Podcasts about plasmid dna

While some of us knew a good bit about mRNA prior to 2020, we all got a crash course on mRNA technology and its prophylactic and therapeutic potential as a result of the COVID pandemic and subsequent SARS CoV-2 vaccine development. In fact, most of us have now received at least one mRNA vaccine at this point. Our guest for this episode, Dr. Christian Cobaugh, Co-founder and CEO of Vernal Biosciences, was a passionate believer in mRNA medicines well before the pandemic. Join us to hear his story and his passion for this technology. He walks us through the molecular methods by which high-purity mRNAs are now made and purified, as well as going into the lipid nanoparticle technology by which they're commonly delivered. As a contract development and manufacturing provider, we get to learn about the state of the market and what clients of their care about today. As a seasoned expert in this space, Christian talks about the future potential of mRNA technology for applications such as personalized cancer vaccines. If you enjoy hearing smart people talk about interesting topics with a passion, you won't want to miss this episode! Subscribe to get future episodes as they drop and if you like what you're hearing we hope you'll share a review or recommend the series to a colleague.  Download Transcripts: Speaking of Mol Bio Podcast | Thermo Fisher Scientific - US Visit the Invitrogen School of Molecular Biology to access helpful molecular biology resources and educational content, and please share this resource with anyone you know working in molecular biology.

The potential of mRNA medicines was postulated for years, but it took the COVID pandemic and emergency use authorizations for that potential to be demonstrated. By now, most of us have received at least one mRNA based vaccine and the platform has been mostly derisked. However, if you're not one of the major players in this space, generating high-purity mRNA, let alone a GMP-grade mRNA-based drug product, can still be quite challenging. Dr. Chrisitan Cobaugh, CEO of Vernal Biosciences in Vermont, has been working in the mRNA field for more than a decade and is passionate about the potential of mRNA medicines. He's also been in the field long enough to know firsthand the challenges of high-purity mRNA and lipid nanoparticle supply. Join us as Christian walks us through his story, the start of Vernal Biosciences, and their progress toward their mission of democratizing access to mRNA technology. Our conversation touches on the molecular biology of making mRNA, and the use of digital PCR and other methods in monitoring development and release of mRNA drug products, and the potential applications of mRNA as a platform (some of which you might not have guessed).Whether you're new to the technology, or have chosen mRNA as a focus area, you're sure to find this conversation engaging and intriguing, and our guest insightful. Visit the Absolute Gene-ius page to learn more about the guests, the hosts, and the Applied Biosystems QuantStudio Absolute Q Digital PCR System. 

Heute: Plasmidgate: weiterer Skandal bei Covid-Impfstoffen ++ Die an Skandalen schon überreiche Geschichte der Covid-Impfstoffe wird um eine weitere Variante bereichert: jetzt kommt mit Plasmidgate ein weiterer Skandal hinzu. Die stellen ein erhebliches Risiko dar, können sie doch durchaus in den Zellkern vordringen und auch Krebserkrankungen auslösen. Damit wird die Verunreinigung der Impfstoffe mit bakterieller Plasmid-DNA bezeichnet. Ein Gespräch mit dem Facharzt für Allgemeinmedizin Dr. Lothar Krimmel ++ Webseite: https://www.tichyseinblick.de

Good day to our listeners! In today's episode, we're bringing you several exciting updates from the world of pharma and biotech. Let's dive right in.First up, we're talking about a game-changing webinar hosted by Cytiva. They're discussing a revolutionary, scalable process for producing clinical-grade plasmid DNA. This method is not only efficient but also reduces the purification steps significantly. If you're interested in the intricacies of genomic medicines, this is something you won't want to miss.In the world of health insurance, there's a buzz about Cigna and Humana potentially merging. This could be a major shake-up for the industry, but it's not without its challenges, especially on the regulatory front. Speaking of challenges, UnitedHealth's Medicare Advantage growth for 2024 could hit a snag, and Providence is still grappling with losses despite their recovery efforts. On a brighter note, Highmark Health's insurance business is making waves with boosted earnings, though Allegheny Health's operational losses are a concern. And in a concerning update, an additional 330,000 Medicare beneficiaries might have been affected by a data breach at a federal contractor. Definitely something to keep an eye on.Now, let's shift gears to A/B testing and experimentation programs. A new report, which analyzed data from over 127,000 experiments, sheds light on why many tests don't hit statistical significance and how we can look beyond revenue to other impactful metrics. If you're into data and analytics, this report is a treasure trove of insights.Patient retention in clinical trials is another hot topic we're covering. It's becoming increasingly clear that a patient-centered approach is key to not just getting patients into trials but keeping them there. The challenges range from the impact of trial locations to the feasibility of travel for patients. It's a complex issue, but solutions like telemedicine and local trial centers could be game-changers.For those interested in oncology, there's an upcoming webinar you won't want to miss. It focuses on making cancer studies more efficient and inclusive. The talk will cover innovative strategies, including the use of real-world data and technology, to improve outcomes in oncology research.Shifting our focus to retail health, we're seeing how on-premise digital screens are transforming patient care and driving revenue growth. These screens aren't just about display; they're a strategic tool for enhancing patient experiences and boosting business performance.And lastly, let's touch on some key developments in the pharmaceutical industry. We're looking at an international tax effort impacting big pharma, the potential Cigna-Humana merger, and some exciting updates from Novartis, BioMarin, Novo Nordisk, GSK, and Regeneron and Sanofi. There's a lot happening, and it's all pointing towards an exciting future for the industry.That's all for today's episode. Thanks for joining us on Pharma and Biotech Daily. Don't forget to subscribe for the latest news and updates in the industry. Have a fantastic day!

No, I will not stop talking about Covid. A professor and PHD says that it is very plausible that these injections cause a risk for future cancers and sustained autoimmune attacks. I believe this is happening, it is clear and it is on purpose. Fact checkers are pretending that the DNA in these injections ust go away in our bodies, but it is not true, not this kind of DNA. Matt Gaetz has a theory for why the Senate is not calling joint hearings with the House and Senate. He says far too many members are bought by Big Pharma, can we argue?What does God's Word say? Revelation 21:8 But as for the cowardly, the faithless, the detestable, as for murderers, the sexually immoral, sorcerers, idolaters, and all liars, their portion will be in the lake that burns with fire and sulfur, which is the second death.”1 John 3:15 Everyone who hates his brother is a murderer, and you know that no murderer has eternal life abiding in him.Exodus 20:13 “You shall not murder.Romans 12:19 Beloved, never avenge yourselves, but leave it to the wrath of God, for it is written, “Vengeance is mine, I will repay, says the Lord.”Episode 1,235 Links:Today, the House will vote on my amendment to DEFUND the creepy HHS “Office of Population Affairs,” which Joe Biden is using to promote abortion and “gender” “transition” procedures with your tax dollarsRep. @MattGaetz Accuses Committee Members of Being 'Bought and Paid for By Big Pharma'Sen. Ron Johnson Shares the Most Censored Chart in Congressional HistoryLadies and gentlemen, @PierrePoilievre, the next great Prime Minister of Canada.Jesse Johnson announces that he's planning to sue the government: “These bastards they literally tried to break me — financially, mentally, and spiritually,” His business in Calgary, Alberta was destroyed by the government for refusing to discriminate based on vaccination status. Today the charges against him were dropped, and he plans to sue for damages, appears to have no regrets, and calls for other restaurants to never discriminate when the next crisis comes. Professor Dr. Phillip Buckhaults, PHD in Biochemistry and Molecular Biology. He does cancer genomics research at Univ of South Carolina. He explains why this matters. 'The Pfizer vaccine is contaminated with plasmid DNA.'Kevin McKernan tells Dr. Craig Wax what the fact checkers fail to mention. "what the fact checkers are failing to mention even though we've put this in front of them multiple times they choose to ignore these citations, is that the DNA in the Pfizer vaccine is very special kind of DNA that has a nuclear targeting sequence in it. This is what's in the SV 40 promoter it's a piece of DNA that transcription factors bind and drag all the DNA attached to it into the nucleus of the cell in fact it's used in gene therapy."John Oliver's writers made him flip-flop on gene-editing in human beings4Patriots https://4Patriots.com/Todd See this week's discounts and deals before they are gone and get free shipping on orders over $97. 4Patriots.com/Todd Alan's Soaps https://alanssoaps.com/TODD Use coupon code ‘TODD' to save an additional 10% off the bundle price. BiOptimizers https://bioptimizers.com/todd Use promo code TODD for 10% off your order plus up to $100 of free product with purchase. Bonefrog https://bonefrogcoffee.com/todd Enter promo code TODD at checkout to receive 10% off your first purchase and save 15% on subscriptions. Bulwark Capital https://KnowYourRiskRadio.com Sign up for the final FREE Live Webinar of the year at KnowYourRiskRadio.com Space is limited. HumanN http://americalovesbeets.com Get a free 30-day supply of Superbeets Heart Chews and a free full-sized bag of Turmeric Chews only at http://americalovesbeets.com SOTA Weight Loss https://sotaweightloss.com SOTA Weight Loss is, say it with me now, STATE OF THE ART! GreenHaven Interactive Digital Marketing https://greenhaveninteractive.com Your Worldclass Website Will Get Found on Google!

Satan's Political Party and their Anti-Christian pruning of societies Plasmid DNA in the Covid injections, cancer and cover-ups4Patriots https://4Patriots.com/Todd See this week's discounts and deals before they are gone and get free shipping on orders over $97. 4Patriots.com/Todd Alan's Soaps https://alanssoaps.com/TODD Use coupon code ‘TODD' to save an additional 10% off the bundle price. BiOptimizers https://bioptimizers.com/todd Use promo code TODD for 10% off your order plus up to $100 of free product with purchase. Bonefrog https://bonefrogcoffee.com/todd Enter promo code TODD at checkout to receive 10% off your first purchase and save 15% on subscriptions. Bulwark Capital https://KnowYourRiskRadio.com Sign up for the final FREE Live Webinar of the year at KnowYourRiskRadio.com Space is limited. HumanN http://americalovesbeets.com Get a free 30-day supply of Superbeets Heart Chews and a free full-sized bag of Turmeric Chews only at http://americalovesbeets.com SOTA Weight Loss https://sotaweightloss.com SOTA Weight Loss is, say it with me now, STATE OF THE ART! GreenHaven Interactive Digital Marketing https://greenhaveninteractive.com Your Worldclass Website Will Get Found on Google!

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 A clean CR passes, no Mayorkas impeachment, fighting and near fisticuffs, secret service shoots at perps and miss plus disturbing revelations about the jab. Don't miss our Patriot, Liberal Lunatic and Outrages of the week! 

Novartis has been active in the contract manufacturing space for twenty years, now under the brand name Global Biotech Cooperations. Novartis Global Biotech Cooperations provides contract manufacturing services across various domains, including Cell & Gene Therapy, Plasmid DNA, RNA, mRNA, SiRNA, Microbial manufacturing, Mammalian cell cultures, Viral vector, and Fill & Finish. In this episode Rathish Ravindrakumar, Business Development Director, Novartis Global Biotech Cooperations, explains how in the last 2-3 years they have been receiving more requests for newer technologies like Cell and Gene Therapy. “These technologies represent both an opportunity and a challenge for some companies to get the required capacity. We can help these companies to manufacture their medicines as we have built our capabilities in these technologies.” Sustainability is high on the Novartis agenda. Rathish highlights how the harmful effects of climate change are now clear to see, and will requires immediate and sustained action by all stakeholders. “Sustainability has become the key issue for all contract manufacturers as pharma companies take proactive steps to deliver value chain emission reductions.” “One of Novartis' business objectives is building the green operations of the future” says Rathish “If you look at recently published analysis, Novartis is already ranked highly for its efforts to reduce greenhouse gas emissions.” As an example of the work Novartis is undertaking, the company recent transitioned to 100% renewable energy in one of its large-scale sites. This is part of a group-wide sustainability journey to be carbon neutral across its own operations by 2025 and across its supply chain by 2030. Full article here

In February, Kevin McKernan, CSO of Medicinal Genomics, was deep sequencing Pfizer mRNA vaccines when he discovered that every sample he tested was contaminated with large quantities of plasmid DNA. A plasmid is a circular piece of DNA integral in the manufacturing process of mRNA vaccines, but it doesn't belong in the final shot going into arms, nor is it a listed component of either the Moderna or Pfizer mRNA vaccines. McKernan re-tested multiple different vaccine samples from multiple different lots using multiple different methods, but consistently got the same result: substantial plasmid DNA contamination in every Pfizer vaccine sample tested. McKernan published his findings but received no coverage by the MSM, nor was he contacted by Pfizer, the CDC, or the FDA. When I interviewed Kevin in April, no other lab in the US had confirmed McKernan's results. Now they have. McKernan's findings have been repeated and confirmed by Dr. Phillip Buckhaults, a cancer genomics expert from the University of South Carolina. On September 18th, Buckhaults testified before the South Carolina Senate Medical Affairs Ad-Hoc Committee on DHEC and presented his findings. There's no longer any doubt that substantial quantities of plasmid DNA contaminated the Pfizer mRNA vaccines injected into humans. The issue that needs to be addressed is: What are the human health effects of being injected with 200-billion plasmid DNA fragments per jab? The MSM, the FDA, the CDC, and Pfizer have been dismissive of the potential public health consequences of injecting hundreds of billions of DNA fragments contained in each jab into the arms of hundreds of millions of humans. Two potential problems identified by both McKernan and Buckhaults is the risk of cancer transformation and immune targeting of cells taking up the vaccine. Today, I cover highlights of that September 18th South Carolina Senate hearing featuring Dr. Phillip Buckhaults discussing the plasmid DNA contamination of the Pfizer vaccines, what it could mean, and what should be done about it.

Official government mouthpieces previously told us that the mRNA jab would only remain in your body for a short period of time. But that was a complete lie according to new data. In fact, we now know the jab contains actual DNA, not just RNA. A recent article written by Dr. Ah Khan Syed highlights this fact and its inherent dangers. -4- The article, Plasmid DNA in Pfizer and Moderna injections can enter the nucleus of our cells in 5 different ways, informs us that DNA is included in the mRNA jab at roughly 30- and then notes that it can clearly gain entrance into the nucleus of our cells. Once there, that DNA remains permanently and creates all sorts of health problems, including -turbo- cancers. Please read the linked article.

In early February, Kevin McKernan, Chief Science Officer of Medicinal Genomics, was deep sequencing Moderna and Pfizer bivalent booster vaccines to help solve a technical problem with his sequencing process when he made an entirely unexpected discovery: the Pfizer and Moderna bivalent booster samples were contaminated with DNA that contained the Spike protein gene within it. Self-replicating DNA plasmids containing the spike protein gene are an integral part of the manufacturing process to make the Pfizer and Moderna COVID vaccines; they are not supposed to be in the final product injected into humans. Kevin repeated the analysis on Pfizer's monovalent vaccine [the original COVID-19 vaccine requiring 3 jabs] using multiple different measuring tools and arrived at the same result: Plasmid DNA with the Spike gene contaminating the vaccine. When he measured the quantity of DNA it was an order of magnitude higher than the acceptable limit specified by the European Medicines Agency [EMA] in some lots. Today, I have Kevin McKernan in the virtual studio to explain his research, what he found, and what it means. #Plasmidgate.

Ankur Gopal struck out into the world but returned home to Kentucky to start his companies. Michael Chambers stayed in North Dakota and built a high-growth company right there in his own backyard. Both of their home states have benefitted.

Brief History of Syphilis Evaluation of the fluorescent treponemal antibody-absorption (FTA-Abs) test specificity Anticardiolipin antibodies in Lyme disease A Brief History of Laboratory Diagnostics for Syphilis Relapsing Fever Syphilis Lyme How the LYMErix Lyme Disease Vaccine was Pulled from the Market Chronic syphilis facts | General center | SteadyHealth.com Distinction between Borrelia and Borreliella is more robustly supported by molecular and phenotypic characteristics than all other neighbouring prokaryotic genera: Response to Margos' et al. "The genus Borrelia reloaded" (PLoS ONE 13(12): e0208432) The History and Evolution of Molecular Diagnostics Ceftriaxone treatment of penicillin resistant neurosyphilis in alcoholic patients Plasmid DNA in Treponema pallidum (Nichols): Potential for Antibiotic Resistance by Syphilis Bacteria Preparation of atypical forms of Treponema pallidum and detection of antibodies to them in the experiment Lupus or syphilis? That is the question! Sexual Transmission of Lyme Borreliosis? The Question That Calls for an Answer A tale of two spirochetes: lyme disease and syphilis Syphilis & Lyme: a comparison Remembering the Tuskegee Syphilis Experiment 45 Years Later: Racism and Medical Denial LYME DISEASE: THE NEW TUSKEGEE EXPERIMENT The Tuskegee Experiment Never Ended: "Why You Can't Get Treated for Lyme Disease" Talky Beat by Twin Musicom is licensed under a Creative Commons Attribution 4.0 license. https://creativecommons.org/licenses/by/4.0/ Source: http://www.twinmusicom.org/song/265/talky-beat Artist: http://www.twinmusicom.org Stream Keep It Easy, All Day by Jeremy T Murphree | Listen online for free on SoundCloud --- Send in a voice message: https://anchor.fm/morgellons/message Support this podcast: https://anchor.fm/morgellons/support

Many Helicobacter pylori (Hp) strains carry cryptic plasmids of different size and gene content, the function of which is not well understood. A subgroup of these plasmids (e.g. pHel4, pHel12), contain a mobilisation region, but no cognate type IV secretion system (T4SS) for conjugative transfer. Instead, certain H. pylori strains (e.g. strain P12 carrying plasmid pHel12) can harbour up to four T4SSs in their genome (cag-T4SS, comB, tfs3, tfs4). Here, we show that such indigenous plasmids can be efficiently transferred between H. pylori strains, even in the presence of extracellular DNaseI eliminating natural transformation. Knockout of a plasmid-encoded mobA relaxase gene significantly reduced plasmid DNA transfer in the presence of DNaseI, suggesting a DNA conjugation or mobilisation process. To identify the T4SS involved in this conjugative DNA transfer, each individual T4SS was consecutively deleted from the bacterial chromosome. Using a marker-free counterselectable gene deletion procedure (rpsL counterselection method), a P12 mutant strain was finally obtained with no single T4SS (P12ΔT4SS). Mating experiments using these mutants identified the comB T4SS in the recipient strain as the major mediator of plasmid DNA transfer between H. pylori strains, both in a DNaseI-sensitive (natural transformation) as well as a DNaseI-resistant manner (conjugative transfer). However, transfer of a pHel12::cat plasmid from a P12ΔT4SS donor strain into a P12ΔT4SS recipient strain provided evidence for the existence of a third, T4SS-independent mechanism of DNA transfer. This novel type of plasmid DNA transfer, designated as alternate DNaseI-Resistant (ADR) mechanism, is observed at a rather low frequency under in vitro conditions. Taken together, our study describes for the first time the existence of three distinct pathways of plasmid DNA transfer between H. pylori underscoring the importance of horizontal gene transfer for this species.

H. pylori kolonisiert die Magenmukosa von etwa 50% der Bevölkerung und ist die Ursache von vielen schweren Erkrankungen, wie z.B. chronischer Gastritis, Magengeschwüren und Magenkrebs. Lange Zeit wurde angenommen, dass das Milieu des Magens aufgrund der dort herrschenden Bedingungen steril sei. H. pylori hat Strategien entwickelt, um dieses Habitat zu besiedeln und stellt den dominierenden Bestandteil der Mikroflora im Magen dar. Eine entscheidende Voraussetzung für die erfolgreiche Kolonisierung ist die genetische Diversität und die damit verbundene Anpassung an die verschiedenen Mikronischen des Magens. Die Pathogenität von H. pylori wird nicht nur durch Toxine vermittelt, sondern resultiert aus der komplexen Interaktion zwischen dem Bakterium, dem Wirt und der Umwelt. Eine wichtige Bedeutung hierbei hat der Austausch von genetischem Material. Während die natürliche Kompetenz eine entscheidende Rolle für die Aufnahme von genetischem Material spielt, wird auch ein konjugativer Mechanismus zum Transfer von DNA diskutiert. Im Rahmen dieser Arbeit wurde erstmals der DNaseI-resistente Transfer der intrinsischen, kryptischen Plasmide pHel4 und pHel12 zwischen H. pylori-Stämmen nachgewiesen. Es konnte gezeigt werden, dass für diesen Mechanismus sowohl die Plasmid-, als auch die chromosomal-kodierten Relaxasen nicht essentiell sind. Möglicherweise wird die Rezirkularisierung der Plasmid-DNA im Rezipienten durch RecA durchgeführt. Um Informationen über die Maschinerie, welche den DNaseI-geschützten Transfer der intrinsischen Plasmide vermittelt zu erhalten, wurden alle in H. pylori P12 identifizierten T4SS mit Hilfe einer Kontraselektionsstrategie sequentiell deletiert und Kokultivierungsexperimente mit den entsprechenden Mutanten durchgeführt. Es konnte gezeigt werden, dass außer dem ComB-System keines der T4SS für den DNA-Transfer zwischen H. pylori-Stämmen essentiell ist. Dieses ist für die Aufnahme von DNA im Rezipienten verantwortlich und spielt auch eine Rolle für den DNA-Export durch den Donor. Das ComB-System stellt somit das entscheidende T4SS für den Transfer von DNA dar und hat eine duale Funktion hinsichtlich Transformation und einem Konjugations-ähnlichen Mechanismus im Donor und Rezipienten. Bemerkenswert ist, dass auch nach Deletion aller T4SS in H. pylori P12 DNA-Transfer stattfindet. Mögliche Kandidaten für einen alternativen DNA-Übertragungsweg, stellen Membranvesikel dar. Darüber hinaus konnte nachgewiesen werden, dass Tfs4 Plasmid-DNA in das umgebende Milieu sekretiert. Durch die Sekretion des Modul-artig aufgebauten kryptischen Plasmids pHel12 kann die Verbreitung von genetischem Material zwischen Stämmen unterstützt werden. Die Unabhängigkeit des DNA-Transfermechanismus von den Relaxasen, sowie die Resistenz gegenüber dem Angriff durch DNaseI lassen einen neuartigen, Konjugations-ähnlichen DNA-Transfermechanismus vermuten, der von der konventionellen Konjugation abgrenzt werden kann. Neben der Charakterisierung der DNA-Transfer-Mechanismen in H. pylori P12 wurden im Rahmen dieser Arbeit auch die kryptischen Plasmide pHel4 und pHel12 und die mögliche Funktion ihrer Genprodukte untersucht. Etwa 50% aller klinischen Isolate enthalten kryptische Plasmide. Ihre Funktion ist bisher allerdings nicht klar. Die Anwesenheit einer mob-Region deutete auf eine konjugative Übertragung der Plasmide hin. Darüber hinaus lässt die Struktur der Plasmide eine Rolle bei der Verbreitung von genetischem Material als Orte des „gene shufflings“ vermuten. Zudem konnten erste Hinweise bezüglich der mit den Plasmiden verbundenen Zytotoxizität bestätigt werden. So beeinflusst die Expression von orf4M aus pHel4 und orf12M aus pHel12 in eukaryotischen Zellen die zelluläre Integrität und führt schließlich zum Zelltod. Die kryptischen Plasmide stellen eine interessante Möglichkeit für H. pylori dar, genetische Information auszutauschen und möglicherweise die Wirtszelle zu beeinflussen.

In this dissertation, systemic delivery of NAs, alone and complexed with polycationic delivery systems, was analysed. The analysis was focused on tolerability and efficiency of the treatment. The in vivo administration is so far hampered by the lack of stable delivery systems that are able to protect their NA cargo in the blood stream and savely lead it to the desired target cells. However, two novel polycationic vectors that had been established in our lab, led to promising results for siRNA delivery in vitro. In the current work we have therefore tried to optimize siRNA / polycationic complexes for in vivo administration. Furthermore, we have used the therapeutically relevant RAN siRNA to influence the growth of subcutaneous Neuro2A tumors in an in vivo model. Another aim was to evaluate different biodegradable polymers, that had been established in our lab, for their efficiency and safety in in vivo application. Polymers were compared with L-PEI as a gold standard regarding efficiency. However, L-PEI can not be degraded within the body and can thereby lead to a long-term toxicity. In contrast, we wanted to show that our biodegradable polymers can reach efficiency levels as high as L-PEI, yet compared with a much better tolerability and the possibility of repeated application. Furthermore we were interested in clarifying the impact of the NA on efficiency and toxicity of the treatment. In this case we have compared plasmid DNA containing bacterially derived CpG motifs, with plasmid DNA that was exempt from CpG sequences. But because plasmids are generated in bacteria and the plasmid purification step leaves behind a certain amount of bacterial genomic DNA, we also wanted to clarify the impact of this (CpG containing) impurities. One approach was to compare the effect of the plasmids alone. For this analysis we had chosen the possibility of hydrodynamic tail vein injection of plasmid solutions in mice. Another approach was to compare the plasmids when they were complexed to L-PEI as a gold standard for polyplex delivery

Im Rahmen dieser Arbeit wurden unterschiedliche Proteomstudien an Halobacterium salinarum durchgeführt, die sich generell in drei unabhängige Themenblöcke unterteilen lassen. Zunächst erfolgte die Erfassung des cytosolischen Proteoms auf standardisierten 2-D Gelen. Hierfür wurde die Auftrennung auf sogenannten Zoom-Gelen aufgrund der engen pI-Verteilung halobakterieller Proteine etabliert. Die erschöpfende Analyse der so aufgetrennten Proteine wurde mit der peptide mass fingerprinting Methode im Hochdurchsatzverfahren durchgeführt. So konnten so 40% des zugänglichen cytosolischen Proteininventars identifiziert und auf Referenz 2-D Gelen kartiert werden. Des Weiteren konnten die massenspektrometrischen Proteinidentifizierungen zur intensiven Verbesserung der in vielen Fällen nicht eindeutigen Genomannotation herangezogen werden. Durch die Korrelationsanalyse von experimentellen und theoretischen pI-Werten konnten weitere Annotationsfehler behoben werden. Das Zusammenspiel von Genomik und Proteomik erlaubte die Identifizierung eines Bereichs ehemaliger Plasmid DNA, die in das Chromosom eingewandert ist. Weiter konnte für ein Plasmid gezeigt werden, dass dieser ehemalig chromosomale DNA und somit eine Reihe wichtiger und essentieller Gene enthält und deshalb eher als zweites Chromosom angesehen werden sollte. Nach der Inventarisierung folgten Analysen zur differentiellen Expression cytosolischer Proteine in Abhängigkeit unterschiedlicher Wachstumszustände. Hierbei wurde das aerobe Wachstum unter Standardbedingungen mit Wachstum unter Mangelbedingungen wie in synthetischem Medium oder unter anaeroben phototrophen Bedingungen verglichen. Bei der Analyse dieser Zustände mittels 2-DE konnten nur wenig regulierte Proteine identifiziert werden. Generell stellte sich die 2-DE aufgrund schwankender Reproduzierbarkeit und des geringen dynamischen Bereichs der gewählten Färbemethode für diese vergleichenden Analysen als wenig geeignet heraus. Die Verwendung isotopenmarkierter Sonden erlaubte hingegen einen detaillierten Einblick in die Regulationen auf Proteomebene unter besagten Bedingungen. Mit dem methodischen Ansatz der 1D-SDS PAGE LC-MALDI-TOF/TOF Analyse konnten von einem beträchtlichen Teil (ca. 40%) des cytosolischen Proteoms Regulationsinformationen erhalten werden. Auch hier zeigte sich, dass unter anaeroben phototrophen Bedingungen überraschend wenige Proteine reguliert werden müssen, damit sich der Organismus auf diese Lebensbedingung einstellen kann. Wachstum unter Nährstoffmangel in synthetischem Medium induziert überwiegend Proteine der Aminosäure- und Nukleotid-Biosynthese sowie Proteine, die am Proteinschutz und der Glykolyse beteiligt sind. Unter Standardwachstum wurden Proteine als induziert identifiziert, die bei nicht ausreichendem Vorhandensein wachstumslimitierend wirken würden. Dies waren hauptsächlich Enzyme der Thiamin und Cobalamin Biosynthese sowie des Peptid-Transportes und -Abbaues. Auch die CO2-Fixierung und die nachfolgende Gluconeogenese sind induziert. Durch die Verwendung unterschiedlicher Proteomik Ansätze konnte am Ende der Großteil der cytosolischen Proteine von H. salinarum identifiziert werden. Es scheinen nahezu alle chromosomalen Proteine auch unter Standard Wachstumsbedingungen expremiert zu werden, was starke Regulationen der Proteinkonzentrationen in Abhängigkeit unterschiedlicher Lebensbedingungen nicht notwendig macht. Der Organismus kann sich so schnell ohne großen energetischen Aufwand an wechselnde Lebensbedingungen anpassen. Im letzten Teil dieser Arbeit wurde das Ausmaß der Proteinphosphorylierung in H. salinarum untersucht. Hierbei konnten zunächst über radioaktive in vivo Markierung phosphattragende Proteine identifiziert werden. Ebenfalls gelang es, die ersten halobakteriellen Serin und Threonin Phosphorylierungsstellen zu identifizieren. Generell zeigte sich jedoch, dass das Ausmaß der Proteinphosphorylierung in H. salinarum eher gering ist. Bei den identifizierten Phosphorylierungen handelte es sich ausschließlich um katalytische Zwischenprodukte. Für die Existenz von regulativen Ser/Thr oder Tyr Proteinphosphorylierungen waren keine eindeutigen Hinweise zu finden. Weiter gelang es, eine Vielzahl von N-terminal acetylierten Proteinen als eine weitere Form der posttranslationalen Modifikationen zu identifizieren.

Im Literaturüberblick der vorliegenden Arbeit wird besonders auf die ökonomische und ökologische Bedeutung der Flusskrebse, den derzeitigen Stand der Forschung die Krebspest betreffend und die aktuelle taxonomische Klassifikation des Krebspesterregers, Aphanomyces astaci, eingegangen. Ziel der vorliegenden Arbeit war es, eine zuverlässige und schnelle Methode zum spezifischen und sensitiven Nachweis von Aphanomyces astaci direkt aus Krebsgewebe zu entwickeln. Zu diesem Zweck wurde zunächst ein kommerzielles Fertigkit (DNeasy® Tissue Kit, Qiagen) als geeignete DNA-Extraktionsmethode ausgewählt. Als Untersuchungsmaterial wurde die Abdominalkutikula von Edelkrebsen (Astacus astacus) eingesetzt. Anhand von Genomanalysen der ITS-Regionen (Genabschnitte, die zwischen den die ribosomale RNA kodierenden Genen liegen) verschiedener Oomycota wurden Oligonukleotid-Primer konstruiert, optimiert und auf ihre Eignung überprüft. Ausgehend von diesen Untersuchungen wurden zwei PCR-Protokolle etabliert: eine Semi-Nested und eine Nested PCR. Die Semi-Nested PCR, die sich für die Routinediagnostik von Aphanomyces astaci als wenig geeignet erwiesen hat, bietet das Potential, als Grundlage für weiterführende Studien an anderen Arten der Gattung Aphanomyces zu dienen. Die Nested PCR mit den äußeren Primern NS 166/NS 681 und den inneren Primern BO 525/BO 640 ergab mit allen 20 untersuchten Aphanomyces astaci-Stämmen ein PCR-Produkt von etwa 115 bp, das mit einer anschließenden Restriktionsenzym-Analyse mit der Endonuklease Hph I verifiziert werden konnte. Die Nested PCR zeigte sich hochspezifisch gegenüber Aphanomyces astaci, während die DNA von anderen bei Krebsen und im Wasser vorkommenden Krankheitserregern und die DNA von Wirtsgewebe nicht amplifiziert wurde, was Grundvoraussetzung für den Einsatz dieser PCR als Standardmethode ist. Die Nachweisgrenze der Nested PCR lag bei Verwendung von Plasmid-DNA bei 1,9 genomischen Einheiten und bei Einsatz von genomischer DNA aus Pilzmyzel bei 1 ag. Als Zusammenfassung 172 Aphanomyces astaci-Sporen in die DNA-Extraktion eingesetzt wurden, lag die Nachweisgrenze bei 1 Spore. Zusätzlich wurden Infektionsversuche durchgeführt, bei denen Edelkrebse mit 10, 100 und 1000 Aphanomyces astaci-Zoosporen/ml infiziert wurden. Ein positives PCR-Ergebnis konnte ab Tag 2 post expositionem (entspricht dem Tag der ersten Probenentnahme) mit der beschriebenen Methode bei allen untersuchten Tieren beobachtet werden. Schließlich wurde eine Validierung der entwickelten Nested PCR unter Einsatz von Wirtskutikula anhand 19 diagnostischer Proben unterschiedlicher Herkunft innerhalb Europas (aus Deutschland, der Schweiz, Österreich, Schweden und England) durchgeführt und die Ergebnisse mittels RFLP verifiziert. Mit der in der vorliegenden Arbeit entwickelten molekularbiologischen Nachweismethode steht ein Verfahren zur Verfügung, das eine zuverlässige Diagnose von Aphanomyces astaci bereits im Anfangsstadium der Infektion direkt aus Krebsgewebe ermöglicht und innerhalb von 12 bis 15 Stunden (Sektion, DNA-Extraktion, Nested PCR, Agarosegel-Elektrophorese und bestätigende Restriktionsenzym-Analyse) durchführbar ist. Diese Methode ist somit ein geeignetes und zuverlässiges Mittel, um in Programmen zur Seuchenbekämpfung der Krebspest (Aphanomyces astaci) eingesetzt zu werden.

Epstein-Barr virus (EBV) is a herpesvirus that transforms B-cells (B-LCL) and has undergone intense scrutiny owing to its association with Burkitt's lymphoma, nasopharyngeal carcinoma, and immunoblastic lymphomas. B-LCL have also proven useful in the study of human immunology. We describe a novel system for inducing efficient foreign gene expression in B-LCL using biotinylated adenovirus as an endosome-disrupting agent. Plasmid DNA is coupled to the exterior of viral particles by streptavidin-polylysine chimeric proteins. Up to 67% of B-LCL may be induced to express foreign genes in vitro in transient expression systems, and gene expression lasts for at least 17 days. We have expressed firefly luciferase, β-galactosidase (β-gal), chloramphenicol acetyltransferase, HIV gag, and env genes, as well as infectious HIV, and the EBV-specific BZLF gene in B-LCL with this system. In vivo delivery of a β-gal reporter gene to B-LCL was documented in a SCID mouse model. Potential applications include study of genetic regulation of EBV infection and transformation events, study of potential gene therapies for EBV-related B-cell tumors, and production of antigen-presenting cells for use in immunologic assays. Because of the high percentage of cells transformed and the length of foreign gene expression, the possibility of examining foreign gene expression in transient assays, without selection for clonal populations, exists.

Sat, 1 Jan 1994 12:00:00 +0100 http://epub.ub.uni-muenchen.de/4060/ http://epub.ub.uni-muenchen.de/4060/1/4060.pdf Cotten, Matt; Baker, A.; Saltik, Mediyha; Wagner, Ernst; Buschle, M. Cotten, Matt; Baker, A.; Saltik, Mediyha; Wagner, Ernst und Buschle, M. (1994): Lipopolysaccharide is a frequent contaminant of plasmid DNA preparations and can be toxic to primary cells in the presence of adenovirus. In: Gene Therapy, Vol. 1: pp. 239-