Podcasts about oct4

Researchers at the Francis Crick Institute in London have used CRISPR-Cas9 "to disrupt the production of a protein called OCT4 that is important for embryo development." The first-of-its-kind study used 58 embryos donated by fertility clinics no longer needed for in-vitro fertilization. But now that they've started down the slippery slope of experimenting on humans, how far will scientists go? SkyWatchTV has been banned by YouTube! Please follow SkyWatchTV on Rumble: www.rumble.com/skywatchtv. Join us in Israel! For details on the SkyWatchTV Israel Tour (new dates: March 20-April 4, 2022), visit www.SkyWatchInIsrael.com.

Researcher and elite athlete Brianna Stubbs is back on the podcast today, checking in before her recent Ironman competition in Santa Cruz, California. Since we last talked with Brianna she’s left HVMN and joined the Buck Institute for Research on Aging as Lead Translational Scientist. There she’ll be studying ketone biology and collaborating on some of the best research being conducted today with the mission of living better, longer. In this interview, Brianna and I talk about some of the latest studies on ketone metabolism, which continues to show promise for athletic performance, cognition, and cardiovascular health. She also notes where the research in this area is lacking and even contradictory. Brianna also shares her personal strategy for dosing the ketone monoester she helped bring to the marketplace.  Here’s the outline of this interview with Brianna Stubbs: [00:00:00] Brianna’s previous podcasts: World Champion Rower and Ketone Monoester Researcher Brianna Stubbs, The D-BHB Ketone Monoester Is Here, Women in Science: Bridging the Gender Gap, and The Latest Research on Exogenous Ketones and Other Performance Enhancers. [00:02:00] Racing Ironman. [00:05:10] Lesley Paterson; Podcast: Off Road Triathlon World Champion Lesley Paterson on FMT and Solving Mental Conundrums. [00:06:57] Dosing the ketone ester during the triathlon. [00:09:55] Ketone ester as a tool to to evaluate perception of exercise; Study: Faull, Olivia Kate, et al. "Beyond RPE: The perception of exercise under normal and ketotic conditions." Frontiers in physiology 10 (2019): 229.  [00:11:09] Lead Translational Scientist at the Buck Institute for Research on Aging. [00:13:01] Dale Bredesen, MD; Eric Verdin, MD. [00:13:36] Review on ketone metabolism: Newman, John C., and Eric Verdin. "Ketone bodies as signaling metabolites." Trends in Endocrinology & Metabolism 25.1 (2014): 42-52. [00:14:56] Jeff Volek, PhD, RD & Stephen Phinney, MD, PhD [00:18:07] STEM-Talk Podcast: Episode 92 with Megan Roberts and Episode 94 with John Newman. [00:19:27] β-Hydroxybutyrate (BHB), as an endogenous histone deacetylase inhibitor; Study: Shimazu, Tadahiro, et al. "Suppression of oxidative stress by β-hydroxybutyrate, an endogenous histone deacetylase inhibitor." Science 339.6116 (2013): 211-214. [00:21:42] HVMN. [00:22:59] Epigenetic effects of ketones. [00:27:27] BHB can affect vascular senescence; Study: Han, Young-min, et al. "β-Hydroxybutyrate prevents vascular senescence through hnRNP A1-mediated upregulation of Oct4." Molecular cell 71.6 (2018): 1064-1078. [00:30:24] BHB inactivates the NLRP3 inflammasome; Study: Youm, Yun-Hee, et al. "The ketone metabolite β-hydroxybutyrate blocks NLRP3 inflammasome–mediated inflammatory disease." Nature medicine 21.3 (2015): 263. [00:31:02] BHB protects against muscle protein wasting; Study: Thomsen, Henrik H., et al. "Effects of 3-hydroxybutyrate and free fatty acids on muscle protein kinetics and signaling during LPS-induced inflammation in humans: anticatabolic impact of ketone bodies." The American journal of clinical nutrition 108.4 (2018): 857-867. [00:32:11] Increased inflammatory response with ketone ester; Study: Neudorf, Helena, et al. "Oral Ketone Supplementation Acutely Increases Markers of NLRP3 Inflammasome Activation in Human Monocytes." Molecular nutrition & food research (2019): 1801171. [00:35:52] Ketogenic diet and exogenous ketones reduce epileptiform spikes associated with Alzheimer’s: Newman, John C., et al. "Ketogenic diet or BHB improves epileptiform spikes, memory, survival in Alzheimer's model." bioRxiv (2017): 136226. [00:36:51] Kenneth Ford, PhD.; Podcast: Optimal Diet and Movement for Healthspan, Amplified Intelligence and More with Ken Ford [00:37:57] Cardiovascular effects of ketone infusions in humans; Studies: 1. Nielsen, Roni, et al. "Cardiovascular effects of treatment with the ketone body 3-hydroxybutyrate in chronic heart failure patients." Circulation 139.18 (2019): 2129-2141. 2. Gormsen, Lars C., et al. "Ketone Body Infusion With 3‐Hydroxybutyrate Reduces Myocardial Glucose Uptake and Increases Blood Flow in Humans: A Positron Emission Tomography Study." Journal of the American Heart Association 6.3 (2017): e005066. [00:38:06] Ketone infusions in a paced model of cardiac failure in dogs. Study: Horton, Julie L., et al. "The failing heart utilizes 3-hydroxybutyrate as a metabolic stress defense." JCI insight 4.4 (2019). [00:43:05] Ketogenic diet and gut health. [00:44:31] Exogenous ketones affect stem cell regeneration and differentiation; Study: Cheng, Chia-Wei, et al. "Ketone Body Signaling Mediates Intestinal Stem Cell Homeostasis and Adaptation to Diet." Cell 178.5 (2019): 1115-1131. (We don’t have access to the Supplementary Methods, which contain Brianna’s favorite molecule!) [00:46:02] Performance enhancing effects of lactate/propionate: Scheiman, Jonathan, et al. "Meta-omics analysis of elite athletes identifies a performance-enhancing microbe that functions via lactate metabolism." Nature Medicine (2019): 1. [00:47:22] Lucy Mailing; Podcast: How to Optimise Your Gut Microbiome. [00:50:24] Improved performance with the ketone monoester; Study: Cox, Pete J., et al. "Nutritional ketosis alters fuel preference and thereby endurance performance in athletes." Cell metabolism 24.2 (2016): 256-268.  [00:50:30] No performance benefit with ketone monoester supplement; Study: Evans, Mark, et al. "No Benefit of Ingestion of a Ketone Monoester Supplement on 10-km Running Performance." Medicine and science in sports and exercise 51.12 (2019): 2506-2515. [00:52:00] Dr. Stephen Cunnane’s studies correlating blood ketone level and changing cognitive function and brain ketone uptake: Cunnane, Stephen C., et al. "Can ketones help rescue brain fuel supply in later life? Implications for cognitive health during aging and the treatment of Alzheimer’s disease." Frontiers in molecular neuroscience 9 (2016): 53; 2. Fortier, Mélanie, et al. "A ketogenic drink improves brain energy and some measures of cognition in mild cognitive impairment." Alzheimer's & Dementia 15.5 (2019): 625-634. [00:52:40] Breath ketone meters. [00:57:03] Find Brianna on Twitter.

Oct4.2016 by Buzz Me In

Post-translational modifications (PTMs) of histones exert fundamental roles in regulating gene expression. During development, groups of PTMs are constrained by unknown mechanisms into combinatorial patterns, which facilitate transitions from uncommitted embryonic cells into differentiated somatic cell lineages. Repressive histone modifications such as H3K9me3 or H3K27me3 have been investigated in detail, but the role of H4K20me3 in development is currently unknown. Here we show that Xenopus laevis Suv4-20h1 and h2 histone methyltransferases (HMTases) are essential for induction and differentiation of the neuroectoderm. Morpholino-mediated knockdown of the two HMTases leads to a selective and specific downregulation of genes controlling neural induction, thereby effectively blocking differentiation of the neuroectoderm. Global transcriptome analysis supports the notion that these effects arise from the transcriptional deregulation of specific genes rather than widespread, pleiotropic effects. Interestingly, morphant embryos fail to repress the Oct4-related Xenopus gene Oct-25. We validate Oct-25 as a direct target of xSu4-20h enzyme mediated gene repression, showing by chromatin immunoprecipitaton that it is decorated with the H4K20me3 mark downstream of the promoter in normal, but not in double-morphant, embryos. Since knockdown of Oct-25 protein significantly rescues the neural differentiation defect in xSuv4-20h double-morphant embryos, we conclude that the epistatic relationship between Suv4-20h enzymes and Oct-25 controls the transit from pluripotent to differentiation-competent neural cells. Consistent with these results in Xenopus, murine Suv4-20h1/h2 double-knockout embryonic stem (DKO ES) cells exhibit increased Oct4 protein levels before and during EB formation, and reveal a compromised and biased capacity for in vitro differentiation, when compared to normal ES cells. Together, these results suggest a regulatory mechanism, conserved between amphibians and mammals, in which H4K20me3-dependent restriction of specific POU-V genes directs cell fate decisions, when embryonic cells exit the pluripotent state.

Die vorliegende Arbeit hatte zum Ziel, die prinzipielle Fragestellung zu beantworten, ob Stammzellen aus humanen Haarfollikeln in ausreichender Menge expandiert werden können und inwieweit eine Differenzierung in neuroendokrine Zellen möglich ist. Es sollte eine Methode zur Isolierung und Langzeitkultivierung von Haarfollikelstammzellen etabliert und optimiert werden, um eine neue Quelle autologer adulter Stammzellen für zelltherapeutische Ansätze zu gewinnen. Durch Verwendung verschiedener Medien und Beschichtungsarten wurde ein Protokoll entwickelt, aus Dispase-verdauten Hautbiopsien Progenitorzellen zu isolieren. Die auf diese Weise expandierten Zellen wurden mit FACS-Analyse, RT-PCR und Immunhistologie charakterisiert. Im letzten Teil der Arbeit wurde durch Zugabe von spezifischen Faktoren die Fähigkeit zur Differenzierung in unterschiedliche Zelltypen untersucht. Nach Austestung verschiedener Zellkulturbedingungen wurde eine neue Population von Zellen aus der Haarfollikelregion isoliert. Diese Zellen, die als hBSCs bezeichnet wurden, waren über mehr als 30 Passagen mit stabilem Phänotyp kultivierbar (Self-Renewal). Zudem waren sie im Colony-Unit-Assay positiv und zeigten die Expression pluripotenter (Oct4) und multipotenter Stammzellmarker (Nestin, BCRP1, Sox2). Die molekulare Signatur der hBSCs zeigt einige Übereinstimmung mit Merkelzellen, neuroektodermalen Zellen der Haut, die eine Rolle als Mechanorezeptoren und neurosekretorische Zellen der Haut spielen. Unter Verwendung von etablierten Protokollen wurde die Fähigkeit der Differenzierung in Adipozyten, Osteoblasten, glatte Muskelzellen, Neuronen und endokrine Zellen untersucht. Der Nachweis der Entwicklung von glatten Muskelzellen, Neuronen und in eingeschränktem Maße auch Adipozyten belegt die Multipotenz der hBSCs. Darüber hinaus besitzen die hBSCs die Fähigkeit, stimulusabhängig Somatostatin zu exprimieren und zu sezernieren. Somit ist es erstmals gelungen, humane adulte Stammzellen/Progenitorzellen mit neuroendokrinen Eigenschaften zu isolieren. Zusammenfassend ist es in der vorliegenden Arbeit gelungen, eine Methode zu etablieren, mittels derer eine neue Population von humanen multipotenten Stammzellen aus der Haarfollikelregion in Langzeitkultur expandiert werden konnte. Die Plastizität der hBSCs und insbesondere die Differenzierung in reife endokrine Zellen muss in weiteren Studien untersucht werden.

Embryonic development represents a sophisticated multistep process. Hereby, specification, patterning and differentiation of cells and tissue need to be extremely well regulated in a temporo-spatial manner. This is based on repression and activation of a vast number of cell-type specific genes, but only a small number of transcription factors seem to be responsible for their regulation. The transcription factor network of Oct4, Sox2 and Nanog are thought to play an essential role in the maintenance of pluripotency and in timing the onset of differentiation. The importance of mouse Oct4 in the regulation of pluripotency is underscored by recent findings providing evidence that Oct4 is essential for reprogramming somatic cells. Nevertheless, little is known on the molecular function of this transcription factor during normogenesis. Given the extra uterine development of the embryos, the well-studied early development and the established manipulation methods like injection of RNA or DNA, Xenopus leavis offers an ideal model organism to study the role of Oct4 homologs in early development. In Xenopus laevis three Oct4 paralogs – Oct25, Oct60 and Oct91 – are known, which are similar in size and have a high sequence homology compared to mammalian Oct4. There are strong evidences that Xenopus Oct proteins and mammalian Oct4 share similar functions. To gain further insights into the function of Oct proteins I generated dominant activating- (VP16-Oct60), dominant repressing- (EnR-Oct60) and hormone inducible (GR-Oct60) transcription factor variants for all three Xenopus Oct proteins. Protein expression was verified in vitro as well as in vivo. Oct60 shows a unique expression pattern among Xenopus Oct proteins: Oct60 is maternally transcribed and its RNA is detectable in mature oocytes. Expression is downregulated in the gastrula, when the expression of other Xenopus POU proteins begins. Therefore, it is one of the earliest genes to be expressed. I decided to concentrate first efforts on Oct60. The transactivating functions of the Oct60 G.o.F. variants were tested in a luciferase assay on two different Oct4 reporter constructs in vivo. Oct60 and VP16-Oct60 acted as strong activators whereas EnR-Oct60 repressed both reporter constructs. By overexpression of Oct60 and its G.o.F. variants, several phenotypes were observed that affected distinct parts of the body. Beside impaired head differentiation, observed by overexpression of VP16-Oct60 and Oct60, a strong hyperpigmentation was observed by injection of EnR-Oct60 and Oct60. Additionally, EnR-Oct60 injected embryos showed hyperpigmented outgrowths in the trunk region. All injected embryos possessed a shortened body axis that was specifically curved depending on the injected mRNA. In situ hybridizations were performed to investigate the molecular mechanism of the observed phenotypic changes. Experiments revealed that all examined constructs promote neuroectodermal fate while repressing mesoderm formation. These results indicate that Oct60 plays an important role in the induction and specification of germ layer formation. By cloning and testing these different G.o.F. variants I accomplished to obtain important tools for further dissecting the molecular function of Oct4 homologs in Xenopus embryos.

Die normale Entwicklung von Embryonen nach somatischem Zellkerntransfer („somatic cell nuclear transfer“, SCNT) hängt unter anderem von der erfolgreichen Reprogrammierung und Aktivierung von Schlüsselgenen ab. Ein Beispiel ist das Gen für den Transkriptionsfaktor Oct4, der im frühen Embryo nachweisbar und als Marker für pluripotente Zellen gilt. Die in klonierten Mausembryonen häufig beobachtete abnormale Expression von Oct4 wird als eine mögliche Ursache für die hohen Verluste und schweren Fehlentwicklungen nach Kerntransfer diskutiert. Beim Rind liegt im Vergleich zur Maus und anderen Spezies die Erfolgsrate am höchsten. Daher ist die Untersuchung der Reprogrammierung von OCT4 nach SCNT in der frühen Embryogenese beim Rind von besonderem Interesse. Um Fragen der epigenetischen Reprogrammierung des Rindergenoms und der Rolle von OCT4 nach SCNT nachzugehen, wurden bovine fetale Fibroblasten stabil mit einem Oct4-EGFP-Reportergenkonstrukt transfiziert. In den unilokulär stabil transfizierten Zellen mit unauffälligem weiblichen Karyotyp war in Analogie zur Inaktivität des endogenen OCT4-Gens in differenzierten Zellklonen keine EGFP-Fluoreszenz nachweisbar. Das Anschalten der Oct4-EGFP-Expression nach SCNT entsprach weitgehend der nach in vitro Fertilisation beobachteten Aktivierung des endogenen OCT4-Gens. In SCNT-Embryonen mit weniger als neun Zellkernen wurde keine EGFP-Fluoreszenz nachgewiesen. In allen Embryonen mit mindestens 17 Zellkernen war das Oct4-EGFP-Reportergenkonstrukt aktiv, was darauf hindeutet, dass Blastomeren nach der vierten Zellteilung den Oct4-Promotor aktivierten. Mittels konfokaler Laser Scanning Mikroskopie wurde an zentralen optischen Schnitten die Intensität der EGFP-Fluoreszenz jedes Embryos gemessen. Im Vergleich mit Tag 4 SCNT-Embryonen war die EGFP-Fluoreszenz in Tag 6 Embryonen deutlich stärker mit erheblichen Unterschieden in der Expressionshöhe zwischen einzelnen Embryonen. Dabei zeigten Embryonen mit einer niedrigeren EGFP-Fluoreszenz im Vergleich zu Embryonen mit stärkerer EGFP-Fluoreszenz einen erheblich höheren Anteil an Zellkernuntergängen (kondensierte und fragmentierte Zellkerne). 34 Tage nach dem Transfer von EGFP-exprimierenden Embryonen auf Empfängertiere wurden drei lebende und morphologisch unauffällige Feten gewonnen. In Fibroblasten, die aus diesen Feten isoliert wurden, war das Reportergenkonstrukt, analog zur normalen Inaktivierung des endogenen OCT4-Gens in differenzierten Zellen, inaktiviert. In SCNT-Embryonen aus den Oct4-EGFP-transgenen Fibroblasten dieser zweiten Generation („second round“ SCNT) wurde das Reportergenkonstrukt erneut regelmäßig aktiviert wie in den SCNT-Embryonen vom Ausgangszellklon („first round“ SCNT). Die Herstellung stabil transfizierter boviner fetaler Fibroblasten mit einer unilokulären Integration des Oct4-EGFP-Reportergenkonstruktes stellt eine wichtige Basis für ein breites Spektrum experimenteller Ansätze zur Aufklärung grundlegender Mechanismen nach Kerntransfer beim Rind dar.