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

IMPORTANT NOTE: due to Dave not paying attention, the networking event this month is on the 23rd at Detroit City Distillery.  We know, it’s always 3rd Thursday… he screwed up, it’s a thing, move on.  That being said, tonight we’re joined by Caston Thomas to chat about our recent appearance on the Internet Advisor show, as well as an upcoming seminar series he’s doing about securing iOT devices and networks that you probably want to pay attention to… Happy patch Tuesday. This a special recording of the IT in the D show recording a day late. But that’s all right. We’ve got a special guest in the studio Caston Thomas in the house. We’re going to be talking about all of the fun stuff that you, a and security that we just got off on. WJR to go home and home are thrilling experience on am radio. Yeah, yeah. We were talking about that and talking about security and property security. Uh, you know what, Dave, you may fire when ready going on. Thanks for hanging out. This is the one and only it in the D show. Broadcasting live here in studio three and podcast. He traded beautiful Royal Oak. Michigan has chaos. May have it happens. They say, is Bob the sales guy? That was Dave the geek. Randy. I do. The Twitters is doing the Twitters. Find a some ways. Find us online it in the D. dot com. Do us a favor, give us a like on the socials and subscribe to us everywhere. Fine podcasts are sold. And so because I’m an idiot and I freely own it and I admit it. Uh, so the meetup for this month was not set up for the third Thursday. It was set up for the fourth Thursday. So it is not this Thursday. It is next Thursday, the 23rd. Uh, it will be at Detroit city distillery. Come on out. Hang. Should be a fun night. Love the venue. Love the liquor. It’s a good time. I’m not gonna lie, it’s big coming. Extremely difficult to book a venue in this town. It is, and we’ve talked about this for two years now. We’re like, you know, places that, you know, we went for, you know, six, seven, eight years when nobody cared about them and nobody went there. All of a sudden now they want, they got the a, they got the no, because they’ve got somebody booked a holiday play there and now they think everybody’s got 1000 bucks an hour. My ass Russell, they booked an M and M video and how they think they can get top dollar, but like I called a, it’s a new retro bowling alley in Royal Oak. That’s shall not be named that has pull in the name. Um, I’m only like 200 bucks an hour just to show up. No food. No, I’m good. Thanks. It’s kind of like, Hey, and I said, we can always just crash a joint. Right? Yeah. But I don’t want to do that because then we, you know, the people ask, the thing is you need to have buy in from the front desk. Like, Hey, I’m here for it in the day. True. Whoa. What? Yeah. Um, so like I go, Hey, there’s going to be like 30 to 50 and flash mobs or so, 2001 we don’t, do they need to die? Well, they know. I say this is the way I set it up. I go, we don’t charge because here’s the thing, I can understand what the venue is when you charge. Like, Hey, I’m going to use your 50 bucks to walk in the door and then I charge you 20 bucks. Like kiss my ass. Yeah. But I’m saying, Hey, we don’t charge cover. We don’t take sponsors. There’s no speakers. Everyone’s on their own for food and whatever they want to order. Um, we just need someplace to hang out. Yeah. Okay. It’ll be 200 bucks an hour. Like for, for, for, for what? Bye-bye. Yeah, exactly. Cause you’re so crowded on a random Thursday night that you can’t use the additional business, you know, then smell you in the window. You know, if you’re, if you’re, if you know someone that’s a bar owner, like literally that would want us reach out to us. Please. We’re glad to hear about it. These phone calls, it’s ridiculous. Like, or we’ll just keep doing all of our events at whiskey in the jar. It’ll be that simple. We’ll just be there every, we don’t have, do we have friends at Ember anymore? No. No. Rusty’s still there. Rusty’s, you’re going to pork around. Um, uh, I don’t know. Maybe I just don’t like that venue that I think it’s, you know, nobody plays pinball, but it’s just cool to have them in the background. Exactly. It’s a fun spot. I’m sure we can find someplace. So the baby, I mean it’s not anywhere convenient, but like dude, that arcade place and Frazier would like cut off their arm to have us come to that. Yeah. And speaking of that like major magic is making a comeback. They are, they’re right there. Right around the corner from me. Here’s an interesting, I never really, I don’t think I ever talked to you about this. Um, when major magics said they were coming back and I happened to pop up my note on, on LinkedIn or Facebook saying, Hey, you know, invested in a brewery in the past didn’t work out. I see you’re coming back. Want to know what you’re doing with it. You wanted to see if you wanted to like maybe adult it. Yeah. Make major magics you know, for the nostalgia crowd. I mean Chuck E cheese serves beer. Yeah. And he’s like, what’s your intention? Like it was really bizarre conversation. Like, I wish I saved it. I wish you could read it. And I’m like, ah, I just want to talk to you. Like maybe we can have a cup of coffee. Well, what, what do you want out of it? Well, I know how much you were looking to. I go, I don’t know what it is yet or what it wants to be, and I can’t really give you a business proposal when nothing’s, I’m not just sitting here on a bag of money looking to do something with like [inaudible] furnace here. I’ve already done that once. That’s why we don’t own a brewery anymore. But then low and behold, maybe like six months ago, four months ago, he’s like, Hey, are you still interested in? And I’m like, ah, ignore like you’re such a weirdo. Like I didn’t want nothing. So now apparently it’s opening in like Chesterfield or something. No, dude, it’s right around the corner from me. Oh, it’s on, it’s on gross back. It’s 15 and gross back. It’s in. So do you remember the, there was the taco bell, there was the abandoned taco bell and then the other building. So the, it’d be a crime. Yeah. So the other building was the old Riviera restaurant and that’s where they was the Ram’s horn. That was the, it was a, it’s called Riviera. No, but it was a rent like long time ago, back in the old taste back when I was dragging the day when I hung out on the East side when I was stumbling at a chaplains and went to get French toast. Uh, ya know, so that’s, yeah apparently in there, I think they’re opening in February if I recall correctly. Like not too long from now. They’re probably just, they pay you. The thing is it’s probably going to be just a Barcade and he’s got like six of the stupid dolls and he’s going to set them up. No, apparently it’s again I’m just going off the articles I read in that kind of stuff cause I was, I was curious cause I like I’ve heard you guys, cause it wasn’t here when I was here, but I’ve heard you guys talk about it enough that I’m just curious to grasp it next to the star theater. And apparently like everything that was in that old one was put into storage and they have pulled everything out of storage and, and loaded up the place except for like skee-ball and like, it smelled like, was like jeepers, it smells like feet and piss. Like it’s not theirs. They should just call it that. Phoenix. Nice pizza. That’s actually probably a website. I’m quite sure. Smell the feet and Piz pizza then in the tokens. Um, but Hey, jumping in, uh, obviously today was patched today. We’re, we’re recording on Tuesday. If you haven’t figured that out yet, today’s patch Tuesday. And if you’re working in a data center tomorrow, it’s gonna be patch Wednesday. What’s, you know what, I’m just gonna throw this out there. Can we talk about the windows? Windows 10 flaw for 10 seconds? Sure. Yeah, I know. Right? So here’s this. No, this is, to me, this is interesting because the NSA came out with this and the NSA hasn’t done this in the past. The NSA has held things for themselves. So apparently there is a pretty wicked windows 10 security patch. Uh, they just basically can expose users to surveillance or, uh, I believe it’s with authentication. Yep. Um, but it’s funny that back in the day, uh, they did, they did not, they held it back. They, they used it because they wanted that access. Right. So, you know, uh, wanna cry an external blue, uh, basically NSA exploited those for years and didn’t say shit. Um, they basically, they develop hacking tools to exploit these holes. Um, and then they gave them to, or they were then found by Russian hacker groups called shadow brokers. Um, eternal blue still use to this day. Well and I think we all know that when we do something fine, but when the Russians do it, no, nah, no, no. Um, but apparently no, this was a even like at work, this is a big deal where it’s all hands on deck first thing in the morning. Uh, security teams, all the server teams. Um, you know, this is, yeah, this is like all hands on deck. So yeah, if you’re in a data center and, uh, you know, tomorrow should be fun. Hey, I mean Sam a broadcaster just updated and added a new notepad function. So, I mean that was pretty cool cause that’s something that everyone’s been dying for. I’m just dried mouse. No to like add notes while you’re, cause you don’t have notepad or WordPad or anything on the computer. You’re running it on, you need a notepad in the stupid, just stupid. Speaking of that, there was um, I was thinking, I don’t know why I think this is funny all the time, but they did doodle for Google. Yeah. And you can, um, so here’s the thing. I don’t know why this is funny. So here’s the thing. I work, we have a thing at work called brilliant ideas and people could submit a brilliant idea and it goes in front of a panel and they either move forward with it and it could be something really simple. Like, Hey, we should do a dial by extension, not by name. Like, or you know what I mean, or whatever. Gotcha. Or five extent, um, or spelled my name anyway, the new hotness now is to do something completely the stupidest thing you could possibly think of and to get the politest answer back. Um, so now, now it’s all about getting eyeballs. So now they have this thing doodle for Google and they want, you have to enter a doodle and then it might get used as a logo. And so I want to do a Dick button. So what you want to or you already have? Cause I haven’t because no I’ve been to be looking at like the butt cheeks. The O’s. No. Oh see rainy. That’s why. That’s why I bring it up. I need better ideas. So there’ll be a panel, there’ll be someone on a panel at Google that is going to get a Dick. But with butt cheeks, his owes that they have to go. Thank you for your submission. I was like I don’t like dragging the Dick boy decided to go in another direction. Oh you know what, I don’t think it doesn’t meet our community standards. Right. You get the nice little legal ease and I don’t without the Wiener too cause I just want the O’s. Cause if the, you know, cause there’s usually a winner in the Dick pot in the back part. Just put little dots on the O’s and make it Google part of Dick buck. Yes. So just, just, but I want him Unix. Yeah. I don’t like drug cause we always got a unit that’s a concerto, you know, come and cut. Don’t know why. It always makes me laugh. Oh my God, this was on a whiteboard next to like the hotel desk with like a dry erase board with markers. Like who thinks while a comic con is going on and people are getting just falling down drunk, let’s leave the markers on the white board overnight. So every time in the morning, I mean giant Dick blended. So I would have to go erase it. And I tried again. I sorta like, Oh I know you did. I don’t know. Like seriously, I’m 46 years old and I still go to friends houses that have alphabets on their fridge and I still have to spell hard. Like I can’t not pass those things by the inner 11 year old game is strong indeed. Indeed. But doodle for Google if you haven’t seen it. Um, let’s do, let’s do a bunch of Dick butts submissions. I’m game all the artists listening. Hello Eric. UTRs. Yeah, you guys all, we’ll talk. We’ll totally rally the troops. So I don’t mind a more serious, serious note. Uh, the deep fake stuff just started. And if they’re kind of creepy and there’s other videos, like when people doing impersonations and they kind of deep fake the face. And then we saw some other stuff with George Lucas. I don’t know that it’s started it like, it’s not that it just started, it just started getting a lot better, right? Words unnoticeable. Well now Facebook announces it’s going to ban deep fakes, but it won’t ban political ads, fake news, any place like it, it’ll let that, well, whatever. Um, and how are they going to know? Well, first thing I said was, cause the whole point of deep fakes is that it’s hard to tell, strengthen his policy towards misleading, manipulated videos. But like again, how do you know it’s like the Joe Rogan when they cut up his pot, the podcast, and he was talking about, uh, gorillas being the, the alter monkeys being the greatest. No, dude, that wasn’t, they didn’t cut it up. That was the AI that generated that. I know. I thought they’d like clipped his voice. No, that was the AI that Lake listened to all of his episodes and then generated a new episode from them all his claims. Right, right. But how can you like, Nope, it’s fake. Like, and that was a thing you couldn’t tell Joe Rogan’s vote. There was like one, like an inflection was weird, but if you do, if you put some time into it, it would sound completely flawless. We’ll do that one with George Lucas. Uh, reacting to the latest movie was his head looked a little weird. Like you could tell something was wrong, but if you’re just sitting there listening to it and only half ass watching it, it looked legit. Or if you scroll down in Facebook puts the little window up in the corner where you just see like three pixels of the video or whatever, but they won’t pull down videos that are, you know, the easy way edited. You know, where if you just slow down the video a little bit to make it seem like a politician is slurring her. Yeah, they won’t pull that down cause that’s not high tech enough of a video. Well I never forget how they do it with pictures. Like I’ll never forget the picture I went like you can see Trump does enough dumb stuff on his own where you don’t need to manipulate him doing dumb stuff usually true. And him and like him and his wife and the Obamas or like going to dinner or going into a thing and Trump rushed front and opened the door for everyone and then walked in last. Okay. Well the picture was, you know, Trump walks like bully, like walked in first and he’s rude like he’s not a gentleman. Well then if you watch the video, like he held the door open and then walked in last and it’s like, it’s out of context. Well that time, by that point it gets to Buzzfeed and it becomes a thing. And if you Google it now, you’d probably, Randy, if you like, if you Google, you’ll probably find that like Trump doesn’t open door for Obama’s and it’s probably, it’s like, I’m sure. Well, like so like, so you’re banning deep pigs but you’re not banning fake political edge. You’re not banning. Yeah, like propaganda, like bullshit. Like where do you draw and who again and how do you, like I said, how are you going to figure out if they’re fake or not? It’s the river. It’s booger yelling and revenge there. It’s too. And who determines the standards? Well, send it. Remember? Do I remember correct? Elected board? No, dude. Remember I got to think it was two years ago we were talking about, okay, so it’s easy enough to fake audio clips. What’s it going to take to prove somebody actually said something? Oh, well video. All right. Now that’s not the answer anymore cause yeah, deep fake video to make them say whatever the hell you want to say. So yeah, we talked about that. We’ve been on, we’ve been doing this for six years. Um, we probably said every word to humanly like, dude, they did the pipe. My AI Brogan was 200 episodes, if I recall correctly. Was that only the toilet word? Tonight’s three 30, he goes for six hours in episode trope when he smokes aardvark esophagus, incandescent. I was like, what are you doing? Just throw some extra words out there for the AI. They would do like discombobulated. David has a vocabulary of like 4,000 words. Bob 140. We can make Bob say anything we want as long as it has to do with fart. Yes. Yeager it’d be be. So we always, we’d like talking about the, the, the mighty, uh, over, uh, the, the company’s worth way too much money. Um, do Uber, Uber, uh, released their first ever, uh, safety report. Uh, this is actually updated version. So we talked about this two months ago when it was first released. So they’ve, they’ve updated it. So they’re just plain plain tag. They did the numbers change their 3045 sexual assaults and nine murders in 2008. Yes. And that includes, they admitted to well, and that includes both passengers and drivers. Right. Um, there was 5,500 other is incidents of groping, unwanted sexual touching in those two years. Uh, basically those attacks were, then they’d have to throw this one out. Those attacks, representative point, uh, three zero, 2%, like 0.0, zero zero 2% of the 1.3 billion rides, theF  facilitated last year. And I the, cause I remember when we talked about it last time, they tried to compare, they tried to make it sound like that number was statistically insignificant. Um, Hey, you can tell I haven’t been really drinking cause I can say it’s statistically insignificant surgery. It’s shorter shoot, traditional shish don’t, uh, know. And, and so they, they put that out there and then the New York subway system, uh, transit authority came out and said, okay, our numbers like a 10th of that. So, and we have, I think it was like they have, God, what was it, 20 X, the passengers and their incident rate was one 10th of that. See, here’s the thing though, that’s apples and oranges because if you look at mass transit, you have 50 people in a subway cars. And you know, it’s a one to one thing in an Uber, usually chances are different than you’re not going to commit a crime when you have 49 people staring at you. I can remember I told this story on the intranet, uh, but so one of the show hosts here, and I got into a rather interesting conversation where he got into an Uber one night after being in a bar. And I’m disclaimer, this is not me. Uh, cause it sounds like it could be me, but it is not me. But a friend of mine totally shows you’re not gonna even tell you who it was. Uh, but he got, he got drunk, uh, got an Uber and passed out. Um, he is a gentleman of color. Uh, and so the driver is so, and he’s looking back. So number one, the driver did not drive him home. The driver drove him to the police station that was less than a half mile from his house and insisted the police come get him out. So he got dragged out of an Uber by three cops. Um, Uber, backseats, my safe place it’s supposed to be. And then like, so the next morning he’s like, do the Uber driver like racked and he put them in a drunk tank? Yeah. Oh yeah. Wouldn’t let him walk home the short leg. Like literally it was like less than a half a mile to his house. Come on. And you give him a five star rating. No. So here’s the best part. So the driver not only did that to him, but drove all over to Helen gone while he was passed out jacking up the fare. Oh shit. So yeah, I mean it’s, it’s one of those Scott, like it doesn’t track you and when you’re separate tomorrow, that’s how he knew, uh, any, any wound up getting like, you know, the, the ride fare refunded and that kind of shit. But I mean that’s, that’s still, dude, that’s an ugly set of circumstances, but it’s just like, can you imagine, can you imagine like getting hauled out of an Uber by three cops cause you passed out. I’m like, dude, I, I done good dude. I like, exactly, this is what I’m supposed to do. Like that’s the thing that like, that always bothers me. Like I was supposed to, who was it? The police? Is it the police chief? Uh, out there in Michigan? They got busted for a DUI and ice. He’s just getting probation. I mean, he, Hey, he was super drunk. I mean, he was, he was like literally more than three times the limit. But Hey, we’re just going to put him on probation and he’s still employed. He’s still the police chief. He used to, yeah. Yeah, we’re a little lenient. Ah. Um, so here, this was an interesting one and I remember we went nuts, what was it, two or three years ago at penguin con when they were implementing our food? They were doing. They were, no, they were, they were doing, they were installing or implement or implants for RFID implants in your hand? Yes. And somebody was actually doing that at penguin cotton and we were like, you are out of your effing mind. A story came out on LinkedIn, basically the title. Would you let your boss chip you? Well, it seems like this accelerates every year because there was like one company in Wisconsin, if I recall correctly, that was doing this like a year ago. Yeah, that sounds familiar. Yeah. And, and but like now apparently it’s becoming more like, it’s, it’s like this is the new company ID badge. So I’m like, so here’s the thing, I’m in a secure building, I have an ID badge, I swipe it to get out. I can’t walk in. Otherwise there’s glass things and they move and then there’s a security person behind the desk and my face comes up on a screen and then they greet me. Good morning Robert. Right? Like I don’t like what else do you want or need? Like what were the point? Are you really going to have dude, what’s the average life expectancy at a job now? Three years? It depends on what you’re doing, but yeah. So what, you’re just going to wind up with scars everywhere from all the various, like is that, is that going to be like, Oh yeah, so this is the scar from when I worked at Cisco. This is the scar from the RV. This is the one. But then they’re talking about, they’re doing it for like offsite construction workers when they get on site. So like they’re going to have sensors so you don’t even have to badge in. So like it’s going to be a smart to know. What, what’s your exit interview like? Is it in an operating room to pull it out? I don’t know. Hey, you’re getting fired. We’re going to cut you open. Well, I remember like five years ago, um, I in Canada and my cousin’s best friend was implementing an app for the snowplow industry. Uh, it puts sensors around parking lots and basically when that, when the, the billing started, as soon as the trucks pulled in and they stopped building bill, you know what I mean? And they would show their path and their trajectory. Right. Um, good. That’s that to me, fine. If you want to track drivers, you want to know, they already do what GPS with with ups and all the FedEx and all that. But like working on site, like could you imagine like, Oh, you’re going off site to the switch data center in grand Rapids. And by the way, um, we’re going to track your every movement while you’re in the data center and when you, when you get there and when you, you know, when you leave, not just badging in or we’re not going to trust you that you, you know, people are either gonna like, I don’t know what they’re going to do. Well, and I’m sorry, but like the weird, like one of the weirder stories that I thought came up was that Airbnb acquired a company, um, that essentially let them now run an AI based on information other than what you give Airbnb. Um, like looking up your job, your social profiles, all this other stuff to determine whether or not you are a quote unquote acceptable fit for the property that you were looking to acquire. Is this China? This isn’t China, is it Danny? I’m just saying like, so you know, and so you know, there’s, there, there according to them, you know, they’re, they’re trying to STEM some of the issues they’ve had where like somebody runs an Airbnb, throws a party, destroys the house or you know, sex workers, you know, porn filming, that kind of stuff. I get it. It’s still creepy as hell. It’s creepy. A sale’s sleeping in someone’s bed with their steak, but like the day before and then you’re going to stay there all weekend. Go to a Marriott. That’s, yeah. I don’t forget crying out loud. We were, we were going down to Florida with friends and they wanted an Airbnb. And I go, Hey, I don’t want to cook breakfast B. I want to get coffee downstairs at like a coffee shop. I don’t want to like have to like go to the grocery store. See, I don’t want to poop on someone else’s stinkbug toilet. Like it’s no for real. Like it’s gross and you’re, you’re wondering if they got hidden cameras and see that that’s always the thing I wonder about when it comes to people staying in Airbnbs carries and shit there, you know what I mean? Cams or whatever. Right. And then all of a sudden now, you know, and you could see it, they could see in the dark. So is there a one over your bed? Uh, you know, if, uh, you know, you’re watching the dirty movie on channel 12, he goes, are you the dirty movie next? Right on channel 11. Right. So very a very minority report ish. The story about Delta and they’re parallel reality. I like this one still very minority report issue. Cause, like we’ve talked about like whenever you can use those powers for good, you can write like, would you, you know, walk into a Kroger and they go, you know, Jaeger Meisters unseal Mr. Walton schpiel ILA eight, right? Like wow, thanks. It’s like the Facebook ads with my stupid tee shirts, right? I’m not, I look at a Facebook ad and like unless it’s a wish ad, nine times out of 10, I’m like God damn what I want it. Right. You know, so, so Delta. So I keep wanting to send a, like a note to the wish team just asking them if they’re okay. Did you see, uh, I posted it on the football forum and it was like a wish thing and I didn’t even realize what was on the right of the cause. It was like a pair of pants and there was like a puff coming [inaudible] and I’m like, what in the world would that be? Is it like carbon filtered pants? But there was Wiener lipsticks to the right of it. Of course. It’s basically your search history Bible. I’m like, no nice clothes. I go, but Dave’s getting at the parallel reality. It’s basically there’s going to be like one giant screen for your departing flights and let’s say like Caston sitting next to me and I’m staring at it and it’s gonna show me my flight and it’s gonna show him his flight at the same time. I, yeah, I got to see, I don’t know how I got to show this in action. Right. And I don’t know how it’s going to work. Um, like I understood like minority report, it was based on basically contact lenses they had in, but right. They scan your Iris basically zap, you know, each individual gets his app while you’re walking through. We’ll see. Um, and they go, they think it’s 10 20 years away, but it’s like literally their PO seeing right now. Yeah. Um, and I don’t know what the big deal is. All I got to do is go like Denver, Denver, Denver, Detroit, 10 40 gate a 42. It’s like, is this a problem for which we need an additional solution? I guess I want to figure it out that I could bring my bottle of water. And that’s what I would have framed it exactly. Is it four, can I give to my four ounce bottle of shampoo going to get thrown away or, uh, so speaking of throwaways and I do love this, apparently there’s a database sitting on a Chinese IP address, uh, with the details of 56 million Americans just hanging out publicly open and accessible. Uh, thanks to a website called check people.com is apparently where it was pilfered from. I love the, uh, the th this is the response. No, the subtext of the, uh, of the headline. If check people could take a look at this, that would be great. That would be great. Yeah. So yeah, it’s apparently it’s names, addresses, employment. Yeah. Good, good, good types. Thanks. Thanks for that. However that happened. That’s a male relatives, criminal records and basically they just skimmed public records. Yeah. Good time. You put it in a database and a white hat hacker. They had to found it. They had to, they had to list that out cause Hey, that’s why I said it’s not the individual breaches you have to worry about. It’s the people that are out there aggregating this. Yeah. It was personal data and metadata. Um, yeah, they were all scraped and, and it’s just sitting out there and uh, yeah. Nice. At this point. It’s funny cause we were talking about like 23 and me and all that crap at work and the guy goes, I don’t do that crap. I go, man, I go, go to activity.google.com. He goes, I delete that stuff. I go, Oh yeah, that’s cause it’s gone. You know. And he’s like, yeah, and Snapchat actually deletes the pictures, you know. But it’s a, it is a sad day though. Um, and I didn’t realize how people, like we know when they forced windows 10 and everybody with windows, windows seven was a good, OSA was, there wasn’t really many bad Microsoft OSS, you know, a change. I hate it. Um, but apparently I always like the, the big bang theory. Sheldon when he was like, when windows eight came out and he was like, windows eight is you, it’s so much more user friendly than windows seven. I don’t like it. Um, but apparently a Microsoft dropping support a seven today and they’re not going to, they as of today, it is end of life. 26% of all PCs 26 that number blew my mind right now because they for, no, because as we sat in WJR on Saturday looking at the windows XP screen, Oh my God, I know windows XP and they were making fun of it and I go that screen right there, I tell CRT monitor, Oh my God. But not because they were forcing 10 upgrades. Yup. So like windows seven 26% I think about that one in four PCs. Yeah. That’s insane. But it’s one of the things, it’s again, if it’s functioning and it works cool, still get windows 10 if you want, if you’re still running windows seven or windows eight you can still get windows 10 it, they stopped pushing it for free, but they’re still supporting it. So we go down the media, download the media creation tool from the Microsoft website, run it, and then check the activation and it’ll use your eight or seven key to activate the windows 10 version. Yep. Yup. Cool. Well, Hey, we’re going to take a speaking of, uh, I, I got nothing. I was gonna use it. Speaking of activating, they’ll try and sense either, uh, speaking of cats, we’re going to take a quick break. We’re going to be back with Cason and Thomas. We’re gonna be talking about, uh, being on WJR this weekend and a bunch of other stuff. This is the it in the D show. They, he’ll be, Hey, welcome back. Segment two, episode three, 30 of the it that he show broadcasting live here. Studio three podcast. He traded beautiful Royal Oak, Michigan. Bob the sales guy, Dave the geek. Randy. I do the Twitters is doing the Twitters. Find us online it in theD  dotcom. You want to know why? Cause we all right, Tina and you, you’re, you’re still not, but we still love you. And as a reminder, again, I am a moron and I scheduled the event for next Thursday, not this Thursday. My apologies. We’ll have that fixed moving forward in future months. But Hey, capital one knows life doesn’t alert you about your credit card. That’s why they created, you know, the capital one assistant to catches things that might look wrong with your credit card, like over tipping duplicate charges or potential fraud. Then sends an alert to your phone and helps you fix it. It’s another way. Capital one is watching out for your money when you’re not capital one. What’s in your wallet? See capital one.com for details that CAAP I T a L O N e.com. But Hey, we are very, very lucky to be joined by the illustrious one, the one and only the 13th time on the it in the D show. I wanted to do like a Rick flair thing, like the 16 time world champion. But Hey Caston Thomas, welcome. Thank you for joining us. Glad to be here. Oh yeah, absolutely. We uh, we haven’t seen you in days. I don’t of fact. No, we haven’t been on jr it feels like a couple of years. Um, it wasn’t that long. I thought it was since the last time we were on, I don’t know, it’s was about a year ago. We were then co-hosting for two and a half years. And this is the first time I’ve been in the studio with you. That’s what I thought we were on once with Jeff and we, uh, were on one trip and now, yeah, I know. Caston’s on 13 times here. We’re on three times on jr. um, it’s probably for the best, let’s be honest. No, you guys were great. No, but so like we always like talking about the differences between podcasting and radio and you know, you kind of cut your teeth, you know, guesting here talking about security and, and all that. You made it all possible guys. That’s in fact, Oh wait, is that jazz music right here? We’ll be back with a quick word from plumbers with a Z in the geek or.studio. I wanted to say, I’m so kicking myself for not saying that. And you’re like, yeah, I’m really proud to be here in the geek or Don studios. Now we might have to do the seven second button on that one. Ah, you just don’t mess with the host. And here’s the thing. You didn’t even, you didn’t even introduce me yet. And I’m already ragging on you cause you’re talking about like a microwave with like a clipboard screen on it. I’m like, yeah, we need to have a conversation about that. By the way. Why go into the long explanation? Because we only had seven minutes on that segment to get real content because you need recipes for Kraft dinner. And I’m like, that’s not it. I’ll talk to you after this riveting. I can’t wait to hear more about the microwave with a clipboard. Yay. CES. Now hang on, hang on. Let me hijack this for just a minute. Uh, and I told you I was going to do it, but you know, our last time together was with foster, uh, coming in and that was a must listen show because it was intense. It was beautiful. Uh, and yet it was still having a little of that irreverence that you guys have and fun. But, uh, I’d like to just stop for a moment and say thank you. I mean, not just for me, but for everybody who’s listening. The impact that it in the D and you, Bob and Dave have had on the it community on Detroit and on all your listeners. I mean, there are guys out in some remote desert listening to this show looking at the beautiful stars because they’re a hundred or a thousand miles away from any city light and you bring sanity into an insane world. Or, wait, did I mean insane? I was like, I don’t, I don’t know that that’s accurate. I don’t, Oh yeah, I’ll tell you. I’ll take it. Yeah. You know, there’s just something that you bring to people’s lives every week. And I enjoy the show and I know thousands if not tens or hundreds of thousands of people, really appreciate the work you do. Thanks, man. I’ll slip you the a hundred hours. No, right. No slipping me anything. Well, here’s the thing, and we’re going to, if we’re going to butter each other’s bread, I mean, uh, I know you’ve only been a part of internet adviser, not for a couple of years, but that you guys are celebrating your 23rd anniversary now you think about and you know, and there’s still like a couple of people that have been on since day one talking about, yeah, Gary Baker’s been on state one talking about technology for 23 years. It’s as long as I’ve been in it. That was before XP. That was before windows 90 that’s, yeah, that’s 1997 so you’re still in the windows 95 brother printer era. Yeah. Elaborate with Dave just said there’s one of our favorite, so the internet adviser shorts, a two hour show on WJR every Saturday an hour now. It’s one hour now, but it used to be a two hour show and the first hour was talking about kind of like what we do, talk about going through the news. Second hour was taking calls and it was fixing people’s stuff and they had the patience of saints. It was the most amazing show of restraint and help knit and helpfulness. Google that, you know. No, cause that was, Oh every answer was let me Google that for you. Yeah, throw it out. That was the guy who [inaudible] my brother printer will not talk to windows. Okay. Printers are 50 bucks. Throw it away by anyone. One bell. We got to fix that. It’s not a correct way. So I started saying in foster love this, I’d say, you know, we’re always smarter after the break. Right. Well we’ll hope, you know, hold on just a minute, we’ll get back to you after the break. And then we had all the answers. It guys don’t know everything. We just know how to Google really, really well. That was maybe one of the best memes with like that stuffed animal. Like it’s like a chimpanzee and it was like doctors, like you can’t go to what the web MD does. Programmers are like, um, but no, two weeks ago my mom calls me after every show and gives me her critique and one one time about, Oh no, you know what, my mother is like stepping back in to leave it to Beaver. She’s that 1950s Southern Belle mother who, you know, I go down to Atlanta and uh, we go into a store and she goes, I raised you better than that. And I’m like, Ooh, sorry. And I take my hat off. Oh geez. Wow. Suddenly I’m 13 again. Sorry Bob. I’m sorry. Sorry. Calls me. And she goes, you’re not going out drinking with those nice boys. Ah, no, no. Mom, go, they went home. I brought a flask, ma. No, but you, you kind of dropped something. I mean like that show is hurt. You said what? 28 States, 38 States, States sun goes down and Canadia and all, all provinces in Canada. [inaudible] pro. Yeah. They cause at sundown many stations shut down. So that’s why the seven 60 band is clear channel across the nation. And I always thought it was fascinating hearing it because we went to Cleveland and you could like where we would go to Cedar point like that. Like I always, for some reason I still have that, like when we used to go up North and the first day she’d pick up as jr we had the blessing of Kettering university on and he goes, it’s really great to be here in the jr studios. He said when I was 12 years old, I erected a 20 foot antenna. I lived about 10 miles outside of Orlando and I would listen to jr at night. Oh my God. So that’s crazy. And another guy came on, he goes, my father is so excited that I’m on the show because he remembers driving to Iowa with me and we drove all the way to Iowa and listen did jr the whole time. That’s what we always talk about. Listening to Ernie Harwell. I like, I probably heard Ernie Harwell speak to me more than my own father. Wow. Like, if you think about it, if you grew up in this town and you’re like baseball, like you would listen to him 162 times for what, two and a half hours a pop do the math like yeah, probably right. It’s a ritual. Yeah, exactly. And it was, it was religion. You know what I mean? So, and everyone’s odd, AM’s dead, no one heard you, you know, and they make fun of us for being on your show. Um, but like there’s, there’s, there’s, we got emails about it. We got picked up followers. There’s a charm to it. It’s, it’s, it’s a thing like I always, uh, I, I don’t think it’ll ever die because someone will do like a ham set up and make, you know, there are other podcast studios out there trying to buy a am radio stations to make that work. So yeah, there’s that. I wanna I still want to, I still have my dream in life cast and I don’t know if we’ve told you this as we went, there was an am radio station for sales, one of those like high school ones that like have like a mile of a reach and I wanted to do the WPI out of the back of somebody’s window, but I want to know non pirate radio. It was a, it was a legit, like it was, we actually got offered to take it over, um, that because we helped them build out a studio there and it was like a secondary education school. Um, and to help them build out the studio with like, you know, some soundproofing and some gear that we had. Um, and they had an old am transmitter that has like a Miley you could like, like [inaudible] for like two miles along one road. You could hear it. We do 38. Yeah, it was not yet, but I want it to be WBRD where every day the bird is the word and I want to play the Trashman Surfin bird on repeat all day. And Java, I think. No, no, no. Just Surfin bird, Java, surf bird Jack or it’d be the, um, the gamut. What was the droid and empire and Hoff, um, the, the probe droid. So, but just to what I originally hijacked to show it, I want to encourage people to get on iTunes, get on Google store, get on YouTube and rate the show. Say thank you. Tell these guys what they mean to you and what the show means to you. Don’t tell us what we mean to you. Might be surprised, but no, really. Um, you know, one of the things that I’ve given a lot of thought about is how do we create compelling radio versus compelling podcasting and we can’t get into the depth and radio. It’s like you said, you know, the jazz music comes on every seven to 10 minutes. And there was a relationship with drew him like that. I had, and I don’t know if it was just the era that I grew up in, like that was like every, it was part of my day every single day. And they actually, they gave them time to talk. I would listen to you guys every morning. You want to get up at fourth I am not getting now. The one thing I never understood is how Mike from Jerome, Mike could laugh at. Like, I’m just like just getting my seven 11 coffee down my down, my pie hole and this guy’s just cracking up laughing at fart jokes. And I’m like, well my eyes are happy. Step one. You would’ve had to gone to bed at eight o’clock the night before, right? Yeah. I mean when foster was doing the 6:00 AM morning show, he was talking about getting up at two 30 in the morning so that he could do a show prep, get into the studio, get everything set up. And it’s just, you know, it’s like taking the night, the night shift. Well, and that’s the thing, like, you know, I having their relationship with drew on Mike, I think people now have relationships with podcasters just cause they’re getting it. They’re getting it when they want, how they want. Right. So like there’s no, I had to, you know, if I wasn’t, if I took the day off that day or if I, you know what I mean? I wouldn’t get to listen to them. Yeah. It was just, it was that easy. I had to be six and 10. I had to be in my car. Usually it was I was going to school or whatever I was doing and I got to listen to them know, otherwise forget it. You know, millennials, you know, get this bad rap for attention span. And I was listening to Jordan Peterson, I think it was, and he was talking about the people who listen to his podcast or watch his two and a half hour videos and he goes, attention spans not the problem. It’s having compelling content. Indeed. You guys have compelling content. When present company did I have short attention span theater? Did it? If I click a video or something like that, man, it’s like the, all that, you know, if you build a great website, they will come. If you make them wait eight seconds, they will leave. I mean, it’s, it’s that simple. Like if I’m bored before you even get started getting rolling. See ya. And if you got a six minute intro, forget it. Four that’s over then. Oh my God, when I got so much grief, uh, when, when Jim Alison was on talking gaming, apparently his wife listened live and she’s like texting them the whole time going like, Jesus Christ. How long do they expect people to listen to this shit? Like she’s like just, just berating us. Like yes Jim, we just added 30 seconds. Exactly. That’s, that’s another 15 seconds that has to get right. That’s the rule. So I mean what are you learning from, I mean from am radio other than like seven seconds. Jazz music, you know, and then you get 12 seconds jazz music. There’s no screw ups. There’s no stopping. There’s no edit. I mean you just got to go and it’s, it’s tough. There’s pressure there. Uh, part of it is, and you’ve got all the FCC rules and restrictions. Well, yeah, not a problem for me because my mom makes me take off the hat. One I was worried about saying suck. Like, I really was, cause you know when we were talking about, uh, yeah, my brother wants to talk to you about that. [inaudible] tell her to watch our TEDx talk and it’s going to wash your mouth out. And no, because that was our thing. Suck. Listen, I, I thought about it, I thought about it. I go, Nope, I’m going to say it. Yeah, no, that’s an okay word. Yeah. Well, because you know, you’re referring to Becky vacuum cleaners and whatever. It’s all context. No, but like, you know, and the thing is too is you’re on payroll. It’s not like, just like you’re letting, it’s not like you’re paying for that time. Right. So, you know, you probably have people listening. You, I don’t know, do you guys have to go through like a a that was good, that wasn’t good. I think at this point, 23 years, you pretty much have carte blanche in there. No, no, no comment. Giga or Dodd didn’t like that bit about podcasts. The one thing I have to consider because sometimes I do, uh, talk about, uh, certain products and things and I have to give a second thought about, you know, am I going to be stepping on a sponsor and getting a call about that? Right? There’s, there’s so much freedom and podcasting and so many ways, uh, it’s difficult at times to, to interact on the radio, but there’s a charm about that real time nature of interacting with people because we do take Colin’s, and that’s part of my, that’s my favorite part of the show is when we actually get to interview or talk to somebody live, you know, they’ve got a problem and it may be really simple, it may be a head scratcher, right? But there’s, there’s something genuine and charming about that that I really enjoy. Well, I mean, we joke about just Google it for crying out loud, but there’s a, there’s kind of like a connection when you can help them. Oh, so two weeks ago, my mother calls in, her review was, you know, that was a very informative show. But what’s 5g again, one more. It’s not her world. It’s one more than four Jima. But what I mean, that’s when four people get in a room full idea. People actually being involved in the real world and they really didn’t even know it. Five G is where the connection to number one warn them about things that they don’t hear enough about. And I remember Nuri talking about having talked to your grandparents about tech day. Yeah, yeah, we’re doing that. Well. So I brought that up with a, there’s another show here that does like a security protection, that kind of stuff. And they started talking about, you know, you know, shredding documents and all that stuff. And I was like, dude, I’m like, cause you don’t know. You don’t even just say like when we’re not, when my wife’s grandparents died, they died within about three months of each other. Um, and cleaning out their house was a nightmare because they literally kept every piece of paper that they had ever gotten from anything ever, including all of their medical stuff, which up until about 20 years ago was just your social security number was everywhere like that. That was all that was there. So they were like literally just crates upon crates, upon crates, upon crates of this shit where we rented an industrial shredder and burned the sucker out a day and a half in and had to be like, Hey, broke it. Sorry. Need another one. Yeah. Amazing. I mean for me, I never thought about it. I’m like, Oh good. That folder of 2012 tech stuff I can actually finally tell us. Yeah. As I say, it sounds like it’s time to just hide it under a pile of leaves and light. The whole thing on fire, dude, like you don’t even know a dude. It would have triggered like a neighborhood fire if we, I mean I seriously, I cannot put into proper context exactly how much paperwork they had stored in their house. That was, I mean he had a garage, but I’ve got this problem. I’ll save things for 20 years and then I throw it out in the wind cleaner. I need it. Does anybody else have that as ask? Ask me for a windows 95 installed city. I have one asked me, ask me for, you know, dos six Oh, installation diskettes asked me if I have anything that would lead to mill serial converter. Right? Yep. Ask me if I have anything that’ll read a 3.44 floppy at this point. Uh, uh, yeah, no, God. I mean that dude, that’s me. Like I’m, I’m a huge fan. It’s, and it’s the, it’s the fear of, you know, in Bob lives, you know, loves to talk about my ego and my hero complex and that kind of stuff. And I admit I do have one, but it’s that. No, no. Well dude, cause it was Jeff who like, and he was like, Hey dude, I know this is a weird request, but my dad’s computer died. You don’t have a windows 95 install CD do yet. I’m like gimme a minute. Yeah. When are you coming over? I think I still have a dos one. I still got my pile. Oh crap. And all these years you haven’t waived a magnet over it accidentally now. No, he talks he, it’s funny he talks about it is I got the Iomega drive too. Oh yeah, I got it. Oh yeah. Here’s the thing. If you put it in a certain place and you never go into that place again and it just sits like that’s my, I think yours is probably organized minded just because I’d even thrown it away. No, not really. No. Just really, I opened a box that I am convinced I have, like I’m convinced the box has moved with me from when I, that the townhouse in DC that I had through every house that I’ve lived here. And I’ve never looked at it because when I opened up this box, dude, my alphanumeric SkyTel pager was in this box. My light just shit that I haven’t even touched or even thought about since I was living in DC. Uh, you know, an old star tack, you know, flip phone. I just, and I’m like, even I was like, why? Why got all my old phones, all my kids, my kids, I was their toy phones. Oh yeah. My Nokia, my flip. Well, I like the nice, you ever have a problem Edward L who’s on her show? He can answer questions on all that. Exactly. Yeah. Start that TARDEC phone, the one that’s connected to the Verizon network, but only show number a Y. Oh, okay. But also, I mean I do, I think it’s cool. I mean that’s a, that’s that’s one of the cause you to you guys, you guys take a a little bit different approach with that kind of stuff than we do. I mean we’re, we’re a little bit more, we’re a lot more irreverent than you are. No, no, but I mean, but that’s, but that’s the thing. I mean that was, that was our taken our approach when we decided that we wanted to do this is, you know, there was already enough and not that you guys were this bad, but there were, there were, there was, there were already enough today on this technology podcast, we’re going to be on the port God Mark corner then Mark. Yeah. Like we’re not, we’re going to review this five port, this Linux distro onco six. Dot. Oh yeah. [inaudible] commandos. Not the Hottie. She once was these guys the wayside. But yes, I mean it’s, it, you know, it is. I mean, and you know, and I do like if I, if I happen to be out driving around and when I know you guys are on, I’ll throw it on and listen. Um, so I mean keep an eye on you and then drive home on the drive home from jr. I was like, Oh, I want to listen to the second hour that I’m doing tech support and it’s Ben Shapiro. And I’m like, Oh, I want, I want to see if anyone was listening to us on there that I know they’re like cringing right now. Call them. Well, you know, we, we try to keep that down-home effect because we know where our audience and you gotta be real, but you also have to know your audience at the same time. And so a little thing that Cal Carson did, you know, one of the coppos was he brought Bob’s daughter up to the microphone and she brought, brought it in and she was really shy and kind of intimidated by all the studio stuff. But that just one, it resonates with our audience. But when you saw the kindness that Cal exhibited with Bob’s daughter, it was just an amazing thing to watch. She nails welcome to the internet advisers show and she doesn’t know her damn name. And I’m like, God, come on kindergarten, you should nail this stuff. All right, so switching gears, uh, there is apparently a workshop that you were doing. Yeah, we wanted to talk about, you know, last time I was on, we were talking about edge computing and uh, sensor-based networks and the way that smart cities and smart cars and smart hospitals and smart factories are building out. Uh, and uh, I’m doing work with this incredible team from [inaudible] solutions group. Well, what’s evolved out of that is we saw a real need for developing a program for securing IOT. And one of the things that, that I’ve been saying over and over and over, almost a mantra is that if we try to apply the same security and support models to IOT that we did to it, we’re going to fail. Catastrophical I says in securing IOT. Isn’t that like an oxymoron at this point in the game? Well it can be, but the difficulty is, is that there’s all this complexity around it. The barn door is open, the horses out, but hasn’t found a gate through the fence yet. A lot of the door horses out, like what are you going to do with it? Yeah, you can assemble them and do nasty things and create DDoS attacks. But the really sophisticated kind of things, the hackers haven’t quite gotten to that point to shut down a massive number of factories or a hospital. We’ve seen incidents of that and occurrences, you know, the Saudi, a refinery got shut down, a manufacturing plant in Germany got totally shut down by malware. And then, uh, you know, you had a couple of outbreaks where not intentionally, but ransomware impacted some really expensive and, uh, really important medical devices over in Europe didn’t spread to the United States. So, uh, we are behind the eight ball. So what we’ve done is we’ve created a half day workshop that we’re going to be going around. I’m going to be going to about 20 cities this year, uh, conducting a half day workshop. The first, uh, the morning’s going to be medical devices. The afternoon’s going to be industrial controls call, internet of industrial things, building automation, those kinds of things because there are step-by-steps and a lot of resources that are out there. I mean, Mayo clinic’s done eight years of securing medical devices. I can’t hear that without going airplane. Give me him on five, hold the mail. There you go. Yeah. The letter that the one guy wrote, there’s a book and he’s like, wrote a, like a really polite dissertate. Like it’s really cool that you guys have a clinic dedicated to the mayonnaise. I just go into Mayo. Yeah, no. So, so we’re taking the best, this is for people who’ve already been or organizations that have already been down this path. We’re packaging it up with the resources, the methodologies, and the construct of a workshop with uh, forms and worksheets and team building exercises so that we can either take in one of these metropolitan based, uh, many people from different organizations, but a slight spin on that is going into a hospital or into, uh, an industrial complex or utility and facilitating, bringing them through the steps to make sure that they’ve checked all the boxes for securing IOT, uh, whether that’s medical devices or industrial or utilities, smart grid, smart network. And so the idea is that we can accelerate the development of, of a comprehensive cybersecurity programs specific to IOT and just a couple of weeks instead of the six months of all the organizational things and defining the structures of why IOT is different, why you support it differently. Because if you’ve got a clinical engineer, a plan engineer who’s got to spend 30 minutes and in order to just to get a device on the network because they’ve got to call the help, right? Got to call the network. You got to provision this guy [inaudible] well is it going to get, is it going to get to the point with IOT that there’s going to be either a standard or a compliance that they need, uh, companies need to comply to cause right now it’s wild, wild West. You know, what come to the early days of the internet, come to find out right here. No, the uh, this has done a lot of good work, uh, particularly in the area of medical devices and organization called MITRE. M. I T R E has uh, I E T F E is an even has a guide on the important factors. These organizations had done a lot of really good work. A lot of it is F the role and conceptual, not real world. We’re trying to bring that real world experience that other organizations have put in. Then our, uh, our expertise in going through this with organizations to put down something that’s efficient and economical. And a buddy of mine who did an IOT security deployment of over a million devices says, yeah, it looked at the NIST requirements. And if we did that in our organization, it would only cost us $8 billion billion. Yeah. [inaudible] the numbers. He’s a numbers guy. Used to work for Google and built data centers, but what’s the in what? And then he had to look at what’s the penalty for not doing it and if it’s less than a billion, that’s the route they were going. Yeah. Yeah. But you know, there aren’t a lot of penalties for this. It’s really looking at the differences in how IOT devices are different than IOT devices. As an example, IOT devices are typically not manned, are they? They may be in remote areas. That idea of being able to patch a thousand devices in an oil pipeline, it ain’t going to happen because if one of the sensors goes down for a couple of minutes, they got to shut down the whole pipe. Right? That’s why you have unpatched devices in industrial controls. That’s why you have all of the sensors that are running, uh, universities and hospitals that haven’t been updated. What about the new consumer Alliance that was just announced though? Can I get home over IP? Apple, Amazon, Google, ZigBee. Gee, I really wish I knew about home. There’s a laser focused, they actually have one called chips chip. You, well they’re not calling it a chip, but everybody else is bringing me punch and John, I’m glad ZigBee still exists. That was like Samsung and I was working with it like an Oh four and I thought I’d died and then Bluetooth became a standard and then, yeah, no I’m glad. I’m happy to hear that cause I used to work in like home club. The home blog Alliance. Yeah, like back in that was Oh four Oh my God. If I’m not mistaken I think Kroger is doing some things with ZigBee throughout all their stores. That may be old information, but yeah. Is that their shopping thing where you go pick up the scanner or is it something these are old brain cells. I’ve already cleared cash. Sorry. Yeah, I was like, Hey, where do people find out more about this seminar and like sign up for it or like is it out yet? Is it, yup. Yup. I’m doing one in Cleveland. We did our first one on medical devices in San Francisco was very well received back at the end of last year. He didn’t step any poop on the street. Did you? A no comment. Okay. I can’t say badly. She totally does. Everyday stepson. Uh, find me on LinkedIn. Caston Thomas sounds like it’s spelled or spelled like it sounds. And uh, uh, you can also go to [inaudible] S G [inaudible] solutions group.com and find out more and uh, click through there. Fill out a form. You can get to me. Awesome internet advisers on a six o’clock on Saturdays on WJR 70, 67. As long as the lions and the Spartans aren’t playing football or basketball, uh, we’re generally generally on live and uh, in color. Nice. Nice. Not really cause it’s a M so you’re not in HTS. Everybody listens to it in the D. just tell your parents to listen. There you go. There you go. Now we’ve got some guys that qualify. We gotta go get some, we get an older crowd that comes around. Yeah. Right. So lucky 13 you know, it’s always a joy to be here with you. Thanks for coming out again man. I appreciate it. Sure. It’s always a good chat. Yeah. Good senior cast. Another always. Hey, we’re going to wrap things up for episode 330 of the it and that he sure would like to thank Caston Thomas for spending time with us. Hosted the internet advisers show and security consultant Xtrordinair on behalf of a Bob David Randi, do us all a favor. Get your trick up, drinks, get your phone numbers. You don’t get to go home. You just got to get the hell out of here. See you next week. Drive careful. Beat it. So you guys.   IT in the D On the web: http://www.ITinTheD.com On Meetup: http://www.meetup.com/ITintheD/ On LinkedIn: https://www.linkedin.com/groups/IT-in-D-91763 On Facebook: http://www.facebook.com/ITintheD On Twitter: http://www.twitter.com/ITintheD Podcast Detroit is at: On the web: http://www.podcastdetroit.com/ On Facebook: https://www.facebook.com/PodcastDetroit On Twitter: https://twitter.com/PodcastDetroit On Soundcloud: https://soundcloud.com/podcastdetroit

(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.