective uptake of inorganic phosphate by cells from the environment depends on specific transport systems. In bacteria, these uptake systems are well characterized and most species contain both secondary transporters as well as primary uptake systems belonging to the ABC-family of transporters. Under phosphate starvation, genes coding for high-affinity phosphate transporters are induced in bacteria as well as in eukarya. In E. coli these are genes of the phosphate-specific transport via Pst or the Glycerol-3-phosphate-specific transporter, Ugp. Archaea possess the PHO stimulon, which induces numerous genes in response to phosphate limitation. So far this stimulon has only been described for Halobacterium salinarum R1. The genome of H. salinarum encodes ABC transporters resembling the Pst and Ugp systems of E. coli which are upregulated under Pi-limited conditions. In this study, the gene expression and function of the phosphate dependent operons pst1, pst2 and ugp of H. salinarum were investigated. First, it was shown that the three operons (pst1, pst2 und ugp) are transcribed as one polycistronic unit. The respective promoters are located upstream of the first ORF (open reading frame) of the operons, and transcription start sites (TSS) were mapped. Two TSSs were found for the pst1 operon, and are utilized in a phosphate dependent manner. Through an unknown regulation mechanism, the cell switches transcription of pst1 mRNA to either a transcript with or without a 60 nt long leader sequence. The transcripts of the pst2 and ugp operons have no 5’UTRs, regardless of phosphate concentration. Using a Ppst1-bgaH reporter system, it was observed that the transcripts with or without a leader sequence have different translation efficiencies. The transcript without a 5‘UTR had a 150-fold higher translation efficiency than the transcript with a 5‘UTR. It was concluded that the expression of the pst1 operon is modulated through a post-transcriptional regulation mechanism. To our knowledge, this is the first identified archaeal protein-coding operon that is transcribed by alternative promoters. The differences in phosphate dependent gene expression of the pst1, pst2 and ugp operons were investigated using the bgaH reporter system. Under phosphate saturated conditions, the expression of the pst2 operon is stronger compared to the expression of the pst1 operon, whereas under phosphate limited conditions, pst1 operon expression is highly induced. This assay system also identified the TATA boxes of the pst1 and pst2 promoters as well as AT-rich motifs, named P boxes. Mutations in the P box, with the consensus ATATWWW , reduced the promoter activity of both the pst1 and pst2 promoters. The results indicated that the TATA-2 box of the pst1 promoter has an impact on the phosphate dependent promoter activity under phosphate limited conditions and could be used as a P box. In the second part of the study, the proposed functions of the transporters Pst1, Pst2 and Ugp and the kinetic parameters were determined. Different knockout mutants were constructed, and these showed that the Pst transporters had different phosphate affinities. It was concluded from the results that the binding proteins PstS1 and PstS2 can interact with both transporters. The phosphate transport systems Pst1 and Pst2 are used differently. Under phosphate saturated conditions the cell operates mainly with the lower-affinity Pst2 transporter. If phosphate becomes limiting in the environment, predominantly the high-affinity transporter Pst1 is induced. This process is regulated on the transcriptional as well as on the post-transcriptional level. Furthermore, it was shown that a deletion of the Pst1 transporters leads to a loss of phosphate-directed chemotaxis under Pi-stress. This result confirms the importance of the Pst1 transporter under phosphate limited conditions. In related studies, growth experiments showed that the Ugp transporter is the only transporter for glycerol-3-phosphate in H. salinarum. In addition, the search for a regulatory protein that is involved in the regulation of the genes of the PHO stimulon did not reveal any likely candidates, but it was shown that deletion of the pst1 operon leads to an induction of the pst2 operon.

microRNAs (miRNAs) are small non-coding RNAs of 21-24 nt in size, which are endogenously expressed in higher eukaryotes and play important roles in processes such as tissue development and stress response and in several diseases including cancers. In mammals, miRNAs guide proteins of the Argonaute family (Ago proteins) to partially complementary sequences typically located in the 3’-untranslated regions (3’-UTRs) of specific target mRNAs, leading to translational repression or mRNA degradation. To gain further insight into the function of human miRNAs, we analyzed the protein as well as the RNA composition of miRNA-Ago protein complexes in molecular detail. To identify novel Ago-interacting proteins, we isolated Ago complexes and investigated them by mass spectrometry and co-immunoprecipitation experiments. We found that trinucleotide repeat-containing 6B (TNRC6B), Moloney leukemia virus 10 (MOV10), RNA binding motif protein 4 (RBM4) and Importin 8 (Imp8) interact with human Ago proteins. Moreover, using RNA interference and EGFP and dual luciferase reporter assays, we demonstrated that these factors are required for miRNA function, indicating that we have identified new components of the miRNA pathway. Intriguingly, depletion of Imp8 does not affect the levels of mature miRNAs or the interaction of miRNAs with Ago proteins, but is required for efficient association of Ago-miRNA complexes with their target mRNAs. Thus, Imp8 is the first factor acting at the level of target mRNA binding, establishing a novel layer of regulation for the miRNA pathway. Imp8 is an Importin-β-like protein, which has previously been implicated in nuclear import of substrate proteins. In line with these results, we demonstrated that a detectable fraction of Ago2 localizes to the nucleus of human cells. Moreover, knockdown of Imp8 by RNAi reduces the nuclear signal of Ago2, suggesting that Imp8 affects the nuclear localization of Ago2. Therefore, our data suggest that Imp8 has a dual function both in the cytoplasmic miRNA pathway and in nuclear transport of Ago proteins. To identify small RNAs, which associate with human Ago proteins, we isolated, cloned and sequenced small RNAs bound to Ago1 and Ago2 complexes. In addition to known miRNAs, we found several small RNAs, which derive from small nucleolar RNAs (snoRNAs). We therefore investigated the function of one particular small RNA, which is derived from the snoRNA ACA45 and showed that it functions like a miRNA. Interestingly, this small RNA is processed by the miRNA maturation factor Dicer, but does not require the microprocessor complex that is essential for processing of primary miRNA transcripts. Thus, we have identified a novel biogenesis pathway of a new class of small RNAs that can function like miRNAs. To experimentally identify mRNAs that are stably associated with miRNA-Ago protein complexes, we isolated and analyzed Ago1 and Ago2-bound mRNAs by cloning and sequencing and by microarray hybridization techniques. Using dual luciferase reporter assays, we demonstrated that many Ago-associated mRNAs are indeed miRNA targets. Therefore, we have developed a method allowing for the identification of miRNA target mRNAs from cell lines or tissues of interest independently of computational predictions. In a project that was independent of our studies on Ago protein complexes, we investigated structural and functional requirements for the activity of small interfering RNAs (siRNAs). siRNAs are small double-stranded RNAs of appr. 21 nt in size, which trigger the sequence-specific endonucleolytic degradation of perfectly complementary target transcripts upon binding to Ago2. However, both single strands of a siRNA duplex can potentially have unwanted “off-target effects” by repressing partially complementary target mRNAs through binding to their 3’-UTRs. We therefore developed a method to selectively inhibit the activity of the siRNA strand that is dispensable for target silencing (“passenger strand”) through chemical modification of its 5’-end. This method could be a useful tool for the design of highly specific siRNAs. Taken together, we have analyzed the composition of Ago-miRNA protein complexes by a variety of methods and identified novel protein factors of the miRNA pathway, a novel class of small RNAs as well as a panel of previously unknown miRNA target mRNAs. The techniques for the purification and the analysis of Ago complexes that were developed in this study will provide useful tools for future analyses of miRNA pathway factors, small RNAs and miRNA target mRNAs from any tissue or cell line of interest.