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In this research paper, researchers demonstrate a promising new treatment option for refractory metastatic gastrointestinal cancers using a combination of two FDA-approved drugs. Researchers Jun Zhang, Lanlan Zhou, Shuai Zhao, and Wafik S. El-Deiry from Fox Chase Cancer Center and Brown University explore the potential of combining TAS102 (trifluridine/tipiracil) and regorafenib as a treatment option for gastrointestinal (GI) cancers. Their research paper, published in Oncotarget's Volume 15 on July 2, 2024, is entitled, “Regorafenib synergizes with TAS102 against multiple gastrointestinal cancers and overcomes cancer stemness, trifluridine-induced angiogenesis, ERK1/2 and STAT3 signaling regardless of KRAS or BRAF mutational status.” The Study The combination of two FDA-approved drugs, TAS102 and regorafenib, has shown promising results in preclinical studies. TAS102 is an oral formulation consisting of trifluridine (FTD) and tipiracil hydrochloride (TPI). It has been approved by the US FDA for the treatment of refractory metastatic colorectal cancer and metastatic gastric cancer. Regorafenib is a multi-target tyrosine kinase inhibitor that inhibits tumor angiogenesis and cell proliferation and is approved for the treatment of gastrointestinal cancers. Recent studies have shown that TAS102, in combination with regorafenib, can lead to improved survival and restrict tumor progression. The combination therapy has been found effective in multiple gastrointestinal cancer cell lines, including colorectal, gastric, and pancreatic cancers. Full blog - https://www.oncotarget.org/2024/08/09/combining-regorafenib-and-tas102-to-target-gastrointestinal-cancers-and-overcome-cancer-stemness/ Paper DOI - https://doi.org/10.18632/oncotarget.28602 Correspondence to - Wafik S. El-Deiry - wafik@brown.edu Video short - https://www.youtube.com/watch?v=tuEmJTkyyGQ Sign up for free Altmetric alerts about this article - https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.28602 Subscribe for free publication alerts from Oncotarget - https://www.oncotarget.com/subscribe/ Keywords - cancer, TAS102, regorafenib, ERK1/2, angiogenesis, microvessel density About Oncotarget Oncotarget (a primarily oncology-focused, peer-reviewed, open access journal) aims to maximize research impact through insightful peer-review; eliminate borders between specialties by linking different fields of oncology, cancer research and biomedical sciences; and foster application of basic and clinical science. Oncotarget is indexed and archived by PubMed/Medline, PubMed Central, Scopus, EMBASE, META (Chan Zuckerberg Initiative) (2018-2022), and Dimensions (Digital Science). To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: Facebook - https://www.facebook.com/Oncotarget/ X - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/@OncotargetJournal LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Spotify - https://open.spotify.com/show/0gRwT6BqYWJzxzmjPJwtVh MEDIA@IMPACTJOURNALS.COM

BUFFALO, NY- July 8, 2024 – A new research paper was published in Oncotarget's Volume 15 on July 2, 2024, entitled, “Regorafenib synergizes with TAS102 against multiple gastrointestinal cancers and overcomes cancer stemness, trifluridine-induced angiogenesis, ERK1/2 and STAT3 signaling regardless of KRAS or BRAF mutational status.” Single-agent TAS102 (trifluridine/tipiracil) and regorafenib are FDA-approved treatments for metastatic colorectal cancer (mCRC). Researchers previously reported that regorafenib combined with a fluoropyrimidine can delay disease progression in clinical case reports of multidrug-resistant mCRC patients. In this new study, researchers Jun Zhang, Lanlan Zhou, Shuai Zhao, and Wafik S. El-Deiry from Fox Chase Cancer Center and Brown University hypothesized that the combination of TAS102 and regorafenib may be active in CRC and other gastrointestinal (GI) cancers and may in the future provide a treatment option for patients with advanced GI cancer. “We investigated the therapeutic effect of TAS102 in combination with regorafenib in preclinical studies employing cell culture, colonosphere assays that enrich for cancer stem cells, and in vivo.” TAS102 in combination with regorafenib has synergistic activity against multiple GI cancers in vitro including colorectal and gastric cancer, but not liver cancer cells. TAS102 inhibits colonosphere formation and this effect is potentiated by regorafenib. In vivo anti-tumor effects of TAS102 plus regorafenib appear to be due to anti-proliferative effects, necrosis and angiogenesis inhibition. Growth inhibition by TAS102 plus regorafenib occurs in xenografted tumors regardless of p53, KRAS or BRAF mutations, although more potent tumor suppression was observed with wild-type p53. Regorafenib significantly inhibits TAS102-induced angiogenesis and microvessel density in xenografted tumors, as well inhibits TAS102-induced ERK1/2 activation regardless of RAS or BRAF status in vivo. TAS102 plus regorafenib is a synergistic drug combination in preclinical models of GI cancer, with regorafenib suppressing TAS102-induced increase in microvessel density and p-ERK as contributing mechanisms. “The TAS102 plus regorafenib drug combination may be further tested in gastric and other GI cancers.” DOI - https://doi.org/10.18632/oncotarget.28602 Correspondence to - Wafik S. El-Deiry - wafik@brown.edu Video short - https://www.youtube.com/watch?v=tuEmJTkyyGQ Sign up for free Altmetric alerts about this article - https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.28602 Subscribe for free publication alerts from Oncotarget - https://www.oncotarget.com/subscribe/ Keywords - cancer, TAS102, regorafenib, ERK1/2, angiogenesis, microvessel density About Oncotarget Oncotarget (a primarily oncology-focused, peer-reviewed, open access journal) aims to maximize research impact through insightful peer-review; eliminate borders between specialties by linking different fields of oncology, cancer research and biomedical sciences; and foster application of basic and clinical science. Oncotarget is indexed and archived by PubMed/Medline, PubMed Central, Scopus, EMBASE, META (Chan Zuckerberg Initiative) (2018-2022), and Dimensions (Digital Science). To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: Facebook - https://www.facebook.com/Oncotarget/ X - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/@OncotargetJournal LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Spotify - https://open.spotify.com/show/0gRwT6BqYWJzxzmjPJwtVh MEDIA@IMPACTJOURNALS.COM

BUFFALO, NY- February 26, 2024 – A new #research paper was #published in Oncotarget's Volume 15 on February 8, 2024, entitled, “GZ17-6.02 interacts with bexarotene to kill mycosis fungoides cells.” In this new study, researchers Michael R. Booth, Laurence Booth, Jane L. Roberts, Cameron West, and Paul Dent from Virginia Commonwealth University and Genzada Pharmaceuticals investigated the therapeutic agent GZ17-6.02, composed of curcumin, harmine and isovanillin. “Combined with our curcumin findings, we believe that isovanillin can complex with curcumin and harmine to create an entity with unique biology when compared to the three individual agents.” GZ17-6.02 has undergone phase I evaluation in patients with solid tumors (NCT03775525) with an RP2D of 375 mg PO BID. The biology of GZ17-6.02 in malignant T cells and in particular those derived from mycosis fungoides (MF) patients, has not previously been studied. The researchers found that GZ17-6.02 alone and in combination with standard-of-care agents was effective in killing MF cells. “All three components are necessary for optimal killing of MF cells.” GZ17-6.02 activated ATM, the AMPK, NFκB and PERK and inactivated ERK1/2, AKT, ULK1, mTORC1, eIF2α, and reduced the expression of BCL-XL and MCL1. GZ17-6.02 increased ATG13 S318 phosphorylation and the expression of Beclin1, ATG5, BAK and BIM. GZ17-6.02 in a dose-dependent fashion enhanced autophagosome formation and autophagic flux, and tumor cell killing. Signaling by ATM and AMPK were both required for efficient killing but not for the dose-response effect whereas ER stress (eIF2α) and macroautophagy (Beclin1, ATG5) were required for both efficient killing and the dose-response. Knock down of the death receptor CD95 reduced killing by ~20% and interacted with autophagy inhibition to further reduce killing, collectively, by ~70%. Inhibition of autophagy and knock down of death-mediators downstream of the mitochondrion, AIF and caspase 3, almost abolished tumor cell killing. Hence in MF cells, the team wrote that GZ17-6.02 is a multi-factorial killer, utilizing ER stress, macroautophagy, death receptor signaling and directly causing mitochondrial dysfunction.” “We discovered that GZ17-6.02 containing harmine, isovanillin and curcumin caused more tumor cell killing than any of the agents individually or in pairs, and that it could interact in an additive fashion with standard of care MF drugs such as bexarotene and vorinostat to cause additional tumor cell death.” DOI - https://doi.org/10.18632/oncotarget.28557 Correspondence to - Paul Dent - paul.dent@vcuhealth.org Sign up for free Altmetric alerts about this article - https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.28557 Subscribe for free publication alerts from Oncotarget - https://www.oncotarget.com/subscribe/ Keywords - cancer, autophagy, ER stress, GZ17-6.02, bexarotene, vorinostat About Oncotarget Oncotarget (a primarily oncology-focused, peer-reviewed, open access journal) aims to maximize research impact through insightful peer-review; eliminate borders between specialties by linking different fields of oncology, cancer research and biomedical sciences; and foster application of basic and clinical science. To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: Facebook - https://www.facebook.com/Oncotarget/ X - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/@OncotargetJournal LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Spotify - https://open.spotify.com/show/0gRwT6BqYWJzxzmjPJwtVh Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

Ketamine is among the most unique pharmaceutical agents on the market. Join us on an exploration of the research on its neuropharmacology and use for depression, and discussion of risks and benefits. References: Article referenced in the beginning: Martinez-Marmol, et al. Hericerin derivatives activates a pan neurotrophic pathway in central hippocampal neurons converging to ERK1/2 signaling enhancing spacial memory. Journal of Neurochemistry. 2023(165);6: 791-808   Ballard E and Zarate C. The role of disassociation in ketamine's anti-depressant effects. Nature Communications. 2020: 6431 Bobo WV, et al. Ketamine for treatment resistant unipolar and bipolar major depression: Critical review and implications for clinical practice. Depression and Anxiety. 6 April 2016. Esketamine [package insert]. Janssen Neuroscience. July 2023. Fava M, et al. Double blind, placebo controlled dose ranging trial of intravenous ketamine as adjunctive therapy in treatment resistant depression (TRD). Molecular Psychiatry. 2020(25): 1592-1603 Gales A, Maxwell S. Ketamine: recent evidence and clinical uses. World Federation of Societies of Anesthesiologists. 12 June 2018. Johnston JN, et al. Ketamine in neuropsychiatric disorders: an update. Neuropsychopharmacology. 2023 Loo CK, et al. Placebo-controlled pilot trial testing dose titration and intravenous, intramuscular, and subcutaneous routes for ketamine in depression. Acta Psychiatria Scandinavica. 30 March 2016. Mandal S, et al. Efficacy of Ketamine therapy in the treatment of depression. Indian Journal of Psychiatry. 2019 Sept-Oct; 61(5). VA/DoD Clinical Practice Guideline. (2022). The Management of Major Depressive Disorder. Washington, DC: U.S. Government Printing Office

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.03.551808v1?rss=1 Authors: Ribeiro, F. C., Cozachenco, D., Argyrousi, E. K., Staniszewski, A., Wiebe, S., Calixtro, J. D., Soares-Neto, R., Al-Chami, A., El Sayegh, F., Bermudez, S., Arsenault, E., Cossenza, M., Lacaille, J.-C., Nader, K., Sun, H., De Felice, F. G., Lourenco, M. V., Arancio, O., Aguilar-Valles, A., Sonenberg, N., Ferreira, S. T. Abstract: Impaired synaptic plasticity and progressive memory deficits are major hallmarks of Alzheimer's disease (AD). Hippocampal mRNA translation, required for memory consolidation, is defective in AD. Here, we show that genetic reduction of the translational repressors, Fragile X messenger ribonucleoprotein (FMRP) or eukaryotic initiation factor 4E (eIF4E)-binding protein 2 (4E-BP2), ameliorated the inhibition of hippocampal protein synthesis and memory impairment induced by AD-linked amyloid-b; oligomers (AbOs) in mice. Furthermore, systemic treatment with (2R,6R)-hydroxynorketamine (HNK), an active metabolite of the antidepressant ketamine, prevented deficits in hippocampal mRNA translation, long-term potentiation (LTP) and memory induced by AbOs in mice. HNK activated hippocampal signaling by extracellular signal-regulated kinase 1/2 (ERK1/2), mechanistic target of rapamycin (mTOR), and p70S6 kinase 1 (S6K1)/ribosomal protein S6 (S6), which promote protein synthesis and synaptic plasticity. S6 phosphorylation instigated by HNK was mediated by mTOR in hippocampal slices, while rescue of hippocampal LTP and memory in HNK-treated AbO-infused mice depended on ERK1/2 and, partially, on mTORC1. Remarkably, treatment with HNK corrected LTP and memory deficits in aged APP/PS1 mice. RNAseq analysis showed that HNK reversed aberrant signaling pathways that are upregulated in APP/PS1 mice, including inflammatory and hormonal responses and programmed cell death. Taken together, our findings demonstrate that upregulation of mRNA translation corrects deficits in hippocampal synaptic plasticity and memory in AD models. The results raise the prospect that HNK could serve as a therapeutic to reverse memory decline in AD. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Det är dags för en forskningsspaning igen tillsammans med Helene Sandström. Helene är vetenskapsjournalist och researcher på tidningen Hälsa och Funktionsmedicin och på läkarklinikerna Funmed. Här är mer detaljer kring studierna vi pratar om i avsnittet:Svamp och ergotionein mot lindrig kognitiv nedsättning Feng L, Cheah IK, Ng MM m.fl. The Association between Mushroom Consumption and Mild Cognitive Impairment: A Community-Based Cross-Sectional Study in Singapore. J Alzheimers Dis. 2019;68(1):197-203. Svamp och ökad nervtillväxt i hjärnan på möss Martínez-Mármol R, Chai Y, Conroy JN, Khan Z, Hong SM, Kim SB, Gormal RS, Lee DH, Lee JK, Coulson EJ, Lee MK, Kim SY, Meunier FA. Hericerin derivatives activates a pan-neurotrophic pathway in central hippocampal neurons converging to ERK1/2 signaling enhancing spatial memory. J Neurochem. 2023 Jun;165(6):791-808.Svamp och cancer Dohlman AB, Klug J, Mesko M, Gao IH, Lipkin SM, Shen X, Iliev ID. A pan-cancer mycobiome analysis reveals fungal involvement in gastrointestinal and lung tumors. Cell. 2022 Sep 29;185(20):3807-3822.e12. Straussman R. Pan-cancer analyses reveal cancer-type-specific fungal ecologies and bacteriome interactions. Cell. 2022 Sep 29;185(20):3789-3806.e17. Weill Cornell Medicine. Fungal Association with Tumors May Predict Worse Outcomes. Pressmeddelande 2022-09-29. Mer läsning om svamp Dahlberg A, Froster A. Svamparnas förunderliga liv. Natur & Kultur 2021. Sheldrake M. Ett sammanvävt liv: hur svamparna förenar vår värld, förändrar våra sinnen och formar vår framtid. Volante 2021.Är du intresserad av forskning, hälsa och nya spännande rön, missa inte att prenumerera på tidningen Hälsa och Funktionsmedicin. Läs mer och prenumerera här: https://www.halsaochfunktionsmedicin.se Hosted on Acast. See acast.com/privacy for more information.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.05.05.539436v1?rss=1 Authors: Smythe, E., Alshahrani, F., Zhu, Z., Ferreira, F., Maib, H., Pruzina, S., Robinson, D., Murray, D. H., McComb, A. Abstract: Epidermal growth factor receptor (EGFR) signalling results in a variety of cell behaviours, including cell proliferation, migration and apoptosis, which depend on cell context. Here we have explored how the Rab5GEF, Rme-6, regulates EGFR signalling by modulating endocytic flux. We demonstrate that Rme-6, which acts early in the endocytic pathway, regulates EGFR trafficking through an endocytic compartment that is competent for ERK1/2 signalling. While overexpression of Rme-6 results in enhanced ERK1/2 nuclear localisation and c-Fos activation, loss of Rme-6 results in aberrant ERK1/2 signalling with increased cytoplasmic ERK1/2 phosphorylation (Thr202/Tyr204) but decreased ERK1/2 nuclear translocation and c-Fos activation, the latter leading to decreased cell proliferation. Phosphorylation of ERK1/2 by protein kinase 2 (CK2) is required for its nuclear translocation and our data support a model whereby Rme-6 provides a scaffold for a population of CK2 which is required for efficient nuclear translocation of ERK1/2. Rme-6 is itself a substrate for CK2 on Thr642 and Ser996 and phosphorylation on these sites can activate its Rab5GEF activity and endocytic trafficking of EGFR. Together our results indicate that that Rme-6 co-ordinates EGFR trafficking and signalling to regulate the assembly and disassembly of an ERK1/2 signalosome. 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/2023.02.19.529100v1?rss=1 Authors: Shakarchy, A., Zarfati, G., Hazak, A., Mealem, R., Huk, K., Avinoam, O., Zaritsky, A. Abstract: Cells dynamically change their internal organization via continuous cell state transitions to mediate a plethora of physiological processes. Understanding such continuous processes is severely limited due to a lack of tools to measure the holistic physiological state of single cells undergoing a transition. We combined live-cell imaging and machine learning to quantitatively monitor skeletal muscle precursor cell (myoblast) differentiation during multinucleated muscle fiber formation. Our machine learning model predicted the continuous differentiation state of single primary murine myoblasts over time and revealed that inhibiting ERK1/2 leads to a gradual transition from an undifferentiated to a terminally differentiated state 7.5-14.5 hours post inhibition. Myoblast fusion occurred ~3 hours after predicted terminal differentiation. Moreover, we showed that our model could predict that cells have reached terminal differentiation under conditions where fusion was stalled, demonstrating potential applications in screening. This method can be adapted to other biological processes to reveal connections between the dynamic single-cell state and virtually any other functional readout. 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/2023.01.20.524871v1?rss=1 Authors: Fojtik, P., Senfluk, M., Holomkova, K., Salykin, A., Gregorova, J., Smak, P., Pes, O., Raska, J., Stetkova, M., Skladal, P., Sedlackova, M., Hampl, A., Bohaciakova, D., Uldrijan, S., Rotrekl, V. Abstract: Precise control of pluripotency is a requirement for the safe and effective use of hPSCs in research and therapies. Here we report that pyruvate dehydrogenase upregulates histone H3 pan acetylation and levels of pluripotency marker NANOG in 5% O2. Pyruvate dehydrogenase (PDH) is an essential metabolic switch and a bottleneck for the glycolytic production of acetyl-CoA. Silencing of gene expression showed that PDH is regulated by the activity of its phosphatase PDP1. We show that PDP1 is sensitive to reactive oxygen species-mediated inactivation, leading to the downregulation of H3 pan acetylation and NANOG levels. Furthermore, we show that FGF2, a cytokine commonly used to maintain pluripotency activates pyruvate dehydrogenase through MEK1/2-ERK1/2 signaling pathway-mediated downregulation of ROS in 5% O2, thus promoting histone acetylation. Our results show the importance of pyruvate dehydrogenase in regulating energy metabolism and its connection to pluripotency. Furthermore, our data highlight the role of reactive oxygen species and redox homeostasis in pluripotency maintenance and differentiation. Highlights- PDP1-induced activation of PDH leads to increased histone H3 pan acetylation and NANOG levels in hPSCs - Reactive oxygen species (ROS) inactivate PDP1 and decrease histone H3 pan acetylation and NANOG levels in hPSCs - MEK1/2-ERK1/2 signaling-mediated downregulation of ROS in 5% O2 activates PDH in hPSCs Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/524871v1_ufig1.gif" ALT="Figure 1" greater than View larger version (39K): org.highwire.dtl.DTLVardef@1d4717forg.highwire.dtl.DTLVardef@678234org.highwire.dtl.DTLVardef@195576borg.highwire.dtl.DTLVardef@19e5684_HPS_FORMAT_FIGEXP M_FIG C_FIG 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/2023.01.12.523781v1?rss=1 Authors: Wang, L., Tang, Z., Deng, Y., Peng, Y., Xiao, Y., Xu, J., Ni, R., Qi, X. Abstract: Alzheimers disease is characterized by abnormal {beta}-amyloid (A{beta}) plaque accumulation, tau hyperphosphorylation, reactive oxidative stress, mitochondrial dysfunction and synaptic loss. Myricetin, a dietary flavonoid, has been shown to have neuroprotective effects in vitro and in vivo. Here, we aimed to elucidate the mechanism and pathways involved in myricetin protective effect on the toxicity induced by the A{beta}42 oligomer. Neuronal SH-SY5Y cells were pretreated with myricetin before incubation with A{beta}42 oligomer. The levels of pre- and post-synaptic proteins, mitochondrial division and fusion proteins, glycogen synthase kinase-3{beta} (GSK-{beta}) and extracellular regulated kinase (ERK) 1/2 were assessed by Western blotting. Flow cytometry assays for mitochondrial membrane potential (JC1) and reactive oxidative stress, as well immunofluorescence staining for lipid peroxidation (4-HNE) and DNA oxidation (8-OHdG), were performed. We found that myricetin prevented A{beta}42 oligomer-induced tau phosphorylation and the reduction in pre/postsynaptic proteins. In addition, myricetin reduced reactive oxygen species generation, lipid peroxidation, and DNA oxidation induced by the A{beta}42 oligomer. Moreover, myricetin prevented the A{beta}42 oligomer-induced reduction in mitochondrial fusion proteins (mitofusin-1, mitofusin-2), fission protein (dynamin-related protein 1) phosphorylation, and mitochondrial membrane potential via the associated GSK-3{beta} and ERK 1/2 signaling pathways. In conclusion, this study provides new insight into the neuroprotective mechanism of myricetin against A{beta}42 oligomer-induced toxicity. 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/2022.11.25.517570v1?rss=1 Authors: Ali, M. K., Liu, Y., Schimmel, K., Juul, N. H., Stockman, C. A., Wu, J. C., Spiekerkoetter, E. Abstract: Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disease that causes arteriovenous vascular malformations (AVMs) in different organs, including the lung. Three genes, ENG (endoglin), ACVRL1 (ALK1) and SMAD4, all members of the TGF-{beta}/BMPR2 signaling pathway, are responsible for over 85% of all HHT cases. However, how these loss-of-function gene mutations lead to AVMs formation and what common downstream signaling they target is unknown. Here, using a combination of siRNA-mediated gene silencing, whole transcriptomic RNA sequencing, bioinformatic analysis, transcriptomic-based drug discovery, endothelial cells functional assays and VEGF signaling analysis, and ex vivo precision cut lung slice (PCLS) cultures approach, we uncovered common downstream transcriptomic gene signatures of HHT-casing genes and identified promising drug for HHT. We found the commonly used BMPR2-signaling downstream target ID1 is not a common downstream target of all the three HHT genes knockdown in human pulmonary microvascular endothelial cells (PMVECs). We identified novel common downstream targets of all the three HHT-causing genes that were enriched for HHT-related biological process and signaling pathways. Among those downstream genes, LYVE1, GPNMB, and MC5R were strong downstream targets that could serve as a better common downstream target than ID1. Furthermore, using the common downstream upregulated genes (HHT disease signature) following HHT gene knockdown, we identified a small molecule drug, Brivanib, that reversed the HHT disease signature, and inhibited VEGF-induced ERK1/2 phosphorylation, proliferation, and angiogenesis in PMVECs and inhibited some of the upregulated HHT disease genes in PCLS. Our findings suggest that Brivanib could be an emerging new drug for HHT. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

El Dr. Jerónimo Rodríguez Cid, oncólogo médico adscrito al Instituto Nacional de Enfermedades Respiratorias en la Ciudad de México, México, junto con la Dra. Florencia Cuadros, oncóloga clínica del Hospital Eva Perón en Rosario, Argentina, nos comentan algunos de los estudios más destacados sobre el tratamiento del melanoma presentados durante el Congreso Anual de ESMO 2022 en París, Francia. Enfermedad temprana SWOG S1801: Estudio fase II, que aleatorizó 1:1 a 313 pacientes con melanomas cutáneos, acrales y mucosos en estadio IIIB-IV (confirmados histológicamente, medibles, clínicamente detectables y resecables) sin metástasis cerebrales, para recibir pembrolizumab como terapia adyuvante (cirugía seguida de 18 dosis del anti-PD-1) o neoadyuvante (3 dosis preoperatorias seguida de cirugía y luego 15 dosis del anti-PD-1). Se permitió la radioterapia después de la cirugía. El objetivo primario fue la supervivencia libre de eventos. IMMUNED: Resultados informados de supervivencia global (SG) del estudio fase II, multicéntrico, aleatorizado 1:1:1, doble ciego, que evalúa el tratamiento de nivolumab adyuvante solo o en combinación con ipilimumab vs. placebo en pacientes ≥18 años con melanoma primario desconocido o cutáneo en estadio IV sin evidencia de enfermedad (los pacientes fueron estratificados por sitio del estudio, sitio de metástasis y estado de PD-L1). El objetivo primario fue supervivencia libre de recaídas en la población con intensión de tratar, el tiempo hasta la progresión, la SG y, como objetivo secundario, la seguridad. Enfermedad avanzada SECOMBIT: Resultados actualizados de SG y una evaluación preliminar de biomarcadores del estudio secuencial, fase II, que aleatorizó a 251 pacientes con melanoma metastásico BRAF V600 no tratado, a tres brazos paralelos: encorafenib + binimetinib como terapia dirigida (brazo A), ipilimumab + nivolumab como inmunoterapia (brazo B) y la estrategia de "sándwich" con la terapia dirigida primero, cambiando a la inmunoterapia después de 8 semanas y volviendo a la terapia dirigida después de la progresión (brazo C). La SG fue el objetivo primario y los secundarios incluyeron la supervivencia libre de progresión total, la tasa de supervivencia a 4 años y una evaluación preliminar de biomarcadores. PIVOT IO 001: Datos de eficacia y seguridad del estudio fase III, aleatorizado 1:1 y abierto que evaluó bempegaldesleukina + nivolumab vs. nivolumab en 783 pacientes con melanoma no tratado previamente, no resecable o metastásico. Los objetivos primarios fueron la tasa de respuesta objetiva, la supervivencia libre de progresión (SLP), ambos por revisión central independiente ciega según RECIST v1.1, y la SG. Abstract LBA40: Estudio fase II, abierto, que aleatorizó a pacientes con melanoma irresecable/metastásico, con mutación BRAF V600 o NRAS, previamente tratado, a recibir múltiples combinaciones del fármaco naporafenib (LXH254). Se exploró la combinación con LTT462 (inhibidor de ERK1/2), con trametinib (inhibidor de MEK1/2) o con ribociclib (inhibidor de CDK4/6). El objetivo primario fue evaluar la eficacia de las combinaciones de naporafenib en función de las tasas de respuesta globales y los objetivos secundarios fueron la seguridad y la tolerabilidad. Abstract LBA3: Estudio fase III, aleatorizado 1:1, multicéntrico y abierto, que evaluó el tratamiento con infiltrado de linfocitos tumorales (TIL, por sus siglas en inglés) vs. ipilimumab, en 168 pacientes con melanoma irresecable en estadio IIIC-IV (86% eran refractarios al tratamiento anti-PD-1). El objetivo primario fue la SLP según RECIST 1.1 y los objetivos secundarios fueron la tasa de respuesta general y completa, la SG y la seguridad. Fecha de grabación: 22 de

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.09.18.508406v1?rss=1 Authors: Tang, Z., Guo, M., Peng, Y., Xiao, Y., Zhang, T., Ni, R., Qi, X. Abstract: Abnormal amyloid-{beta} (A{beta}) abnormal accumulation and oxidative stress play important roles in Alzheimer' disease (AD). Quercetin has been reported to possess antioxidant and anti-inflammatory properties, and thus of therapeutic interests for neurodegenerative disorders. In the present study, we aimed to characterize the mechanisms by which quercetin exerts neuroprotective effects in murine neuroblastoma N2a cells stably expressing human Swedishh mutant amyloid precursor protein (APP). Quercetin treatment exhibited low cytoxicity, attenuated APP expression and APP-induced oxidative neurotoxicity in N2a/APP cells. We found that quercetin effected via the down-regulation of phospho-extracellular signal regulated protein kinase (p-ERK1/2) pathway and up-regulation of phospho-protein kinase B (p-AKT) pathway in N2a/APP cells. In addition, quercetin ameliorated the elevated levels of reactive oxygen species using DCFH-DA flow-cytometry in N2a/APP cells, lipid peroxidation using (4-HNE), and DNA oxidation (8-OHdG assays). Quercetin ameliorated the loss of mitochondrial membrane potential using JC-1 fluorescence assay in N2a/APP cells in a dose-dependent mannor. In conclusion, we domenstrated the neuroprotective effects of quercetin against the APP expression induced oxidative neurotoxicity, impairment of mitochondrial function and oxidative stress through inactivation of the ERK1/2 signaling pathway and activation of AKT signaling pathways. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.09.12.507553v1?rss=1 Authors: Franco-Garcia, A., Guerrero-Bautista, R., Hidalgo, J., Milanes, M. V., Gomez-Murcia, V., Nunez, C. Abstract: Stress is an important trigger of relapses in cocaine use. These relapses engage the activity of memory-related nuclei, such as the basolateral amygdala (BLA) and the dentate gyrus (DG). Further, preclinical research signals D3 receptor (D3R) antagonists as promising therapeutic tools to attenuate cocaine reward and relapse. Therefore, we assessed the effect of SB-277011-A, a D3R antagonist, in the activity of Akt/mTOR and MEK/ERK1/2 pathways in these areas during the reinstatement of cocaine-induced conditioned place preference (CPP) evoked by psychological (restraint) and physiological (tail pinch) stress. Both stimuli reactivated the extinguished cocaine-CPP, but only restrained animals decreased their locomotor activity during reinstatement. Moreover, p-Akt, p-mTOR and p-ERK1/2 activity in the BLA and DG of restrained animals decreased during the reactivation of cocaine memories, contrasting to tail-pinched mice. While D3R blockade prevented stress-induced CPP reactivation and plasmatic corticosterone enhancement, SB-277011-A distinctly modulated Akt, mTOR and ERK1/2 activities in the BLA and DG based on the stressor and the dose of antagonist. Corticosterone may be partially responsible for these variations as we found high correlations among its levels and mTOR and/or Akt activity in the BLA and DG of restrained animals receiving SB-277011-A. Besides, locomotor activity of animals receiving 48 mg/kg of the antagonist highly correlated with p-mTOR/mTOR and p-ERK1/2 /ERK1/2 in the BLA during restraint- and tail pinch-induced relapse in cocaine-CPP, respectively. Hence, our study endorses D3R antagonists as therapeutic tools to prevent stress-induced relapses in drug use through a complex balance of Akt/mTOR and MEK/ERK1/2 pathways in memory-processing brain nuclei. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.09.05.506638v1?rss=1 Authors: Uzay, B., Hokelekli, F. O., Yilmaz, M., Esen, E. C., Basar, K., Bahadir-Varol, A., Ayhan, Y., Dalkara, T., Eren-Kocak, E. Abstract: Fibroblast growth factor-2 (FGF2) is involved in the regulation of affective behavior and shows antidepressant effects through Akt and ERK1/2 pathways. NUDT6 is a protein encoded from the antisense strand of the FGF2 gene and its role in the regulation of affective behavior is unclear. Here, we show that increasing NUDT6 expression in the hippocampus results in depression-like behavior in rats without changing FGF2 levels or activating its downstream effectors, Akt and ERK1/2. Instead, NUDT6 acts by inducing inflammatory signaling, specifically by increasing S100A9 levels, activating NF-kB, and rising microglia number along with a reduction in neurogenesis. Conversely, inhibition of hippocampal NUDT6 expression by shRNA results in antidepressant effects and increases neurogenesis without altering FGF2 levels. Together these findings suggest that NUDT6 may play a role in major depression by inducing a proinflammatory state and serve as a novel therapeutic target for antidepressant development. The opposite effects of NUDT6 and FGF2 on depression-like behavior may serve as a mechanism to fine-tune affective behavior. Our findings open up new venues for studying the differential regulation and functional interactions of sense and antisense proteins in neural function and behavior as well as in neuropsychiatric disorders. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.08.26.505502v1?rss=1 Authors: Wei, X., Cheng, Z., Chen, W., Guo, H., Liu, Z., Hu, T., Zhang, Y., Cai, Q., Ge, F., Fan, Y., Chang, J., Guan, X. Abstract: Cocaine abuse during adolescence increases the risk for developing drug addiction in later life, but the underlying molecular mechanism remains unclear. Here, adolescent cocaine-exposed (ACE) male mice models were established by giving once-daily intraperitoneal injections of 15 mg/kg cocaine to mice during adolescence (P28-P42). We found that ACE mice exhibited a higher sensitivity to subthreshold dose of cocaine (1 mg/kg) in adulthood, accompanied with triggered activities and dendritic spine density of pyramidal neuron, increased Dusp1 gene, as well as reduced protein levels and activity of dual specificity phosphatase 1 (DUSP1) in mPFC. Specific overexpression of DUSP1 on mPFC glutamatergic neurons efficiently blocked cocaine-preferred behaviors and reduced mPFC activity, while knockdown of DUSP1 maintained cocaine-preferred behaviors and increased mPFC activity in ACE mice. MAPK-related signals, especially ERK1/2, might underlie the mediating effects of DUSP1. Collectively, these findings suggested that targeting mPFC DUSP1 may represent a promising therapeutic strategy for the treatment and attenuation of addiction susceptibility, particularly in addicts with a history of adolescent drugs exposure. Copy rights belong to original authors. Visit the link for more info Podcast created by PaperPlayer

Videos :  2. Fake Cases: The Fraudulent PCR Test Is at the Heart of This Entire Plandemic – Dr. Reiner Fuellmich With Judy Mikovits & More   3.  Over 17,000 Physicians and Scientists Agree: “There Is No Medical Emergency” – Dr. Robert Malone   4. Honest Government Ad | Julian Assange   Cranberry juice may slash cardiometabolic risk factors: RCT study USDA Agriculture Research Center, April 30, 2022 Daily consumption of a low-calorie cranberry juice may improve certain risk factors of heart disease, including blood pressure and triglycerides, says a new study from the Agricultural Research Service at the USDA. Eight weeks of supplementing the diet with a cranberry juice containing 173 mg of phenolic compounds per serving was associated with significant reductions in C-reactive protein (CRP), diastolic blood pressure, and blood sugar levels, according to findings published in the Journal of Nutrition . While the majority of the science supporting the health benefits of cranberries is for urinary tract health, a growing body of data supports the cardiovascular potential of the berries. For example, a study by scientists at the Mayo Clinic and College of Medicine found that two glasses of cranberry juice a day may protect against the development of hardening of the arteries. Writing in the European Journal of Nutrition (Vol. 52, pp 289-296), the Mayo Clinic researchers reported that no effect was observed on the function of the cells lining the arteries (endothelial cells), but cranberry juice may reduce the number of endothelial cells that produce a compound called osteocalcin, which has been linked to hardening of the arteries.     Vitamin D toxicity rare in people who take supplements, researchers report Mayo Clinic, April 30, 2022   Over the last decade, numerous studies have shown that many Americans have low vitamin D levels and as a result, vitamin D supplement use has climbed in recent years. In light of the increased use of vitamin D supplements, Mayo Clinic researchers set out to learn more about the health of those with high vitamin D levels. They found that toxic levels are actually rare. A vitamin D level greater than 50 nanograms per milliliter is considered high. Vitamin D levels are determined by a blood test called a serum 25-hydroxyvitamin D blood test. A normal level is 20-50 ng/mL, and deficiency is considered anything less than 20 ng/mL, according the Institute of Medicine (IOM). The researchers analyzed data collected over 10 years from patients in the Rochester Epidemiology Project, a National Institutes of Health-funded medical records pool , one of the few places worldwide where scientists can study virtually an entire geographic population to identify health trends. Of 20,308 measurements, 8 percent of the people who had their vitamin D measured had levels greater than 50 ng/mL, and less than 1 percent had levels over 100 ng/mL. "We found that even in those with high levels of vitamin D over 50 ng/mL, there was not an increased risk of hypercalcemia, or elevated serum calcium, with increasing levels of vitamin D," says study co-author Thomas D. Thacher, M.D., a family medicine expert at Mayo Clinic. Only one case over the 10-year study period was identified as true acute vitamin D toxicity; the person's vitamin D level was 364 ng/mL. The individual had been taking 50,000 international units (IU) of vitamin D supplements every day for more than three months, as well as calcium supplements. The IOM-recommended upper limit of vitamin D supplementation for people with low or deficient levels is 4,000 IU a day.     Reducing sedentary time mitigates the risk of type 2 diabetes and cardiovascular diseases University of Turku (Finland), May 2, 2022 A new study suggests that reducing daily sedentary time can have a positive effect on the risk factors of lifestyle diseases already in three months. Spending just one hour less sitting daily and increasing light physical activity can help in the prevention of these diseases. In an intervention study of the Turku PET Centre and the UKK Institute in Finland, the researchers investigated whether health benefits can be achieved by reducing daily sedentary time during a three-month intervention period. The research participants were sedentary and physically inactive working-age adults with an increased risk of type 2 diabetes and cardiovascular diseases. The intervention group managed to reduce sedentary time by 50 minutes per day on average, mainly by increasing the amount of light- and moderate-intensity physical activity. In the three-month period, the researchers observed benefits in health outcomes related to blood sugar regulation, insulin sensitivity and liver health in the intervention group.     Study Finds Cannabis May Be A “Miracle” Treatment For Autistic Kids Shaare Zedek Medical Center (Israel), April 26, 2022 Autism could now be added to the lengthy and perpetually-expanding list of afflictions and symptoms treatable with the one product of nature shamefully prohibited by the federal government — the “miracle” palliative, cannabis. In a recent article titled, “Marijuana may be a miracle treatment for children with autism,” Israeli researchers began a new study comprised of 120 children ranging in age from five to 29 years, who have been diagnosed with mild to severe autism. Study participants are given one of two cannabis oil treatments or a placebo, drops of which can be mixed into a meal — none contain high levels of THC, the ingredient which gives users a ‘high.' Myriad scientific studies and innumerable anecdotal cases have proven cannabis to treat everything from PTSD to ADHD, various cancers to the painful pressure of glaucoma — but the plant's miraculous quality has been most apparent in treating severe seizures of childhood epilepsy. Now, it appears, cannabis — specifically, the non-psychoactive compound, cannabidiol or CBD — may offer improved quality of life for children with autism, and the families providing their care. In an observational study, the doctor found 70 patients with autism experienced positive results from cannabis — so the clinical trial was launched for in-depth study.     Resveratrol and pinostilbene provide neuroprotectoin against age-related deficits. Duquesne University, April 27, 2022 According to news, research stated, "Age-related declines in motor function may be due, in part, to an increase in oxidative stress in the aging brain leading to dopamine (DA) neuronal cell death. In this study, we examined the neuroprotective effects of natural antioxidants resveratrol and pinostilbene against age-related DAergic cell death and motor dysfunction using SH-SY5Y neuroblastoma cells and young, middle-aged, and old male C57BL/6 mice." The news reporters obtained a quote from the research from Duquesne University, "Resveratrol and pinostilbene protected SH-SY5Y cells from a DA-induced decrease in cell viability. Dietary supplementation with resveratrol and pinostilbene inhibited the decline of motor function observed with age. While DA and its metabolites (DOPAC and HVA), dopamine transporter, and tyrosine hydroxylase levels remain unchanged during aging or treatment, resveratrol and pinostilbene increased ERK1/2 activation in vitro and in vivo in an age-dependent manner. Inhibition of ERK1/2 in SH-SY5Y cells decreased the protective effects of both compounds." "These data suggest that resveratrol and pinostilbene alleviate age-related motor decline via the promotion of DA neuronal survival and activation of the ERK1/2 pathways." Study sheds light on the benefits of exercise in fatty liver disease University of Eastern Finland, May 3, 2022 Exercise supports the treatment of non-alcoholic fatty liver (NAFLD) disease by impacting on several metabolic pathways in the body, a new study from the University of Eastern Finland shows Regular high-intensity interval training (HIIT) exercise over a period of 12 weeks significantly decreased the study participants' fasting glucose and waist circumference, and improved their maximum oxygen consumption rate and maximum achieved workload. These positive effects were associated with alterations in the abundance of a number of metabolites. In particular, exercise altered amino acid metabolism in adipose tissue. The study was published in Scientific Reports. Exercise had a beneficial effect on fasting glucose concentrations, waist circumference, maximum oxygen consumption rate, and maximum achieved workload. These factors were also associated with many of the observed alterations in the abundance of various metabolites in the exercise intervention group. The most significant alterations were observed in amino acids and their derivatives, lipids, and bile acids. In particular, exercise increased the levels of amino acids, which are the building blocks of proteins, in adipose tissue. According to the researchers, their higher accumulations in adipose tissue may be associated with improved lipid and glucose metabolism, as well as with reduced insulin resistance. The levels of various gut microbial metabolites were altered as a result of exercise, which is suggestive of changes in the composition of gut microbes, or in their function. Among these metabolites, increased amount of indolelactic acid, for example, can strengthen the intestinal mucosa, immune defense, and glucose balance.    

This week's show features a panel discussion between authors Adrian Wells and Hyeon Chang Kim as they discuss their articles "Improving the Effectiveness of Psychological Interventions for Depression and Anxiety in Cardiac Rehabilitation PATHWAY—A Single-Blind, Parallel, Randomized, Controlled Trial of Group Metacognitive Therapy" and "Associations of Ideal Cardiovascular Health and Its Change During Young Adulthood With Premature Cardiovascular Events: A Nationwide Cohort Study." Dr. Carolyn Lam: Welcome to Circulation on the Run, your weekly podcast summary and backstage pass to the journal and its editors. We're your co-hosts. I'm Dr. Carolyn Lam, Associate Editor from the National Heart Center and Duke National University of Singapore. Dr. Greg Hundley: And I'm Dr. Greg Hundley, also your co-host. And Associate Editor, Director of the Pauley Heart Center, VCU Health in Richmond, Virginia. Dr. Carolyn Lam: Greg, we're starting off the month with double features, and these are just so interesting. The first paper talks about psychological interventions for depression and anxiety in cardiac rehabilitation. And the next talks about ideal cardiovascular health and its change during young adulthood and how that relates to premature cardiovascular events. Cool, huh? Dr. Greg Hundley: Absolutely. Well, Carolyn. How about we grab a cup of coffee and start discussing some of the other articles in the issue? And I could go first. Carolyn, the first article that I've got is from Mrs. Elizabeth Jordan from Ohio State University Wexner Medical Center. And it really pertains to cardiomyopathies. And remember, Carolyn, classically, we categorize hypertrophic, dilated, and arrhythmogenic right ventricular cardiomyopathy. And each has a signature genetic theme. Hypertrophic cardiomyopathy and ARVC are largely understood as genetic diseases of sarcomere or desmosome proteins. But in contrast, there are over 250 genes spanning more than 10 gene ontologies that have been implicated in dilated cardiomyopathy. And therefore, it really represents a very complex and diverse genetic architecture. So to clarify this, a systematic curation of evidence to establish the relationship of genes with dilated cardiomyopathy was conducted by an international panel with clinical and scientific expertise in dilated cardiomyopathy genetics. And they evaluated evidence supporting monogenic relationships of genes with idiopathic dilated cardiomyopathy. Dr. Carolyn Lam: Oh, wow. That sounds like a lot of work. And what did they find, Greg? Dr. Greg Hundley: Right, Carolyn. So in the curation of 51 genes, 19 had high evidence. 12 are definitive strong, and seven moderate. And notably, these 19 genes only explain the minority of cases, leaving the remainder of dilated cardiomyopathy genetic architecture really incompletely addressed. And clinical genetic testing panels include most high evidence genes. However also, the panel noted that genes lacking robust evidence are very commonly observed clinically. Dr. Greg Hundley: So Carolyn, the take home message from this international panel is that while dilated cardiomyopathy genetic testing panels include an average of about 60 genes, when curating published evidence for dilated cardiomyopathy, only 19 have really emerged as high levels of evidence. And then in this study, 51 genes were evaluated. And the 19 genes appraised as high evidence were recommended to be routinely used in the genetic evaluation of dilated cardiomyopathy. And one more point. Rare variants from genes without moderate, strong, or definitive evidence should not be used in clinical practice to predict dilated cardiomyopathy risk most importantly when also you're screening at risk family members. Dr. Carolyn Lam: Wow. Very nice. Stunning numbers. Well, my paper is identifying a novel therapeutic target in pulmonary arterial hypertension. Do you want to know what that is? Dr. Greg Hundley: Ah, yes, Carolyn. Very interesting. So what is it? Dr. Carolyn Lam: It's switch-independent 3A. Which is an epigenetic modifier, which is drastically down-regulated in pulmonary arterial hypertension patients and rodent models of pulmonary arterial hypertension. And strongly associated with decreased bone morphogenic protein receptor type two, or BMPR2 expression. So this switch-independent 3A overexpression up-regulated BMPR2 expression by modulating critical epigenetic pathways and decreasing a specific transcription factor binding to the BMPR2 promoter in pulmonary vascular smooth muscle cells. Furthermore, aerosolized lung-targeted gene transfer of adeno-associated virus zero type one and containing switch-independent 3A reversed and prevented pulmonary arterial hypertension phenotype in preclinical animal models. So this beautiful study, from Dr. Hadri from Icahn School of Medicine at Mount Sinai in New York and colleagues, really suggests that switch-independent 3A can be a clinically relevant molecule for the treatment of pulmonary arterial hypertension. Dr. Greg Hundley: Wow, Carolyn. Really nice. Very intricate science for the study of pulmonary hypertension. Well, my next paper actually comes to us from Dr. Joe Hill and colleagues at UT Southwestern Medical Center. And Carolyn, as we know, cardiac hypertrophy is an independent risk factor for heart failure. Of course, the leading cause of morbidity and mortality globally. And the calcineurin NFAT, or nuclear factor of activated T-cells pathway, and the MAP kinase ERK, or extra cellular signal regulated kinase pathway, contributes to the pathogenesis of cardiac hypertrophy as an interdependent network of signaling cascades. However, Carolyn, how these pathways interact really remains unclear. And so Dr. Hill and colleagues engineered a cardiomyocyte-specific ETS2, a member of the E26 transformation specific sequence or ETS domain family knockout mouse, and investigated the role of ETS2 in cardiac hypertrophy. Primary cardiomyocytes were also used to evaluate ETS2 function in cell growth. Dr. Carolyn Lam: Wow. Okay. So what were the results, Greg? Dr. Greg Hundley: Right, Carolyn. Three main findings. First, ETS2 is activated by ERK1/2, or extracellular signal-regulated kinase 1/2, in both hypertrophied murine hearts and in human dilated cardiomyopathy. Second, ETS2 is required for both pressure overload, and calcineurin induced cardiac hypertrophy responses involving signaling cascades distinct from, but interdependent with ERK1/2 signaling. And third, this group discovered that ETS2 synergizes with NFAT to transactivate RCAN1-4, an established downstream target of NFAT, or nuclear factor of activated T-cells. And they identified an MIR-223 as a novel transcriptional target of NFAT ETS2 in cardiomyocytes. Dr. Carolyn Lam: Wow. Wow. That sounds like a lot of detailed work. Could you tell us what the clinical implications are, Greg? Dr. Greg Hundley: You bet, Carolyn. So in aggregate, these findings unveil a previously unrecognized molecular interaction between two conical hypertrophic signaling pathways, MAP kinase-driven hypertrophy, and calcineurin driven hypertrophy. And therefore, as pathological cardiac hypertrophy is an established risk factor for heart failure development, this unveiling of novel signaling mechanisms really is of potential clinical relevance. Dr. Carolyn Lam: Thanks, Greg. Well, let's round up with what else there is in this week's issue. There's a Frontiers paper by Dr. Chris Granger. And it's a big call to action to the cardiology community, to incorporate SGLT2 inhibitors and GLP-1 receptor agonists for cardiovascular and kidney disease risk reduction. There's a Joint Opinion piece from the American Heart Association, World Heart Federation, American College of Cardiology, and European Society of Cardiology on, “The Tobacco Endgame: Eradicating a Worsening Epidemic,” by Dr. Elkind. Dr. Greg Hundley: Oh great, Carolyn. Well, I've got an On My Mind piece from Professor Bhatt. And it's entitled, “Does SGLT1 inhibition Add Benefit to SGLT2 Inhibition in Type 2 Diabetes Mellitus?” And next, Dr. Viskin has an ECG Challenge entitled, “Long QT Syndrome and Torsade de Pointes Ultimately Treated With Quinidine, The Concept of Pseudo Torsade de Pointes.” And then finally, there's a Letter to the Editor by Dr. Lu regarding the article, “Association of Body Mass Index and Age with Morbidity and Mortality in Patients Hospitalized with COVID-19, Results from the American Heart Association COVID-19 Cardiovascular Disease Registry.” Well, Carolyn, I can't wait to get on to this double feature. Dr. Carolyn Lam: Me too. Let's go. Dr. Greg Hundley: Welcome, listeners, to our feature discussion today. And again, we're going to create today a forum, because we have two very interesting papers to present during this timeframe. Our first is going to come to us from Dr. Adrian Wells from University of Manchester. And our second paper will come to us from Dr. Hyeon Chang Kim from Yonsei University. I want to welcome you both, gentlemen. And Adrian, I would like to start with you. Tell us a little bit about the background related to your study. And then what was the hypothesis that you wanted to address? Dr. Adrian Wells: Okay, well thank you for inviting me to take part in this podcast. Following cardiac events, around one in three individuals will develop significant anxiety and depression symptoms. And we know that anxiety and depression can have an impact on prognosis, quality of life, future outcomes. Psychological treatment isn't routinely offered in cardiac rehabilitation for anxiety and depression, despite the fact that we identified that many of our patients felt that they would benefit from a psychological intervention to address these issues. And they felt that their needs were not really being met. So our primary question was, can we improve psychological outcomes in patients with cardiovascular disease? Dr. Greg Hundley: Very nice. And Adrian, what was your study population? And also, what was your study design? Dr. Adrian Wells: So we selected patients who entered cardiac rehabilitation in the UK. So these are patients with acute coronary syndrome, revascularization, stable heart failure, heart transplantation, and so on. And so, a wide group of individuals. We recruited 332 patients, all of whom had had anxiety and depression scores of eight or more. So these were people showing mild to severe levels of psychological distress. We conducted a two arm single blind randomized controlled trial, with 332 patients who were randomly allocated to one of these two conditions. And we assessed anxiety and depression symptoms before treatment at four months and at 12 months. Dr. Greg Hundley: Describe a little bit some of the specifics of your intervention. And then what did you find? Dr. Adrian Wells: We use relatively recent new treatment called metacognitive therapy. And this was delivered in a group format over six sessions. And we trained cardiac rehabilitation staff, nurse consultants, physiotherapists, in the delivery of this intervention. Metacognitive therapy works on helping patients discover unhelpful patterns of thinking, such as worrying and ruminating ,and excessive threat monitoring. And to reduce those patterns of thinking that contribute to anxiety, depression, and poor adaptation following stressful life experiences. Dr. Greg Hundley: And what did you find? Dr. Adrian Wells: Well, what we found was that the addition of metacognitive therapy to treatment to usual cardiac rehabilitation, significantly improved outcomes at four months and 12 months. What was striking about this was that our effect sizes were modest and moderate to large. They seem to be larger than those obtained in other studies or psychological treatments. And of note, the treatment seemed to impact well on both anxiety and depression symptoms. Whereas other types of intervention evaluated in the past have tended to treat the depression, but not so much the anxiety. Dr. Greg Hundley: Very good. So it sounds like a group-based intervention. And I'm assuming maybe participants interacted not only with your staff, but with one another. How would you put your results really in the context with other research that's going on in this space? Dr. Adrian Wells: Well, there have been a number of studies in the past that have looked at individual and group-based treatments, and patient preference for different types of intervention. I think this is the first study to use a clear manualized intervention that's based on the psychological theory of mechanisms that contribute to the maintenance of psychological problems. Obviously, this tended to use more prescriptive interventions like anxiety management, stress management, taking techniques from a range of different sources. So I think there's a difference of conceptual basis to this kind of intervention. And it's something that is highly manualized and structured, and in fact can be delivered by a range of different healthcare professionals. Dr. Greg Hundley: Very nice. And also during cardiovascular rehab. Correct? Dr. Adrian Wells: Absolutely, yeah. During cardiac rehab. One interesting finding... And we were a little concerned that this might adversely affect attendance at cardiac rehab. But we found that the treatment was well tolerated, and it didn't have any negative impact on attendance at these other sessions. Dr. Greg Hundley: Excellent. Well, congratulations on this new finding. Well, listeners, we're next going to turn to Dr. Hyeon Chang Kim from Yonsei University in Korea. And Yong-Chan, could you describe for us also the background related to your study, and the hypothesis that your research wanted to test? Dr. Hyeon Chang Kim: Thank you for inviting me to this wonderful discussion. South Korea is among the countries with the lowest cardiovascular mortality in the world. And the rate is even decreasing. However, cardiovascular risk factor is worsening. Especially in younger generation in Korea. So these young people may not have a very high cardiovascular risk, but I wanted to know the potential impact of worsening cardiovascular risk profile in this younger Korean generation. And furthermore, I wanted to know how much we can lead youth cardiovascular risk by improving their cardiovascular health profile. Dr. Greg Hundley: Very nice. And so tell us about your study design and what was the study population, related to your study? Dr. Hyeon Chang Kim: My study is basically based on the national health checkup program and national health insurance claim database. In Korea, adults over the age of 20 and employed workers of all ages are required to take general health checkup every two years. The participation rate is between 70 and 80%. So we identified three and a half million adults, age 20 to 39 years, who complete the health checkup. And cardiovascular health scores was calculated as the number of ideal cardiovascular health component, which include non-smoking, moderate physical activity three times a week, body mass index below 2030, normal blood pressure, normal cholesterol and normal fasting glucose. So the score can range from zero to six. And higher score meaning better cardiovascular health. Our outcomes were myocardial infarction, stroke, heart failure, and cardiovascular deaths in about 16 years. In addition, we also evaluate the risk of cardiovascular disease. According to two year change in how the vascular health score using repeated health checkup data. Dr. Greg Hundley: Very nice. So evaluating a set of behavioral patterns and risk factors in younger individuals, and then predicting what their longer term adverse cardiovascular outcomes would be. So what did you find? Dr. Hyeon Chang Kim: So even in this relatively low risk population, better cardiovascular health score was associated with significantly lower cardiovascular risk. About 20% reduction per one point higher score. And more importantly, people with improving cardiovascular score over two years showed leading toward cardiovascular risk. Even if their baseline cardiovascular health score was very low. Dr. Greg Hundley: Really unique findings. Tell us about the impact of your results relative to other studies published in this space. And was this also.... This was unique, because it's an Asian population, Dr. Hyeon Chang Kim: Asian population. And we are among the very low risk population. And even in this low risk population, cardiovascular health score was... Fear can be a good predictor of cardiovascular risk. And compared to many Western countries, we have very low cardiovascular risk. And our population was younger than most other studies. So we can provide some evidence that even in the higher risk population, they can do much better, based on our study. Another important thing, we can check the impact of a changing cardiovascular score, even in the younger generation. Dr. Greg Hundley: Very good. And just as a frame of reference for our listeners. Give us some characteristics, if you wouldn't mind, on what really constitutes practically a low risk score, versus what would constitute a high risk score Dr. Hyeon Chang Kim: In this younger Korean population, their cigarette smoking, and their obesity, and physical inactivity are the most common causes of worsening cardiovascular profile. And the behavioral risk factor also can attack the blood glucose and cholesterol blood pressure. So in this younger generation, they're keeping the good behavior. Past behavior is very important and it's beneficial in the very long-term. Dr. Greg Hundley: Very nice, well listeners. We're going to turn to our experts here. Two very interesting studies. And ask them both, what do they think is the next study that needs to be performed in their respective areas of research? So Yong-Chan, we'll start with you. Since we just discussed your paper. What do you think is the next study to be performed really in this sphere of research. Dr. Hyeon Chang Kim: Korea is a relatively low cardiovascular risk, has a very small size, and no racial diversity. But even in this country, disparity and inequality in cardiovascular health is becoming an important issue. So I want to identify subcultural relatively poor cardiovascular health among younger population. And also I want to find ways to improve their cardiovascular score. The conventional approaches, such as education and mass campaign, are less effective oppose this younger adults have a poor socioeconomic status. So, we may need to develop newer target-specific strategies to improve their cardiovascular health. Dr. Greg Hundley: Good. And Dr. Wells, our agent will turn next to you. What do you see is the next area of investigation or research study that needs to be performed in your sphere of interests? Dr. Adrian Wells: Well, I think the next step is to look at rollout of this intervention. Is that feasible, and how acceptable is this to cardiac services? In fact, the National Institute of Health Research have just awarded us some funding to examine feasibility and barriers to implementation in the healthcare system. In addition to that, we're beginning to examine the effects of metacognitive therapy with other health conditions, such as cancer in children and adolescents. Dr. Greg Hundley: Nice. Well listeners, we have had just a wonderful discussion today from both Dr. Adrian Wells from University of Manchester. Who brought to us combining a group-mediated, psychological stress-reducing, anxiety-reducing, intervention to the cardiac rehab sphere. And how impactful that was in reducing both anxiety, and overall depressive symptoms. And then also exciting research from Dr. Hyeon Chang Kim from South Korea. Identifying for us that in Asian population, as well as what we know in other races, those individuals in their twenties to thirties with favorable lifestyle habits, have reduced cardiovascular risk much later in life. Dr. Greg Hundley: Well, on behalf of both Carolyn and myself, we want to wish you a great week. And we'll catch you next week on the run. Dr. Greg Hundley: This program is copyright of the American Heart Association, 2021. The opinions expressed by speakers in this podcast are their own, and not necessarily those of the editors, or of the American Heart Association. For more, visit ahajournals.org.  

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.09.289306v1?rss=1 Authors: Noor, S. I., Hoffmann, M., Rinis, N., Bartels, M. F., Winterhalter, P., Hoelscher, C., Henning, R., Himmelreich, N., Thiel, C., Ruppert, T., Rapp, E., Strahl, S. Abstract: Defects in protein O-mannosylation lead to severe congenital muscular dystrophies known as -dystroglycanopathy. A hallmark of these diseases is the loss of the O-mannose-bound matriglycan on -dystroglycan, which leads to a reduction in cell adhesion to the extracellular matrix. Mutations in protein O-mannose {beta}1,2-N-acetylglucosaminyltransferase 1 (POMGNT1), which is crucial for the elongation of O-mannosyl glycans, are mainly associated with muscle-eye-brain (MEB) disease. In addition to defects in cell-extracellular matrix adhesion, aberrant cell-cell adhesion has occasionally been observed in response to defects in POMGNT1. However, direct molecular mechanisms are largely unknown. We used POMGNT1 knock-out HEK293T cells and fibroblasts from a MEB patient to gain a deeper insight into the molecular changes in POMGNT1 deficiency. A combination of biochemical and molecular biological techniques with proteomics, glycoproteomics and glycomics revealed that a lack of POMGNT1 activity strengthens cell-cell adhesion. We demonstrate that the altered intrinsic adhesion properties are due to an increased abundance of N-cadherin (N-Cdh). In addition, site-specific changes in the N-glycan structures in the extracellular domain of N-Cdh were detected, which positively impact on homotypic interactions. We found that in POMGNT1 deficient cells ERK1/2 and p38 signaling pathways are activated and transcriptional changes that are comparable to the epithelial-mesenchymal transition (EMT) are triggered, defining a possible molecular mechanism underlying the observed phenotype. Our study indicates that changes in cadherin-mediated cell-cell adhesion and other EMT-related processes may contribute to the complex clinical symptoms of MEB or -dystroglycanopathy in general, and suggests a previously underestimated impact of changes in O-mannosylation on N-glycosylation. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.31.276253v1?rss=1 Authors: Blackwood, C. A., McCoy, M. T., Ladenheim, B., Cadet, J. L. Abstract: To identify signaling pathways activated by oxycodone self-administration (SA), Sprague-Dawley rats self-administered oxycodone for 20 days using short-access (ShA, 3 h) and long-access (LgA, 9 h) paradigms. Animals were euthanized two hours after SA cessation and dorsal striata were used in post-mortem molecular analyses. LgA rats escalated their oxycodone intake and separated into lower (LgA-L) or higher (LgA-H) oxycodone takers. LgA-H rats showed increased striatal protein phosphorylation of ERK1/2 and MSK1/2. Histone H3, phosphorylated at serine 10 and acetylated at lysine 14 (H3S10pK14Ac), a MSK1/2 target, showed increased abundance only in LgA-H rats. RT-qPCR analyses revealed increased AMPA receptor subunits, GluA2 and GluA3 mRNAs in the LgA-H rats. GluA3, but not GluA2, expression correlated positively with changes in pMSK1/2 and H3S10pK14Ac. Our findings indicate that escalated oxycodone SA results in MSK1/2-dependent histone phosphorylation, which promoted increases in striatal gene expression. Our observations offer novel avenues for pharmacological interventions against oxycodone addiction. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.22.262923v1?rss=1 Authors: Bronfman, F. C., Moya-Alvarado, G. Abstract: Brain-Derived Neurotrophic Factor (BDNF) is broadly expressed in many circuits of the central nervous system (CNS). It binds TrkB and p75 to trigger different signaling pathways, including ERK1/2 and PI3K-mTOR, to induce dendritic growth and synaptic plasticity. When binding to BDNF, TrkB and p75 are endocytosed to signaling endosomes to continue signaling inside the cell. Whether BDNF/TrkB-p75 signaling endosomes in axons are regulating long-distance signaling in cell bodies to modify neuronal morphology is unknown. Here, we studied the functional role of BDNF signaling endosomes in long-distance regulation of dendritic growth using compartmentalized cultures of rat and mouse cortical neurons derived from p75exonIII knock-out or TrkBF616A knock-in mice. By applying BDNF to distal axons, we showed the capacity of axonal BDNF to increase dendritic arborization in cell bodies. This process depended on TrkB activity, but not p75 expression. In axons, BDNF/TrkB co-localized with Rab5 endosomes and increased active Rab5. Also, dynein was required for BDNF long-distance signaling, consistent with sorting and transport of signaling endosomes. Using neurons derived from TrkBF616A knock-in mice and the 1NM-PP1 inhibitor, we were able to demonstrate that TrkB receptors activated in the axons by BDNF, were required in the neuronal cell body to increase TrkB activity and phosphorylation of CREB. Also, we were able to visualize endosomes containing activated TrkB. PI3K activity was not required in the axons for dynein dependent BDNF responses. However, dendritic arborization induced by axonal BDNF signaling required both nuclear CREB and PI3K activation in cell bodies. Consistently, axonal BDNF increased protein translation in cell bodies and CREB and PI3K and mTOR activity were required for this process. Altogether, these results show that BDNF/TrkB signaling endosomes generated in axons allows long-distance control of dendritic growth coordinating both transcription and protein translation. Our results suggest a role of BDNF-TrkB signaling endosomes wiring circuits in the CNS. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.19.255844v1?rss=1 Authors: Winter, J., Meyer, M., Berger, I., Peters, S., Royer, M., Bianchi, M., Stang, S., Langgartner, D., Reber, S. O., Kuffner, K., Schmidtner, A. K., Hartmann, F., Bludau, A., Bosch, O. J., Slattery, D. A., van den Burg, E. H., Neumann, I. D., Jurek, B. Abstract: Recently, oxytocin (OXT) has generated considerable interest as potential treatment for psychiatric disorders, including general anxiety disorder or autism spectrum disorder. Therefore, knowledge on the involved molecular processes downstream of OXT receptor (OXTR) activation is indispensable. We reveal that alternative splicing of corticotropin releasing factor receptor 2a (CRFR2a) parallels increased anxiety-like behavior following chronic OXT treatment, contrasting the well-known anxiolysis of acute OXT. In detail, chronic OXT shifts the splicing ratio between membrane-bound (mCRFR2a) and soluble CRFR2a (sCRFR2a) in favor of the latter via ERK1/2-MEF2A signaling. Targeted manipulations of Crfr2a splicing mimic the effect of chronic OXT, confirming its role in the regulation of anxiety-like behavior. Furthermore, chronic OXT triggers cytoplasmic distribution and extracellular release of sCRFR2a into the cerebrospinal fluid, with sCRFR2a levels positively correlating with anxiety-like behavior. Concluding, the dichotomy between anxiolytic mCRFR2a and anxiogenic sCRFR2a is the basis for the deleterious effects of chronic OXT on anxiety. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.11.246504v1?rss=1 Authors: Campbell, A., Ferraz Franco, C., Su, L.-I., Perkins, S., Jones, A. R., Brownridge, P. J., Perkins, N. D., Eyers, C. E. Abstract: Different types of DNA damage can initiate phosphorylation-mediated signalling cascades that result in stimulus specific pro- or anti-apoptotic cellular responses. Amongst its many roles, the NF-{kappa}B transcription factor RelA is central to these DNA damage response pathways. However, we still lack understanding of the co-ordinated signalling mechanisms that permit different DNA damaging agents to induce distinct cellular outcomes through RelA. Here, we use label-free quantitative phosphoproteomics to examine the temporal effects of exposure of U2OS cells to either etoposide (ETO) or hydroxyurea (HU) by monitoring the phosphorylation status of RelA and its protein binding partners. Although few stimulus-specific differences were identified in the constituents of phosphorylated RelA interactome after exposure to these DNA damaging agents, we observed subtle, but significant, changes in their phosphorylation states, as a function of both type and duration of treatment. The DNA double strand break (DSB)-inducing ETO invoked more rapid, sustained responses than HU, with regulated targets primarily involved in transcription, cell division and canonical DSB repair. Kinase substrate prediction of ETO-regulated phosphosites suggest abrogation of CDK1 and ERK1 signalling, in addition to the known induction of ATM/ATR. In contrast, HU-induced replicative stress mediated temporally dynamic regulation, with phosphorylated RelA binding partners having roles in rRNA/mRNA processing and translational initiation, many of which contained a 14-3-3{varepsilon} binding motif, and were putative substrates of the dual specificity kinase CLK1. Our data thus point to differential regulation of key cellular processes and the involvement of distinct signalling pathways in modulating DNA damage-specific functions of RelA. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.10.197368v1?rss=1 Authors: Martina A. Maibach, Ester Piovesana, Julia Kaiser, Mea M. Holm, Zorica Ristic, Michael Maurer, Martina E. Schwab Abstract: Nogo-A is a well-characterized myelin-associated membrane protein that restricts fibre growth and the regenerative capacity of the adult central nervous system after injury. To date Nogo-A post-receptor signalling pathway research focused on the RhoA/ROCK cascade, which can lead to growth cone collapse and neurite retraction. Much less is known about continued intracellular Nogo-A signalling mediating long-term neurite outgrowth inhibition resulting from transcriptional and translational changes. Here, we propose a simple but highly reproducible in vitro assay to study Nogo-A related signaling and neurite outgrowth inhibition in general. Furthermore, we identified ERK1/2 as downstream effector of Nogo-A, partially mediating its neurite outgrowth inhibition. We describe ERK1/2 dependent changes of translational events such as elevation of RhoA levels within the growth cone, which may potentiate the cells' responses to Nogo-A. We also observed Nogo-A dependent upregulation of the JAK/STAT pathway inhibitors SOCS3 and KLF4 and downregulation of insulin mediated phosphorylation of AKT, indicating direct negative crosstalk between Nogo-A signalling and the growth promoting JAK/STAT and AKT/mTORC1 pathways.Competing Interest StatementThe authors have declared no competing interest. Copy rights belong to original authors. Visit the link for more info

Evidence suggests that THC and CBD each act on specific receptors in the brain to cause different effects on Biomarkers, other neurotransmitter systems and molecular pathways. In fact, CBD can even prevent many of the side effects associated with potent cannabis (high THC cannabis) such as psychosis, paranoia, and in some cases can act as a treatment for those diagnosed with schizophrenia. In this episode of First-Person Science, PhD Candidate & Vanier Scholar Roger Hudson speaks about his recently published manuscript in the Journal Of Neuroscience, discussing how THC causes adverse psychiatric side-effects in some users, and the science of how cannabidiol (CBD) can counteract these side effects. "Cannabidiol (CBD) Counteracts the Psychotropic Side-Effects of Delta-9-Tetrahydrocannabinol (THC) in the Ventral Hippocampus through Bidirectional Control of ERK1–2 Phosphorylation", published in The Journal of Neuroscience [2019 Oct;39(44):8762–8777] / DOI:10.1523/JNEUROSCI.0708-19.2019. https://www.ncbi.nlm.nih.gov/pubmed/31570536Produced by Roger Hudson, PhDc 

Dr Carolyn Lam:                Welcome to Circulation on the Run, your weekly podcast summary and backstage pass to the journal and its editors. We're your co-hosts, I'm Dr Carolyn Lam, Associate Editor from the National Heart Center and Duke National University of Singapore. Dr Greg Hundley:             And I'm Dr Greg Hundley: Hundley, Associate Editor from the Pauley Heart Center in Richmond, Virginia at VCU Health. Well Carolyn, our featured article this week addresses the age at which to initiate clinical screening of relatives for hypertrophic cardiomyopathy. Our guidelines suggest screening of relatives from age ten and onwards but data are lacking to substantiate this suggestion. I look forward to the authors' discussion of their findings regarding initiation of screening in children. For now though, do you have an article that you'd like to share? Dr Carolyn Lam:                You bet, Greg. So, the first paper I chose really demonstrates that patients inducible pluripotent stem cells or IPSC cardio derived myocytes can be used as a disease modeling platform to delineate the functional mechanisms that underlie cardiac hypertrophy and in this particular case they looked at Noonan Syndrome and showed that how these techniques can be subsequently used to identify novel molecular and genetic therapeutic targets. So, Greg, here's your quiz. The genetics of Noonan Syndrome. Dr Greg Hundley:             I remember it was on our board exam. Dr Carolyn Lam:                Let me tell you about it. So more than 90% of patients with Noonan Syndrome have a mutation in the hinge region CR2 domain of Raf-1 and they exhibit severe hypertrophic cardiomyopathy for which there is no treatment. Authors, Dr Jaffrey from Cornell University and Dr Kontaridis from Masonic Medical Research Institute in Utica in New York and their colleagues used Noonan Syndrome Raf-1 patient and CRISPR corrected IPSC cardiomyocytes to recapitulate the Noonan Syndrome cardiac phenotype.                                                 These Noonan Syndrome IPSC derived cardiomyocytes exhibited the same hypertrophy and myofibrillar disarray that's really observed in Noonan Syndrome patient hearts, so mechanistically the authors showed that activation of mitogen-activated protein kinase or mech-1 or -2, but not the extracellular regulated kinase, which is ERK1 or 2 triggered abnormal cardiomyocytes structure and conversely ERK5 mediated increased cell size in these Noonan Syndrome mutant IPSC derived cardiomyocytes.                                                 RNA sequencing further identified genes dysregulated in the Noonan Syndrome cardiomyocytes that may underlie hypertrophic cardiomyopathy downstream if the mech-1 or -2 and ERK5 genes. Dr Greg Hundley:             So, Carolyn, that's a lot of genetic information, so what can I take home as I think about this further and what may come down the line as we manage patients with Noonan Syndrome? Dr Carolyn Lam:                Thanks, Greg. The real take home message is that these pathways could serve as novel therapeutic targets to treat hypertrophic cardiomyopathy in patients with Noonan Syndrome and Raf-1 mutations. Overall, the elucidation of rare disease mechanism of hypertrophic cardiomyopathy may further unravel and reveal causes of other more common idiopathic congenital disorders and hypertrophic diseases. Dr Greg Hundley:             Oh, very good. Well, I'm going to switch gears and talk a little bit about infective endocarditis prophylaxis and this article comes from Pallavi Garg at the London Health Scientist Center. Carolyn, as you may recall, given the lack of proven efficacy and concerns about the perceived risks of antibiotic prophylaxis like development of antibiotic resistance, the American Heart Association in 2007 and the European Society of Cardiology in 2009 published revised guidelines recommending cessation of antibiotic prophylaxis prior to dental procedures for patients at moderate risk of infective endocarditis while continuing the practice in high risk patients. This Canadian study was conducted from 2002 to 2014 among all adults and those at high and moderate risk for infective endocarditis and they were stratified by age. Prescriptions for antibiotic prophylaxis were obtained from the Ontario Drug Benefit Database for adults 65 and older and outcomes regarding antibiotic prophylaxis prescription rates and the incidents of infective endocarditis related hospitalization were assessed. Dr Carolyn Lam:                 Ooh, interesting. What did they find? Dr Greg Hundley:             The authors found a sustained reduction in antibiotic prophylaxis prescriptions among individuals at moderate risk for infective endocarditis that coincided with the change in guidelines. In contrast, while there was a decreasing trend in antibiotic prophylaxis among individuals at high risk of infective endocarditis and a minimal drop following the guidelines released, the overall rates of prophylaxis prescribing in this group continued to climb since early 2007, and collectively, these findings suggest that appropriate uptake of the revised AHA guidelines occurred.                                                 Furthermore, over the thirteen-year study period, the authors identified an increase in hospitalizations for new episodes of endocarditis approximately three years after the AHA guidelines were revised. This timeline along with the rise of endocarditis incidents in both the high and moderate risk groups suggests that this observed increase in endocarditis is likely unrelated to the change in the prescribing practice of antibiotic prophylaxis. This conclusion is further supported by the overall decrease in endocarditis cases attributable to streptococcal infections over time, a finding contrary to what might be expected as a result of the reduction in antibiotic prophylaxis. Dr Carolyn Lam:                Oh, very interesting, Greg. At first a little bit scary and then after when you described it more, it does seem a little bit more reassuring. Very interesting. Well, thank you. My next paper deals with functional tricuspid regurgitation, which as you know is really common in heart failure with reduced ejection fraction or HFrEF and mostly consequent to pulmonary hypertension. However, what is the access mortality associate with functional tricuspid regurgitation in HFrEF? Well, this paper from Dr Maurice Serrano from Mayo Clinic and colleagues looked at all Mayo Clinic patients from 2003 to 2011 diagnosed with heart failure stage B and C and an ejection fraction less than 50% who had functional tricuspid regurgitation grading and systolic pulmonary artery pressure measured by Doppler echocardiography.                                                 Now among more than 13,000 patients meeting these inclusion criteria, functional tricuspid regurgitation was detected in 88%. Functional tricuspid regurgitation was independently associated with more dyspnea, more impaired kidney function, and lower cardiac output. For the long term outcomes, the higher the degree of functional tricuspid regurgitation compared with a group with trivial tricuspid regurgitation was independently associated with a higher mortality hazard. The five year survival was substantially lower with increasing severity of tricuspid regurgitation so it was 68% on average for trivial functional tricuspid regurgitation versus 34% for severe functional tricuspid regurgitation.                                                 Importantly, this access mortality observed with moderate or severe functional tricuspid regurgitation was independent of pulmonary hypertension and any other clinical characteristics. Dr Greg Hundley:             Hmm, interesting but Carolyn, wouldn't we expect this? Dr Carolyn Lam:                You know what, you may expect it, but this is really the largest series, I think, that has shown this and shown this in the systematic way that functional tricuspid regurgitation in and of itself may play an important pathophysiologic role and thus, may represent a potential therapeutic target in HFrEF. In other words, the present study really advocates for a trial to test treating functional tricuspid regurgitation in patients with HFrEF. Dr Greg Hundley:             Oh wow, you really put that in great perspective, Carolyn. Well, your reward is   going to be a quiz. Dr Carolyn Lam:                 Oh my gosh, Greg. Dr Greg Hundley:             We're going to talk about ... Dr Carolyn Lam:                 What now? Dr Greg Hundley:             Caveolin-1, an atherogenesis and nitric oxide and this is from Professor Carlos Fernandez Hernando at the Yale University School of Medicine. Okay, multiple choice. What are caveolae? Now I'm going to give you some choices, you get to pick A. Are they crypts within the walls of vessels. B. Crypts within the membranes of endothelial cells. Or C. Crypts within the border zones of infarcts. Dr Carolyn Lam:                Wait a minute, Greg. I'm not even sure we're pronouncing it the same. You're asking about caveolae like ... Potato potata. They're invaginations of cell membranes, that's all I know. Dr Greg Hundley:             Oh wow, fantastic. This study focused on the effect of Caveolin-1, a protein integral to the formation of caveolae. The investigators found in a series of mouse experiments that A. The athero-protection observed in mice lacking Caveolin-1 is independent of endothelial nitric oxide synthase activation and nitric oxide production. B. Endothelial Caveolin-1 controls lipoprotein infiltration in vascular inflammation in early stage atherosclerotic lesion. C. Endothelial Caveolin-1 promotes pro-atherogenic matrix deposition leading to endothelial cell activation in atheroprone regions of the aorta and finally, D. Atheroprone regions of the aorta are characterized by increased intracellular and basolateral caveolae distribution in endothelial cells compared to athero-resistant areas. Dr Carolyn Lam:                Wow, I like the way you broke that down into four points, but could you summarize what it means clinically? Dr Greg Hundley:             Yeah, so I think if you had to summarize all of this in a sentence, perhaps the suppression of Caveolin-1 expression in endothelial cells might prevent the progression and promote the regression of atherosclerosis so in the future perhaps an interesting target to treat atherosclerosis. Well, now Carolyn, I guess we should proceed to that talk with our featured discussion. Dr Carolyn Lam:                 Absolutely. Thanks, Greg.                                                 Hypertrophic cardiomyopathy is an inheritable myocardial disease with age-related penetrance. Current guidelines recommend that clinical screening of relatives start from the age of ten years onwards by the European Society of Cardiology and twelve years onwards by the American College of Cardiology or American Heart Association. There are of course caveats for earlier screening but the clinical value of this approach has really not been systematically evaluated. That is until today's feature paper and we are so pleased to be here discussing it. This is Greg Hundley and Carolyn Lam and we're your co-hosts for Circulation on the Run. So happy to welcome Dr Juan Pablo Kaski who's the corresponding author of today's feature paper from Great Ormand Street Hospital in London and we also have Dr Gerald Greil, Associate Editor from UT Southwestern.                                                 Welcome, everyone. Juan, if you don't mind, could you start by summarizing this very important study of yours? Dr Juan Pablo Kaski:        Thank you very much. Hypertrophic cardiomyopathy is a genetic muscle condition that is characterized by hypertrophy and is most commonly inherited as a dominant trait. Previous studies have suggested that in familial disease at least ventricular hypertrophy doesn't usually present until adolescence and this has led to the current guidelines which do not recommend routine screening of children below the age of twelve according to the American guidelines below the age of ten and the European guidelines for hypertrophy cardiomyopathy but own clinical experience was different and suggested that perhaps sarcomeric disease and familial disease could present in younger children, so what we aimed to do with this study was to assess the validity of this approach and tried to assess the yield of clinical screening in children from families of hypertrophic cardiomyopathy. Dr Juan Pablo Kaski:        We took our collective experience in our institution over a period of many years and recruited just on the 1,200 consecutive children all aged less than eighteen years at the time of initial assessment coming from just under 600 families and these were children who were referred for clinical screenings because a first degree relative had been diagnosed with hypertrophic cardiomyopathy. What we found was that in 5% of these children and in fact, in 8% of the families that we screened, we were able to pick up early phenotypic features of hypertrophic cardiomyopathy. In 72% of patients, we made a diagnosis before the age of twelve, so before current clinical screening guidelines we'd recommend and importantly, a third of these patients during follow up had a change in their management as a result of the diagnosis. Their medication was commenced, they underwent procedures or implantations of defibrillators. Dr Greg Hundley:             Juan, this is Greg Hundley and I was wondering when did the participants that were enrolled experience events? Did those that were say under fourteen or even under twelve, did they experience events relative to those that were a little older? Dr Juan Pablo Kaski:        The events that our participants experienced were relatively few. Many of these occurred during the childhood age but some occurred once the children had transitioned into the adult age. We did look to see whether there was any difference in terms of early diagnosis and subsequent events, but we didn't find anything, we didn't identify two separate populations in that respect. Dr Greg Hundley:             And then did you perform genetic analyses? I know you described phenotypic characterization of the patient population but how about genetically? What results did you find there? Dr Juan Pablo Kaski:        The main aim of the study really was to determine a yield of clinical screenings, so this is a reflection of a real-world practice where genetic testing may not necessarily be routinely available. Having said that, we did have genetic data in a third of our families and in fact, in maybe 70% of those children who made clinical diagnosis of hypertrophic cardiomyopathy was made and what we find in those individuals who have undergone genetic testing is that the vast majority of those had mutations in sarcomeric protein genes and pathogenic or likely pathogenic variants in sarcomeric genes in just under 70% and these were well characterized mutations that are very similar to those that are seen in adolescence or adult onset hypertrophic cardiomyopathy.                                                 I think what was interesting about these genetic results is that we seem to have identified a population of early onset sarcomeric disease that genetically appears to be indistinguishable from a sort of later onset adult disease but with the clinical presentation and natural history curve shifted somewhat to the left. Dr Greg Hundley:             Gerald, just switching over, can you tell us some of your thoughts about how the results of this study will impact clinical practice, both in the European countries as well as U.S.? Dr Gerald Greil:                 I mean, I was obviously delighted to see the study being submitted to circulation because there's a very important message particularly for pediatric cardiologists which is potentially influencing the guidelines and Dr Kaski may comment on this as well as the next step meaning that it seems like screening patients older than ten or twelve years and once again, there's a slight discrepancy between the European and U.S. guidelines, seems to be ... Can be questioned and potentially we should screen these patients earlier.                                                 Another amplification of this study is that we should think about how much genetic screening can be an essential tool in our methods in looking at these patients and I want to point out that because of these discrepancies we also initiated an editorial letter for this publication done by Dr Ommen and by Dr Mital kind of pointing out there needs a lot of work to be done maybe even including rewriting the current guidelines.                                                 There's another paper that came out recently in European Society Cardiology, the European Heart Journal about a similar topic so it's something which is very, very heavily discussed in our community. We think how we are looking at these patients and how we're following them up. Dr Greg Hundley:             What would you suggest are next steps for the world community in this space in regards to modifying those guidelines? Dr Gerald Greil:                 I think there's now enough literature around which suggests that we should look at these patients earlier and screen them earlier on both sides in European, in the U.S., in the Asian world, and ideally these two groups should sit together and write combined guidelines. It's still interesting that the European and U.S. guidelines are slightly different in that we're talking about a similar group of patients, so I'm very, very delighted to see that this is coming up in the national literature as a new topic and I think everything is open now to rethink this topic and rewrite these guidelines. Dr Greg Hundley:             Do you think prospective studies would be necessary because I believe, and Dr Kaski please weigh in here, this was a retrospective review, and do you think there could have been triggering circumstances that prompted early screening? I mean, would a next step be some sort of prospective registry? Dr Gerald Greil:                 I mean definitely that's the next step. I think we have enough data material around once again to rethink the strategy which age these patients should be looked at. A prospective registry and Dr Kaski can probably comment on it better than I can, I think that something which is a logical next step and there may be even something being on the way to make this happen. Dr Juan Pablo Kaski:        I agree. I think further validation and confirmation of these data from prospective studies would be extremely helpful. I think one of the things that we need to bear in mind is the potential cost implications of expending screening to ever increasing populations and so perhaps an additional further step would be to try to refine the screening tools so that we are able to identify clinical by a chemical of those individuals who are more likely to present in childhood and perhaps set a target screening towards that population.                                                 I can just go back to one of your sort of previous points also about a potential bias and it is true that these patients were referred for clinical screening at a time when clinical recommendations do not suggest that this is necessary and although we didn't specifically in this cohort look at those that would have fulfilled current early screening criteria, the vast majority of the patients were asymptomatic at the time that they were referred. We also looked to see whether there was any link between those individuals who had a family history of early onset disease and an early diagnosis, and that was the only factor that came up as potentially significant so perhaps the current guidelines that do recommend considering earlier screenings if there's a family history of childhood disease are still applicable. Dr Carolyn Lam:                That was just an amazing interview, by the way. I've learned so much and thank you so much for publishing this very important paper with us.                                                 You've been listening to Circulation on the Run. Don't forget to tune in again next week. This program is Copyright American Heart Association 2019.  

Background Tendons are dense connective tissues subjected periodically to mechanical stress upon which complex responsive mechanisms are activated. These mechanisms affect not only the development of these tissues but also their healing. Despite of the acknowledged importance of the mechanical stress for tendon function and repair, the mechanotransduction mechanisms in tendon cells are still unclear and the elucidation of these mechanisms is a key goal in tendon research. Tendon stem/progenitor cells (TSPC) possess common adult stem cell characteristics, and are suggested to actively participate in tendon development, tissue homeostasis as well as repair. This makes them an important cell population for tendon repair, and also an interesting research target for various open questions in tendon cell biology. Therefore, in our study we focused on TSPC, subjected them to five different mechanical protocols, and investigated the gene expression changes by using semi-quantitative, quantitative PCR and western blotting technologies. Results Among the 25 different genes analyzed, we can convincingly report that the tendon-related genes - fibromodulin, lumican and versican, the collagen I-binding integrins - α1, α2 and α11, the matrix metalloproteinases - MMP9, 13 and 14 were strongly upregulated in TSPC after 3 days of mechanical stimulation with 8% amplitude. Molecular signaling analyses of five key integrin downstream kinases suggested that mechanical stimuli are mediated through ERK1/2 and p38, which were significantly activated in 8% biaxial-loaded TSPC. Conclusions Our results demonstrate the positive effect of 8% mechanical loading on the gene expression of matrix proteins, integrins and matrix metalloproteinases, and activation of integrin downstream kinases p38 and ERK1/2 in TSPC. Taken together, our study contributes to better understanding of mechanotransduction mechanisms in TPSC, which in long term, after further translational research between tendon cell biology and orthopedics, can be beneficial to the management of tendon repair.

Die Familie A der G-Protein-gekoppelten Rezeptoren (GPCRs) bildet die größte und vielfältigste aller Transmembranrezeptorfamilien. Ihre Mitglieder spielen eine wesentliche Rolle in fast allen (patho)physiologischen Prozessen. Nach Agonistenbindung aktivieren GPCRs, wie ihr Name andeutet, heterotrimere G-Proteine aber auch G-Protein-unabhängige Signalwege. Die verschiedenen aktiven G-Proteinuntereinheiten (Gα-GTP und βγ) induzieren nach Dissoziation vom Rezeptor entsprechende Signalkaskaden z.B. über Phospholipase A und Cβ. Um eine Fehlregulation zellulärer Prozesse z.B. durch „Überstimulation“ zu verhindern, unterliegen GPCRs strengen Regulationsmechanismen, die ihre Fähigkeit zur Signaltransduktion und ihre Verfügbarkeit an der Zelloberfläche bestimmen. Die Bradykininrezeptoren B1 und B2 (B1R, B2R) gehören zur Familie A der GPCRs, also zu den Rhodopsin-ähnlichen GPCRs, und werden durch die pro-inflammatorischen Peptide desArg9-Bradykinin/desArg10-Kallidin (DABK/DAK) bzw. Bradykinin (BK)/Kallidin aktiviert. Im Gegensatz zum konstitutiv exprimierten B2R, der nach Stimulation schnell desensitisiert und internalisiert wird, erfolgt eine B1R-Expression fast ausschließlich unter pathophysiologischen Bedingungen über Induktion durch Zytokine. Nach Stimulation wird der B1R nicht internalisiert, sondern verbleibt an der Zelloberfläche. Beide Rezeptoren koppeln sowohl an Gαq/11 als auch an Gαi und aktivieren somit weitgehend identische Signalwege [vor allem Phospholipase Cβ (PLCβ) und „mitogen activated protein kinase“ (MAPK)-Kaskaden]. Durch ihre - besonders im Hinblick auf ihre Internalisierungs-eigenschaften - konträre Regulation, stellen die Bradykininrezeptoren ein interessantes Modell zur Untersuchung regulatorischer Mechanismen und deren Einflüsse auf die Signalübertragung von GPCRs dar. Beide Bradykininrezeptoren spielen bei inflammatorischen Prozessen eine Rolle. Sie fördern die Ausschüttung pro-inflammatorischer Zytokine und rekrutieren Immunzellen. Während entzündlicher Ereignisse kommt es zu erhöhter Zytokinfreisetzung z.B. von Interleukin-1β (IL-1β) und dadurch zur de novo Synthese von B1R. Pro-inflammatorische Zytokine wie IL-1β, die zur B1R-Expression führen, induzieren unter anderem aber auch einen Anstieg der Körpertemperatur (Fieber), eine häufige Begleiterscheinung inflammatorischer Vorgänge. Trotz des bekannten Zusammenhangs zwischen Inflammation und erhöhter Temperatur war über den Einfluss eines Temperaturanstiegs auf Membranrezeptoren und ihre Signalvermittlung auf zellulärer Ebene bisher nur sehr wenig bekannt. In dieser Arbeit wurde - unseres Wissens nach - erstmals auf die Temperatur als regulatorische Komponente für GPCR-vermittelte Signalübertragung eingegangen. Am Beispiel der Bradykininrezeptoren wurde gezeigt, dass die Stärke der Signalübertragung von GPCRs signifikant durch eine Temperaturerhöhung von 37°C auf 41°C beeinflusst werden kann. Hierbei war jedoch zwischen einer Temperaturabhängigkeit des Signalwegs selbst und einer rezeptorspezifischen Temperatursensitivität zu unterscheiden. So wurde die Aktivierung von ERK1/2 unter pathophysiologisch erhöhter Temperatur (41°C; normale Körpertemperatur: 37°C) signifikant gesteigert, unabhängig davon ob sie durch B1 oder B2 Rezeptoren stimuliert wurde. Die gesteigerte Aktivität PLCβ-vermittelter Signalkaskaden bei 41°C konnte hingegen auf eine nur für den B1R spezifische Temperaturabhängigkeit zurückgeführt werden. Diese Beobachtung zusammen mit der Tatsache, dass die B1R-Expression unter pathophysiologischen Bedingungen besonders induziert wird, deutet darauf hin, dass der B1R in Kombination mit Fieber eine verstärkte Wirkung im Organismus haben könnte. Ob diese Heilungs-fördernd oder -abträglich wirkt, müsste noch genauer untersucht werden. Neben dem Einfluss der Temperatur wird die Signalübertragung der GPCRs durch die jeweiligen Rezeptorkonformationen und die sich daraus ergebenden Funktionsunterschiede bestimmt. Die Familie A der GPCRs wird durch einige hoch konservierte Strukturmerkmale wie die E/DRY-Sequenz mit R3.50 in der dritten Transmembrandomäne (TM) oder die NPXXY-Sequenz am Ende der siebten TM charakterisiert. Publizierte Ergebnisse deuten darauf hin, dass bovines Rhodopsin durch eine Salzbrücke zwischen R3.50135 (TM3) und E6.30247 (TM6), auch „ionic lock“ genannt, im inaktiven Zustand gehalten wird. Der B2R ist einer der wenigen Peptid-GPCRs, der ein Glutamat an Position 6.30 (E6.30238) trägt, und eignete sich daher zur Untersuchung der Anwesenheit und Funktion eines möglichen „ionic lock“ auch in „nicht-Rhodopsin“-GPCRs. Für alle bisher entsprechend untersuchten GPCRs ist bekannt, dass R3.50 für eine effiziente G-Protein-Aktivierung unabdingbar ist (selbiges wurde in der vorliegenden Arbeit auch für den B2R bestätigt). Die funktionelle Analyse eines „ionic lock“ anhand einer R3.50 Mutation und G-Protein-abhängiger Kaskaden ist deshalb nicht möglich. Die Rolle eines „ionic lock“ im Hinblick auf G-Protein-unabhängige Mechanismen wie die Rezeptorinternalisierung, einem wichtigen Regulationsschritt für die meisten GPCRs, wurde bisher jedoch noch nicht untersucht. In der vorliegenden Arbeit wurde erstmals gezeigt, dass die Rezeptorendozytose durch Mutation von R3.50128 zu Alanin (R3.50128A), im Gegensatz zur G-Protein-Aktivierung, nicht zum Erliegen kommt. Die mutierten Rezeptorkonstrukte wiesen sogar ein konstitutives Internalisierungsverhalten auf. Dies verwies auf unterschiedliche Funktionen dieser Aminosäure bei der G-Protein-vermittelten Signaltransduktion und bei der Rezeptorinternalisierung. Ein Aufbrechen des möglichen „ionic lock“ durch Mutation von E6.30238 zu Alanin oder Arginin resultierte ebenfalls in konstitutiv internalisierenden Rezeptorkonstrukten. Im Gegensatz zur Endozytose zeigten diese Mutanten zwar keine konstitutive Signalübertragung, wurden aber auch durch prinzipiell als Antagonisten klassifizierte Verbindungen effizient aktiviert. Diese Ergebnisse legen einen mehrstufigen Aktivierungsprozess nahe, dessen Stufen sich durch verschiedene Rezeptorkonformationen mit unterschiedlichen Interaktionsmöglichkeiten für die G-Protein-Rekrutierung/Aktivierung oder mit der Internalisierungsmaschinerie [GPCR-Kinasen (GRKs), Arrestine] auszeichnen. Der wechselseitige Austausch der beiden hoch konservierten Aminosäuren R3.50128 und E6.30238 ermöglichte wahrscheinlich die Bildung eines inversen „ionic lock“, wodurch normales B2R-Verhalten wieder hergestellt wurde. Diese Arbeit zeigt somit erstmals, dass ein Aufbrechen eines möglichen „ionic lock“ in einem Peptidrezeptor unterschiedliche Auswirkungen für die Prozesse der G-Protein-Aktivierung und der Rezeptorendozytose haben kann. Dadurch wird die Annahme bestärkt, dass es bei einem GPCR mehrere aktive Konformationen geben kann, die unterschiedliche Affinitäten gegenüber regulatorischen Proteinen (GRKs, Arrestinen) oder Effektoren (G-Proteinen, Arrestinen) aufweisen und dadurch differenziert zelluläre Signale auslösen können. Die Aufklärung der unterschiedlichen Aktivierungsmechanismen von GPCRs in Kombination mit der Herstellung von Verbindungen z.B. sogenannten „small molecule compounds“, die bestimmte Rezeptorkonformationen mit ihren signalspezifischen Eigenschaften stabilisieren können, wäre möglicherweise für die Entwicklung von Agonisten oder Antagonisten, die nur ganz bestimmte Signalwege modulieren und so eine optimierte therapeutische Anwendung erlauben, hilfreich.

Video Podcast (CC)Aired date: 5/30/2012 3:00:00 PM Eastern Time

Audio PodcastAired date: 5/30/2012 3:00:00 PM Eastern Time

Guido Tarone, Molecular Biotechnology Center, University of Turin, Turin - ITALY, speaks on "Melusin and IQGAP1 are key players in ERK1/2 signaling and adaptive cardiac hypertrophy" . This seminar has been recorded by ICGEB

Die vorliegende Dissertation befasst sich mit den durch S-Lost-verursachten Symptomen in der Haut, die zunächst durch starke Blasenbildung und später durch eine verzögerte Wund-heilung charakterisiert sind. Bei S-Lost handelt es sich um einen chemischen Kampfstoff, der erstmals im ersten Weltkrieg zum Einsatz kam und bis heute in vielen internationalen Kon-flikten großen Schaden anrichtete, obwohl der Gebrauch schon 1925 durch die Genfer Konvention verboten wurde. Aktuell stellt S-Lost zudem eine Bedrohung durch terroristische Aktivitäten dar. Da für S-Lost-induzierte Verletzungen bislang keine spezifisch wirksamen Behandlungs-methoden verfügbar sind, besteht großes Interesse an der Aufklärung der dem Krankheitsbild zugrunde liegenden molekularen Pathomechanismen, um daraus Rückschlüsse auf besser ge-eignete therapeutische Maßnahmen ziehen zu können. In unseren ersten Experimenten wurden als mögliche Auslöser der Blasenbildung die Expression und Sekretion ausgewählter Matrix-Metalloproteinasen (MMPs) und deren endogenen Inhibitoren, den tissue inhibitors of matrix metalloproteinases (TIMPs) in einem 3D-Haut-modell und in verschiedenen Zelltypen der Haut (Keratinozyten, Fibroblasten, mikrovaskuläre Endothelzellen, mesenchymale Stammzellen, monozytäre Zellen, PMN-Granulozyten) sowohl in Mono- als auch in Mischkultur untersucht. Unter Verwendung von molekularbiologischen und proteinbiochemischen Methoden wie qRT-PCR, Zymographie und Western Blot gelang der Nachweis, dass MMPs - insbesondere MMP-9 - nach Exposition der Zellen (v.a. Fibroblasten und monozytäre Zellen) mit S-Lost deutlich hochreguliert wurden. Zu Erhärtung der Annahme, dass MMP-9 durch Degradation der Basalmembran zwischen Epidermis und Dermis zur Blasenbildung beiträgt, konnte als Pathomechanismus nun erstmals eine parakrine Stimulation von Fibroblasten durch S-Lost-behandelte Keratinozyten identifiziert werden, als deren Folge eine vermehrte MMP-9-Sekretion resultierte. Darüber hinaus zeigte sich in weiteren Versuchen unter Verwendung des sog. Scratch-Assays und eines Transwell-basierten Invasionsassays, dass das Migrations- und Invasionsverhalten der Fibroblasten in Gegenwart des konditionierten Mediums der S-Lost-behandelten Keratinozyten positiv beeinflusst wurde. Aus klinischer Sicht sprechen diese Erkenntnisse für neue therapeutische Ansätze, die darauf beruhen sollten, die S-Lost-induzierte, auf proteolytischer Aktivität basierende Blasenbildung der Haut durch Applikation spezifischer MMP-Inhibitoren zu behandeln. In einem weiteren Projekt wurde die verzögerte Wundheilung als spätes Symptom der S-Lost-Vergiftung auf zellulärer Ebene untersucht, bei der eine eingeschränkte Re-Epithelialisierung der betroffenen Hautstellen beobachtet wird. Sowohl für den Prozess der Wundheilung als auch für die stetige Erneuerung der Haut werden epidermale Stammzellen benötigt, die für die Bildung von Keratinozyten verantwortlich sind. Diese unipotenten Progenitorzellen befinden sich in der basalen Schicht der Epidermis und sind in der Lage zu proliferieren und anschließend terminal zu differenzieren. Um eine Beeinflussung dieser Prozesse durch S-Lost zu untersuchen, wurden primäre unreife Keratinozyten (NHEK) verwendet und hinsichtlich ihres Differenzierungspotenzials untersucht. Dabei erwies sich S-Lost als potenter Induktor der Differenzierung von NHEK, was durch Bestimmung der Expression typischer Markerproteine wie Keratin-1, Involucrin und Loricrin gezeigt wurde. Die Induktion des Reifungsprozesses war sowohl von einem Rückgang der Proliferation als auch von einer verminderten Migrationsrate der Zellen begleitet. Die eingehende Analyse von mitogen-activated protein kinase (MAPK)-Signaltransduktionswegen führte zu der Erkenntnis, dass die Aktivitäten von p38 und ERK1/2 gegenteilige Rollen im Differenzierungsprozess einnehmen. Studien mit spezifischen Inhibitoren der MAPK be¬legten, dass p38 für den Reifungsvorgang in NHEK essentiell ist, während ERK1/2 diesem entgegen wirkt. So konnte durch Blockade von p38 die von S-Lost ausgelöste Differenzierung der Zellen verhindert werden. Ebenso war es durch diese Behandlung möglich, die von S-Lost stark beeinträchtige Migrationsfähigkeit der Keratinozyten wiederherzustellen, welche mit einer erhöhten MMP-1-Expression einherging. Davon abgeleitet erscheint es therapeutisch sinnvoll, selektive p38-Inhibitoren für die Behandlung von Wundheilungsstörungen nach Exposition der Haut mit S-Lost einzusetzen. Zusammenfassend erbrachten unsere Studien also den Nachweis, dass der S-Lost-induzierten Blasenbildung (als frühes Symptom) die spezifische Induktion der MMP-9 zugrunde liegt. Darüber hinaus konnte erstmals eine verfrühte Differenzierung in unreifen Keratinozyten der Haut (als mögliche Ursache für die verzögerte Wundheilung) nachgewiesen werden, wobei die MAPK p38 bei der Initiierung des Prozesses von entscheidender Bedeutung ist. Aufgrund dieser Resultate empfiehlt sich eine kombinierte Applikation von Inhibitoren der Aktivitäten von MMP-9 und p38, wobei der Einsatz jedoch zeitlich abgestimmt erfolgen sollte, um pathologische Effekte (Blasenbildung bzw. Differenzierungsinduktion in Keratinozyten) zu blockieren, ohne die positiven Auswirkungen von MMP-9 und p38 auf die Heilung (Migration von Immunzellen und Keratinozyten bzw. Reepithelialisierung) zu hemmen.

In the present study, the equine histamine 4 receptor (eH4R) was cloned, sequenced and pharmacologically characterized. The findings were compared to those, obtained with the human H4R (hH4R). Due to its expression in cells of the immune system, the eH4R provides a promising target for the development of novel therapeutic strategies in allergic diseases, such as Recurrent Airway Obstruction (RAO) and allergic dermatitis in the horse. To clone the eH4R, mRNA was isolated from horse white blood cells and cDNA was synthesized by reverse transcription. Specific primers were used to amplify the eH4R sequence, which was then cloned into pJET1.2/blunt vectors. The open reading frame is 1185 bp long and codes for a 394 amino acid protein which shows 72,9 % homology to the human receptor. The cDNA sequence was published in the NCBI GenBank under the accession number HM015200. To pharmacologically and functionally characterize the eH4R and hH4R, their cDNAs were subcloned into the expression vector pcDNA3.1 and either transfected transiently into COS-7 cells or stably into HEK293 cells. Binding-characteristics were examined by homologous und heterologous competition experiments using the antagonist 3H-pyrilamine or the agonist 3H-histamine as radioligand. High affinity binding of histamine could only be detected in hH4R, but not in eH4R transfected COS-7 cells. Nevertheless, histamine was able to inhibit cAMP-production in stably transfected HEK293 cells via the eH4R and the hH4R. The eH4R expressed in HEK293 cells is coupled to the stimulation of ERK1/2, while the hH4R shows already high constitutive activity. The antagonists JNJ7777120, Thioperamide, Pyrilamine and Diphenhydramine display considerable species-specific differences concerning the affinities between eH4R and hH4R and also vary in their intrinsic activities. Thioperamide, known for its inverse agonism at the hH4R showed agonist behaviour in ERK1/2 regulation. In contrast, the non-selective antagonist Diphenhydramin showed inverse agonist behaviour, which was more pronounced at the hH4R than the eH4R. These findings suggest that there are considerable pharmacological and functional differences between the cloned eH4R and hH4R.

Introduction: alpha(1)-Adrenoceptors are considered critical for the regulation of prostatic smooth muscle tone. However, previous studies suggested further alpha(1)-adrenoceptor functions besides contraction. Here, we investigated whether alpha(1)-adrenoceptors in the human prostate may activate extracellular signal-regulated kinases (ERK1/2). Methods: Prostate tissues from patients undergoing radical prostatectomy were stimulated in vitro. Activation of ERK1/2 was assessed by Western blot analysis. Expression of ERK1/2 was studied by immunohistochemistry. The effect of ERK1/2 inhibition by U0126 on phenylephrine-induced contraction was studied in organ-bath experiments. Results: Stimulation of human prostate tissue with noradrenaline (30 mu M) or phenylephrine (10 mu M) resulted in ERK activation. This was reflected by increased levels of phosphorylated ERK1/2. Expression of ERK1/2 in the prostate was observed in smooth muscle cells. Incubation of prostate tissue with U0126 (30 mu M) resulted in ERK1/2 inhibition. Dose-dependent phenylephrine-induced contraction of prostate tissue was not modulated by U0126. Conclusions: alpha(1)-Adrenoceptors in the human prostate are coupled to ERK1/2. This may partially explain previous observations suggesting a role of alpha(1)-adrenoceptors in the regulation of prostate growth. Copyright (C) 2011 S. Karger AG, Basel

Der Typ-2-Diabetes mellitus ist durch eine chronische Hyperglykämie charakterisiert, welche auf eine Kombination aus peripherer Insulinresistenz und Dysfunktion der insulinproduzierenden β-Zellen des endokrinen Pankreas zurückzuführen ist. Um eine bessere Prävention und Therapie dieser häufig vorkommenden Stoffwechselerkrankung zu ermöglichen, war es das Ziel der vorliegenden Dissertation, neue Aspekte der β-Zelldysfunktion in der Pathogenese des Typ-2-Diabetes mellitus aufzuklären. Im ersten Teil dieser Arbeit sollten auf zellulärer Ebene am Typ-2-Diabetesmodell der db/db-Maus neue Erkenntnisse über die sequenzielle Abfolge von Ereignissen gewonnen werden, welche sich im endokrinen Pankreas bei der Entwicklung eines Typ-2-Diabetes abspielen. Durch den direkten Vergleich diabetesresistenter db/db-Mäuse mit C57BL/6J-Hintergrund (db/db B6) und diabetessuszeptibler db/db-Mäuse mit C57BLKS/J-Hintergrund (db/db BKS) wurde erstmalig gezeigt, dass beide Mausstämme eine ähnlich ausgeprägte Insulinresistenz aufweisen und zunächst in der Lage sind, den dadurch erhöhten Insulinbedarf effektiv zu kompensieren. Der sich ab einem Alter von 9 Wochen bei db/db BKS-Mäusen manifestierende Typ-2-Diabetes ist auf einer altersbedingten, inadäquaten Expansion der β-Zellmasse begründet, welche aus einer abnehmenden β-Zellhyperproliferation und einer signifikant gestei-gerten Apoptose der β-Zellen resultiert. Da kompensatorische Defizite der β-Zellmasse möglicherweise auch die humane Typ-2-Diabetesentstehung entscheidend beeinflussen, ist bei prädisponierten Personen eine frühzeitige therapeutische Unterstützung der β-Zellmasse als erfolgversprechende Maßnahme zur Prävention eines Typ-2-Diabetes mellitus vorstellbar. Im zweiten Abschnitt der vorliegenden Arbeit sollten neue Gene identifiziert werden, die eine regulatorische Funktion in den β-Zellen einnehmen und deshalb mögliche Angriffspunkte für neue Therapieformen der β-Zelldysfunktion beim Typ-2-Diabetes darstellen. In Voruntersuchungen wurden die Proteine OPG (Osteoprotegerin) und SOCS2 („suppressor of cytokine signaling 2“) zur genaueren Analyse ausgewählt. Die Hypothese einer positiven Regulatorfunktion von OPG in den pankreatischen β-Zellen konnte zunächst durch die Feststellung einer zeitlichen Korrelation zwischen der β-zellspezifischen OPG-Expression und den kompensatorischen β-Zellveränderungen während der murinen Schwangerschaft bekräftigt werden. In vitro-Versuche an den Insulinomzelllinien Ins-1E und MIN6 sowie an isolierten C57BL/6-Pankreasinseln ergaben, dass das Sekretionsprotein OPG keinen signifikanten Einfluss auf die glukosestimulierte Insulinsekretion und Proliferation von β-Zellen ausübt. OPG wird jedoch durch Zytokine in Ins-1E-Zellen induziert und schützt diese Zellen partiell vor einer IL-1β-induzierten Apoptose. Somit kann eine Rolle von OPG als autokriner Überlebensfaktor pankreatischer β-Zellen postuliert werden. Dieser protektive Effekt geschieht vermutlich unabhängig von den klassischen OPG-Liganden RANKL und TRAIL über eine Inhibierung der IL-1β-induzierten MAP-Kinasen JNK1/2, p38 und ERK1/2. Die physiologische Funktion von SOCS2 wurde sowohl in vivo an SOCS2-Knockout-Mäusen als auch in vitro an Ins-1E-Zellen und isolierten Pankreasinseln untersucht. Dabei konnte kein signifikanter Einfluss von SOCS2 auf die GH-induzierte β-Zellproliferation und die zytokininduzierte Apoptose der β-Zellen demonstriert werden. Unter Anwendung glukosestimulierter Insulinsekretionsversuche und Glukosetoleranztests wurde des Weiteren nachgewiesen, dass SOCS2 weder in vitro noch in vivo die Funktion von β-Zellen signifikant beeinflusst. Auch ein Effekt von SOCS2 auf die Insulinsensitivität konnte an SOCS2-Knockout-Mäusen ausgeschlossen werden. Die erhaltenen Ergebnisse zeigen, dass SOCS2 - im Gegensatz zu SOCS1 und SOCS3 - keine nicht-kompensierbaren Effekte auf die Physiologie pankreatischer β-Zellen und auf den Glukosemetabolismus ausübt. Erklärt werden kann dies wahrscheinlich durch die hohe Redundanz innerhalb der SOCS-Proteinfamilie. Zusammenfassend tragen die in dieser Arbeit gewonnenen Ergebnisse zu einem besseren Verständnis der pankreatischen β-Zellphysiologie und der Pathogenese des Typ-2-Diabetes mellitus bei und ermöglichen es dadurch, die diesbezügliche Entwicklung präventiver und therapeutischer Maßnahmen voranzutreiben.

Hypertone Kochsalzlösung können die Adhärenz von neutrophilen Granulozyten and das Endothel verhindern und auf diese Weise das Gewebe vor Ischämie bedingten Zellschäden schützen. In dieser Arbeit wurde hypertone Natriumchloridlösung mit anderen hypertonen Flüssigkeiten (Cholinchlorid bzw. Mannit) verglichen. Nur für die hypertone Natriumchloridlösung konnte ein Hemmeffekt auf die fMLP-induzierte numerische und funktionelle Hochregulation der ß2-Integrine gezeigt werden. Weiterhin wurde untersucht welche Effekte hypertone NaCl bzw. ChCl-Lösungen auf die bindungsfähigkeit des fMLP and dessen Rezeptor besitzt, sowie die Wirkung dieser Flüssigkeiten auf die Signalwege der PKC, der Calcium/Kalmodulin-Kinase sowie der MAPKinase p38 und ERK1/2. Es wurden ebenfalls die Effekte dieser Lösungen auf das Zellvolumen analysiert und die Wirkung von Amilorid auf die fMLP induzierte Hochregulation der ß2-Integrine untersucht.

This thesis applies quantitative mass spectrometry to research topics in relation to cancer. Proteome-wide quantification at the protein expression level and phosphorylation level were achieved. The technologies developed and used here cover the latest improvements in instrumentation in mass spectrometry, strategies in phosphopeptide enrichment in large scale, algorithms in data analysis and their streamlined implementation, and data mining in downstream bioinformatics. For each of the projects described in this thesis, proteome mapping routinely resulted in identification and quantitation of around 4,000 proteins and phosphoproteome mapping often lead to quantitation of more than 5,000 phosphorylation sites. This ‘systems-wide’ quantitation of the proteome and phosphoproteome is a completely novel development, which has not been used in cancer related topics before. Three major biology topics are studied in this thesis. In the first project, the phosphoproteome of a mouse liver cancer cell line Hepa1-6 was analyzed in-depth, by using phosphatase inhibitors (calyculin A, deltamethrin, and Na-pervanadate) to boost phosphorylation. The characterization of the phosphoproteome revealed a broad spectrum of cellular compartmentalization and biological functions. Quantitation of phosphatase inhibitor treatment using the Stable Isotope Labeling by Amino Acids in Cell culture (SILAC) method revealed the quantitative effects of these inhibitor compounds on the whole phosphoproteome. To our surprise, these three broadband phosphatase inhibitors displayed very different efficiency, with tyrosine phosphorylation significantly boosted but serine/threonine phosphorylation much less affected. Additionally, a method to estimate an upper bound of the stoichiometry of phosphorylation was introduced by comparing phosphorylation in three SILAC conditions: non-treated cells, stimulated cells (e.g. with insulin), and only phosphatase inhibitor treated cells. The methods developed here can be used directly in development of drugs directed against kinases and phosphatases, key regulators in cancer and other diseases. The second project continues with the application of phosphoproteomics techniques. Kinase inhibitors influence cellular signal transduction processes and therefore are of great potential in rescuing aberrant cellular signaling in tumors. In fact they constitute a significant portion of drug developing programs in pharmaceutical industry. With the aim of quantifying the effect of kinase inhibitors over the entire signaling network, the second project first set out to study two very commonly used kinase inhibitor compounds for MAPKs: U0126 and SB202190. Their effect on epidermal growth factor (EGF) signal transduction was quantified and compared using the HeLa cell system. The study confirmed that the MAPK cascades are the predominant signaling branches for propagating the EGF signaling at early time points of stimulation. These large scale examinations also suggest that U0126 and SB202190 are quite specific inhibitors for MAPKs as the majority of regulated phosphopeptides appears to belong to the MAPK pathways. In the second part of the project, the effect on phosphoproteome changes of the chemical compound dasatinib, which was demonstrated to effectively inhibit the constitutively activated fusion protein BCR-ABL and was recently approved for chronic myelogenous leukemia (CML) therapy, was quantified in the human CML cell line K562. Bioinformatic analysis revealed that the most influenced signal transduction branch was the Erk1/2 cascade. Overall more than 500 phosphorylation sites were found to be regulated by dasatinib, the vast majority not described in the literature yet. The third project compared the proteomes of mouse hepatoma cell line Hepa1-6 with the non-transformed mouse primary hepatocytes. This was performed by combining the SILAC heavy labeled form of Hepa1-6 with the primary hepatocytes. To characterize the features of these two proteomes, quantitation information (i.e. protein ratios between the two cell types) was used to divide all proteins into five quantiles. Each quantile was clustered according to the Gene Ontology and KEGG pathway databases to assess their enriched functional groups and signaling pathways. To integrate this information at a higher level, hierarchical clustering based on the p-value from the first Gene Ontology and KEGG clustering was performed. Using this improved bioinformatic algorithm for data mining, the proteomic phenotypes of the primary cells and transformed cells are immediately apparent. Primary hepatocytes are enriched in mitochondrial functions such as metabolic regulation and detoxification, as well as liver functions with tissue context such as secretion of plasma and low-density lipoprotein (LDL). In contrast, the transformed cancer cell line Hepa1-6 is enriched in cell cycle and growth functions. Interestingly, several aspects of the molecular basis of the “Warburg effect” described in many cancer cells became apparent in Hepa1-6, such as increased expression of glycolysis markers and decreased expression of markers for tricarboxylic acid (TCA) cycle. Studies in this thesis only provide examples of the application of mass spectrometry-based quantitative proteomics and phosphoproteomics in cancer research. The connection to clinical research, especially the assessment of drug effects on a proteome wide scale, is a specific feature of this thesis. Although this development is only in its infancy, it reflects a trend in the quantitative mass spectrometry field. We believe that more and more clinical related topics can and will be studied by these powerful methods.

The Nbs1 protein (nibrin, p95) is a member of the DNA repair/checkpoint complex Mre11/Rad50/Nbs1 (MRN), which plays a critical role in the cellular responses to DNA damage, cell cycle checkpoints, and telomere and genome stability. Many transgenic models in mice and clinical symptoms of NBS patients have clearly shown that Nbs1 exerts pleiotropic actions in growth and development of mammals. However, the molecular role of Nbs1 in mitogenic signaling pathways which could explain the growth retardation, developmental defects and impaired proliferation capacity of NBS patient cells has not been demonstrated, so far. This study shows that after repression of endogenous Nbs1 levels using short interference RNA, hTERT-immortalized RPE cells exhibit decreased proliferation ability and poor response to IGF-1 stimulation. After release from G1 arrest, NBS1 siRNA-transfected cells display disturbances in periodical oscillations of cyclin E and A, and delayed cell cycle progression. Remarkably, lower phosphorylation levels of c-Raf, and diminished activity of ERK1/2 in response to IGF-1 suggest a link between NBS1, IGF-1 signaling, and Ras/Raf/MEK/ERK cascade. The functional relevance of NBS1 in mitogenic signaling and initiation of cell cycle progression are demonstrated in NBS1 siRNA-transfected cells where IGF-1 has a limited capacity to induce expressions of FOS and CCND1. The impact of NBS1 on the IGF-1 signaling cascade is finally identified by the reduction of IGF1R, SOS1 and SOS2 expression in NBS1 siRNA-transfected cells. The disturbed IGF-1 signaling, a consequence of diminished expression of the key components of the cascade, results in a failure of IGF-1 to rescue NBS1 siRNA-transfected cells from gamma radiation-induced cell death. In conclusion, this study provides the first evidence that, by modulating the IGF-1 signaling cascade, NBS1 has a functional role in the promotion of cell cycle progression, cell proliferation, and cellular radio-resistance in addition to its well known function for proper DNA double strand break signaling.

Endothelial cell survival is indispensable to maintain endothelial integrity and initiate new vessel formation. We investigated the role of SHP-2 in endothelial cell survival and angiogenesis in vitro as well as in vivo. SHP-2 function in cultured human umbilical vein and human dermal microvascular endothelial cells was inhibited by either silencing the protein expression with antisense-oligodesoxynucleotides or treatment with a pharmacological inhibitor (PtpI IV). SHP-2 inhibition impaired capillary-like structure formation (p < 0.01; n = 8) in vitro as well as new vessel growth ex vivo (p < 0.05; n = 10) and in vivo in the chicken chorioallantoic membrane (p < 0.01, n = 4). Additionally, SHP-2 knock-down abrogated fibroblast growth factor 2 (FGF-2)-dependent endothelial proliferation measured by MTT reduction ( p ! 0.01; n = 12). The inhibitory effect of SHP-2 knock-down on vessel growth was mediated by increased endothelial apoptosis ( annexin V staining, p ! 0.05, n = 9), which was associated with reduced FGF-2-induced phosphorylation of phosphatidylinositol 3-kinase (PI3-K), Akt and extracellular regulated kinase 1/2 (ERK1/2) and involved diminished ERK1/2 phosphorylation after PI3-K inhibition (n=3). These results suggest that SHP-2 regulates endothelial cell survival through PI3-K-Akt and mitogen-activated protein kinase pathways thereby strongly affecting new vessel formation. Thus, SHP-2 exhibits a pivotal role in angiogenesis and may represent an interesting target for therapeutic approaches controlling vessel growth. Copyright (C) 2007 S. Karger AG, Basel.

Endothelial cell survival is indispensable to maintain endothelial integrity and initiate new vessel formation. We investigated the role of SHP-2 in proliferation, survival and sprouting of human microvascular- and umbilical vein endothelial cells (HMEC, HUVEC) using antisense oligonucleotides (AS-ODN) and a pharmacological SHP-2 inhibitor (PtpI IV). Knock-down of SHP-2 decreased bFGF and PDGF dependent endothelial cell proliferation (p

Regulation of adenylate cyclases and extracellular signal-regulated protein kinases by delta-opioid receptors in HEK293 cells Stimulation of G protein coupled opioid receptors result in both inhibition of adenylate cyclases and stimulation of extracellular signal-regulated protein kinases ERK1/2. As regulation of cellular effectors may be accomplished by various G proteins as well as by the different G protein subunits (G alpha, G betagamma), delta-opioid receptors were thus examined for activating different G proteins underlying different regulation of these cellular effectors. In transfected HEK293 cells, activation of delta-opioid receptors by peptidergic opioids (DADLE, DPDPE) and alkaloids (etorphine, morphine) brought about concentration-dependent inhibition of adenylate cyclases and stimulation of the ERKs, respectively. Since the high-affinity opioids DADLE, DPDPE and etorphine accomplished regulation of respective effector molecules already at nanomolar ligand concentrations, a 1000-fold higher dose of low-affinity agonist morphine was required for both inhibition of adenylate cyclases and ERK activation. However, for all tested opioids, a higher EC50 could have been determined for inhibition of adenylate cyclases than for stimulation of the ERKs. Thus, adenylate cyclases expressed in HEK cells seems to be more sensitive to delta-opioid receptor activation than the ERKs. As previously shown, exposure of HEK-DOR cells to pertussis toxin (PTX) resulted in incomplete inhibition of adenylate cyclases by DADLE and DPDPE, whereas etorphine and morphine totally lost their ability to inhibit the cyclases under these conditions. In contrast, activation of ERKs by all tested opioids was abolished by PTX treatment. However, PTX also blocked ERK activation by Gq-coupled receptors and receptor tyrosine kinases, both regulating ERKs independent from PTX-sensitive Gi proteins. Thus, PTX is suggested to inhibit ERK activation also independent from affecting G protein activation. Since inhibition of G alpha q subunits by the G alpha q-binding protein EBP50 did not affect effector regulation, inhibition of G alpha q and G alpha 12 mediated signaling by neomycin and 1-butanol brought about partial blockade of ERK activation by all tested opioids. Exposure of HEK-DOR cells with neomycin and 1-butanol together even totally blocked ERK activation by respective opioids. In contrast, inhibition of G alpha 11 signaling partially blocked inhibition of adenylate cyclases by DADLE and DPDPE, whereas regulation of the cyclases by the alkaloids was not affected under this condition. Since inhibition of G betagamma signaling by phosducin did not affect regulation of adenylate cyclases and ERKs by opioids, delta-opoioid receptors are supposed to regulate these cellular effectors by G alpha subunits. Although the tested cellular effectors are regulated differently, inhibition of adenylate cyclases seems to support activation of ERKs, since simultaneous stimulation of the cyclases by forskolin impairs ERK activation by DPDPE and etorphin. In contrast, activation of ERKs did not affect regulation of the cyclases by delta-opioid receptors. Together the findings let suppose that different G alpha subunits might be involved in regulation of adenylate cyclases and ERKs by delta-opioid receptors. Since stimulation of delta-receptors might be supposed to bring about inhibition of adenylate cyclases by G alpha i and G alpha 11 subunits, alkaloids seems to regulate cyclases by G alpha i subunits. In contrast, both peptide and alkaloid opioids seem to stimulate ERKs by G alpha 11- and G alpha 12-mediated signaling.

In dieser Arbeit wurde die biologische Funktion der PTP-Meg2 in der zellulären Signaltransduktion untersucht. Analysen mittels c-DNA-Filter, „Real Time PCR” und Immunblot zeigen eine ubiquitäre Expression der PTP-Meg2 auf ähnlichem, jedoch geringem Niveau in fast allen untersuchten Krebszelllinien unterschiedlicher Gewebeherkunft, wobei die Expressions-stärke nicht in direktem Zusammenhang mit krebsrelevanten Eigenschaften wie Invasivität und Metastasierung steht. Die induzierte Differenzierung von MCF 7-Zellen durch Natriumbutyrat steigert die Meg2-Expression um das 5-fache, wogegen die Differenzierung von SW948- und SK-N- SH-Zellen mit TPA bzw. Retinolsäure die Meg2-Expression reprimiert. Zellfraktionierung und Immunfluoreszenz zeigen eine primär zytosolische, aber partiell auch vesikuläre bzw. strukturierte Lokalisation der PTP-Meg2, für welche die CRALBP-Domäne der PTP-Meg2 mitverantwortlich ist. Untersuchungen der endogenen Meg2-Aktivität nach Immunpräzipitation und in vitro Phosphatasetests zeigen eine erhöhte Phosphataseaktivität nach Stimulation von Zellen mit FCS, EGF und LPA, wogegen TPA stark inhibierenden wirkt. Aktivitätsstudien mit GST-Meg2-Fusionsproteinen zeigen, dass die CRALBP-Domäne die Meg2-Phosphataseaktivität negativ reguliert. Im Protein-Lipid-Overlay interagiert PTP-Meg2 mit PI(3)P, PI(4)P, PI(5)P und Phosphatidylserin. Eine Interaktion mit PI(4)P führt zu einer erhöhten Meg2 Aktivität. Pervanadat-Stimulation von Zellen führt zu einer Tyrosinphosphorylierung sowie einer Mobilitätsänderung der PTP-Meg2, was auch mit einer katalytisch inaktiven Meg2-Mutante beobachtet wurde. PTP-Meg2 interagiert in vitro und in Koexpressionsstudien mit dem EGF-Rezeptor in Abhängigkeit von dessen Aktivierung. Eine physiologische Relevanz konnte nicht gezeigt werden. Die Depletion der PTP-Meg2 durch spezifische siRNA führt zu einer erhöhten Tyrosinphosphorylierung einiger, noch zu identifizierender Proteine. PTP-Meg2 vermindert, die inaktive PTP-Meg2CS-Mutante erhöht die durch v-ErbB und EGF-Rezeptor, nicht aber die durch HER2 und v-Ki-Ras induzierte Transformation von NIH3T3-Zellen im Focusbildungstest. Zudem bewirkt PTP-Meg2CS, mit Ausnahme der v-Ki-Ras infizierten Zellen, eine leicht erhöhte ERK1/2-Aktivität. Ferner stimuliert PTP-Meg2 die Migration von NIH3T3-Zellen im Wundheilungsexperiment. Ein Einfluss auf die basale und durch Stimuli induzierte Proliferation von Zellen in Wachstumstests wurde nicht beobachtet. Ein durch siRNA-vermittelter Meg2- „knockdown“ führte zur Induktion bzw. Repression der Expression von Genen, wie z.B. einiger Liganden, Caveolin-2, Nck und Rock, was auf eine Beteiligung der PTP-Meg2 an der Regulation von Signalwegen kleiner GTPasen bzw. von endo- sowie exocytotischen Prozessen schließen lässt.

Die vorliegende Arbeit wurde durch den SFB 369 (Teilprojekt B7) unterstützt. Sie sollte auf Basis der bereits vorliegenden Erkenntnisse klären, welcher Mechanismus der Apoptoseinduktion durch Ajoen in HL-60 Zellen zu Grunde liegt. Dirsch et al. 5 zeigten bereits 1998, dass Ajoen Apoptose in humanen akut-myeloischen Leukämiezellen (HL-60) induziert. Weiterhin zeigte sich eine dosis- und zeitabhängige Produktion der reaktiven Sauerstoffspezies (ROS). N-Acetylcystein (NAC), ein Antioxidans, verhinderte partiell die Ajoeninduzierte ROS-Produktion und die Apoptose. Auf dieser Grundlage konnten folgende Ergebnisse erarbeitet werden: 1) Ajoen verursacht in HL-60 Zellen die Aktivierung der Caspase-3 sowie der Caspase-8. Eine generelle Aktivierung von Caspasen ist für die Ajoen-induzierte Apoptose nötig, da der Breitbandcaspaseinhibitor z-VAD-fmk die durch Ajoen provozierte DNA-Fragmentation völlig verhindert. 2) Die Ajoen-induzierte Apoptose wird nicht durch den Todesrezeptor CD95 vermittelt. Dafür sprechen folgende Ergebnisse: a) Unsere HL-60 Zellen exprimieren den CD95-Rezeptor, jedoch kann der natürliche CD95-Ligand (CD95L) keine Apoptose hervorrufen. Wahrscheinlich ist der CD95-Rezeptor inaktiv. b) Außerdem kann für die Caspase-8, die für die Signalweiterleitung vom CD95-Rezeptor u.a. mit verantwortlich ist, durch Einsatz eines spezifischen Caspase-8 Inhibitors keine Bedeutung für die Ajoeninduzierten Apoptose gezeigt werden. c) CD95-resistente Jurkat Zellen (JurkatR) sind auf Ajoen genauso empfindlich wie die Kontrollzellen. 3) Es konnte bewiesen werden, dass Ajoen Apoptose über den mitochondrialen Signalweg induziert: a) Ajoen verursacht sowohl den Verlust des mitochondrialen Membranpotentials der inneren Membran als auch eine Cytochrom c- Freisetzung aus dem intermembranären Spalt. b) Die Ajoen-induzierte Apoptose hängt von der provozierten mitochondrialen Dysfunktion ab: HL-60 Zellen, die das anti-apoptotische Protein Bcl-xL überexprimieren, sind vor Apoptose geschützt. c) Die höhere Sensitivität der HL-60/neo bzw. die niedrigere Sensitivität der HL-60/bcl-xL Zellen auf Ajoen konnte auch morphologisch durch TEM-Untersuchungen untermauert werden. Zusammenfassend konnte also für die Ajoen-induzierte Apoptose eine von Mitochondrien abhängige Signalweiterleitung gezeigt werden. 4) Untersuchungen zur Frage, wie es zur Ajoen-induzierten mitochondrialen Dysfunktion kommt, brachten folgende Erkenntnisse: a) Eine Aktivierung von Caspasen ist für die Auslösung der mitochondrialen Ereignisse nicht notwendig. Die abgeschwächte und verzögerte Caspaseaktivierung in HL-60/bcl-xL Zellen beweist: Caspasen werden „downstream“ der mitochondrialen „Aktivierung“ gespalten. b) Die ROS-Entstehung ist ein Ereignis vor („upstream“) der mitochondrialen Dysfunktion. c) Die folgerichtige Untersuchung der MAPK JNK, p38 und ERK1/2 (die Aktivierung des Transkriptionsfaktors AP-1 war bereits bekannt), ergab deren Aktivierung, jedoch ist diese für die Signalvermittlung nicht nötig. Nur für die ERK1/2 Kinase konnte eine direkte Beteiligung, und zwar als „survival“-Faktor, festgestellt werden, während die Akt als „survival“- Faktor keine Bedeutung hat. Ergebnisübersicht: CD95 Caspase-8 ROS p 38 MAPK ERK1/2 JNK Akt DNA- Fragmentierung Apoptose Caspase-3 Caspase-8 Aktivierung Caspase-3- ähnlicherCaspasen z-VAD-fmk Cytochrom c- Freisetzung z-VAD-fmk AP-1 Extrinsischer intrinsischer Signalweg Signalweg Bcl-xL Ajoen Abb. 61: Ergebnisübersicht Rot:-- Involvierung im Signalweg, blau:-- kein kausaler Zusammenhang gegeben; –I = Hemmung.