Podcasts about overexpression

This month on Episode 71 of Discover CircRes, host Cindy St. Hilaire highlights three articles featured in the March 28th and April 11th issues of Circulation Research. This Episode also includes a discussion with Dr Magali Noval Rivas and Dr Prasant Jena from Cedars-Sinai Medical Center about their study, Intestinal Microbiota Contributes to the Development of Cardiovascular Inflammation and Vascular in Mice.   Article highlights: Han, et al. PRL2 Dephosphorylates AMPKα2 in Heart Perelli, et al. TAX1BP3 and Arrhythmogenic Cardiomyopathy Lalaguna, et al. Overexpression of Wild Type TMEM43 Improves ARVC5 Liu, et al. NR4A1 Inhibits Platelet Function

Featuring perspectives from Dr Justin F Gainor and Dr Karen Reckamp, including the following topics: Introduction: Bringing Research into Practice (0:00) EGFR Activating Mutations (14:01) Exon 20 Insertion Mutations (38:06) MET Exon 14 Alterations (41:49) HER2 Mutations and Overexpression (50:27) CME information and select publications

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.18.549510v1?rss=1 Authors: Fletcher-Jones, A., Spackman, E., Craig, T. J., Nakamura, Y., Wilkinson, K. A., Henley, J. M. Abstract: Endocannabinoid signalling mediated by cannabinoid type 1 receptors (CB1Rs) is critical for homeostatic neuromodulation of both excitatory and inhibitory synapses. This requires highly polarised axonal surface expression of CB1R, but how this is achieved remains unclear. We previously reported that the H9 domain in the intracellular C-terminus of CB1R contributes to polarised surface expression by an unknown mechanism. Here we show the H9 domain binds to the endocytic adaptor protein SGIP1 to promote CB1R expression in the axonal membrane. Overexpression of SGIP1 increases CB1R axonal surface localisation but has no effect on CB1R lacking the H9 domain (CB1R{Delta}H9). Conversely, SGIP1 knockdown reduces axonal surface expression of CB1R but does not affect CB1R{Delta}H9. Furthermore, SGIP1 knockdown diminishes CB1R-mediated inhibition of presynaptic Ca2+ influx in response to neuronal activity. Together, these data advance mechanistic understanding of endocannabinoid signalling by demonstrating that SGIP1 interaction with H9 underpins axonal CB1R surface expression to regulate presynaptic responsiveness. 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/2023.07.15.549173v1?rss=1 Authors: Kumar, V., Bermea, K. C., Kumar, D., Singh, A., Verma, A., Kaileh, M., Sen, R., Lakatta, E. G., Adamo, L. Abstract: Mice with cardiac-specific overexpression of adenylyl cyclase (AC) type 8 (TGAC8) are under a constant state of severe myocardial stress and have been shown to have a remarkable ability to adapt to this stress. However, they eventually develop accelerated cardiac aging and cardiac fibrosis, and experience reduced longevity. Here we show that young (3-month-old) TGAC8 animals are characterized by a broad and extensive inflammatory state, that precedes the development of cardiac fibrosis. We demonstrate that activation of ACVIII in the cardiomyocytes results in cell-autonomous RelA-mediated NF-{kappa}B signaling. This is associated with non-cell-autonomous activation of proinflammatory and age-associated signaling in myocardial endothelial cells, increases in serum levels of inflammatory cytokines, changes in myocardial immune cells, and changes in the size or composition of lymphoid organs. Finally, we provide evidence suggesting that ACVIII-driven RelA activation in cardiomyocytes might be mediated by calcium-Protein Kinase A (PKA) signaling. Our findings highlight a novel mechanistic connection between cardiomyocyte stress, myocardial para-inflammation, systemic inflammation, and aging, and therefore point to novel potential therapeutic targets to reduce age-associated myocardial deterioration. 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/2023.07.09.548214v1?rss=1 Authors: Crowther, A., Kashem, S., Jewell, M. E., Chang, H. L., Casillas, M. R., Midavaine, E., Rodriguez, S., Braz, J., Kania, A., Basbaum, A. Abstract: Mouse models that combine tetracycline-controlled gene expression systems and conditional genetic activation can tightly regulate transgene expression in discrete cell types and tissues. However, the commonly used Tet-Off variant, tetracycline transactivator (tTA), when overexpressed and fully active, can lead to developmental lethality, disease, or more subtle behavioral phenotypes. Here we describe a profound itch phenotype in mice expressing a genetically encoded tTA that is conditionally activated within the Phox2a lineage. Phox2a; tTA mice develop intense, localized scratching and regional skin lesions that can be controlled by the tTA inhibitor, doxycycline. As gabapentin, but not morphine, completely relieved the scratching, we consider this phenotype to result from chronic neuropathic itch, not pain. In contrast to the Phox2a lineage, mice with tTA activated within the Phox2b lineage, which has many similar areas of recombination within the nervous system, did not recapitulate the scratching phenotype. In Phox2a-Cre mice, but not Phox2b-Cre, intense Cre-dependent reporter expression was found in skin keratinocytes localized to the area of scratching-induced skin lesions. Most interestingly, topical application of the DREADD agonist, CNO, administered repeatedly over two months, which chronically induced Gi signaling in keratinocytes, completely reversed the localized scratching and skin lesions. Furthermore, ablation of TRPV1-expressing, primary afferent neurons reduced the scratching with a time course comparable to that produced by Gi-DREADD inhibition. These temporal properties suggest that the neuropathic itch condition arises not only from localized keratinocyte activation of peripheral nerves but also from a persistent, gabapentin-sensitive state of central sensitization. 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/2023.06.24.546411v1?rss=1 Authors: Yang, N. V., Rogers, S., Guerra, R., Pagliarini, D. J., Theusch, E., Krauss, R. M. Abstract: Background: Statins are the drugs most commonly used for lowering plasma low-density lipoprotein (LDL) cholesterol levels and reducing cardiovascular disease risk. Although generally well tolerated, statins can induce myopathy, a major cause of non-adherence to treatment. Impaired mitochondrial function has been implicated as a cause of statin-induced myopathy, but the underlying mechanism remains unclear. We have shown that simvastatin downregulates transcription of TOMM40 and TOMM22, genes that encode major subunits of the translocase of outer mitochondrial membrane (TOM) complex which is responsible for importing nuclear-encoded proteins and maintaining mitochondrial function. We therefore investigated the role of TOMM40 and TOMM22 in mediating statin effects on mitochondrial function, dynamics, and mitophagy. Methods: Cellular and biochemical assays and transmission electron microscopy were used to investigate effects of simvastatin and TOMM40 and TOMM22 expression on measures of mitochondrial function and dynamics in C2C12 and primary human skeletal cell myotubes. Results: Knockdown of TOMM40 and TOMM22 in skeletal cell myotubes impaired mitochondrial oxidative function, increased production of mitochondrial superoxide, reduced mitochondrial cholesterol and CoQ levels, disrupted mitochondrial dynamics and morphology, and increased mitophagy, with similar effects resulting from simvastatin treatment. Overexpression of TOMM40 and TOMM22 in simvastatin-treated muscle cells rescued statin effects on mitochondrial dynamics, but not on mitochondrial function or cholesterol and CoQ levels. Moreover, overexpression of these genes resulted in an increase in number and density of cellular mitochondria. Conclusion: These results confirm that TOMM40 and TOMM22 are central in regulating mitochondrial homeostasis and demonstrate that downregulation of these genes by statin treatment mediates disruption of mitochondrial dynamics, morphology, and mitophagy, effects that may contribute to statin-induced myopathy. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

A new research paper was published in Oncotarget's Volume 14 on April 10, 2023, entitled, “A phase I trial of riluzole and sorafenib in patients with advanced solid tumors: CTEP #8850.” Overexpression of metabotropic glutamate receptor 1 (GRM1) has been implicated in the pathogenesis of multiple cancers. Riluzole, an inhibitor of glutamate release, showed synergistic antitumor activity in combination with the multi-kinase inhibitor sorafenib in preclinical models. In a new phase I trial, researchers Kristen R. Spencer, Daniella E. Portal, Joseph Aisner, Mark N. Stein, Jyoti Malhotra, Weichung Shih, Nancy Chan, Ann W. Silk, Shridar Ganesan, Susan Goodin, Murugesan Gounder, Hongxia Lin, Jiadong Li, Robert Cerchio, Christina Marinaro, Suzie Chen, and Janice M. Mehnert from Rutgers University, Dana-Farber Cancer Institute, and the Perlmutter Cancer Center of NYU Langone Health identified the toxicity profile, dose-limiting toxicities, maximum tolerated dose (MTD), and pharmacokinetic and pharmacodynamic properties of riluzole combined with sorafenib in patients with advanced cancers. “Riluzole functions as an inhibitor of GRM1 signaling through antagonism of glutamate release, and sorafenib is a multi-kinase inhibitor targeting both the MAPK and PI3K/AKT pathways through the inhibition of RAF1, ARAF and, to a lesser extent BRAF, as well as a set of tyrosine kinases including VEGFR. Our phase I study determined the tolerable dose of this combination and investigated its biologic effects.” Patients with refractory solid tumors were enrolled utilizing a 3+3 dose-escalation design. Riluzole was given at 100 mg PO BID in combination with sorafenib, beginning at 200 mg PO daily and escalating in 200 mg increments per level in 28-day cycles. Restaging evaluations were performed every 2 cycles. In total, 35 patients were enrolled over 4 dose levels. The MTD was declared at dose level 3 (riluzole: 100 mg PO BID; sorafenib: 400 mg AM/200 mg PM). Pharmacokinetic analyses did not reveal definitive evidence of drug-drug interactions. Consistent decreases in phospho-forms of ERK and AKT in tumor tissue analyses with accompanying decrease in GRM1 expression and increase in pro-apoptotic BIM suggest target engagement by the combination. Best responses included a partial response in 1 (2.9%) patient with pancreatic acinar cell carcinoma with a KANK4-RAF1 fusion, and stable disease in 11 (36%) patients. “Combination therapy with riluzole and sorafenib was safe and tolerable in patients with advanced solid tumors. The partial response in a patient with a RAF1 fusion suggests that further exploration in a genomically selected cohort may be warranted.” DOI: https://doi.org/10.18632/oncotarget.28403 Correspondence to: Janice M. Mehnert - Janice.Mehnert@nyulangone.org Sign up for free Altmetric alerts about this article - https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.28403 Subscribe for free publication alerts from Oncotarget - https://www.oncotarget.com/subscribe/ Keywords - GRM1, riluzole, sorafenib, phase I, clinical trial About Oncotarget Oncotarget is a primarily oncology-focused, peer-reviewed, open access journal. Papers are published continuously within yearly volumes in their final and complete form, and then quickly released to Pubmed. On September 15, 2022, Oncotarget was accepted again for indexing by MEDLINE. Oncotarget is now indexed by Medline/PubMed and PMC/PubMed. To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - 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/ MEDIA@IMPACTJOURNALS.COM

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.04.08.535951v1?rss=1 Authors: Ling, Y. H., Chen, Y. Y., Leung, K. N., Chan, K. M., Liu, W. K. Abstract: CCHCR1 (coiled-coil alpha-helical rod protein 1) is a candidate gene of psoriasis and was recently identified as a novel component in P-bodies, the site for regulating mRNA turnover, and a protein in centrosomes. Little is known about its transcriptional regulation or its functions in cell cycle progression. In this study, CCHCR1 was found in a close (287-bp) head-to-head orientation with its neighboring gene, TCF19. The transcription of the CCHCR1/TCF19 gene pair was controlled by a shared bidirectional promoter and was induced at the G1/S transition of the cell cycle. The 287-bp intergenic sequence was sufficient for the G1/S expression of both genes, but the expression of CCHCR1 was further promoted by the presence of exon 1 of TCF19 and CCHCR1. The expression of the CCHCR1/TCF19 gene pair was dependent on the E2F1 transcription factor. E2F1 binding sites were predicted in the CCHCR1/TCF19 bidirectional promoter by in silico analysis. Overexpression of E2F1 induced the expression of CCHCR1 and TCF19. In addition, E2F1 knockdown inhibited both CCHCR1 and TCF19 expression. Knockdown of CCHCR1 or TCF19 reduced cell count but only depletion of CCHCR1 significantly induced p21 expression, implying that CCHCR1 and TCF19 may both regulate cell growth but in divergent pathways. Taken together, we revealed a bidirectional regulation of the CCHCR1/TCF19 gene pair in the G1/S transition and provide a new perspective to understand the role of CCHCR1 as a candidate gene of psoriasis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Dr. Claudia Benavente studied Molecular Biotechnology Engineering at Universidad de Chile where her interest in pursuing cancer research first started. To further her studies, she came to the US to pursue a doctoral degree in Cancer Biology at The University of Arizona as a Fulbright scholar. She then moved on as a postdoctoral fellow to St. Jude Children's Research Hospital in Memphis, TN where she became familiar with childhood solid tumors. Professor Benavente is currently an Associate Professor at the Departments of Pharmaceutical Sciences and Developmental and Cell Biology and a member of the Chao Family Comprehensive Cancer Center at the University of California, Irvine. Dr. Benavente's research focuses on understanding how pediatric tumors form to design new ways to treat them. Her research aims to understand how genes are normally controlled in developing tissues and how epigenetic processes are perturbed to facilitate cancers to arise. This information guides her in the development of new therapies. --- What We Do at MIB Agents: PROGRAMS: ✨ End-of-Life MISSIONS ✨ Gamer Agents ✨ Agent Writers ✨ Prayer Agents ✨ Healing Hearts - Bereaved Parent Support ✨ Ambassador Agents - Peer Support ✨ Warrior Mail ✨ Young Adult Survivorship Support Group ✨  EDUCATION for physicians, researchers and families: ✨ OsteoBites, weekly webinar & podcast with thought leaders and innovators in Osteosarcoma ✨ MIB Book: Osteosarcoma: From our Families to Yours ✨ RESEARCH: Annual MIB FACTOR Research Conference ✨ Funding $100,000 annually for OS research ✨ MIB Testing & Research Directory ✨ The Osteosarcoma Project partner with Broad Institute of MIT and Harvard ... Kids are still dying with 40+ year old treatments. Help us MakeItBetter. 

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.31.535068v1?rss=1 Authors: Shi, Q., Zheng, L. Abstract: Long-term space flight will lead to bone loss and osteoblasts dysfunction. The underlying mechanism is still far to reveal. Frizzled-9 (Fzd9) is a Wnt receptor which is essential to osteoblasts differentiation and bone formation. Here we investigate whether Fzd9 plays a role in simulated microgravity (SMG) induced osteoblasts dysfunction. After 1-3 days of SMG, the osteogenic markers were decreased which accompanied the decline of Fzd9 expression. Fzd9 also decreased in the femur of the rats after 3 weeks of hindlimb unloading. Overexpression of Fzd9 will counteract SMG-induced osteoblasts dysfunction. However, Fzd9 overexpression did not affect SMG induced pGSK3 and -catenin expression or sublocalization. Overexpression of Fzd9 regulates the phosphorylation of Akt and ERK, as well as induces F-actin polymerization to form the actin cap, presses the nuclei, and increases the nuclear pore size, which promotes nuclear translocation of YAP. Our study provides mechanistic insights into the role of Fzd9 regulates YAP in SMG-mediated osteoblasts dysfunction and indicates Fzd9 as a potential target to restore osteoblast function in bone diseases and space flight. 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/2023.03.24.534091v1?rss=1 Authors: Krüger, J., Lerche, H. Abstract: Objective: KCNQ5 encodes the voltage-gated potassium channel KV7.5, a member of the KV7 channel family, which conducts the M-current. This current was shown to be a potent regulator of neuronal excitability by mediating the medium and slow afterhyperpolarization. Recently, we have identified five loss-of-function variants in KCNQ5 in patients with genetic generalized epilepsy. Using the most severe dominant-negative variant p.(Arg359Cys) (R359C), we set out to investigate pharmacological therapeutic intervention by KV7 channel openers on channel function and neuronal firing. Methods: Whole-cell patch clamp recordings were conducted in human embryonic kidney cells to investigate the immediate effect of retigabine, gabapentin and intracellular application of zinc on the R359C variant in absence and presence of KV7.5-WT subunits. Transfected primary hippocampal cultures were used to examine the effect of R359C on neuronal firing and whether this effect could be reversed by drug application. Results: Retigabine and gabapentin both increased R359C-derived K+ current density and M-current amplitudes in both homomeric and heteromeric mutant KV7.5 channels. Retigabine was most effective in restoring K+ currents. Ten M retigabine was sufficient to reach the level of WT currents without retigabine, whereas 100 M of gabapentin showed less than half of this effect and application of 50 M zinc only significantly increased M-current amplitude in heteromeric channels. Overexpression of KV7.5-WT potently inhibited neuronal firing by increasing the M-current, and medium afterhyperpolarization, whereas R359C overexpression had the opposite effect. All three aforementioned drugs reversed the effect of R359C reducing firing to nearly normal levels at high current injections. Significance: Our study shows that a dominant-negative complete loss-of-function variant in KV7.5 leads to largely increased neuronal firing indicating a neuronal hyperexcitability. KV7 channel openers, such as retigabine or gabapentin, could be treatment options for otherwise pharmacoresistant epilepsy patients carrying loss-of-function variants in KCNQ5. 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/2023.03.21.533604v1?rss=1 Authors: Hoyer-Fender, S., Otto, M. Abstract: Primary cilia are essential sensory organelles that develop when an inhibitory cap consisting of CP110 and other proteins is eliminated. Degradation of CP110 by the ubiquitin-dependent proteasome pathway mediated by NEURL4 and HYLS1 removes the inhibitory cap. Here, we investigated the suitability of rapamycin-mediated dimerization for centriolar recruitment and asked whether the induced recruitment of NEURL4 or HYLS1 to the centriole promotes primary cilia development and CP110 degradation. We used rapamycin-mediated dimerization with ODF2 to induce their targeted recruitment to the centriole. We found decreased CP110 levels in transfected cells, but independent of rapamycin-mediated dimerization. By knocking down ODF2, we show that ODF2 controls CP110 levels. Overexpression of ODF2 is not sufficient to promote the formation of primary cilia, but overexpression of NEURL4 or HYLS1 is. Co-expression of ODF2 and HYLS1 resulted in the formation of tube-like structures, indicating an interaction. Thus, ODF2 controls primary cilia formation by negatively regulating the concentration of CP110 levels. Our data suggest that ODF2 most likely acts as a scaffold for the binding of proteins such as NEURL4 or HYLS1 to mediate CP110 degradation. 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/2023.03.16.532898v1?rss=1 Authors: Watanuki, S., Kobayashi, H., Sugiura, Y., Yamamoto, M., Karigane, D., Shiroshita, K., Sorimachi, Y., Koide, S., Oshima, M., Nishiyama, A., Murakami, K., Miho, H., Tamaki, S., Yamamoto, T., Yabushita, T., Tanaka, Y., Honda, H., Okamoto, S., Goda, N., Tamura, T., Nakamura-Ishizu, A., Suematsu, M., Iwama, A., Suda, T., Takubo, K. Abstract: Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define metabolic differences between steady-state and stress conditions in HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of adenosine triphosphate (ATP) levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells. 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/2023.03.14.532132v1?rss=1 Authors: Downton, P., Bagnall, J. S., England, H., Spiller, D. G., Humphreys, N., Jackson, D. A., Paszek, P., White, M. R. H., Adamson, A. D. Abstract: Cells respond to inflammatory stimuli such as cytokines by activation of the nuclear factor-kappaB (NF-kappaB) signalling pathway, resulting in oscillatory translocation of the transcription factor p65 between nucleus and cytoplasm to mediate immune response. We investigate the relationship between p65 and inhibitor-kappa Balpha (IkappaBalpha) protein levels and dynamic properties of the system, and how this interaction impacts on the expression of key inflammatory genes. Using bacterial artificial chromosomes, we developed new cell models of kappaBalpha-eGFP protein overexpression in a native genomic context. We find that cells with high levels of the negative regulator IkappaBalpha remain responsive to inflammatory stimuli and maintain dynamics for both p65 and kappaBalpha. In contrast, canonical target gene expression is dramatically reduced by overexpression of IkappaBalpha, but can be partially rescued by overexpression of p65. Treatment with leptomycin B to promote nuclear accumulation of IkappaBalpha also suppresses canonical target gene expression, suggesting a mechanism in which nuclear IkappaBalpha accumulation prevents productive p65 interaction with promoter binding sites. This causes reduced target promoter binding and gene transcription, which we validate by chromatin immune precipitation and in primary cells. Overall, we show how inflammatory gene transcription is modulated by the expression levels of both IkappaBalpha and p65, and that transcription can be partially decoupled from p65 protein dynamics. This results in an anti-inflammatory effect on transcription, demonstrating a broad mechanism to modulate the strength of inflammatory response. 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/2023.03.05.531163v1?rss=1 Authors: Mascanzoni, F., Ayala, I., Iannitti, R., Luini, A., Colanzi, A. Abstract: The decision to enter mitosis requires not only the control of DNA replication but also additional and crucial preparatory steps such as, for example, partial disassembly of the Golgi complex during G2. The Golgi complex is fundamental for the processing and sorting of proteins and lipids in the secretory pathway. It is organized as stacks of cisternae laterally connected by tubules to form a continuous Golgi ribbon. During G2, the Golgi ribbon is unlinked into isolated stacks in preparation for cell division. This structural reorganization is necessary for entry into mitosis, indicating that a "Golgi mitotic checkpoint" controls the correct segregation of this organelle. To understand the physiological significance of the pre-mitotic Golgi unlinking, we devised a strategy to accumulate cells in G2 with an intact Golgi ribbon and then induce entry into mitosis. Here, we show that forcing the entry of cells into mitosis with an intact Golgi ribbon causes remarkable cell division defects, including spindle multipolarity and binucleation, favoring cell transformation. We also find that the cells entering mitosis with an intact Golgi ribbon show reduced levels at the centrosome of the kinase Aurora-A, a pivotal regulator of spindle formation. Overexpression of Aurora-A rescues spindle formation, indicating that the Golgi-dependent Aurora-A recruitment has a crucial role in spindle formation. Thus, our results show that alterations of the pre-mitotic Golgi segregation have profound consequences on the fidelity of the mitotic process, representing potential risk factors for cell transformation and cancer development. 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/2023.03.01.530580v1?rss=1 Authors: Singh, S., Gaur, A., Kumari, R., Prakash, S., Kumari, S., Chaudhary, A. D., Sharma, R. K., Prasun, P., Pant, P., Thum, T., Jagavelu, K., Bharati, P., Hanif, K., Chitkara, P., Kumar, S., Mitra, K., Gupta, S. K. Abstract: Regulation of RNA stability and translation by RNA-binding proteins (RBPs) is a crucial process altering gene expression. Musashi family of RBPs comprising Msi1 and Msi2 are known to control RNA stability and translation. However, despite the presence of MSI2 in the heart, its function remains entirely unknown. Here, we aim to explore the cardiac functions of MSI2. We confirmed the presence of MSI2 in the adult mouse, rat heart, and neonatal rat cardiomyocytes. Furthermore, Msi2 was significantly enriched in the heart's cardiomyocyte fraction. Next, using RNA-seq data and isoform-specific PCR primers, we identified, Msi2 isoforms 1, 4, and 5 and two novel putative isoforms labeled as Msi2 isoforms 6 and 7 to be expressed in the heart. Overexpression of Msi2 isoforms led to cardiac hypertrophy in cultured cardiomyocytes. Additionally, Msi2 was also found to be significantly increased in a pressure-overload model of cardiac hypertrophy. To validate the hypertrophic effects, we selected isoforms 4 and 7 due to their unique alternative splicing patterns. AAV9-mediated overexpression of Msi2 isoforms 4 and 7 in murine hearts led to cardiac hypertrophy, dilation, heart failure, and eventually early death, confirming a pathological function for Msi2. Using global proteomics, gene ontology, transmission electron microscopy, and transmembrane potential measurement assays increased MSI2 was found to cause mitochondrial dysfunction in the heart. Mechanistically, we identified Cluh and Smyd1 as direct downstream targets of Msi2. Overexpression of Cluh or Smyd1 inhibited Msi2-induced hypertrophy and mitochondrial dysfunction in cardiomyocytes. Collectively, we show that Msi2 induces hypertrophy, mitochondrial dysfunction, and heart failure. 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/2023.02.23.529672v1?rss=1 Authors: Yang, Y., Yu, C., Le, Y., Gong, W., Ju, J., Zhang, G., Ji, P., Zuo, R., Liu, Z., Zhang, P., Hou, R., Fu, Y. Abstract: Proliferation and migration of epidermal stem cells (EpSCs) are essential for epithelialization during skin wound healing. Angiopoietin-like 4 (ANGPTL4) has been reported to play an important role in wound healing, but the mechanisms involved are not fully understood. Here we investigate the contribution of ANGPTL4 to full-thickness wound re-epithelialization and the underlying mechanisms using Angptl4 knockout mice. Immunohistochemical staining reveals that ANGPTL4 is significantly upregulated in the basal layer cells of the epidermis around the wound during cutaneous wound healing. ANGPTL4 deficiency impairs wound healing. H & E staining shows that ANGPTL4 deficiency significantly reduces the thickness, length and area of regenerated epidermis postwounding. Immunohistochemical staining for markers of EpSCs (alpha 6 integrin and beta 1 integrin) and cell proliferation (PCNA) shows that the number and proliferation of EpSCs in the basal layer of the epidermis are reduced in ANGPTL4-deficient mice. In vitro studies show that ANGPTL4 deficiency impedes EpSC proliferation, causes cell cycle arrest at the G1 phase and reduced the expression of cyclins D1 and A2, which can be reversed by ANGPTL4 overexpression. ANGPTL4 deletion suppresses EpSC migration, which is also rescued by ANGPTL4 overexpression. Overexpression of ANGPTL4 in EpSCs accelerates cell proliferation and migration. Collectively, our results indicate that ANGPTL4 promotes EpSCs proliferation by upregulating cyclins D1 and A2 expression and accelerating cell cycle transition from G1 to S phase, and ANGPTL4 promotes skin wound re-epithelialization by stimulating EpSC proliferation and migration. Our study reveals a novel mechanism underlying EpSC activation and re-epithelialization during cutaneous wound healing. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

A new research paper was published in Oncotarget's Volume 14 on January 12, 2023, entitled, “Targeting CD74 in B-cell non-Hodgkin lymphoma with the antibody-drug conjugate STRO-001.” Overexpression of CD74, a type II transmembrane glycoprotein involved in MHC class II antigen presentation, has been reported in many B-cell non-Hodgkin lymphomas (NHLs) and in multiple myeloma (MM). STRO-001 is a site-specific, predominantly single-species antibody-drug conjugate (ADC) that targets CD74 and has demonstrated efficacy in xenograft models of MM and tolerability in non-human primates. In this new study, researchers Xiaofan Li, Cristina Abrahams, Abigail Yu, Millicent Embry, Robert Henningsen, Venita DeAlmeida, Shannon Matheny, Toni Kline, Alice Yam, Ryan Stafford, Trevor Hallam, Mark Lupher, and Arturo Molina from Sutro Biopharma reported the results of preclinical studies designed to elucidate the potential role of STRO-001 in B-cell NHL. “In order to explore the potential of STRO-001 in NHL, in the present study we investigated CD74 expression in cell types found in bone marrow, evaluated its cytotoxicity in NHL cell lines, and assessed its antitumor efficacy and toxicity in xenograft models of NHL.” STRO-001 displayed nanomolar and sub-nanomolar cytotoxicity in 88% (15/17) of cancer cell lines tested. STRO-001 showed potent cytotoxicity on proliferating B cells while limited cytotoxicity was observed on naïve human B cells. A linear dose-response relationship was demonstrated in vivo for DLBCL models SU-DHL-6 and U2932. Tumor regression was induced at doses less than 5 mg/kg, while maximal activity with complete cures were observed starting at 10 mg/kg. In MCL Mino and Jeko-1 xenografts, STRO-001 starting at 3 mg/kg significantly prolonged survival or induced tumor regression, respectively, leading to tumor eradication in both models. “In summary, high CD74 expression levels in tumors, nanomolar cellular potency, and significant anti-tumor in DLBCL and MCL xenograft models support the ongoing clinical study of STRO-001 in patients with B-cell NHL.” DOI: https://doi.org/10.18632/oncotarget.28341 Correspondence to: Xiaofan Li - xli@sutrobio.com, Arturo Molina - amolina@sutrobio.com Keywords: CD74, antibody-drug conjugate, non-Hodgkin lymphoma, xenograft models, STRO-001 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, visit Oncotarget.com and connect with us on social media: Twitter – https://twitter.com/Oncotarget Facebook – https://www.facebook.com/Oncotarget YouTube – https://www.youtube.com/@OncotargetJournal Instagram – https://www.instagram.com/oncotargetjrnl/ LinkedIn – https://www.linkedin.com/company/oncotarget/ Pinterest – https://www.pinterest.com/oncotarget/ LabTube – https://www.labtube.tv/channel/MTY5OA SoundCloud – https://soundcloud.com/oncotarget For media inquiries, please contact: media@impactjournals.com.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.12.20.521329v1?rss=1 Authors: Doggett, K., Keating, N., Dehkhoda, F., Bidgood, G. M., Leong, E., Kueh, A., Nicola, N. A., Kershaw, N. J., Babon, J. J., Alexander, W. S., Nicholson, S. E. Abstract: Suppressor Of Cytokine Signaling (SOCS) 1 is a critical negative regulator of cytokine signaling and required to protect against an excessive inflammatory response. Genetic deletion of Socs1 results in unrestrained cytokine signaling and neonatal lethality, characterised by an inflammatory immune infiltrate in multiple organs. Overexpression and structural studies have suggested that the SOCS1 kinase inhibitory region (KIR) and Src homology 2 (SH2) domain are important for interaction with and inhibition of the receptor-associated JAK1, JAK2 and Tyk2 tyrosine kinases, which initiate downstream signaling. To investigate the role of the KIR and SH2 domain in SOCS1 function, we independently mutated key conserved residues in each domain and analysed the impact on cytokine signaling, and the in vivo impact on SOCS1 function. Mutation of the SOCS1-KIR or SH2 domain had no impact on the integrity of the SOCS box complex, however, mutation within the phosphotyrosine binding pocket of the SOCS1-SH2 domain specifically disrupted SOCS1 interaction with phosphorylated JAK1. In contrast, mutation of the KIR did not affect the interaction with JAK1, but did prevent SOCS1 inhibition of JAK1 autophosphorylation. In human and mouse cell lines, both mutants impacted the ability of SOCS1 to restrain cytokine signaling, and crucially, Socs1-R105A and Socs1-F59A mice displayed a neonatal lethality and excessive inflammatory phenotype similar to SOCS1 null mice. This study defines a critical and non-redundant role for both the KIR and SH2 domain in endogenous SOCS1 function. 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/2022.12.15.520620v1?rss=1 Authors: Yakout, D. W., Shroff, A., Allen, Z. D., Nazarko, T. Y., Mabb, A. M. Abstract: Tauopathies are neurodegenerative disorders characterized by the deposition of aggregates of the microtubule-associated protein tau, a main component of neurofibrillary tangles. Alzheimers disease (AD) is the most common type of secondary tauopathy. While stabilizing microtubules is a well-established role for tau, tau is also localized at postsynaptic sites and can disrupt synaptic plasticity when knocked out or overexpressed. A major gap in understanding tau functions is identifying the intracellular mechanisms through which tau modulates synaptic function. Here, we found that overexpression of the 0N4R isoform of tau in HEK 293 cells decreased the stability of the activity-regulated cytoskeleton-associated protein (Arc), an immediate early gene that plays a key role in synaptic plasticity, learning and memory. Importantly, tau-induced Arc degradation was found to be isoform-specific in that overexpression of the 0N3R tau isoform had no effect. Tau-dependent reduction of Arc required proteasome activity, yet was independent of Arc ubiquitination. Surprisingly, tau-induced Arc removal required the endophilin-binding domain of Arc. Overexpression of 0N4R tau in primary hippocampal neurons led to Arc instability exclusively in neuronal dendrites, which was coupled to increases in the expression of dendritic and somatic surface GluA1-containing AMPA receptors. Interestingly, these effects on Arc stability and GluA1 localization were not observed in the commonly studied tau mutant, P301L. Our findings implicate isoform- and domain-specific effects of tau in regulating Arc stability and AMPA receptor targeting, which may in part explain the deficits in synaptic plasticity that are observed in select types of tauopathies. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

https://psychiatry.dev/wp-content/uploads/speaker/post-11188.mp3?cb=1671138210.mp3 Playback speed: 0.8x 1x 1.3x 1.6x 2x Download: SELENBP1 overexpression in the prefrontal cortex underlies negative symptoms of schizophrenia – PubMed Soojin Kim et al. PNAS. 2022. The selenium-binding proteinFull EntrySELENBP1 overexpression in the prefrontal cortex underlies negative symptoms of schizophrenia – PubMed

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.30.518472v1?rss=1 Authors: Bravo-Plaza, I., Tagua, V. G., Arst, H. N., Alonso, A., Pinar, M., Monterroso, B., Galindo, A., Penalva, M. A. Abstract: Uso1/p115 and RAB1 tether ER-derived vesicles to the Golgi. Uso1/p115 contains a globular-head-domain (GHD), a coiled-coil (CC) mediating dimerization/tethering and a C-terminal region (CTR) interacting with golgins. Uso1/p115 is recruited to vesicles by RAB1. Paradoxically, genetic studies placed Uso1 acting upstream of, or in conjunction with RAB1 (Sapperstein et al., 1996). We selected two missense mutations in uso1 resulting in E6K and G540S substitutions in the GHD permitting growth of otherwise inviable rab1-deficient Aspergillus nidulans. Remarkably, the double mutant suppresses the complete absence of RAB1. Full-length Uso1 and CTR{Delta} proteins are dimeric and the GHD lacking the CC/CTR is monomeric irrespective of whether they carry or not E6K/G540S. Microscopy showed recurrence of Uso1 on puncta (60 sec half-life) colocalizing with RAB1 and less so with early Golgi markers Sed5 and GeaA/Gea1/Gea2. Localization of Uso1 but not of Uso1E6K/G540S to puncta is abolished by compromising RAB1 function, indicating that E6K/G540S creates interactions bypassing RAB1. By S-tag-coprecipitation we demonstrate that Uso1 is an associate of the Sed5/Bos1/Bet1/Sec22 SNARE complex zippering vesicles with the Golgi, with Uso1E6K/G540S showing stronger association. Bos1 and Bet1 bind the Uso1 GHD directly, but Bet1 is a strong E6K/G540S-independent binder, whereas Bos1 is weaker but becomes as strong as Bet1 when the GHD carries E6K/G540S. AlphaFold2 predicts that G540S actually increases binding of GHD to the Bos1 Habc domain. In contrast, E6K seemingly increases membrane targeting of an N-terminal amphipathic a-helix, explaining phenotypic additivity. Overexpression of E6K/G540S and wild-type GHD complemented uso1{Delta}. Thus, a GHD monomer provides the essential Uso1 functions, demonstrating that long-range tethering activity is dispensable. Therefore, when enhanced by E6K/G540S, Uso1 binding to Bos1/Bet1 required to regulate SNAREs bypasses both the contribution of RAB1 to Uso1 recruitment and the reported role of RAB1 in SNARE complex formation (Lupashin and Waters, 1997), suggesting that the latter is consequence of the former. 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/2022.11.25.517917v1?rss=1 Authors: Villadiego, J., Garcia-Swinburn, R., Garcia-Gonzalez, D., Lebron-Galan, R., Murcia-Belmonte, V., Garcia-Roldan, E., Suarez-Luna, N., Nombela, C., Marchena, M., de Castro, F., Toledo-Aral, J. J. Abstract: The development and survival of dopaminergic neurons are influenced by the fibroblast growth factor (FGF) pathway. Anosmin-1 (A1) is an extracellular matrix protein that acts as a major regulator of this signaling pathway, controlling FGF diffusion, and receptor interaction and shuttling. Furthermore, overexpression of A1 in vivo gives rise to higher number of dopaminergic neurons in the olfactory bulb. Here, using A1 overexpressing mice (A1-mice), we studied the effects of A1 on different populations of catecholaminergic neurons in the central (CNS) and the peripheral nervous systems (PNS). A1 overexpression increases the number of dopaminergic SNpc neurons and alters the striosome/matrix organization of the striatum. Interestingly, these numerical and morphological changes in the nigrostriatal pathway of A1-mice do not confer an altered susceptibility to experimental MPTP-parkinsonism with respect to wild type controls. Moreover, the study of the effects of A1 overexpression was extended to different dopaminergic tissues associated with the PNS, detecting a significant reduction in the number of dopaminergic chemosensitive carotid body glomus cells in A1-mice. Overall, these analyses confirm A1 as a principal regulator of the FGF pathway in the development and survival of dopaminergic neurons in different nuclei of the mammalian nervous system. 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/2022.11.14.516471v1?rss=1 Authors: Dou, D., Smith, E. M., Evans, C. S., Boecker, C. A., Holzbaur, E. L. F. Abstract: Gain-of-function mutations in the LRRK2 gene cause Parkinson's disease (PD), increasing phosphorylation of RAB GTPases through hyperactive kinase activity. We found that LRRK2-hyperphosphorylated RABs disrupt the axonal transport of autophagosomes by perturbing the coordinated regulation of cytoplasmic dynein and kinesin motors. In iPSC-derived human neurons, knock-in of the strongly-hyperactive LRRK2-p.R1441H mutation caused striking impairments in autophagosome transport, inducing frequent directional reversals and pauses. Knock-out of the opposing Protein Phosphatase 1H (PPM1H) phenocopied the effect of hyperactive LRRK2. Overexpression of ADP-ribosylation factor 6 (ARF6), a GTPase that acts as a switch for selective activation of dynein or kinesin, attenuated transport defects in both p.R1441H knock-in and PPM1H knock-out neurons. Together, these findings support a model where a regulatory imbalance between LRRK2-hyperphosphorylated RABs and ARF6 induces an unproductive "tug-of-war" between dynein and kinesin, disrupting processive autophagosome transport. This disruption may contribute to PD pathogenesis by impairing the essential homeostatic functions of axonal autophagy. 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/2022.11.01.514746v1?rss=1 Authors: Lemerle, E., Laine, J., Moulay, G., Bigot, A., Labasse, C., Madelaine, A., Canette, A., Aubin, P., Vallat, J.-M., Romero, N. B., Bitoun, M., Mouly, V., Isabelle, M., Cadot, B., Picas, L., Vassilopoulos, S. Abstract: Excitation-contraction coupling requires a highly specialized membrane structure, the triad, composed of a plasma membrane invagination, the T-tubule, surrounded by two sarcoplasmic reticulum terminal cisternae. Although the precise mechanisms governing T-tubule biogenesis and triad formation remain largely unknown, studies have shown that caveolae participate in T-tubule formation and mutations of several of their constituents induce muscle weakness and myopathies. Here, we demonstrate that, at the plasma membrane, caveolae composed of caveolin-3 and Bin1 assemble into ring-like structures from which emerge tubes enriched in the dihydropyridine receptor. Overexpression of Bin1 lead to the formation of both rings and tubes and we show that Bin1 forms scaffolds on which caveolae accumulate to form the initial T-tubule. Cav3 deficiency caused by either gene silencing or pathogenic mutations cause defective ring formation and perturbed Bin1-mediated tubulation that may explain defective T-tubule organization in mature muscles. Our results uncover new pathophysiological mechanisms that may prove relevant to myopathies caused by Cav3 or Bin1. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.31.512457v1?rss=1 Authors: Sneyers, F., Kerkhofs, M., Welkenhuyzen, K., Speelman-Rooms, F., Shemy, A., Voet, A. R., Eelen, G., Dewerchin, M., Tait, S., Ghesquiere, B., Bootman, M. D., Bultynck, G. Abstract: Intracellular Ca2+ signals control several physiological and pathophysiological processes. The main tool to chelate intracellular Ca2+ is intracellular BAPTA (BAPTAi), usually introduced into cells as a membrane-permeant acetoxymethyl ester (BAPTA-AM). We previously demonstrated that BAPTAi enhanced apoptosis induced by venetoclax, a Bcl-2 antagonist, in diffuse large B-cell lymphoma (DLBCL). These findings implied a novel interplay between intracellular Ca2+ signaling and anti-apoptotic Bcl-2 function. Hence, we set out to identify the underlying mechanisms by which BAPTAi enhances cell death in B-cell cancers. We observed that BAPTAi induced apoptosis in lymphoma cell models that were highly sensitive to S63845, an Mcl-1 antagonist. BAPTAi provoked a rapid decline in Mcl-1-protein levels by inhibiting mTORC1-driven MCL-1 translation. Overexpression of nondegradable Mcl-1 rescued BAPTAi-induced cell death. We further examined how BAPTAi diminished mTORC1 activityand found that BAPTAi impaired glycolysis by directly inhibiting 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) activity, an up to now unappreciated effect of BAPTAi. All aforementioned effects of BAPTAi were also elicited by a BAPTAi analog with low affinity for Ca2+. Thus, our work reveals PFKFB3 inhibition as an unappreciated Ca2+-independent mechanism by which BAPTAi impairs cellular metabolism and ultimately the survival of Mcl-1-dependent cancer cells. Our work has two important implications. First, direct inhibition of PFKFB3 emerged as a promising target in cancer treatment. Second, cellular effects caused by BAPTAi are not necessarily related to Ca2+ signaling. Our data support the need for a reassessment of the role of Ca2+ in cellular processes when findings were based on the use of BAPTAi. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.26.513832v1?rss=1 Authors: Jiang, Z., Wang, J., Luo, B., Bai, F., Qin, Y., Wei, H., Zhang, S., Wei, J., Ding, G., Ma, L., He, S., Chen, R., Wang, L., Xu, H., Wang, X., Chen, G., Lei, W. Abstract: Alzheimer's disease (AD) is one of the most burdening diseases of the century with no disease-modifying treatment yet. Non-human primates (NHPs) share genetic, anatomical and physiological similarities with humans, making them an ideal model for investigating the pathogenesis and therapeutics of AD. However, the applications of NHPs in AD research have been hindered by the paucity of spontaneous or induced monkey models for AD due to their long generation time, ethical considerations and technical challenges in making genetically modified monkeys. Here we developed an AD-like NHP model by overexpressing human tau in bilateral hippocampi of adult rhesus macaque monkeys. We evaluated the pathological features of these monkeys with immunostaining, cerebrospinal fluid (CSF) analysis, magnetic resonance imaging (MRI), positron emission tomography (PET) scan, and behavioral tests. We demonstrated that after hippocampal overexpression of human tau, the rhesus macaque monkeys displayed multiple pathological features of AD, including neurofibrillary tangle formation, neuronal loss, hippocampal atrophy, neuroinflammation, A{beta} clearance deficit, blood vessel damage and cognitive decline. This work establishes a human tau-induced AD-like NHP model that may facilitate mechanistic studies and therapeutic treatments for AD. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.17.512492v1?rss=1 Authors: Martin, S. D., Connor, T., Sanigorski, A., McEwen, K. A., Henstridge, D. C., Nijagal, B., De Souza, D. P., Tull, D., Meikle, P., Kowalski, G. M., Bruce, C., Gregorevic, P., Febbraio, M., Collier, F., Walder, K. R., McGee, S. Abstract: Lipotoxicity, the accumulation of lipids in non-adipose tissues, alters the metabolic transcriptome and mitochondrial metabolism in skeletal muscle. The mechanisms involved remain poorly understood. Here we show that lipotoxicity increased histone deacetylase 4 (HDAC4) and histone deacetylase 5 (HDAC5), which reduced the expression of metabolic genes and oxidative metabolism in skeletal muscle. This metabolic reprogramming was linked with reduced expression of p53-dependent genes that mediate apoptosis and ferroptosis, which preserved cell viability in response to lipotoxicity. Mechanistically, impaired mitochondrial metabolism reduced acetylation of p53 at K120, a modification required for transcriptional activation of apoptosis, while redox drivers of ferroptosis were also reduced. Overexpression of loss-of-function HDAC4 and HDAC5 mutants in skeletal muscle of obese db/db mice enhanced oxidative capacity, increased apoptosis and ferroptosis and reduced muscle mass. This study identifies HDAC4 and HDAC5 as repressors of the oxidative state of skeletal muscle, and that this metabolic reprogramming, considered deleterious for normal metabolism, is critical to preserve muscle integrity in response to lipotoxicity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

This month on Episode 38 of Discover CircRes, host Cynthia St. Hilaire highlights original research articles featured in the Jue 24th, July 8th and July 22nd issues of the journal. This episode also features an interview with the 2022 BCBS Outstanding Early Career Investigator Award finalists, Dr Hisayuki Hashimoto, Dr Matthew DeBerge and Dr Anja Karlstadt.   Article highlights:   Nguyen, et al. miR-223 in Atherosclerosis.   Choi, et al. Mechanism for Piezo1-Mediated Lymphatic Sprouting   Kamtchum-Tatuene, et al.  Plasma Interleukin-6 and High-Risk Carotid Plaques   Li, et al. 3-MST Modulates BCAA Catabolism in HFrEF   Cindy St. Hilaire:        Hi, and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire, from the Vascular Medicine Institute at the University of Pittsburgh. And today I'm going to be highlighting articles from our June 24th, July 8th and July 22nd issues of Circulation Research. I'm also going to have a chat with the finalists for the 2022 BCBS Outstanding Early Career Investigator Award, Dr Hisayuki Hashimoto, Dr Matthew DeBerge and Dr Anja Karlstadt.   Cindy St. Hilaire:        The first article I want to share is from our June 24th issue and is titled, miR-223 Exerts Translational Control of Proatherogenic Genes in Macrophages. The first authors are My-Anh Nguyen and Huy-Dung Hoang, and the corresponding author is Katey Rayner and they're from the University of Ottawa. A combination of cholesterol accumulation in the blood vessels and subsequent chronic inflammation that's derived from this accumulation drive the progression of atherosclerosis. Unfortunately, current standard medications tackle just one of these factors, the cholesterol. And this might explain why many patients on such drugs still have vascular plaques. In considering treatments that work on both aspects of the disease, meaning lipid accumulation and inflammation, this group investigated the micro RNA 223 or miR-223, which is a small regulatory RNA that has been shown to suppress expression of genes involved in both cholesterol uptake and inflammatory pathways in both liver and immune cells.   Cindy St. Hilaire:        The team showed that mouse macrophages deficient in miR-223, exhibited increased expression of pro-inflammatory cytokines and reduced cholesterol efflux compared with control cells. Overexpression of miR-223 had the opposite effects. Furthermore, atherosclerosis prone mice, whose hematopoietic cells lacked miR-223, had worse atherosclerosis with larger plaques and higher levels of pro-inflammatory cytokines than to control animals with normal levels of miR-223. These findings highlight miR-223's dual prompt, antiatherogenic action, which could be leveraged for future therapies.   Cindy St. Hilaire:        The second article I want to share is from our July 8th issue of Circulation Research and is titled, Piezo1-Regulated Mechanotransduction Controls Flow-Activated Lymph Expansion. The first author is Dongwon Choi and the corresponding author is Young-Kwon Hong, and they're from UCLA.   As well as being super highways for immune cells, lymph vessels are drainage channels that help maintain fluid homeostasis in the tissues. This network of branching tubes grows as fluids begin to flow in the developing embryo. This fluid flow induces calcium influx into the lymphatic endothelial cells, which in turn promotes proliferation and migration of these cells, leading to the sprouting of lymph tubules. But how do LECs, the lymphatic endothelial cells, detect fluid flow in the first place? Piezo1 is a flow and mechanosensing protein known for its role in blood vessel development and certain mutations in Piezo1 cause abnormal lymphatic growth in humans.   Cindy St. Hilaire:        This script found that Piezo1 is expressed in the embryonic mouse LECs and that the suppression of Piezo1 inhibits both flow activated calcium entry via the channel ORAI1, as well as downstream target gene activation. Overexpression of Piezo1, by contrast, induced the target genes. The team went on to show that mice lacking either Piezo1 or ORAI1 had lymphatic sprouting defects and that pharmacological activation of Piezo1 in mice enhanced lymphogenesis and prevented edema after tail surgery. Together, the results confirmed Piezo1's role in flow dependent lymphatic growth and suggest it might be a target for treating lymphedema.   Cindy St. Hilaire:        The third article I want to share is also from our July 8th issue and is titled, Interleukin-6 Predicts Carotid Plaque Severity, Vulnerability and Progression. The first and corresponding author of this study is Joseph Kamtchum-Tatuene from University of Alberta.   Excessive plasma cholesterol and systemic inflammation are contributing factors in atherosclerosis. While traditional remedies have been aimed at lowering patient's lipid levels, drugs that tackle inflammation are now under investigation, including those that suppress Interleukin-6, which is an inflammatory cytokine implicated in the disease. Focusing on carotid artery disease, this group conducted a prospective study to determine whether IL-6 levels correlated with disease severity. 4,334 individuals were enrolled in the cardiovascular health study cohort. They had their blood drawn and ultrasounds taken at the start of the study and five years later. This group found IL-6 was robustly correlated with and predicted plaque severity independent of other cardiovascular risk factors. This study also determined that an IL-6 blood plasma level of 2.0 picograms/mls, identified individuals with the highest likelihood of plaque, vulnerability and progression. This threshold value could be used to select patients who might benefit from novel IL-6 lowering medications.   Cindy St. Hilaire:        The last article I want to share is from our July 22nd issue of Circulation Research and is titled, Mitochondrial H2S Regulates BCAA Catabolism in Heart Failure. The first author is Zhen Li, and the corresponding author is David Lefer from Louisiana State University. Hydrogen sulfide, or H2S, is a compound that exerts mitochondrial specific actions that include the preservation of oxidative phosphorylation, mitochondrial biogenesis and ATP synthesis, as well as inhibiting cell death. 3-mercaptopyruvate sulfurtransferase, or 3-MST, is a mitochondrial H2S producing enzyme, whose functions in cardiovascular disease are not fully understood.   Cindy St. Hilaire:        This group investigated the global effects of 3-MST deficiency in the setting of pressure overload induced heart failure. They found that 3-MST was significantly reduced in the myocardium of patients with heart failure, compared with non failing controls. 3-MST knockout mice exhibited increased accumulation of branch chain amino acids in the myocardium, which was associated with reduced myocardial respiration and ATP synthesis, exacerbated cardiac and vascular dysfunction, and worsened exercise performance, following transverse aortic constriction. Restoring myocardial branched-chain amino acid catabolism, or administration of a potent H2S donor, ameliorated the detrimental effects of 3-MST deficiency and heart failure with reduced injection fraction. These data suggest that 3-MST derived mitochondrial H2S, may play a regulatory role in branch chain amino acid catabolism, and mediate critical cardiovascular protection in heart failure.   Cindy St. Hilaire:        Today, I'm really excited to have our guests, who are the finalists for the BCVS Outstanding Early Career Investigator Awards. Welcome everyone.   Hisayuki Hashimoto:   Thank you.   Anja Karlstaedt:          Hi.   Hisayuki Hashimoto:   Hi.   Matthew DeBerge:      Hello. Thank you.   Cindy St. Hilaire:        So the finalists who are with me today are Dr Hisayuki Hashimoto from Keio University School of Medicine in Tokyo, Japan, Dr Matthew Deberge from Northwestern University in Chicago and Dr Anja Karlstaedt from Cedar Sinai Medical Center in LA. Thank you again. Congratulations. And I'm really excited to talk about your science.   Hisayuki Hashimoto:   Thank you. Yes. Thanks, first of all for this opportunity to join this really exciting group and to talk about myself and ourselves. I am Hisayuki Hashimoto, I'm from Tokyo, Japan. I actually learned my English... I went to an American school in a country called Zaire in Africa and also Paris, France because my father was a diplomat and I learned English there. After coming back to Japan, I went to medical school. During my first year of rotation, I was really interested in cardiology, so I decided to take a specialized course for cardiology. Then I got interested in basic science, so I took a PhD course, and that's what brought me to this cardiology cardiovascular research field.   Matthew DeBerge:      So I'm currently a research assistant professor at Northwestern University. I'm actually from the Chicagoland area, so I'm really excited to welcome you all to my hometown for the BCVS meeting.   Cindy St. Hilaire:        Oh, that's right. And AHA is also there too this year. So you'll see a lot of everybody.   Matthew DeBerge:      I guess I get the home field advantage, so to speak. So, I grew up here, I did my undergrad here, and then went out in the east coast, Dartmouth College in New Hampshire for my PhD training. And actually, I was a viral immunologist by training, so I did T cells. When I was looking for a postdoctoral position, I was looking for a little bit of something different and came across Dr Edward Thorpe's lab at Northwestern university, where the interest and the focus is macrophages in tissue repair after MI. So, got into the macrophages in the heart and have really enjoyed the studies here and have arisen as a research assistant professor now within the Thorpe lab. Now we're looking to transition my own independent trajectory. Kind of now looking beyond just the heart and focusing how cardiovascular disease affects other organs, including the brain. That's kind of where I'm starting to go now. Next is looking at the cardiovascular crosstalk with brain and how this influences neuroinflammation.   Anja Karlstaedt:          I am like Hisayuki, I'm also a medical doctor. I did my medical training and my PhD in Berlin at the Charité University Medicine in Berlin, which is a medical faculty from Humboldt University and Freie University. II got really interested in mathematical modeling of complex biological systems. And so I started doing my PhD around cardiac metabolism and that was a purely core and computationally based PhD. And while I was doing this, I got really hooked into metabolism. I wanted to do my own experiments to further advance the model, but also to study more in crosstalk cardiac metabolism. I joined Dr Heinrich Taegteyer lab at the University of Texas in the Texas Medical Center, and stayed there for a couple of years. And while I was discovering some of the very first interactions between leukemia cells and the heart, I decided I cannot stop. I cannot go back just after a year. I need to continue this project and need to get funding. And so after an AHA fellowship and NIHK99, I am now here at Cedars Sinai, an assistant professor in cardiology and also with a cross appointment at the cancer center and basically living the dream of doing translational research and working in cardio-oncology.   Cindy St. Hilaire:        Great. So, Dr Hashimoto, the title of your submission is, Cardiac Reprogramming Inducer ZNF281 is Indispensable for Heart Development by Interacting with Key Cardiac Transcriptional Factors. This is obviously focused on reprogramming, but why do we care about cardiac reprogramming and what exactly did you find about this inducer ZNF281?   Hisayuki Hashimoto:   Thank you for the question. So, I mean, as I said, I'm a cardiologist and I was always interested in working heart regeneration. At first, I was working with pluripotent stem cells derived cardiomyocyte, but then I changed my field during my postdoc into directly programming by making cardiomyocyte-like cells from fiberblast. But after working in that field, I kind of found that it was a very interesting field that we do artificially make a cardiomyocyte-like cell. But when I dissected the enhanced landscape, epigenetic analysis showed that there are very strong commonalities between cardiac reprogramming and heart development. So I thought that, hey, maybe we can use this as a tool to discover new networks of heart development. And the strength is that cardiac reprogramming in vitro assay hardly opens in vivo assay, so it's really time consuming. But using dark programming, we can save a lot of time and money to study the cardiac transitional networks. And we found this DNF281 from an unbiased screen, out of 1000 human open reading frames. And we found that this gene was a very strong cardiac reprogramming inducer, but there was no study reporting about any functioning heart development. We decided to study this gene in heart development, and we found out that it is an essential gene in heart development and we were kind of able to discover a new network in heart development.   Cindy St. Hilaire:        And you actually used, I think it was three different CRE drivers? Was that correct to study?   Hisayuki Hashimoto:   Ah, yes. Yeah.   Cindy St. Hilaire:        How did you pick those different drivers and what, I guess, cell population or progenitor cell population did those drivers target?   Hisayuki Hashimoto:   So I decided to use a mesodermal Cre-driver, which is a Mesp1Cre and a cardiac precursor Cre-driver, which is the Nkx2-5 Cre and the cardiomyocyte Cre, which is the Myh6-Cre. So three differentiation stages during heart development, and we found out that actually, DNF281 is an essential factor during mesodermal to cardiac precursor differentiation state. We're still trying to dig into the molecular mechanism, but at that stage, if the DNF281 is not there, we are not able to make up the heart.   Cindy St. Hilaire:        That is so interesting. Did you look at any of the strains that survived anyway? Did you look at any phenotypes that might present in adulthood? Is there anything where the various strains might have survived, but then there's a kind of longer-term disease implicating phenotype that's observed.   Hisayuki Hashimoto:   Well, thank you for the question. Actually, the mesodermal Cre-driver knocking out the DNF281 in that stage is embryonic lethal, and it does make different congenital heart disease. And they cannot survive until after embryonic day 14.5. The later stage Nkx2-5 Cre and Myh6-Cre, interestingly, they do survive after birth. And then in adult stage, I did also look into the tissues, but the heart is functioning normally. I haven't stressed them, but they develop and they're alive after one year. It looks like there's really no like phenotype at like the homeostatic status.   Cindy St. Hilaire:        Interesting. So it's kind of like, once they get over that developmental hump, they're okay.   Hisayuki Hashimoto:   Exactly. That might also give us an answer. What kind of network is important for cardiac reprogramming?   Cindy St. Hilaire:        So what are you going to do next?   Hisayuki Hashimoto:   Thank you. I'm actually trying to dig into the transitional network of what kind of cardiac transitional network the ZNF281 is interacting with, so that maybe I can find a new answer to any etiology of congenital heart disease, because even from a single gene, different mutation, different variants arise different phenotypes in congenital heart disease. Maybe if I find a new interaction with any key cardiac transitional factors, maybe I could find a new etiology of congenital heart disease phenotype.   Cindy St. Hilaire:        That would be wonderful. Well, best of luck with that. Congratulations on an excellent study. Hisayuki Hashimoto:   Thank you.   Cindy St. Hilaire:        Dr DeBerge, your study was titled, Unbiased Discovery of Allograft Inflammatory Factor-1 as a New and Critical Immuno Metabolic Regulatory Node During Cardiac Injury. Congrats on this very cool study. You were really kind of focused on macrophages in myocardial infarction. And macrophages, they're a Jeckel Hyde kind of cell, right? They're good. They're bad. They can be both, almost at the same time, sometimes it seems like. So why were you interested in macrophages particularly in myocardial infarction, and what did you discover about this allograft inflammatory factor-1, or AIF1 protein?   Matthew DeBerge:      Thank you. That's the great question. You really kind of alluded to why we're interested in macrophages in the heart after tissue repair. I mean, they really are the central mediators at both pro-inflammatory and anti-inflammatory responses after myocardial infarction. Decades of research before this have shown that inflammation has increased acutely after MI and has also increased in heart failure patients, which really has led to the development of clinical efforts to target inflammatory mediators after MI. Now, unfortunately, the results to target inflammation after MI, thus far, have been modest or disappointing, I guess, at worst, in the respect that broadly targeting macrophage function, again, hasn't achieved results. Again, because these cells have both pro and anti-inflammatory functions and targeting specific mediators has been somewhat effective, but really hasn't achieved the results we want to see.   Matthew DeBerge:      I think what we've learned is that the key, I guess, the targeting macrophage after MI, is really to target their specific function. And this led us to sort of pursue novel proteins that are mediating macrophage factor function after MI. To accomplish this, we similarly performed an unbiased screen collecting peri-infarct tissue from a patient that was undergoing heart transplantation for end stage heart failure and had suffered an MI years previously. And this led to the discovery of allograft inflammatory factor-1, or AIF1, specifically within cardiac macrophages compared to other cardiac cell clusters from our specimen. And following up with this with post-mortem specimens after acute MI to show that AIF1 was specifically increased in macrophages after MI and then subsequently then testing causality with both murine model of permanent inclusion MI, as well as in vitro studies using bone marrow drive macrophages to dig deeper mechanistically, we found that AIF1 was crucial in regulating inflammatory programing macrophages, which ultimately culminated in worse in cardiac repair after MI.   Cindy St. Hilaire:        That's really interesting. And I love how you start with the human and then figure out what the heck it's doing in the human. And one of the things you ended up doing in the mouse was knocking out this protein AIF1, specifically in macrophage cells or cells that make the macrophage lineage. But is this factor in other cells? I was reading, it can be intracellular, it can be secreted. Are there perhaps other things that are also going on outside of the macrophage?   Matthew DeBerge:      It's a great question. First, I guess in terms of specificity, within the hematopoietic compartment, previous studies, as well as publicly available databases, have shown that AIF1 is really predominantly expressed within macrophages. We were able to leverage bone marrow chimera mice to isolate this defect to the deficiency to macrophages. But you do bring up a great point that other studies have shown that AIF1 may be expressed in other radio-resistant cell populations. I mean, such as cardiomyocytes or other treatable cells within the heart. We can't completely rule out a role for AIF1 and other cell populations. I can tell you that we did do the whole body knockout complementary to our bone marrow hematopoetic deficient knockouts, and saw that deficiency of AIF1 within the whole animal, recapitulate the effects we saw within the AIF1 deficiency within hematopoietic department.   Matthew DeBerge:      It was encouraging to us that, again, the overall role of AIF1 is pro-inflammatory after MI.   Cindy St. Hilaire:        I mean, I know it's early days, but is there a hint of any translational potential of these findings or of this protein?   Matthew DeBerge:      Yeah, I think so. To answer your question, we were fortunate enough to be able to partner with Ionis that develops these anti-sensible nucleotides so that we could specifically target AIF1 after the acute phase during MI. We saw that utilizing these anti-sensible nucleotides to deplete AIF1, again, within the whole mouse, that we were able to reduce inflammation, reduce in heart size and preserve stock function. I think there really is, hopefully a therapeutic opportunity here. And again, with it being, perhaps macrophage specific is, even much more important as we think about targeting the specific function of these cells within the heart.   Cindy St. Hilaire:        Very cool stuff. Dr Karlstaedt, the title of your submission is, ATP Dependent Citrate Lyase Drives Metabolic Remodeling in the Heart During Cancer. So this I found was really interesting because you were talking about, the two major killers in the world, right? Cardiovascular disease and cancer, and you're just going to tackle both of them, which I love. So obviously this is built on a lot of prior observations about the effects of cancer on cardiac metabolic remodeling. Can you maybe just tell us a little bit about what is that link that was there and what was known before you started?   Anja Karlstaedt:          Yeah. Happy to take that question. I think it's a very important one and I'm not sure if I will have a comprehensive answer to this, because like I mentioned at the beginning, cardio-oncology is a very new field. And the reason why we are starting to be more aware of cancer patients and their specific cardiovascular problems is because the cancer field has done such a great job of developing all these new therapeutics. And we have far more options of treating patients with various different types of cancers in particular, also leukemias, but also solid tumors. And what has that led to is an understanding that patients survive the tumors, but then 10, 20 years later, are dying of cardiovascular diseases. Those are particular cardiomyopathies and congestive heart failure patients. What we are trying, or what my lab is trying to do, is understanding what is driving this remodeling. And is there a way that we can develop therapies that can basically, at the beginning of the therapy, protect the heart so that this remodeling does not happen, or it is not as severe.   Anja Karlstaedt:          Also, identifying patients that are at risk, because not every tumor is created equally and tumors are very heterogeneous, even within the same group. To get to your question, what we found is, in collaboration actually with a group at Baylor College of Medicine, Peggy Goodell's group, who is primarily working on myeloid malignancies, is that certain types of leukemias are associated with cardiomyopathies. And so when they were focusing on the understanding drivers of leukemia, they noticed that the hearts of these animals in their murine models are enlarged on and actually developing cardiomyopathies. And I joined this project just very early on during my postdoc, which was very fortunate and I feel very lucky of having met them. What my lab is now studying here at Cedars is how basically those physiological stress and mutations coming from the tumors are leading to metabolic dysregulation in the heart and then eventually disease.   Anja Karlstaedt:          And we really think that metabolism is at the center of those disease progressions and also, because it's at the center, it should be part of the solution. We can use it as a way to identify patients that are at risk, but also potentially develop new therapies. And what was really striking for us is that when we knock down ACLY that in a willdtype heart where the mouse doesn't have any tumor disease, ACLY actually is critically important for energy substrate metabolism, which seems counterintuitive, because it's far away from the mitochondria, it's not part of directly ADP provision. It's not part of the Kreb cycle. But what we found is that when we knock it out using a CRISPR-Cas9 model, it leads to cardiomyopathy and critically disrupts energy substrate metabolism. And that is not necessarily the case when the mouse has leukemia or has a colorectal cancer, which upregulated in the beginning, this enzyme expression. And so we have now developed models that show us that this could be potentially also therapeutic target to disrupt the adverse remodeling by the tumor.   Cindy St. Hilaire:        That is so interesting. So one of the things I was thinking about too is we know that, I mean, your study is showing that, the tumor itself is causing cardiac remodeling, but we also know therapies, right? Radiation, chemotherapy, probably some immune modulatory compounds. Those probably do similar, maybe not exactly similar, but they also cause, adverse cardiac remodeling. Do you have any insights as to what is same and what is different between tumor driven and therapy driven adverse remodeling?   Anja Karlstaedt:          So we do not know a lot yet. It's still an open question about all the different types of chemotherapeutics, how they are leading to cardio toxicities. But what we know, at least from the classic anti-cyclic treatments, is right now at the core, the knowledge is that this is primarily disrupting cardiac mitochondrial function. And through that again, impairing energy provision and the interaction, again, with the immune system is fairly unknown, but we know through studies from Kathryn Moore and some very interesting work by Rimson is that myocardial infarction itself can lead to an increase in risk for tumor progression. And what they have shown as independent of each other, is that the activation of the immune system in itself can lead to an acceleration of both diseases, both the cardiac remodeling, and then also the tumor disease. We don't fully understand which drivers are involved, but we do know that a lot of the cardiomyopathies on cardiotoxicities that are chemotherapeutically driven, all have also metabolic component.   Cindy St. Hilaire:        Nice. Thank you. When I prepare for these interviews, I obviously read the abstracts for the papers, but I found myself also Googling other things after I read each of your abstracts. It was a rabbit hole of science, which was really exciting.                               I now want to transition to kind of a career angle. You all are obviously quite successful, scientifically, at the bench, right? But now you are pivoting to a kind of completely opposite slash new job, right? That of, independent researcher. I would love to hear from each of you, if there was any interesting challenge that you kind of overcame that you grew from, or if there was any bit of advice that you wish you knew ahead of time or anything like that, that some of our trainee listeners and actually frankly, faculty who can pass that information onto their trainees, can benefit from.   Anja Karlstaedt:          I think the biggest challenge for me in transitioning was actually the pandemic. Because I don't know how it was for Hisa and Matt, but trying to establish a lab, but also applying for faculty position during a major global pandemic, is challenging is not quite something that I expected that would happen. And so I think saying that and looking more conceptually and philosophically at this as, you can prepare as much as you want, but then when life just kicks in and things happen, they do happen. And I think the best is to prepare as much as you can. And then simply go with the flow. Sometimes one of my mentors, Dave Nikon, mentioned that to me when I was applying for faculty positions, it's sometimes good to just go with the flow. And as a metabolism person, I absolutely agree. And there are some things that you can do as a junior investigator.   Anja Karlstaedt:          We need to have a good network. So just very important to have good mentors. I was blessed with have those mentors, Peggy Goodell's one of them, Heinrich Taegtmeyer was another. And now with this study that we are publishing, Jim Martin and Dave Nikon were incredible. Without them, this study wouldn't have been possible and I would not be here at Cedars.   Anja Karlstaedt:          You need to reach out to other people because those mentors have the experience. They have been through some of this before. Even if they have never had a major event, like COVID-19 in their life before, because none of us had before, they had other experiences and you can rely on them and they set you then up for overcoming these challenges. And the other thing I would say, is put yourself out there, go and talk to as many people as possible or set conferences, present a poster, not only talks. Don't be disappointed if you don't get a talk, posters are really great to build this network and find other people that you probably wouldn't have encountered and apply for funding. Just again, put yourself out there and try to get the funding for your research. Even if it's small foundations, it builds up over time and it is a good practice to then write those more competitive grants.     Cindy St. Hilaire:        Dr Hashimoto, would you like to go next?   Hisayuki Hashimoto:   Just my advice is that, could be like a culture of difference, but in east Asia, like in Japan, we were taught to, do not disturb people, don't interrupt people and help people. But I realized that I wasn't really good at asking for help. After I am still not like fully independent, but I do have my own group and I have to do grant writing. I still work at the bench and then have to teach grad students, doing everything myself. I just realized it's just impossible. I didn't have time. I need like 48 hours a day. Otherwise, you won't finish it. I just realized that I wasn't really good at asking for help. So my advice would be, don't hesitate to ask for help. It's not a shame. You can't do everything by just yourself. I think, even from the postdoc, even from grad school, I think, ask for help and then get used to that. And then of course, help others. And that is the way I think to probably not get overwhelmed and not stress yourself. Science should be something fun. And if you don't ask for help and if you don't help someone, I think you are losing the chance of getting some fun part from the science.   Cindy St. Hilaire:        That's great advice. I really like that, especially because I find at least, I started my lab seven years ago now. And I remember the first couple months/year, it was extremely hard to let go, right? Like I taught my new people how to do the primary cell culture we needed, but I was terrified of them doing it wrong or wasting money or making too many mistakes. But you realize, you got to learn to trust people. Like you said, you got to learn to ask for help. And sometimes that help is letting them do it. And you doing, you're being paid now to write grants and papers. That's a big brain, you're not paid to do the smaller things. That's really great advice. I like that. Thank you. Dr DeBerge, how about you?   Matthew DeBerge:      So I guess towards a bit of life advice, I think two obvious things is one, be kind, science is hard enough as it is. So I think we should try to lift each other up and not knock each other down. And along those lines as the others have alluded to as well, one of the mantras we sort of adapted on the lab, is a rising tide raises all ships, this idea that we can work together to elevate each other's science and really, again, collaborate.   Towards the career side of things I'll just touch on, because I guess one thing I'll add, there's more than one path, I guess, to achieving your goals. I've been fortunate enough to have an NIH post-doctoral fellowship and had an AHA career development award, but I'm not a K99 recipient. Oftentimes, I think this is the golden ticket to getting the faculty job, so I'm trying to, I guess, buck trend, I just submitted an RO1. So fingers crossed that leads to some opportunity.   Even beyond academia, I'm not certain how much everyone here is involved in science Twitter, it's really become a thing over the last couple years, but I think, kind of the elephant in the room is that academia, it's really hard on the trainees nowadays to have a living wage, to go through this. I mean, I'm really excited to see my, fellow finalists here are starting their own groups and stuff, but for many, that's not the reality for many, it's just not financially feasible. So I think, kind of keeping in mind that there's many, many alternative careers, whether it's industry, whether it's consulting, science writing, etcetera, going back to what Dr Hash says, find what you love and really pursue that with passion.   Cindy St. Hilaire:        I think it's something only, I don't know, five to 10% of people go into or rather stay in academia. And that means, 90 to 95% of our trainees, we need to prepare them for other opportunities, which I think is exciting, because it means it can expand our network for those of us in academia.   Anja Karlstaedt:          I think right now it's even worse because it's about 2% of old postdocs that are actually staying and becoming independent researchers, independent or tenure track or research track. And I think I second, as what Matt said, because I play cello. I do music as a hobby and people always ask me if I'm a musician. And at the beginning I felt like, no, of course not. I'm not like Yoyo Ma. I'm just playing, it's a hobby. And then I, that got me thinking. I was like, no, of course you are because there's so many different types. And what we need to understand is that scientists, like you are always a scientist. It doesn't matter if you are working at Pfizer or if you are working at a small undergrad institution and you're teaching those next generation scientists, you are still scientist and we all need those different types of scientists because otherwise, if everybody is just a soloist, you are never going to listen to symphony. You need those different people and what we need to normalize beyond having those different career paths, is also that people are staying in academia and becoming those really incredible resources for the institutions and labs, quite frankly, of being able to retain those technologies and techniques within an institution. And I think that's something to also look forward to, that even if you're not the PI necessarily, you're the one who is driving those projects. And I hope to pass this on at some point also to my trainees that they can be a scientist, even if they're not running a lab and they become an Institute director and that's also critically important.   Cindy St. Hilaire:        There's lots of ways to do science. Thank you all so much for joining me today. Either waking up at 5:00 AM or staying up past midnight, I think it is now in Japan or close to it. So Matt and I kind of made it out okay. It's like 8:00 or 9:00 AM.   Matthew DeBerge:      Thank you.   Hisayuki Hashimoto:   My apologies for this time zone difference.   Cindy St. Hilaire:        I'm very glad to make it work. Congratulations to all of you, your presentations. I forget which day of the week they are on at BCVS, but we are looking forward to the oral presentations of these and congratulations to all of you. You are amazing scientists and I know I'm really looking forward to seeing your future work so best of luck.   Matthew DeBerge:      Thank you.   Hisayuki Hashimoto:   Thank you.   Anja Karlstaedt:          Thank you so much.   Cindy St. Hilaire:        That's it for the highlights from the June 24th, July 8th and July 22nd issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle at CircRes and hashtag Discover CircRes. Thank you to our guests. The BCVS Outstanding Early Career Investigator Award Finalists, Dr Hisayuki Hashimoto, Dr Matthew DeBerge and Dr Anja Karlstaedt. This podcast is produced by Ashara Ratnayaka, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy text for the highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire. And this is Discover CircRes, you're on the go source for the most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association, 2022. 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 information visit ahajournals.org.  

Race Oncology Ltd's Phillip Lynch speaks with Proactive during the ASX Small and Mid-Cap Conference. They're a precision oncology company with a cancer drug called Zantrene®. It's a potent inhibitor of the Fatso/Fat mass and obesity associated (FTO) protein. Overexpression of FTO has been shown to be the genetic driver of a diverse range of cancers.

Dr. Dae Joon Kim and Liza Morales from the Department of Biomedical Sciences, School of Medicine, University of Texas Rio Grande Valley, Edinburg, TX, detail a priority research paper they co-authored that was published by Oncotarget in Volume 8, Issue 53, entitled, “UVB-induced nuclear translocation of TC-PTP by AKT/14-3-3σ axis inhibits keratinocyte survival and proliferation.” DOI - https://doi.org/10.18632/oncotarget.21794 Correspondence to - Dae Joon Kim - dae.kim@utrgv.edu Abstract Understanding protein subcellular localization is important to determining the functional role of specific proteins. T-cell protein tyrosine phosphatase (TC-PTP) contains bipartite nuclear localization signals (NLSI and NLSII) in its C-terminus. We previously have demonstrated that the nuclear form of TC-PTP (TC45) is mainly localized to the cytoplasm in keratinocytes and it is translocated to the nucleus following UVB irradiation. Here, we report that TC45 is translocated by an AKT/14-3-3σ-mediated mechanism in response to UVB exposure, resulting in increased apoptosis and decreased keratinocyte proliferation. We demonstrate that UVB irradiation increased phosphorylation of AKT and induced nuclear translocation of 14-3-3σ and TC45. However, inhibition of AKT blocked nuclear translocation of TC45 and 14-3-3σ. Site-directed mutagenesis of 14-3-3σ binding sites within TC45 showed that a substitution at Threonine 179 (TC45/T179A) effectively blocked UVB-induced nuclear translocation of ectopic TC45 due to the disruption of the direct binding between TC45 and 14-3-3σ. Overexpression of TC45/T179A in keratinocytes resulted in a decrease of UVB-induced apoptosis which corresponded to an increase in nuclear phosphorylated STAT3, and cell proliferation was higher in TC45/T179A-overexpressing keratinocytes compared to control keratinocytes following UVB irradiation. Furthermore, deletion of TC45 NLSII blocked its UVB-induced nuclear translocation, indicating that both T179 and NLSII are required. Taken together, our findings suggest that AKT and 14-3-3σ cooperatively regulate TC45 nuclear translocation in a critical step of an early protective mechanism against UVB exposure that signals the deactivation of STAT3 in order to promote keratinocyte cell death and inhibit keratinocyte proliferation. Sign up for free Altmetric alerts about this article - https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.21794 Keywords - TC-PTP, nuclear translocation, AKT, 14-3-3σ, keratinocytes About Oncotarget Oncotarget is a peer-reviewed, open access biomedical journal covering research on all aspects of oncology. To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/c/OncotargetYouTube/ LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Oncotarget is published by Impact Journals, LLC: https://www.ImpactJournals.com Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

有許多醫學文獻證實抗發炎藥物長期使用會降低癌症發生率， 2012年一篇名為「選擇性和非選擇性環氧酶抑制劑減少癌症風險」摘要如下，醫學文獻查詢可找到更多論文。 Reduction in cancer risk by selective and nonselective cyclooxygenase-2 (COX-2) inhibitors Abstract We conducted a series of epidemiologic studies to evaluate the chemopreventive effects of aspirin, ibuprofen, and selective cyxlooxygenase-2 (COX-2) inhibitors (coxibs) against cancers of the breast, colon, prostate, and lung. Composite results across all four cancer sites revealed that regular intake of 325 mg aspirin, 200 mg ibuprofen, or standard dosages of coxibs (200 mg celecoxib or 25 mg rofecoxib) produced risk reductions of 49%, 59%, and 64%, respectively. Use of coxibs for at least 2 years was associated with risk reductions of 71%, 70%, 55%, and 60% for breast cancer, colon cancer, prostate cancer and lung cancer, respectively. Effects of ibuprofen were similar to selective coxibs, and slightly stronger than aspirin. These observed effects are consistent with the relative COX-2 selectivity of ibuprofen, coxibs, and aspirin. Acetaminophen, an analgesic without COX-2 activity, had no effect. Overexpression of COX-2 and increased prostaglandin biosynthesis correlates with carcinogenesis and metastasis at most anatomic sites. These results indicate that regular intake of nonselective or selective COX-2 inhibiting agents protects against the development of major forms of cancer. 各位朋友，若您有醫美、減肥、健康相關問題，請直接到「景升診所」官網任一頁面留言，或用下列方法與我們聯絡： 直接撥打24小時專線 +886-931919066 Line id=“Gscline" WeChat id=“Gscline" 哈囉，大家好: 我們有幾個不同的頻道： Grand Health 大健康 （健康加財富、知足就是福）https://goo.gl/6EGLMd Grand Beauty 大醫美 （好好愛自己、就從現在起）https://goo.gl/g1E1rq Grand Touring 大旅遊 （大叔向前跑、永遠沒煩惱）https://goo.gl/7HN4bk 歡迎大家欣賞，喜歡就請按個讚，想獲取最新訊息就按「訂閱」吧！ 我們會提供更多更新的知識和訊息給大家。 感謝以下單位的幫助： ✪景升診所 醫美中心 https://www.gscline.com ✪愛瘦美官網 https://www.isome.com.tw ✪邱醫師醫話園 https://www.okclinic.gscline.com ✪隆乳 https://www.gscline.com/ifatgraft/breast-adsc-htm ✪減肥 https://www.gscline.com/islimcenter-htm/fat-htm ✪自體脂肪隆乳 https://www.gscline.com/ifatgraft/breast-adsc-htm ✪瘦臉 https://www.gscline.com/ilipolysis-htm/fll-face-htm ✪瘦手臂 https://www.gscline.com/ilipolysis-htm/fll-arm-htm ✪瘦小腹 https://www.gscline.com/ilipolysis-htm/fll-abdomen-htm ✪瘦腿 https://www.gscline.com/ilipolysis-htm/fll-leg-htm ✪瘦大腿 https://www.gscline.com/ilipolysis-htm/fll-thigh-htm ✪抽脂 https://www.gscline.com/liposuction-new-technique/fat_liposuction-htm ✪男性女乳 https://www.gscline.com/ihair-htm/fll-gynecomastia-htm ✪狐臭 https://www.gscline.com/ihair-htm/laserhyperhidrosis-htm

Hear from NEB Scientists Mehmet Berkman and Paul Riggs about the lessons that New England Biolabs has learned about protein expression and purification over the last 40 years.

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.27.357780v1?rss=1 Authors: Pinkerton, M., Ruetenik, A., Bazylianska, V., Nyvltova, E., Barrientos, A. Abstract: Human neurodegenerative proteinopathies are disorders associated with abnormal protein depositions in brain neurons. They include polyglutamine (polyQ) conditions such as Huntington's disease (HD) and alpha-synucleinopathies such as Parkinson's disease (PD). Overexpression of NMNAT/Nma1, an enzyme in the NAD+ biosynthetic salvage pathway, acts as a powerful suppressor of proteotoxicities in yeast, fly, and mouse models. Screens in yeast models of HD and PD in our lab allowed us to identify three additional enzymes of the same pathway that achieve similar protection against proteotoxic stress: Npt1, Pnc1, and Qns1. Here, we report that their ability to maintain proteostasis is independent of their catalytic activity. Furthermore, we show that, under proteotoxic stress, the four proteins are recruited as molecular chaperones with holdase and foldase activities. In yeast cells, the NAD+ salvage proteins act by preventing misfolding and, together with the Hsp90 chaperone, promoting the refolding of extended polyQ domains or alpha-synuclein. For Nma1, we demonstrate that its foldase activity maps to the C-terminal domain of the protein. The proteostasis activity of Nma1, Npt1, Pnc1, and Qns1 does not require cellular protein quality control systems such as the proteasome or autophagy. We conclude that the entire salvage NAD+ biosynthetic pathway links NAD+ metabolism and proteostasis and emerges as a target for therapeutics to combat age-associated neurodegenerative proteotoxicities. Our observations also illustrate the existence of an evolutionarily conserved strategy of repurposing or moonlighting housekeeping enzymes under stress conditions. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.12.336883v1?rss=1 Authors: Yeates, C. J., Frank, C. A. Abstract: Synapses and circuits rely on homeostatic forms of regulation in order to transmit meaningful information. The Drosophila melanogaster neuromuscular junction (NMJ) is a well-studied synapse that shows robust homeostatic control of function. Most prior studies of homeostatic plasticity at the NMJ have centered on presynaptic homeostatic potentiation (PHP). PHP happens when postsynaptic muscle neurotransmitter receptors are impaired, triggering retrograde signaling that causes an increase in presynaptic neurotransmitter release. As a result, normal levels of evoked excitation are maintained. The counterpart to PHP at the NMJ is presynaptic homeostatic depression (PHD). Overexpression of the Drosophila vesicular glutamate transporter (VGlut) causes an increase in the amplitude of spontaneous events. PHD happens when the synapse responds to the challenge by decreasing quantal content during evoked neurotransmission, again, resulting in normal levels of postsynaptic excitation. We hypothesized that there may exist a class of molecules that affects both PHP and PHD. Impairment of any such molecule could hurt the ability of a synapse to respond to any significant homeostatic challenge. We conducted an electrophysiology-based screen for blocks of PHD. While we did not observe a block of PHD in the genetic conditions screened, we instead found loss-of-function conditions that led to excess depression, i.e., a substantial deficit in evoked amplitude when combined with VGlut overexpression. The conditions causing this phenotype included a double heterozygous loss-of-function condition for genes encoding the inositol trisphosphate receptor (IP3R, itpr) and ryanodine receptor (RyR). IP3Rs and RyRs gate calcium release from intracellular stores. Pharmacological agents targeting IP3R and RyR recapitulated the genetic losses of these factors, as did lowering calcium levels from other sources. Our data are consistent with the idea that some factors required for homeostatic potentiation are also required for the synapse to achieve appropriate levels of homeostatic depression. Loss of such factors may disorient compensatory plasticity signals. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.09.289835v1?rss=1 Authors: Zolotarov, Y., Ma, C., Gonzalez-Recio, I., Hardy, S., Franken, G., Uetani, N., Latta, F., Kostantin, E., Boulais, J., Thibault, M.-P., Cote, J.-F., Diaz Moreno, I., Diaz Quintana, A., Hoenderop, J. G. J., Martinez-Cruz, L. A., Tremblay, M. L., de Baaij, J. H. F. Abstract: Cyclin M (CNNM1-4) proteins maintain cellular and body magnesium (Mg2+) homeostasis. Using various biochemical approaches, we have identified members of the CNNM family as direct interacting partners of ADP-ribosylation factor-like protein 15 (ARL15), a small GTP-binding protein. ARL15 interacts with CNNMs at their carboxyl-terminal conserved cystathionine-{beta}- synthase (CBS) domains. In silico modeling of the interaction using the reported structures of both CNNM2 and ARL15 supports that the small GTPase specifically binds the CBS1 domain. Immunocytochemical experiments demonstrate that CNNM2 and ARL15 co-localize in the kidney, with both proteins showing subcellular localization in the Golgi-apparatus. Most importantly, we found that ARL15 is required for forming complex N-glycosylation of CNNMs. Overexpression of ARL15 promotes complex N-glycosylation of CNNM3. Mg2+ uptake experiments with a stable isotope demonstrate that there is a significant increase of 25Mg2+ uptake upon knockdown of ARL15 in multiple kidney cancer cell lines. Altogether, our results establish ARL15 as a novel negative regulator of Mg2+ transport by promoting the complex N-glycosylation of CNNMs. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.20.259994v1?rss=1 Authors: Seif, E., Kang, J. J., Sasseville, C., Senkovitch, O., Kaltashov, A., Boulier, E. L., Kapur, I., Kim, C. A., Francis, N. Abstract: Polycomb Group (PcG) proteins organize chromatin at multiple scales to regulate gene expression. A conserved Sterile Alpha Motif (SAM) in the Polycomb Repressive Complex 1 (PRC1) subunit Polyhomeotic (Ph) is important for chromatin compaction and large-scale chromatin organization. Like many SAMs, Ph SAM forms helical head to tail polymers, and SAM-SAM interactions between chromatin-bound Ph/PRC1 are believed to compact chromatin and mediate long-range interactions. To understand mechanistically how this occurs, we analyzed the effects of Ph SAM on chromatin in vitro. We find that incubation of chromatin or DNA with a truncated Ph protein containing the SAM results in formation of concentrated, phase-separated condensates. Condensate formation depends on Ph SAM, and is enhanced by but not strictly dependent on, its polymerization activity. Ph SAM-dependent condensates can recruit PRC1 from extracts and enhance PRC1 ubiquitin ligase activity towards histone H2A. Overexpression of Ph with an intact SAM increases ubiquitylated H2A in cells. Thus, phase separation is an activity of the SAM, which, in the context of Ph, can mediate large-scale compaction of chromatin into biochemical compartments that facilitate histone modification. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.13.249201v1?rss=1 Authors: Moharir, S. C., Raghawan, A. K., Swarup, G. Abstract: Optineurin (OPTN), a cytoplasmic adaptor protein involved in cargo selective autophagy of bacteria, damaged mitochondria and mutant protein aggregates, is frequently seen in pathological structures containing protein aggregates, associated with several neurodegenerative diseases. However, the function of OPTN in these protein aggregates is not known. Here, we have explored the role of OPTN in mutant protein aggregation and in cytoprotection from toxicity of mutant proteins. Mutant huntingtin (mHtt) and mutant ataxin-3 (mAtax-3) showed reduced formation of aggregates in Optn-/- mouse embryonic fibroblasts as compared with wild type cells. Co-expression of OPTN enhanced aggregate formation by mHtt and mAtax-3 in Optn-/- cells. C-terminal domain of OPTN (412-577 amino acids) was necessary and sufficient to promote aggregate formation by these mutant proteins. The E478G mutant of OPTN, defective in ubiquitin-binding and autophagy, was also able to promote aggregation of mHtt and mAtax-3. OPTN and its C-terminal domain form a complex with the chaperone HSP70 known to promote mutant protein aggregation. Overexpression of mHtt or mAtax-3 induced more cell death in Optn-/- cells compared with wild type cells. Importantly, compared to wild type cells, Optn-deficient cells having mHtt or mAtax-3 aggregates showed higher level of cell death in neuronal (N2A) and non-neuronal cells. Our results show that OPTN promotes formation of mutant huntingtin and mutant ataxin-3 aggregates, and this function of OPTN might be mediated through interaction with HSP70 chaperones. Our results also show that OPTN reduces cytotoxicity caused by these mutant protein aggregates. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.31.230656v1?rss=1 Authors: Campos, A. C., Scarante, F. F., Martin, S., Lazzarin, M., Hermes, D., De Souza-Silva, M. A., Sanchez, A., Pardo, L. A., Stühmer, W., Del-Bel, E. A. Abstract: Although sex differences in memory tasks dependent on hippocampal function have been described in several species, including rodents and humans, the exact mechanisms involved remain debatable. The function of the small-conductance Ca2+-activated K+ channel type 3 has been associated with cognitive deficits, and its overexpression in male mice (T/T) induces shrinkage of the hippocampus. Here we describe that opposite to the observation in males, in female mice, SK3-induced-reduction in the volume of the hippocampal formation does not interfere with working and social memory performance. Male, but not female T/T mice showed decreased adult hippocampal neurogenesis and down-regulation of the expression of the genes related to Akt/mTOR and MAP kinase pathways. T/T male mice exhibit impaired estrogen and Neurogulin 1 signaling. An increased number of filopodia spines is observed in the dentate gyrus (DG). Our results suggest a fine-tune modulation of SK3 expression participates in the sex-dependent function of the hippocampus via estrogen signaling and neuroplasticity in the DG. Our results reinforce the importance of testing male and female mice while conducting experiments with transgenic mice. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.29.224972v1?rss=1 Authors: Malhotra, I., Oyarzun, B., Mognetti, B. Abstract: Nuclear molecules control the functional properties of the chromatin fiber by shaping its morphological properties. The biophysical mechanisms controlling how bridging molecules compactify the chromatin are a matter of debate. On the one side, bridging molecules could cross-link faraway sites and fold the fiber through the formation of loops. Interacting bridging molecules could also mediate long-range attractions by first tagging different locations of the fiber and then undergoing microphase separation. Using a coarse-grained model and Monte Carlo simulations, we study the conditions leading to compact configurations both for interacting and non-interacting bridging molecules. In the second case, we report on an unfolding transition at high densities of the bridging molecules. We clarify how this transition, which disappears for interacting bridging molecules, is universal and controlled by entropic terms. In general, chains are more compact in the case of interacting bridging molecules since, in this case, interactions are not valence-limited. However, this result is conditional on the ability of our simulation methodology to relax the system towards its ground state. In particular, we clarify how, unless using reaction dynamics that change the length of a loop in a single step, the system is prone to remain trapped in metastable, compact configurations featuring long loops. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.23.158014v1?rss=1 Authors: Ravel-Godreuil, C., Massiani-Beaudoin, O., Mailly, P., Prochiantz, A., Joshi, R. L., Fuchs, J. Abstract: Heterochromatin disorganization is a key hallmark of aging and DNA methylation state is currently the main molecular predictor of chronological age. The most frequent neurodegenerative diseases like Parkinson disease and Alzheimer's disease are age-related but how the aging process and chromatin alterations are linked to neurodegeneration is unknown. Here, we investigated the consequences of viral overexpression of Gadd45b, a multifactorial protein involved in active DNA demethylation, in the midbrain of wild-type mice. Gadd45b overexpression induces global and stable changes in DNA methylation, particularly on gene bodies of genes related to neuronal functions. DNA methylation changes were accompanied by perturbed H3K9me3-marked heterochromatin and increased DNA damage. Prolonged Gadd45b expression resulted in dopaminergic neuron degeneration accompanied by altered expression of candidate genes related to heterochromatin maintenance, DNA methylation or Parkinson disease. Gadd45b overexpression rendered midbrain dopaminergic neurons more vulnerable to acute oxidative stress. Heterochromatin disorganization and DNA demethylation resulted in derepression of mostly young LINE-1 transposable elements, a potential source of DNA damage, prior to Gadd45b-induced neurodegeneration. Our data implicate that alterations in DNA methylation and heterochromatin organization, LINE-1 derepression and DNA damage can represent important contributors in the pathogenic mechanisms of dopaminergic neuron degeneration with potential implications for Parkinson disease. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.12.149062v1?rss=1 Authors: da Rocha, J. F., Bastos, L., Domingues, S. C., Bento, A. R., Konietzko, U., da Cruz e Silva, O. A. B., Vieira, S. I. Abstract: The amyloid precursor protein (APP) is a transmembrane glycoprotein central to Alzheimer's disease (AD) with functions in brain development and plasticity, including in neurogenesis and neurite outgrowth. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) are well described neurotrophic and neuromodulator EGFR ligands, both implicated in neurological disorders like Schizophrenia and AD. Here we show that APP interacts with these two EGFR ligands and characterize the effects of APP-EGF interaction in ERK activation and neuritogenesis. HB-EGF was identified as a novel APP interactor in a yeast two-hybrid screen of a human brain cDNA library. Yeast co-transformation and co-immunoprecipitation assays confirmed APP interaction with HB-EGF. Moreover, co-immunoprecipitation also revealed that APP binds to cellular pro-EGF. Overexpression of HB-EGF in HeLa cells, or exposure of SH-SY5Y cells to EGF, both resulted in increased APP protein levels. EGF and APP were also observed to synergistically activate the ERK signaling pathway, crucial for early neuronal differentiation. Immunofluorescence analysis of cellular neuritogenesis in conditions of APP overexpression and EGF exposure, confirmed a synergistic effect in promoting the number and the mean length of neurite-like processes per cell. Synergistic ERK activation and neuritogenic effects were completely blocked by the EGFR inhibitor PD 168393, implying EGF-induced activation of EGFR as part of the mechanism. This work shows novel APP protein interactors and provides a major insight into the APP-driven mechanisms underlying neurite outgrowth and neuronal differentiation, with potential relevance for AD and for adult neuroregeneration. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.08.139675v1?rss=1 Authors: Kalogeropulou, A. F., Lis, P., Polinski, N. K., Alessi, D. R. Abstract: Mutations that enhance LRRK2 protein kinase activity cause inherited Parkinsons disease. LRRK2 phosphorylates a group of Rab GTPase proteins, including Rab10, within the effector-binding switch-II motif. Previous work has indicated that the PARK16 locus, which harbors the gene encoding for Rab29, is mutated in Parkinsons, and that Rab29 operates in a common pathway with LRRK2. Co-expression of Rab29 and LRRK2 stimulates LRRK2 activity by recruiting LRRK2 to the surface of the trans-Golgi network. Pathogenic mutations including LRRK2[R1441C] promote GTP-binding are more readily activated by Rab29. As previous work was based on overexpression approaches, we were curious to define the importance of endogenous Rab29 in regulating basal LRRK2 activity. We report that knock-out of Rab29 does not influence endogenous LRRK2 activity, based on assessment of Rab10 phosphorylation, in wildtype LRRK2, LRRK2[R1441C] as well as in VPS35[D620N] knock-in mouse tissues and embryonic fibroblasts. We also generated a transgenic mouse model that moderately overexpresses Rab29, and found that this was not sufficient to stimulate basal LRRK2 activity. Our data suggest that the bulk of the basal LRRK2 activity measured in whole cell and tissue extracts is not controlled by Rab29. LRRK2 is not associated with the Golgi unless Rab29 is highly overexpressed, which could account for the lack of effect that Rab29 knock-out or moderate overexpression has on basal LRRK2 activity. Further work is required to establish how basal LRRK2 activity is regulated, and whether other Rab proteins control basal LRRK2 by targeting it to diverse membranes. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.07.087221v1?rss=1 Authors: Lim, T. K. Y., Ruthazer, E. S. Abstract: Partial phagocytosis - called trogocytosis - of axons by microglia has been documented in ex vivo preparations but has yet to be observed in vivo. Fundamental questions regarding the mechanisms that modulate axon trogocytosis as well as its function in neural circuit development remain unanswered. Here we used 2-photon live imaging of the developing Xenopus laevis retinotectal circuit to observe axon trogocytosis by microglia in vivo. Amphibian regulator of complement activation 3 (aRCA3) was identified as a neuronally expressed, synapse-associated complement inhibitory molecule. Overexpression of aRCA3 enhanced axonal arborization and inhibited trogocytosis, while expression of VAMP2-C3, a complement-enhancing fusion protein tethered to the axon surface, reduced axonal arborization. Depletion of microglia also enhanced axonal arborization and reversed the stereotypical escape behaviors to dark and bright looming stimuli. These findings demonstrate that microglia remodel axon morphology through the complement system and that neurons may control this process through expression of complement inhibitory proteins. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.05.31.126607v1?rss=1 Authors: PAREEK, G., Pallanck, L. J. Abstract: The m-AAA proteases play a critical role in the proteostasis of the inner mitochondrial membrane proteins, and mutations in the genes encoding these proteases cause severe incurable neurological diseases. To further explore the biological role of the m-AAA proteases and the pathological consequences of their deficiency, we used a genetic approach in the fruit fly Drosophila melanogaster to inactivate the ATPase family gene 3-like 2 (AFG3L2) gene, which encodes a component of the m-proteases. We found that null alleles of Drosophila AFG3L2 die early in development, but partial inactivation of AFG3L2 using RNAi extended viability to the late pupal and adult stages of development. Flies with partial inactivation of Afg3l2 exhibited marked behavioral defects, neurodegeneration, mitochondrial morphological alterations, and diminished respiratory chain (RC) activity. Further work revealed that reduced RC activity was a consequence of widespread defects in mitochondrial gene expression, including diminished mitochondrial transcription, translation and impaired mitochondrial ribosome biogenesis. These defects were accompanied by the compensatory activation of the mitochondrial unfolded protein response (mito-UPR) and accumulation of unfolded mitochondrial proteins, including proteins involved in transcription. Overexpression of the mito-UPR components partially rescued the Afg3l2-deficient phenotypes, indicating that sequestration of essential components of the mitochondrial gene expression into aggregates partly accounts for these defects. However, Afg3l2 also co-sediments with the mitochondrial ribosome biogenesis machinery, suggesting an additional novel role for Afg3l2 in ribosome biogenesis. Our work suggests that strategies designed to modify mitochondrial stress pathways and mitochondrial gene expression could be therapeutic in the diseases caused by mutations in AFG3L2. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.05.21.103036v1?rss=1 Authors: Liu, X., Blazejewski, S. M., Bennison, S. A., Toyooka, K. Abstract: GSTP proteins are metabolic enzymes involved in removal of oxidative stress and intracellular signaling and also have inhibitory effects on JNK activity. However, the functions of Gstp proteins in the developing brain are unknown. In mice, there are three Gstp proteins, Gstp1, 2 and 3, while there is only one GSTP in humans. By RT-PCR analysis, we found that Gstp1 was expressed beginning at E15.5 in the cortex, but Gstp2 and 3 started expressing at E18.5. Gstp 1 and 2 knockdown caused decreased neurite number in cortical neurons, implicating them in neurite initiation. Using in utero electroporation to knockdown Gstp1 and 2 in layer 2/3 pyramidal neurons in vivo, we found abnormal swelling of the apical dendrite at P3 and reduced neurite number at P15. Using time-lapse live imaging, we found that the apical dendrite orientation was skewed compared to the control, but these defects were ameliorated. Overexpression of Gstp 1 or 2 resulted in changes in neurite length, suggesting a role in neurite elongation. We explored the molecular mechanism and found that JNK inhibition rescued reduced neurite number caused by Gstp knockdown, indicating that Gstp regulates neurite formation through JNK signaling. Thus, we found novel functions of Gstp proteins in neurite initiation during cortical development. Furthermore, the overexpression experiments suggest different functions of Gstp1 and 2 in neurite elongation. Since previous studies have shown the potential implication of Gstp in Autism Spectrum Disorder, our findings will attract more clinical interests in Gstp proteins in neurodevelopmental disorders. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.05.11.089078v1?rss=1 Authors: De Miranda, B. R., Rocha, E. M., Castro, S., Greenamyre, J. T. Abstract: Dopaminergic neurons of the substantia nigra are selectively vulnerable to mitochondrial dysfunction, which is hypothesized to be an early and fundamental pathogenic mechanism in Parkinson's disease (PD). Mitochondrial function depends on the successful import of nuclear-encoded proteins, many of which are transported through the TOM20-TOM22 outer mitochondrial membrane import receptor machinery. Recent data suggests that post-translational modifications of -synuclein promote its interaction with TOM20 at the outer mitochondrial membrane and thereby inhibit normal protein import, which leads to dysfunction and death of dopaminergic neurons. As such, preservation of mitochondrial import in the face of -synuclein accumulation might be a strategy to prevent dopaminergic neurodegeneration, however, this is difficult to assess using current in vivo models of PD. To this end, we established an exogenous co-expression system, utilizing AAV2 vectors to overexpress human -synuclein and TOM20, individually or together, in the adult Lewis rat substantia nigra in order to assess whether TOM20 overexpression attenuates -synuclein-induced dopaminergic neurodegeneration. Twelve weeks after viral injection, we observed that AAV2-TOM20 expression was sufficient to prevent loss of nigral dopaminergic neurons caused by AAV2-Syn overexpression. The observed TOM20-mediated dopaminergic neuron preservation appeared to be due, in part, to the rescued import of nuclear-encoded mitochondrial electron transport chain proteins that were inhibited by -synuclein overexpression. In addition, TOM20 overexpression rescued the import of the chaperone protein GRP75/mtHSP70/mortalin, a stress-response protein involved in -synuclein-induced injury. Collectively, these data indicate that TOM20 expression prevents -synuclein-induced mitochondrial dysfunction, which is sufficient to rescue dopaminergic neurons in the adult rat brain. Copy rights belong to original authors. Visit the link for more info

Hear from NEB Scientists Mehmet Berkman and Paul Riggs about the lessons that New England Biolabs has learned about protein expression and purification over the last 40 years.

Episode 268 looks at Rev-ERB over expression as a whole and how the synthetic ligand SR9009 plays a role in that expression. I wanted to cover the nuclear receptor superfamily, specifically Rev-ERB alpha and beta, first so you can understand why SR has a lot of validity. I then moved into its practical application for males and females as well as remembering to take acute ENVIRONMENT into the equation! Hope you guys enjoy the episode!    •••SUPPORT OUR SPONSOR••• (COACHING) Alex - www.theprepcoach.com   (FREE OPEN FORUM w/ EXCLUSIVE VIDEOS) http://www.theprepcoachforum.com (SUPPLEMENTS) www.projectad.me___use discount code “BFR25” to save off your order! (RESEARCH CHEMS) www.maresearchchems.com___use discount code “alex15” to save off your order! (SPECIALTY SUPPS) www.masupps.com___use discount code “alex20” to save off your order! (INJECTABLE L-CARNITINE) www.synthetek.com___use discount code “alexkikel” to save off your order! (BULK SUPPLEMENTS) www.truenutrition.com___use discount code “AXK5” to save off your order!   •••FIND THE EPISODES••• ITUNES:https://itunes.apple.com/us/podcast/beastfitness-radios-podcast/id1065532968 LIBSYN:http://beastfitnessradio.libsyn.com VIMEO: www.vimeo.com/theprepcoach    •••PREP COACH APPAREL••• https://teespring.com/stores/the-prep-coach-apparel

Meet The Professors: Key Questions and Emerging Research in the Management of Lymphoma, Chronic Lymphocytic Leukemia and Multiple Myeloma — Part 2: A special video supplement to a CME symposium held at the 2019 ASCO Annual Meeting featuring Dr Raje’s comments on the application of emerging research to patient care: Updated risk-stratification model incorporating the revised IMWG diagnostic criteria for smoldering multiple myeloma (MM)  (0:00) Role of imaging in the monitoring and management of MM (5:07) Perspective on the progression-free survival improvement with lenalidomide compared to observation alone for patients with asymptomatic high-risk smoldering MM treated on the Phase III ECOG-E3A06 trial (6:49) Results of the Phase III CASSIOPEIA study evaluating the addition of daratumumab to bortezomib/thalidomide/dexamethasone for newly diagnosed MM in patients eligible for autologous stem cell transplant (ASCT) (9:19) Emerging data from the Phase III MAIA trial evaluating lenalidomide/dexamethasone (Rd) with or without daratumumab for patients with newly diagnosed MM who are not candidates for ASCT (11:32) Role of MRD assessment in clinical decision-making (15:09) Results of the Phase III FORTE trial: Efficacy of carfilzomib/Rd (KRd) with or without ASCT for newly diagnosed MM according to risk status (19:31) Status of the ongoing Phase III ECOG-E1A11 (ENDURANCE) trial: Bortezomib/Rd versus KRd followed by limited or indefinite maintenance lenalidomide for symptomatic newly diagnosed MM (22:53) Clinical experience with and mitigation of differing side effects of bortezomib and carfilzomib (24:29) Appropriate use of oral ixazomib as maintenance therapy after ASCT (26:24) Dose, duration and tolerability of maintenance lenalidomide (29:49) Therapeutic options for patients with MM experiencing disease relapse on maintenance lenalidomide  (33:12) COLUMBA trial: Efficacy and safety of subcutaneous versus intravenous administration of daratumumab for patients with R/R MM (35:36) Similarities and differences between the anti-CD38 antibodies daratumumab and isatuximab (37:47) Melflufen: Novel mechanism of action and structural comparison to standard melphalan (39:47) Overexpression of Bcl-2 in patients with t(11;14) MM and rationale for the use of venetoclax  (41:42) Activity and tolerability of anti-B-cell maturation antigen (BCMA) CAR T-cell therapy in patients with MM (45:40) Use of a bispecific T-cell engager to target BCMA and CD3 in patients with MM; early efficacy and safety data with AMG 420 (49:55) Incidence and management of cytokine release syndrome and neurotoxicity associated with anti-BCMA CAR T-cell therapy for MM (52:39) Future directions for CAR T-cell therapy in MM (55:09) CME information and select publications  

How does an engineer approach microbial genetics? cworks with microbes of all kinds to optimize metabolic and agricultural systems. Here he discusses his work with Rhodobacter to make biofuels and for membrane protein expression, with Agrobacterium and plant pathogenic viruses to make drought-resistant plants, and with Clostridium and yeast cocultures for lignocellulose digestion. Take the listener survey at asm.org/mtmpoll Full shownotes at asm.org/mtm Links for this Episode: Wayne Curtis Lab site at Penn State University PLoS One: Molecular Cloning, Overexpression, and Characerization of a Novel Water Channel protein from Rhodobacter sphaeroides Protein Expression and Purification: Advancing Rhodobacter sphaeroides as a Platform for Expression of Functional Membrane Proteins Biotechnology for Biofuels: Consortia-Mediated Bioprocessing of Cellulose to Ethanol with a symbiotic Clostridium phytofermentans/Yeast Co-Culture HOM Tidbit: Genentech “Cloning Insulin” blog HOM Tidbit: Genentech press release announcing insulin cloning  

En el program de esta semana hablamos sobre Pseudomonas aeruginosa y el rol en el tratamiento que tendría el ceftolozano / tazobactam  sobre todo el lo referente a cepas multiresistentes y como los diversos mecanismos de resistencia que este tipo de bacterial puede manifestar pueden afectar la sensibilidad a dicho antimicrobiano. Referencias: A.P Magiorakos y colaboradores. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012; 18 (3) 268-81. Gabriel Cabot y colaboradores. Pseudomonas aeruginosa Ceftolozane-Tazobactam Resistance Development Requires Multiple Mutations Leading to Overexpression and Structural Modification of AmpC. Antimicrob Agent Chemother 2014; June 58 (6): 3091-9. Pablo A. Fraile-Ribot y colaboradores. Mechanisms leading to in vivo ceftolozane/tazobactam resistance development during the treatment of infections caused by MDR Pseudomonas aeruginosa. J Antimicrob Chemother 2017; 73: 658-663.  Yu Mi Wi y colaboradores. Activity of Ceftolozane-Tazobactam against Carbapenem- Resistant, Non-Carbapenemase-Producing Pseudomonas aeruginosa and Associated Resistance Mechanisms. Antimicrob Agents Chemother 2017; 62 (1).  Andrew Walkty y colaboradores. In vitro activity of ceftolozane/tazobactam versus antimicrobial non-susceptible Pseudomonas aeruginosa clinical isolates including MDR and XDR isolates obtained from across Canada as part of the CANWARD study, 2008–16. J Antimicrob Chemother 2018; 73: 703-708. Tommaso Giani y colaboradores. Italian nationwide survey on Pseudomonas aeruginosa from invasive infections: activity of ceftolozane/tazobactam and comparators, and molecular epidemiology of carbapenemase producers. J Antimicrob Chemother 2018; 73: 664-671. David M. Livermore y colaboradores. Activity of ceftazidime/avibactam against problem Enterobacteriaceae and Pseudomonas aeruginosa in the UK, 2015–16. J Antimicrob Chemother 2018. 73: 648-657.   La Frase de la Semana:   Y esta semana no puede ser sino de Stephen Hawkings debido a queel británico murió este martes (la madrugada del miércoles en Inglaterra), a sus 76 años. Fue uno de los físicos más importantes de la cosmología moderna y se destacó por su teoría sobre agujeros negros y por sus esfuerzos matemáticos para unificar la teoría general de la relatividad de Einstein con los conocimientos emergentes de la física cuántica. La frase dice:  “El peor enemigo del conocimiento no es la ignorancia, es la ilusión del conocimiento”        

Full text - http://bit.ly/2yVSSPa Oncotarget | Interview with Dr. Jaclyn Hechtman from the Department of Pathology Memorial Sloan Kettering Cancer Center, New York, NY, USAtalking about their Cover paper - Volume 7 Issue 32 - Oncotarget -"Recurrent, truncating SOX9 mutations are associated with SOX9 overexpression, KRAS mutation, and TP53 wild type status in colorectal carcinoma" Facebook - http://bit.ly/2xznxjV Twitter - http://bit.ly/2xzWvsu LinkedIn - http://bit.ly/2xzJ6kc Pintrest - http://bit.ly/2xzX8SS Reddit - http://bit.ly/2hoxI0N www.Oncotarget.com

Commentary by Dr. Valentin Fuster

Dr Harris talks to ecancertv at SABCS 2015, about his research on APOBEC3B and its correlation to drug resistance in breast cancer. APOBEC, particularly APOBEC3B is a brand new source of mutation in cancer, he observes. Infection with the human papillomavirus (HPV) has been shown to account for upregulation of APOBEC3B in cervical and head/ and neck cancers but it is not clear how it becomes changed in on-viral malignancies such as breast cancer. Dr Harris discusses data that show that upregulation in breast cancer may occur via the PKC-NFκB pathway. These data establish the first mechanistic link between APOBEC3B and a common signal transduction pathway, he says, suggesting that using inhibitors that target this pathway may be of benefit.

Idiopathic pulmonary fibrosis (IPF) is an irreversible and progressive disease of the lungs, which is characterized by aberrant tissue remodeling and massive deposition of extracellular matrix proteins. This process is mainly conducted by myofibroblasts, an activated fibroblast phenotype. During the pathogenesis of IPF, the fine alveolar structure is destroyed and gas exchange declines, finally resulting in organ failure. So far, pharmacological treatment options are very limited and lung transplantation still remains the only curative therapy. Pathologic tissue remodeling in IPF is closely connected to altered cell and protein homeostasis. The ubiquitin-proteasome system is critical for degradation of polyubiquitinated proteins in a spatially and timely controlled manner, thereby regulating protein levels. The proteasome is a multicatalytic enzyme complex consisting of a barrel shaped 20S catalytic core particle (CP) and one or two 19S regulatory particles (RP), thus forming active 26S/30S proteasomes. Dysregulation of the proteasome has been reported for several chronic diseases of the heart, brain, and also lung. Furthermore, inhibition of the proteasome has been shown to provide antifibrotic effects in different organs, including the lung. As nothing is known about proteasome function in the pathogenesis of IPF, the first aim of the present study was to analyze proteasomal regulation during tissue remodeling and myofibroblast differentiation. For that, lung fibroblasts were treated with transforming growth factor-β (TGF-β) and proteasome activity as well as composition was examined. For in vivo testing, the bleomycin mouse model of lung fibrosis was used and human lung tissue of IPF patients was analyzed. It was found that induction of myofibroblast differentiation by TGF-β mediated assembly of 19S RPs with 20S CPs, thereby forming 26S/30S complexes, which was critically dependent on the regulatory particle non ATPase 6 subunit (Rpn6). In addition, silencing of Rpn6 in primary human lung fibroblasts counteracted TGF β induced myofibroblast differentiation. During bleomycin-induced fibrotic remodeling of mouse lungs, increased formation of 26S/30S proteasomes was accompanied by augmented expression of Rpn6 in fibrotic lungs. Here, Rpn6 was highly expressed in hyperplastic alveolar epithelial cells and Clara cells. Overexpression of Rpn6 was also observed in myofibroblasts and hyperplastic bronchiolar basal cells of fibrotic lung tissue of IPF patients and accompanied by enhanced polyubiquitination of proteins. As therapeutic application of proteasome inhibitors in pulmonary fibrosis showed controversial results including beneficial antifibrotic effects but also toxicity, the second aim of this study was to test whether site specific inhibition of the proteasome, using the novel second generation inhibitor oprozomib, provides antifibrotic effects in the absence of systemic side effects after local pulmonary application. Oprozomib was compared to the FDA-approved proteasome inhibitor bortezomib and tested on the human alveolar epithelial cancer cell line A549 and on primary mouse alveolar epithelial type II cells regarding its cytotoxic effects. Oprozomib was less toxic than bortezomib and provided high selectivity for the chymotrypsin-like active site of the proteasome. In primary mouse lung fibroblasts, oprozomib showed significant antifibrotic effects like reduction of collagen I and α-smooth muscle actin expression at non-toxic doses. When applied locally into the lungs of healthy mice via instillation, oprozomib was well tolerated and effectively reduced pulmonary proteasome activity. In bleomycin-challenged mice, however, locally applied oprozomib resulted in accelerated weight loss and increased mortality. Furthermore, oprozomib failed to reduce fibrosis in these mice, but rather augmented fibrotic lung remodeling in bleomycin-challenged animals. To conclude, this study identified a novel mechanism for fibrotic remodeling of the lungs involving 26S/30S proteasome activation via Rpn6 upon TGF-β-mediated myofibroblast differentiation. Increased levels of Rpn6 and polyubiquitinated proteins in IPF lungs further suggest an important contribution of the ubiquitin-proteasome system to the pathogenesis of this disease. Inhibition of the proteasome with the novel site-specific proteasome inhibitor oprozomib provided low toxicity and antifibrotic effects in alveolar epithelial cells and pulmonary fibroblasts. These results could not be confirmed in pulmonary fibrosis of bleomycin-treated mice, as oprozomib treatment showed high toxicity in fibrotic animals. In light of these data, current proteasome inhibitors, which block the catalytic core, might be too toxic as therapeutic agents for the treatment of fibrotic lung diseases. However, interference with the formation of 26S/30S proteasomes, as shown by Rpn6 knockdown, might provide a novel concept for therapeutic regulation of proteasome activity in lung fibrosis.

Sat, 31 Jan 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18066/ https://edoc.ub.uni-muenchen.de/18066/1/Porada_Sabrina.pdf Porada, Sabrina ddc:590, ddc

Wed, 9 Jul 2014 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17697/ https://edoc.ub.uni-muenchen.de/17697/1/Guo_Yang.pdf Guo, Yang

Background: The receptor for activated C-kinase 1 (RACK1) is a conserved protein belonging to the WD40 repeat family of proteins. It folds into a beta propeller with seven blades which allow interactions with many proteins. Thus it can serve as a scaffolding protein and have roles in several cellular processes. Results: We identified the product of the Dictyostelium discoideum gpbB gene as the Dictyostelium RACK1 homolog. The protein is mainly cytosolic but can also associate with cellular membranes. DdRACK1 binds to phosphoinositides (PIPs) in protein-lipid overlay and liposome-binding assays. The basis of this activity resides in a basic region located in the extended loop between blades 6 and 7 as revealed by mutational analysis. Similar to RACK1 proteins from other organisms DdRACK1 interacts with G protein subunits alpha, beta and gamma as shown by yeast two-hybrid, pull-down, and immunoprecipitation assays. Unlike the Saccharomyces cerevisiae and Cryptococcus neoformans RACK1 proteins it does not appear to take over G beta function in D. discoideum as developmental and other defects were not rescued in G beta null mutants overexpressing GFP-DdRACK1. Overexpression of GFP-tagged DdRACK1 and a mutant version (DdRACK1mut) which carried a charge-reversal mutation in the basic region in wild type cells led to changes during growth and development. Conclusion: DdRACK1 interacts with heterotrimeric G proteins and can through these interactions impact on processes specifically regulated by these proteins.

Dr. Louise Rodino-Klapac Discusses Alpha 7 Integrin As A Therapeutic Approach to Muscular Dystrophy Guest: Louise Rodino-Klapac, PhD, principal investigator, Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital. Access an abstract of this month’s featured research article: AAV-mediated Overexpression of Human a7 Integrin Leads to Histological and Functional Improvement in Dystrophic Mice. Mol Ther. 2013 Mar;21(3):520-5. doi: 10.1038/mt.2012.281. Epub 2013 Jan 15.

Dr. Louise Rodino-Klapac Discusses Alpha 7 Integrin As A Therapeutic Approach to Muscular Dystrophy Guest: Louise Rodino-Klapac, PhD, principal investigator, Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital. Access an abstract of this month’s featured research article: AAV-mediated Overexpression of Human a7 Integrin Leads to Histological and Functional Improvement in Dystrophic Mice. Mol Ther. 2013 Mar;21(3):520-5. doi: 10.1038/mt.2012.281. Epub 2013 Jan 15.

Prof Untch discusses treating HER 2, over-expressing tumours, at EBCC 8 in Vienna, and recent developments using combination treatments with trastuzumab, pertuzumab and other HER2 blocking agents. It has been found that with treatment administered before surgery, efficacy is high and the tumour is eradicated from breast and lymph nodes. With data from a recent meta-analysis, predictions can now be made on the outcome of patients in HER2+ cases with HER2 blocking agents, chemotherapy and surgery.

Overexpression or mutation of alpha-Synuclein is associated with protein aggregation and interferes with a number of cellular processes, including mitochondrial integrity and function. We used a whole-genome screen in the fruit fly Drosophila melanogaster to search for novel genetic modifiers of human A53T] alpha-Synuclein-induced neurotoxicity. Decreased expression of the mitochondrial chaperone protein tumor necrosis factor receptor associated protein-1 (TRAP1) was found to enhance age-dependent loss of fly head dopamine (DA) and DA neuron number resulting from A53T] alpha-Synuclein expression. In addition, decreased TRAP1 expression in A53T] alpha-Synuclein-expressing flies resulted in enhanced loss of climbing ability and sensitivity to oxidative stress. Overexpression of human TRAP1 was able to rescue these phenotypes. Similarly, human TRAP1 overexpression in rat primary cortical neurons rescued A53T] alpha-Synuclein-induced sensitivity to rotenone treatment. In human (non) neuronal cell lines, small interfering RNA directed against TRAP1 enhanced A53T] alpha-Synuclein-induced sensitivity to oxidative stress treatment. A53T] alpha-Synuclein directly interfered with mitochondrial function, as its expression reduced Complex I activity in HEK293 cells. These effects were blocked by TRAP1 overexpression. Moreover, TRAP1 was able to prevent alteration in mitochondrial morphology caused by A53T] alpha-Synuclein overexpression in human SH-SY5Y cells. These results indicate that A53T] alpha-Synuclein toxicity is intimately connected to mitochondrial dysfunction and that toxicity reduction in fly and rat primary neurons and human cell lines can be achieved using overexpression of the mitochondrial chaperone TRAP1. Interestingly, TRAP1 has previously been shown to be phosphorylated by the serine/threonine kinase PINK1, thus providing a potential link of PINK1 via TRAP1 to alpha-Synuclein.

Signaling through PAS kinases bypasses the pro-growth requirement of Tor2 in yeast.

Eny2, the mammalian ortholog of yeast Sus1 and drosophila E(y)2, is a nuclear factor that participates in several steps of gene transcription and in mRNA export. We had previously found that Eny2 expression changes in mouse pancreatic islets during the metabolic adaptation to pregnancy. We therefore hypothesized that the protein contributes to the regulation of islet endocrine cell function and tested this hypothesis in rat INS-1E insulinoma cells. Overexpression of Eny2 had no effect but siRNA-mediated knockdown of Eny2 resulted in markedly increased glucose and exendin-4-induced insulin secretion from otherwise poorly glucose-responsive INS-1E cells. Insulin content, cellular viability, and the expression levels of several key components of glucose sensing remained unchanged; however glucose-dependent cellular metabolism was higher after Eny2 knockdown. Suppression of Eny2 enhanced the intracellular incretin signal downstream of cAMP. The use of specific cAMP analogues and pathway inhibitors primarily implicated the PKA and to a lesser extent the EPAC pathway. In summary, we identified a potential link between the nuclear protein Eny2 and insulin secretion. Suppression of Eny2 resulted in increased glucose and incretin-induced insulin release from a poorly glucose-responsive INS-1E subline. Whether these findings extend to other experimental conditions or to in vivo physiology needs to be determined in further studies.

The transcription factor HNF1B, encoded by the TCF2 gene, plays an important role in the organogenesis of vertebrates. In humans, heterozygous mutations of HNF1B are associated with several diseases, such as pancreatic β-cell dysfunction leading to maturity-onset diabetes of the young (MODY5), defective kidney development, disturbed liver function, pancreas atrophy, and malformations of the genital tract. The African claw frog Xenopus laevis is an excellent model to study the processes involved in embryogenesis and organogenesis, as it can be manipulated easily with a series of methods. In the present study, we overexpressed HNF1β mutants in the developing Xenopus embryo to assess their roles during organogenesis, particularly in the developing pronephric kidney. Towards this goal, we developed a heat-shock inducible binary Cre/loxP system with activator and effector strains. Heat-shock activation of the mutant HNF1B variants P328L329del and A263insGG resulted in malformations of various organs and the affected larvae developed large edemas. Defects in the pronephros were primarily confined to malformed proximal tubules. Furthermore, the expression of the proximal tubule marker genes tmem27 and slc3a1, both involved in amino acid transport, was affected. Both P328L329del and A263insGG downregulated expression of slc3a1. In addition, P328L329del reduced tmem27 expression while A263insGG overexpression decreased expression of the chloride channel clcnk and the transcription factor pax2. Overexpression of two mutant HNF1B derivatives resulted in distinct phenotypes reflected by either a reduction or an enlargement of pronephros size. The expression of selected pronephric marker genes was differentially affected upon overexpression of HNF1B mutations. Based on our findings, we postulate that HNF1B mutations influence gene regulation upon overexpression in specific and distinct manners. Furthermore, our study demonstrates that the newly established Cre/loxP system for Xenopus embryos is an attractive alternative to examine the gene regulatory potential of transcription factors in developing pronephric kidney as exemplified here for HNF1B.

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.

T cells directed against brain antigens are generally held to play a crucial role in the initiation of multiple sclerosis (MS). This was deduced from experimental autoimmune encephalomyelitis (EAE). In this model for MS, T cells reactive for myelin antigens induced a severe paralytic disease upon transfer to healthy syngeneic recipients. Intriguingly, the disease does not start immediately upon transfer of the pathogenic effector T cells. Instead, as earlier studies have shown, the effector T cells attack their target organ only after having migrated in the periphery through secondary lymphoid organs. The aim of the project was to characterize the functional properties of these migrating encephalitogenic T cells during the course of EAE and to identify biological pathways which determine their migratory behaviour and pathogenic potential. To this end, average linkage hierarchical clustering, pathway and gene ontology (GO) analyses of transcriptomes from cultured and ex vivo-isolated myelin basic protein-reactive T cells (TMBP cells) were performed. At the time of transfer, encephalitogenic T cells in vitro are maximally activated, i.e. they exhibit a prominent upregulation of cell cycle genes such as cyclin A2 (CCNA2) and cyclin B2 (CCNB2) among others. In contrast, T cells isolated from spleen 3 days post transfer, downregulated activation markers such as interleukin 2 receptor (IL2R) and interferon γ (IFNγ), and at the same time upregulated migration specific genes such as CC-chemokine receptor 1 (CCR1), CC-chemokine receptor 2 (CCR2) and CC-chemokine receptor 5 (CCR5). Hierarchical cluster analysis revealed that several transcription regulators known for inhibiting cell cycle progression such as krüppel-like factor 4 (KLF4), B-cell translocation gene 2 (BTG2) and transducer of ERBB2, 1 (TOB1) were clustered together with cell cycle and migration genes. Overexpression of KLF4 in T cells not only inhibited G1/S phase progression of the cell cycle but additionally induced upregulation of CCR2 and CCR5. A novel tetraspan membrane protein called epithelial membrane protein (EMP1), was found to be up regulated in ex vivo-isolated effector T cells. Overexpression of EMP1 in encephalitogenic T cells influenced the migratory behaviour of effector T cells both in vitro and in vivo. EMP1 enhanced T cell motility within the extracellular matrix milieu in vitro and promoted T cell migration from the connective tissue to lymph nodes in vivo resulting in an accelerated onset of EAE. In conclusion, gene expression profiling of encephalitogenic T cells revealed interesting genome wide transcriptomic changes and established a correlation between cell cycle progression and cell migration. As a result, in silico analysis put forth several interesting candidate genes that hold promise as potential targets for therapeutic intervention.

The crista junction is a tubular element of the mitochondrial inner membrane connecting inner boundary membrane and cristae membrane. The molecular basis of its formation was so far largely unknown. The lack of the mitochondrial inner membrane protein mitofilin results in the absence of crista junctions in HeLa-cells. Moreover, deletion of Fcj1, the orthologue of mitofilin in S. cerevisiae, leads to concentric stacks of cristae membranes and the virtual absence of crista junctions. The aim of the present work was to investigate the function of Fcj1 as well as the molecular basis of the formation of cristae and crista junctions in S. cerevisiae. Fcj1 undergoes homotypic interactions and is part of a stable oligomeric protein complex. Using immuno-EM, Fcj1 has been shown to be specifically enriched at crista junctions. All other proteins, which have been studied so far, such as subunits Su e and Su g of the F1FO-ATP-Synthase, are rather underrepresented in this region. Overexpression of Fcj1 increases the number of crista junctions, enlarges the crista junction diameter and leads to internal branching of cristae. Upon downregulation of Fcj1, the mitochondrial ultrastructure progressively changes reflecting the deletion phenotype more and more. Considering the fact that the decrease of the number of crista junctions occurs at an early time point, Fcj1 seems directly involved in the formation of crista junctions. In order to investigate if cristae form a detached compartment in the absence of crista junctions, the accessibility of the intracristal space for the protein-modifying substance AMS was examined. In fcj1 mitochondria the modification of cytochrome c was temporally delayed and incomplete in comparison to wild-type. Hence, metabolite exchange between intracristal space and cristae space seems impaired though not entirely impossible in the absence of crista junctions. This might be due to some extent of fusion and fission tracing back to protein or lipid exchange within the inner membrane of mitochondria. Cryo-EM tomograms revealed regular zipper-like arrangements of F1FO-ATP-Synthase particles. An inverse correlation of the amount of Fcj1 and the oligomeric state of F1FO-ATP-Synthase was observed upon biochemical analysis: in fcj1 mitochondria, F1FO-ATP-Synthase oligomers have a higher molecular weight and are more stable than in wild-type mitochondria, whereas in mitochondria of cells overexpressing Fcj1 they are smaller and less stable. Subunits Su e and Su g are essential for the stabilization of dimers and oligomers of the F1FO-ATP-Synthase. As the influence of Su e/Su g on the formation of crista junctions has not been investigated so far, the mitochondrial ultrastructure of both deletion strains was analyzed concerning this matter. Enlargement of crista junctions and internal branching of cristae similar to the overexpression phenotype of Fcj1 have been observed in both strains. Furthermore, crista tips are virtually absent. Apart from this functional interaction, Fcj1 and Su e/Su g have also been shown to interact genetically. Based on the gained insights on the roles of Fcj1 and Su e/Su g in cristae formation the following model is proposed: the modulation of the inner membrane of mitochondria is based on an antagonistic behavior of Fcj1 and Su e/Su g. Fcj1 introduces negative membrane curvatures, possibly by impairing the oligomerisation of the F1FO-ATP-Synthase. The presence of Su e/Su g, in contrast, promotes positive curvatures of cristae membranes by stabilizing dimers and oligomers of the F1FO-ATP-Synthase. Taken together, relative amounts of Fcj1 and Su e/Su g locally modulate the oligomeric state of the F1FO-ATP-Synthase and thereby enable the formation of crista junctions and crista tips in mitochondria.

Sat, 13 Feb 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/11347/ https://edoc.ub.uni-muenchen.de/11347/1/Grzech_Marjeta.pdf Grzech, Marjeta ddc:590, dd

Filamin and Cortexillin are F-actin crosslinking proteins in Dictyostelium discoideum allowing actin filaments to form three-dimensional networks. GAPA, an IQGAP related protein, is required for cytokinesis and localizes to the cleavage furrow during cytokinesis. Here we describe a novel interaction with Filamin which is required for cytokinesis and regulation of the F-actin content. The interaction occurs through the actin binding domain of Filamin and the GRD domain of GAPA. A similar interaction takes place with Cortexillin I. We further report that Filamin associates with Rac1a implying that filamin might act as a scaffold for small GTPases. Filamin and activated Rac associate with GAPA to regulate actin remodelling. Overexpression of filamin and GAPA in the various strains suggests that GAPA regulates the actin cytoskeleton through interaction with Filamin and that it controls cytokinesis through association with Filamin and Cortexillin.

Background Cumulative light exposure is significantly associated with progression of age-related macular degeneration (AMD). Inhibition of vascular endothelial growth factor is the main target of current antiangiogenic treatment strategies in AMD. However, other growth factors, such as platelet-derived growth factor (PDGF) and placenta growth factor (PlGF), have a substantial impact on development of AMD. Previous reports indicate that sorafenib, an oral multikinase inhibitor, might have beneficial effects on exudative AMD. This study investigates the effects of sorafenib on light-induced overexpression of growth factors in human retinal pigment epithelial (RPE) cells. Methods Primary human RPE cells were exposed to white light and incubated with sorafenib. Viability, expression, and secretion of VEGF-A, PDGF-BB, and PlGF and their mRNA were determined by reverse transcription-polymerase chain reactions, immunohistochemistry and enzyme-linked immunosorbent assays. Results Light exposure decreased cell viability and increased expression and secretion of VEGF-A, PDGF-BB and PlGF. These light-induced effects were significantly reduced when cells were treated with sorafenib at a dose of 1 mu g/ml. Conclusion The results show that sorafenib has promising properties as a potential antiangiogenic treatment for AMD.

Background: Curcumin induces apoptosis in many cancer cells and it reduces xenograft growth and the formation of lung metastases in nude mice. Moreover, the plant derived polyphenol has been reported to be able to overcome drug resistance to classical chemotherapy. These features render the drug a promising candidate for tumor therapy especially for cancers known for their high rates concerning therapy resistance like melanoma. Results: We show here that the melanoma cell line M14 is resistant to Curcumin induced apoptosis, which correlates with the absence of any effect on NF kappa B signaling. We show that CXCL1 a chemokine that is down regulated in breast cancer cells by Curcumin in an NF kappa B dependant manner is expressed at variable levels in human melanomas. Yet in M14 cells, CXCL1 expression did not change upon Curcumin treatment. Following the hypothesis that Curcumin is rapidly removed from the resistant cells, we analyzed expression of known multi drug resistance genes and cellular transporters in M14 melanoma cells and in the Curcumin sensitive breast cancer cell line MDA-MB-231. ATP-binding cassette transporter ABCA1, a gene involved in the cellular lipid removal pathway is over-expressed in resistant M14 melanoma as compared to the sensitive MDA-MB-231 breast cancer cells. Gene silencing of ABCA1 by siRNA sensitizes M14 cells to the apoptotic effect of Curcumin most likely as a result of reduced basal levels of active NF kappa B. Moreover, ABCA1 silencing alone also induces apoptosis and reduces p65 expression. Conclusion: Resistance to Curcumin thus follows classical pathways and ABCA1 expression should be considered as response marker.

Fri, 19 Oct 2007 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/9088/ https://edoc.ub.uni-muenchen.de/9088/1/Le_Bris_Anna.pdf Le Bris, Anna ddc:500, ddc:540, Fakultät für Chemie und Pharmazie

Insulin-like growth factors (IGF) are not only mediators of metabolic actions, but also regulate cell growth, differentiation and apoptosis. IGF-II is of particular interest because of its mitogenic effects. It is known that endothelial progenitor cells (EPC) improve myocardial function after acute myocardial infarction. The aim of this study was to investigate whether overexpression of IGF-II in EPC further contributes to improvement in left ventricular function after myocardial infarction. Human CD34+ cells from cord blood were isolated and cultured in adequate cell medium. Early passage EPC were transduced ex vivo by a retroviral vector expression of IGF-II (EPC-IGF-II) or empty vector (EPC-pLXSN) and expanded up to 46 population doublings. Expression levels were confirmed by RT-PCR. Athymic, nude rats were thoracotomized and ligation of the LAD (left anterior descending artery) was performed for 30 minutes before reperfusion was initiated. Vector only transduced EPC or EPC-IGF-II cells were injected immediately after reperfusion in the border of the infarct zone. Serial echocardiographic measurements were performed to analyze left ventricular ejection fraction (EF) up to 2 weeks after myocardial infarction when animals were mercy killed. Transplantation of EPC-derived cells improved left ventricular function after experimental myocardial infarction from 47,3 ± 1,8 % (EF of the control group, n = 11) to 51,4 ± 0,7 % EF 2 weeks after infarction (p < 0,05, n = 9). Overexpression of IGF-II further improved left ventricular ejection fraction to 53,6 ± 0,5 % EF at 2 weeks as compared to empty vector transduced cells (p < 0,05, n = 10). In vitro experiments revealed that IGF-II dose-dependently enhanced the proliferation capacity of H9C2 rat cardiomyoblasts measured by a BrdU incorporation assay. Immunhistological analysis of proliferating cells in the myocardium showed an increased number of Ki67+ cells within the infarct zone 7 days after transplantation of IGF-II overexpressing cells as compared to empty vector transduced cells. It was shown that transplantation of IGF-II overexpressing EPC impaired the infarction size by nearly 20 % in comparison to EPC-pLXSN (p < 0,05). Thus, transplantation of IGF-II overexpressing EPC in acute myocardial infarction may improve myocardial function by enhancing the proliferation capacity of resident cardiomyocyteprogenitors.

ABCG2 is a transporter protein that has the ability to efflux many drugs and fluorescent dyes. Primitive haematopoietic stem cells highly express ABCG2 and the expression level decreases as these cells differentiate indicating a possible role of this transporter in HSC. In the present study, we have analyzed the role of ABCG2 in early haematopoietic stem cells by constitutively expressing ABCG2 in human CB derived CD133+ cells. This constitutive expression of ABCG2 demonstrated an enhancement of primary CFCs in vitro, including the most primitive clonogenic cells the CFU-GEMM (n=12, p

Phosphate regulating Hormone with homologies to Endopeptidases on the X- chromosome (PHEX, formerly identified as PEX) is the gene responsible for the hereditary disease X-linked hypophosphatemic rickets (XLH) and affects one in twenty thousand people, making it the most common form of rickets. A homologous disease has also been identified in Mus musculus and given the label Hyp for Hypophosphatemia. The cause of both diseases is an inactivation of the carboxy terminal end of the gene through mutation or deletion. It has been demonstrated that PHEX affects the pathway or regulatory elements for the expression of the renal sodium dependant phosphate transporter, NPT2a, and therefore phosphate resorption in the kidney. In a separate regulatory pathway PHEX affects the mineralization of osteoid, the scaffolding of hard bone. In this thesis, I have created and analyzed transgenic mouse strains overexpressing hPHEX. The transgenic animals were classified by PCR and PHEX was pinpointed by in situ hybridization to be expressed in trabecular and cortical bone as expected. Phenotypical analysis of transgenic animals demonstrated that biochemical measurements were not affected by the presence of the transgene under the control of a ubiquitous promoter. The transgenic hPHEX animals were crossed with Hyp mice to establish whether a rescue or partial rescue of the mutant phenotype was possible. Phenotypical analysis of the rescue mice indicated an improvement in body weight and bone morphology, including mineralization, over the mutant hyp mice, while most biochemical parameters remained unchanged.

The OSTL gene is localized at the band q23 in the chromosome 6. Its localization corresponds to a translocation breakpoint between chromosomes 6 and 12, the t(6;12)(q23;p13), that was characterized in our group in an acute lymphoblastic leukemia cell line. This translocation involves the ETV6 (translocation ETs leukemia) gene localized in chromosome 12 with the STL (six twelve leukemia gene) gene localized in chromosome 6. The STL gene shares the first exon with a novel gene, that we named OSTL (opposite STL), but they are transcribed in opposite directions. Since the fusion gene ETV6/STL encodes only for a very small protein which lacks any known functional domain, we speculate that the main leukemogenic effect of this translocation is the deregulation of OSTL. OSTL has a RING-Finger motif that is highly conserved between species and has a significant homology with other genes in human as well as C. elegans, D. melanogaster, and S. cerevisiae. OSTL showed a very specific expression pattern during the mouse embryogenesis. The aim of this project was the functional characterization of OSTL, with special emphasis in normal hematopoiesis and leukemogenesis. Therefore we have sequenced the whole human and mouse OSTL cDNA by using OSTL cDNA clones from the RZPD (“Resource Zentrum Primäre Datenbank”) in Berlin. These sequences encode for a 307 (mouse) and a 275 (human) amino acids length protein. The protein length differences between human and mouse are explained because of the existence of alternative spliced exons. The homology between human and mouse sequence is 99% at the protein level. The expression of GFP-OSTL fusion protein in mouse fibroblast cell line enable us to observe the subcellular localization of OSTL protein. GFP-OSTL is localized mainly in the cytoplasm, showing small spots, probably in the mitochondrial region. In a mouse multiple tissue Northern blot, we could show that OSTL is expressed in testis, ovary and liver. In an human multiple tissue Northern, OSTL expression was observed in skeletal muscle, testis, ovary, heart, placenta, pancreas and prostate. Northern blotting with different human cell lines revealed expression of OSTL in three EBV (Epstein Barr Virus) transformed lymphoblastoid cell lines (LCL B, LCL D, and LCL R) and in one NHL (Non-Hodgkin Lymphoma) cell line (Karpas 422). In Reverse Transcriptase PCR experiments using B cell in different maturation stages, the expression of OSTL was observed in naive, memory B and plasma cells, and in leukemic patient samples, expression was observed in several AML and ALL cDNAs. Whole mount in situ hybridization experiments were performed to investigate the temporo-spatial expression pattern of OSTL during mouse embryogenesis. There was distinct expression of Ostl in the somites (myotome), first and second branchial arches, optic and otic vesicles, in the hair follicles of the vibrissae, and limb buds in mouse embryos of embryonal days 9.5 to 14.5. This expression pattern suggests an important role for Ostl in the early development of these structures. Aiming to find protein interaction partners of OSTL, we performed a Yeast Two Hybrid assay using a Hela cDNA library. Among others we found interaction of OSTL with the antiapoptotic protein, HAX-1 (HS1-associated protein X-1), that is involved in the regulation of B-cell signal transduction, and interaction with the pro-apoptotic protein, SIVA. SIVA was originally identified as an interaction partner of CD27 (TNFRSF7), a member of the TNF-receptor superfamily, which is expressed in B cells. These interactions were confirmed by in vitro (cotransformation in yeast, CoIP) and in vivo (colocalization of these proteins in mammalian cells and CoIP) assays. Overexpression of Ostl in primary mouse hematopoietic cells followed by injection of the cells into lethally irradiated mice resulted in a T-Acute-Lymphoblastic-Leukemia (T-ALL) phenotype. In summary, our experiments could demonstrate that OSTL is important in B cell development and signaling and deregulation of this gene can contribute to the development of hematologic malignancies.

ATP synthase is one of the major photosynthetic complexes that represents one of the smallest molecular motors known in nature. The rotating γ subunit is a key feature of this enzyme. It contains features specific for the chloroplast ATP synthase. In this work the γ subunit has been functionally analyzed in Arabidopsis thaliana. - The nuclear gene atpC1 encoding the γ subunit of the plastid ATP synthase has been inactivated by T-DNA insertion mutagenesis. In the seedling-lethal dpa1 mutant the absence of detectable amounts of the γ subunit destabilizes the entire ATP synthase complex and consequently photophosphorylation is abolished. However, in vivo protein labelling analysis suggests that assemαβ bly of the ATP synthase and subunits into the thylakoid membrane still occurs in dpa1. Further effects of the mutation include an increased light sensitivity of the plants and an altered photosystem II activity. A high non-photochemical quenching develops with increasing actinic light intensity. It has been shown that a high proton gradient is responsible for most quenching (qE). The photoprotective role of qE was further demonstrated in the double mutant dpa1 x psbS in which PsbS, essential factor for qE, is missing. - The expression of a second gene copy, atpC2, is unaltered in dpa1 and is not sufficient to compensate for the lack of atpC1 expression. The two proteins, AtpC1 and AtpC2, share less similarity than AtpC1 of Arabidopsis with γ subunits of other plant species suggesting that the γ subunits so far isolated in other plant species are AtpC1 orthologs. It has been established that AtpC2 is also imported into the chloroplast. Therefore, it is likely that the chloroplast ATP synthase complexes contain both atpC1 and atpC2 encoded γ subunits. However, the atpC2 gene is expressed more than hundred times at a lower level than atpC1 and array data show the differential and tissue specific expression of the two genes. The function of AtpC2 could not be revealed by inactivating the gene. Overexpression of atpC2 in dpa1 generated viable lines with an ATP synthase complex containing only γ2, although wild type phenotype is not completely restored. The second part of this work regarded the optimization of conditions for plastid transformation in Arabidopsis thaliana. An efficient and fast regeneration system from cotyledon protoplasts was established for Arabidopsis thaliana accessions C24, Columbia, and Wassilewskija. Culture conditions and media compositions were optimized for the development of protoplasts embedded in thin alginate layers. The protocol is reproducible, efficient, extremely fast, and regenerated plants are fertile. Thus, this cotyledon-based system could prove useful for studying plant cell and molecular biology in A. thaliana. - The sul gene appeared to be a potential novel candidate as selectable marker for plastid transformation. However, genetic and molecular studies demonstrated that sul can not be used for this purpose. On the other hand a new function of sul appeared. The gene could be the missing marker for mitochondria transformation in higher plants.

Insulin-like growth factor I (IGF-I) and -II (IGF-II) are single chain peptides produced by many tissues, functioning in an endocrine, autocrine or paracrine fashion to regulate cellular proliferation, survival and differentiation. IGF actions are initiated upon binding to the insulin-like growth factor I receptor (IGF-IR) and are modulated through interactions with a family of six secreted IGF-binding proteins (IGFBP-1 to -6). IGF-I is necessary for normal growth and differentiation during both, embryonic and postnatal development. IGF-II is a stimulator of fetal growth but its functions in the postnatal period are still unclear. Notably, expression of IGF-II is shut down shortly after birth in rodents (but not in humans). Previous studies in phosphoenolpyruvate-carboxykinase (PEPCK)-IGF-II transgenic mice demonstrated that overexpression of IGF-II resulted in disproportionate growth of specific organs but a significant increase in body size was not observed. Homozygous IGF-I deficient mice were shown to be severely retarded in growth. The aim of this study was to test whether elevated levels of circulating IGF-II can rescue the dwarfism in IGF-I deficient mice and thereby function as a stimulator of postnatal growth in the absence of IGF-I. For this purpose, we crossed heterozygous IGF-I deficient mice [I+/- IIwt] with heterozygous IGF-I deficient mice carrying PEPCK-IGF-II transgenes [I+/- IItg]. The resulting offspring comprised six different groups: homozygous IGF-I knockout and PEPCK-IGF-II wildtype mice [I-/- IIwt], homozygous IGF-I knockout and PEPCK-IGF-II transgenic mice [I-/- IItg], animals lacking one IGF-I allele and wildtype for the PEPCK-IGF-II transgene [I+/- IIwt], lacking one IGF-I allele and harbouring the PEPCK-IGF-II transgene [I+/- IItg], wildtype for the IGF-I mutation and carrying the PEPCK-IGF-II transgene [I+/+ IItg], and completely wildtype [I+/+ IIwt]. The genotype of all mice was determined by PCR. Body weight of mice was recorded daily until the age of 8 weeks. The nose-rump length (NRL) and the weights of individual organs and of the carcass were recorded and the femurs and lumbar vertebras prepared for further investigations. At an age of 8 weeks, mean serum concentrations of IGF-I were beyond detection level in [I-/- IIwt] and [I-/- IItg] mice, intermediate in [I+/- IIwt] and [I+/- IItg] animals and highest in [I+/+ IIwt] and [I+/+ IItg] mice. IGF-II levels were significantly increased in animals harbouring the PEPCK-IGF-II transgene ([I-/- IItg], [I+/- IItg], and [I+/+ IItg]) when compared to their wildtype counterparts ([I-/- IIwt], [I+/- IIwt], and [I+/+ IIwt]). This reflected the genotype, demonstrating the appropriateness of our experimental model. Analysis of body weight data from day 3-4 after birth until day 60 revealed that in the absence of IGF-I, elevated levels of IGF-II have no effect on body weight gain. The same was found for the nose-rump length and the carcass. The weight of specific organs, however, was altered. Compared to the wildtype counterparts ([I-/- IIwt]), the relative kidney weight in [I-/- IItg] mice was significantly increased. IGF-I is known to play an important role in bone growth and in cancellous bone homeostasis. Investigations of geometric and structural bone parameters showed that in the presence or absence of IGF-I, an increase in the circulating levels of IGF-II was without effect on the skeleton and could not substitute for the skeletal functions of IGF-I in IGF-I-ablated mice. Homozygous IGF-I deficient mice are known to have elevated levels of growth hormone (GH). To demonstrate that the lack of effect on growth in our [I-/- IItg] animals was not due to a loss of these elevated GH-levels, a GH-Western immunoblot was performed, revealing that, despite elevated levels of IGF-II, increased levels of GH were still present in [I-/- IItg] animals. Evaluation of the serum levels of IGFBPs by Western ligand blot analysis demonstrated that IGFBP-1 and IGFBP-4 levels were similar in all groups, whereas the levels of IGFBP-2 and IGFBP-3 were strongly reduced in [I-/- IIwt] animals. In the presence of IGF-II ([I-/- IItg]), they were partially restored but the amounts were still smaller than in the IGF-I wildtype animals ([I+/+ IIwt] and [I+/+ IItg]). In summary, these results show that under our experimental conditions, IGF-II is not able to rescue the postnatal growth deficit of IGF-I knockout mice and apparently does not exert a negative feedback on the secretion of growth hormone. However, it could be demonstrated, that the IGFs have differentiated effects on the regulation of the expression/stability of individual IGFBPs.

The cells of the mammalian central nervous system (CNS) arise from multipotential precursor cells. The mechanisms that drive precursor cells toward a distinct cell fate are not well understood. Since transcription factors are known to control fate decisions, I attempted to determine the role of transcription factors Emx1, Emx2 and Pax6 that are particularly interesting since they specify area identities in the mouse telencephalon. To analyze their roles in precursor cells I chose gain-of-function experiments. Overexpression of these transcription factors showed that Emx2, Emx1 and Pax6 affect precursor cells in a region-specific manner. Emx2 transduction increases proliferation by promoting symmetric cell divisions, whereas blockade of endogenous Emx2 by antisense Emx2 mRNA limits the number and fate of progenitors generated by an individual cortical precursor cell. In the Emx2-/- asymmetrical cell divisions are increased in the cerebral cortex in vivo. In contrast to Emx2 Pax6 decreases proliferation. Pax6 deficient cells show more symmetrical cell divisions while Pax6 promotes asymmetric cell divisions in vitro. Emx2 endows in vitro cortical precursor cells with the capacity to generate multiple cell types, including neurons, astrocytes and oligodendrocytes. Emx1 keeps cells in an undifferentiated cell type, while Pax6 increases the proportion of neurons and can also convert astrocytes to neurons. The bHLH transcription factors Olig2 and Mash1 are up-regulated upon Emx2-transduction whereas Pax6 negatively influences those transcription factors and specifically up-regulates Ngn2. Thus, Emx2 is the first cell-intrinsic determinant able to instruct CNS precursors towards a multipotential fate. These results demonstrated an important role of Pax6 as intrinsic fate determinant of the neurogenic potential of glial cells. Taken together, Emx2 and Pax6 have opposing roles in cell proliferation, mode of cell division and cell fate.

BRG1 is a conserved subunit of the SWI/SNF family of ATP dependent chromatin remodeling complexes. These complexes play an important role in the transcription of various genes by making promoters accessible to the transcription machinery. Mutations in BRG1 have been connected to various cancers. In addition, a BRG1 knock-out in mice is lethal at the periimplantation stage, while BRG1 heterozygote mice are predisposed to exencephaly and tumors of epithelial origin, showing the importance of BRG1 in normal development and disease. In this study, I used Xenopus laevis to study the role of BRG1 because this system allows manipulation of endogenous protein levels by the use of antisense oligonucleotide mediated knock-down as well as interference analysis at early stages of development by overexpression of wild type and dominant negative protein variants. Since BRG1 is conserved among all vertebrates, I initially studied the role of BRG1 in Xenopus development by overexpression of wild type and dominant negative human BRG1. Overexpression of dominant negative human BRG1 gave a ventralized phenotype suggesting a role of BRG1 in dorsal-ventral patterning. The specificity of phenotypes was confirmed by using wild type human BRG1. On the other hand, overexpression of wild type and dominant negative variants of human BRM showed no developmental phenotypes. Prompted by these results, a frog brg1 cDNA was cloned by searching the Xenopus laevis EST database, using human BRG1 as a query. In addition, monoclonal antibodies specific to xBRG1 were raised and characterized. The expression pattern of Xbrg1 was found to be ubiquitous until gastrula stage and is tissue specific from neurula stage onwards. A Xenopus homologue of INI1, a subunit of SWI/SNF chromatin-remodeling complex, was cloned using database search. The expression pattern of Xini1 was found to be similar to Xbrg1. Using site directed mutagenesis, a dominant negative construct of xBRG1 was made by mutating the conserved lysine into arginine (K770R). Loss and gain of function studies showed that BRG1 is involved in AP axis formation during Xenopus development. The gain of function studies were done by overex-pressing wild type and dominant negative xBRG1, while loss of function studies were done using highly specific antisense morpholino oligos. Specificity of morpholino treatment was further proven by the rescue of ventralized phenotypes of morphant embryos by overexpression of human BRG1. It was found that BRG1 knock-down affects several tissues as assessed by in-situ hybridization using tissue specific markers. To determine the molecular explanation for these pleiotropic effects, several genes involved in early patterning of Xenopus embryo during organizer formation were analyzed. The analysis was done using whole mount in-situ hybridization, revealing the spatial gene expression pattern. This analysis revealed that BRG1 mostly affects WNT signaling dependent genes required for dorsal mesoderm formation while leaving pan-mesodermal genes unaffected. Furthermore the genetic interaction of BRG1 with the WNT pathway was confirmed by epistasis experiments showing that overexpression of β-CATENIN can rescue the xBrg1 antisense morpholino oligos dependent ventralized phenotypes as well as formation of secondary axis by overexpression of β-CATENIN could be prevented by BRG1 knock-down. Since the whole embryo represents a complex situation whereby many signaling pathways interact with each other and influence the outcome, the animal cap system was used to analyze the effect of BRG1 on various signaling pathways by analyzing corresponding direct target genes. Animal cap assays showed that the effect of BRG1 is signal specific. Moreover, among the affected signaling pathways, BRG1 knock-down affected only specific genes. These results showed that the BRG1 effect is gene and signal specific. The importance of WNT signaling has also been shown in cancer as well as in haematopoietic and embryonic stem cell self renewal. Given the importance of the WNT signaling, the role of BRG1 on the WNT signaling pathway was further investigated. Treatment of animal cap cells with various doses of Wnt8 mRNA showed the differential requirement of the WNT signal for maximal stimulation of direct target genes. The direct target genes of the WNT pathway showed various degrees of reduction in their maximal stimulation upon BRG1 protein knock-down. The requirement of BRG1 for proper stimulation of the WNT target genes was further confirmed by overexpression of xBRG1 under sub-optimal conditions of WNT stimulation. A major conclusion from these experiments is that BRG1 protein defines signaling thresholds for WNT-mediated activation of target genes. This implies that chromatin remodeling complexes are part of the machinery, which translates inductive signals into spatial gene expression domains.

Fri, 11 Feb 2005 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/3404/ https://edoc.ub.uni-muenchen.de/3404/1/Fisch_Thomas_M.pdf Fisch, Thomas Martin

The centrosome is the major microtubule organizing centre (MTOC) in animal cells. Most microtubules (MTs) emanate from the centrosome, where gamma-tubulin ring complexes (gammaTuRCs) act as templates for MT nucleation. During interphase, the centrosome organizes a MT array that imparts shape and polarity to the cell and is essential for intracellular transport and positioning of organelles such as the Golgi apparatus. During mitosis, centrosomes ensure bipolarity and correct orientation of the spindle by forming the spindle poles. In order to switch from the interphasic to the mitotic state, the centrosome undergoes a structural reorganization, termed maturation, which is mainly characterized by an increase in MT nucleation activity. A full appreciation of how centrosomes contribute to cellular functions requires the isolation and characterization of unknown centrosome-associated molecules. Here we describe the identification and characterization of a novel centrosomal component, the human protein Nlp (ninein-like protein) related to the previously characterized MT-anchoring protein ninein. In the first part of the present thesis we describe the identification of Nlp as a novel centrosomal substrate of Polo-like kinase 1 (Plk1), an important regulator of mitosis whose activity is required for centrosome maturation. Nlp interacts with two distinct gammaTuRC components, gamma-tubulin and hGCP4, and stimulates MT nucleation. Plk1 phosphorylates Nlp and disrupts its centrosomal association. Overexpression of an Nlp mutant lacking Plk1 phosphorylation sites induces defects in mitotic spindle formation. We propose that Nlp acts as a gammaTuRC binding protein (GTBP), contributing to the MT nucleation activity of the centrosome during interphase. At the onset of mitosis, the displacement of Nlp from the centrosome triggered by Plk1 phosphorylation could represent an important step in the maturation process which allows the centrosome to switch from the interphasic to the mitotic state. Thus, we conclude that Nlp, as well as the related protein ninein, plays an important role in MT organization. However the function of these two proteins possibly diverged during evolution: whilst Nlp gained a more prominent role in MT nucleation, ninein became principally involved in MT anchoring. In the second part of this thesis we report the initial characterization of the molecular mechanisms underlying the ability of Nlp and ninein to induce the fragmentation of the Golgi apparatus when overexpressed in human cells. We show that the ability of these two centrosomal proteins to affect the organization of the Golgi clearly depends on their capacity to associate with the cytoplasmic dynein-dynactin complex, a molecular motor complex primarly involved in the maintainance of Golgi architecture. We propose that the excess of Nlp and ninein could induce the disruption of the Golgi apparatus by sequestering the dynein-dynactin complexes. Future investigations should be aimed at understanding whether the dissociation of the Golgi apparatus from the centrosome induced by the excess of Nlp and ninein could interfere with cell migration and cell polarization processes, which require a highly coordinated action of these two organelles. Cell migration and cell polarization represent critical events for immune responses as well as for embryonic development, invasive growth and metastasis. Thus, our findings raise the interesting possibility that an upregulation in the expression levels of structural centrosomal proteins could represent the molecular basis for developmental disorders and malfunctioning of the immune system and, on the other hand, modulate the acquisition of invasive properties by neoplastic cells.

The reaction product of phospholipase D (PLD), phosphatidic acid (PA), was found to stimulate phosphatidylinositol-4-phosphate-5-kinase (PIP-5-kinase) activity in vitro. In the present study, we have examined wether PLD affects the synthesis of phosphatidylinositol 4,5-bisphosphate (PIP2) by PIP-5-kinase. Overexpression of PLD isoforms in HEK-293 cells led to an increase in PIP-5-kinase activity and to elevated PIP2 levels in intact cells. As both PLD and PIP-5-kinase are stimulated by the GTPases Arf1 and RhoA, we investigated in the following, if PLD is involved in the regulation of PIP2 synthesis by these GTPases. Both PLD1- and PLD2-induced PIP2 synthesis was completely blocked by coexpression of catalytically inactive Arf1 T31N. Reversely, the effect of constitutive active Arf1 Q71L was fully inhibited by catalytically inactive PLD constructs. Whereas the effects of Arf1 Q71L and wild-type PLD2 were additive, coexpression of Arf1 Q71L with wild-type PLD1 led to a synergistic increase in PIP-5-kinase activity. Previously, we have shown that RhoA regulates the activity of PLD and PIP-5-kinase by its downstream effector Rho-kinase. Expression of small amounts of inactive PLD1, but not of PLD2, nearly completely abolished Rho-kinase-stimulated PIP-5-kinase activity. Also expression of a non-phosphorylatable mutant of cofilin, which participates in the signalling cascade from RhoA via Rho-kinase and LIM-kinase to PLD1, suppressed the stimulating effect of Rho-kinase on PIP2 synthesis. These findings suggest that PLD1 is involved in the stimulation of PIP-5-kinase by Arf1 as well as by RhoA and Rho-kinase. After sucrose density gradient centrifugation of HEK-293 cell lysates, we isolated two separate PIP2 pools. PLD1 and Arf1 selectively control the non-caveolar PIP2 pool in the high density fraction, whereas PLD2 affected PIP2 in both pools. In summary, these data suggest that particularly PLD1, apparently by the production of PA, functions as a physiological regulator of PIP-5-kinase that controls the synthesis of cellular PIP2 downstream to Arf1 and RhoA.

Overexpression of proto-oncogene c-jun and constitutive activation of the Jun NH2-terminal kinase (JNK) signaling pathway have been implicated in the leukemic transformation process. However, c-jun expression has not been investigated in acute myeloid leukemia (AML) cells containing the most common chromosomal translocations. t(8;21) is one of the most common AML-associated translocation and results in the AML1-ETO fusion protein. Overexpression of AML1-ETO in NIH3T3 cells leads to increased phosphorylation of Ser63 in c-Jun, which is generally JNK dependent. The role of the JNK signaling pathway for the functional properties of AML1-ETO is, however, unknown. In the present study we found high expression levels of c-jun mRNA in t(8;21), t(15;17) or inv(16) positive patient cells by microarray analysis. Within t(8;21) positive patient samples, there was a correlation between AML1-ETO and c-jun mRNA expression levels. In myeloid U937 cells, c-jun mRNA and c-Jun protein expression levels increased upon induction of AML1-ETO. AML1-ETO transactivated the human c-jun promoter through the proximal AP-1 site via activating the JNK signaling pathway. JNK targets c-Jun and ATF-2, which also bind to the proximal AP-1 site in U937 cells, were also phosphorylated upon AML1-ETO induction. Furthermore, AML1-ETO induction increased the DNA binding capacity of c-Jun and ATF-2 to the proximal AP-1 site of the c-jun promoter, which might result in their enhanced transactivation capacities. Interference with JNK and c-Jun activation by using JIP-1 or a JNK inhibitor reduced the transactivation capacity of AML1-ETO on the c-jun promoter and the pro-apoptotic function of AML1-ETO in U937 cells. AML1-ETO seems to activate the JNK signaling pathway by inducing the expression of a cytoplasmic factor, possibly G-CSF, because supernatant of AML1-ETO expressing cells was sufficient to induce phosphorylation of JNK and c-Jun in wildtype U937 cells. This data demonstrates a novel mechanism of how AML1-ETO might exert positive effects on target gene expression and identifies the proto-oncogene c-jun as a common target gene in AML patient cells.

The transcription factor C/EBPa is crucial for the differentiation of granulocytes. Conditional expression of C/EBPa triggers neutrophilic differentiation and C/EBPa can block TPA induced monocytic differentiation of bipotential myeloid cells. In C/EBPa knockout mice, no mature granulocytes are present. A dramatic increase of c-jun mRNA in C/EBPa knockout mice fetal liver was observed. c-jun, a component of the AP-1 transcription factor complex and a co-activator of the transcription factor PU.1, is important for monocytic differentiation. Here we report that C/EBPa downregulates c-jun expression to drive granulocytic differentiation. Ectopic increase of C/EBPa expression decreases c-jun mRNA level, and the human c-jun promoter activity is downregulated 8 fold in presence of C/EBPa. C/EBPa and c-jun interact through their leucine zipper domains and this interaction prevents c-jun from binding to DNA. This results in downregulation of c-jun’s capacity to autoregulate its own promoter through the proximal AP-1 site. Overexpression of c-jun prevents C/EBPa induced granulocytic differentiation. c-jun expression was higher in AML M2 patients with dominant negative C/EBPa mutations in comparison to AML M2 patients without C/EBPa mutations. Thus, we propose a model in which C/EBPa needs to downregulate c-jun expression and transactivation capacity for promoting granulocytic differentiation.

AML1-ETO is a fusion protein encoded by the translocation t(8;21) and found in 15% of acute myeloid leukemia patients. Here, we report a negative functional impact of AML1-ETO on the transcriptional activity of PU.1, an important transcription factor for normal myeloid differentiation. We have demonstrated that AML1-ETO interacts with PU.1 by immunoprecipitation assay in Kasumi-1 cells having t(8;21). On mapping the region of interaction in PU.1, we found that AML1-ETO binds to the b3b4 region in the DNA binding domain of PU.1 and displaces the co-activator c-Jun from PU.1, thus downregulating PU.1’s transcriptional activity. In doing so AML1-ETO does not change the DNA binding capacity of PU.1. The expression levels of PU.1 target genes in acute myeloid leukemia (AML)-M2 patients with t(8;21) were lower than in patients without t(8;21). Conditional expression of AML1-ETO causes proliferation in mouse bone marrow cells and inhibits PU.1 induced differentiation in HL60 cells. Overexpression of PU.1 differentiates AML1-ETO carrying Kasumi-1 cells to the monocytic lineage. Thus, PU.1’s function is downregulated in presence of AML1-ETO in acute myeloid leukemia, whereas overexpression of PU.1 can restore normal differentiation.

The work presented in this dissertation gave the following results: (i) Sequence homologous of the membrane proteins Lag1p and Dgt1p exist all over the kingdom of eucaryotes. All of them share a highly conserved motif with two hydrophilic residues within a transmembrane domain. A limited proteolytic digestion showed, that the N-terminus of Dgt1p is orientated to the cytosolic side. Together with the in silicio analysis, the proteins of the LAG family have six transmembrane domains, a cytosolic C-terminus and an ER lumenal loop of ca. 45 amino acids. (ii) Different mutants of Dgt1p showed, that the C-terminus is essential for the function of the protein. The soluble N-terminus or its dilysine motif, normally mediating retrieval of ER resident proteins that escaped to the Golgi, have not obvious importance for Dgt1p. (iii) Lag1p and Dgt1p fulfill their function in the membrane of the ER and are no constitutive parts of COPII transport vesicles. Moreover, both proteins interact with each other and therefore appear to form a heteropolymeric complex in the ER membrane. (iv) The synthesis rates of complex sphingolipids are drastically reduced in the absence of LAG1 and DGT1. Consequently, lag1Ddgt1D cells have a decreased portion of sphingolipids compared to the wild type. The lipid defekt is not due to lacking phosphoinositolation of ceramide, since the catalytic activity of the corresponding enzyme was proven in vivo.(v) The synthesis block in lag1Ddgt1D cells leads to an accumulation of sphingoid bases. In this connection it was shown, that Lag1p and Dgt1p are essential for the incorporation of exogenous dihydrosphingosine into ceramide, whereas in their absence endogenous dihydrosphingosine is converted to ceramide with a merely decreased rate. The elongation of fatty acids to C26 is not reduced in lag1Ddgt1D cells. Lipid extracts of fumonisin B1 treated wild type cells resemble that of the double mutant, whereas the latter is not influenced by a toxin based inhibition of ceramide synthase. Overexpression of LAG1 and DGT1 does not lead to an overproduction of ceramide or sphingolipids. However, a direct in vitro assay for ceramide synthase activity finally proved the essential requirement of the reaction for Lag1p and Dgt1p. (vi) The overproduction of the ceramidases Ypc1p and Ydc1p in lag1Ddgt1D cells increases the synthesis rates as well as the effective amounts of complex sphingolipids. The defective incorporation of exogenous dihydrosphingosine into ceramide was, however, not reverted. The additional deletion of YPC1 and YDC1 in the absence of LAG1 and DGT1 decreased the amounts of inositol containing sphingolipids beyond detectable levels and, therefore, confirmed the essential requirement of Lag1p and Dgt1p for the ceramide synthase reaction. (vii) The survival of lag1Ddgt1D cells compared to wild type cells after short heat shock is drastically impaired. Moreover, the ability of yeast to form single cells on agar plates directly contributes to the temperature and the amount of ceramide available. As a consequence of the data obtained, the proteins Lag1p and Dgt1p are the first to be characterized as essential parts of the ceramide synthase reaction.