المؤلفون: Tadao Nagasaki, Alexander J. Schuyler, Jinming Zhao, Svetlana N. Samovich, Kazuhiro Yamada, Yanhan Deng, Scott P. Ginebaugh, Stephanie A. Christenson, Prescott G. Woodruff, John V. Fahy, John B. Trudeau, Detcho Stoyanovsky, Anuradha Ray, Yulia Y. Tyurina, Valerian E. Kagan, Sally E. Wenzel

المصدر: The Journal of Clinical Investigation, Vol 132, Iss 1 (2022)

مصطلحات موضوعية: Metabolism, Pulmonology, Medicine

الوصف: Altered redox biology challenges all cells, with compensatory responses often determining a cell’s fate. When 15 lipoxygenase 1 (15LO1), a lipid-peroxidizing enzyme abundant in asthmatic human airway epithelial cells (HAECs), binds phosphatidylethanolamine-binding protein 1 (PEBP1), hydroperoxy-phospholipids, which drive ferroptotic cell death, are generated. Peroxidases, including glutathione peroxidase 4 (GPX4), metabolize hydroperoxy-phospholipids to hydroxy derivatives to prevent ferroptotic death, but consume reduced glutathione (GSH). The cystine transporter SLC7A11 critically restores/maintains intracellular GSH. We hypothesized that high 15LO1, PEBP1, and GPX4 activity drives abnormal asthmatic redox biology, evidenced by lower bronchoalveolar lavage (BAL) fluid and intraepithelial cell GSH:oxidized GSH (GSSG) ratios, to enhance type 2 (T2) inflammatory responses. GSH, GSSG (enzymatic assays), 15LO1, GPX4, SLC7A11, and T2 biomarkers (Western blot and RNA-Seq) were measured in asthmatic and healthy control (HC) cells and fluids, with siRNA knockdown as appropriate. GSSG was higher and GSH:GSSG lower in asthmatic compared with HC BAL fluid, while intracellular GSH was lower in asthma. In vitro, a T2 cytokine (IL-13) induced 15LO1 generation of hydroperoxy-phospholipids, which lowered intracellular GSH and increased extracellular GSSG. Lowering GSH further by inhibiting SLC7A11 enhanced T2 inflammatory protein expression and ferroptosis. Ex vivo, redox imbalances corresponded to 15LO1 and SLC7A11 expression, T2 biomarkers, and worsened clinical outcomes. Thus, 15LO1 pathway–induced redox biology perturbations worsen T2 inflammation and asthma control, supporting 15LO1 as a therapeutic target.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/1558-8238Test

الوصول الحر: https://doaj.org/article/45e6d435807b449cbb11a73fc2795bf4Test

المؤلفون: Scott P. Ginebaugh, Yomna Badawi, Tyler B. Tarr, Stephen D. Meriney

المصدر: Biomolecules, Vol 12, Iss 6, p 740 (2022)

مصطلحات موضوعية: active zone, neuromuscular junction, Lambert-Eaton myasthenic syndrome, computational modeling, Microbiology, QR1-502

الوصف: The mouse neuromuscular junction (NMJ) has long been used as a model synapse for the study of neurotransmission in both healthy and disease states of the NMJ. Neurotransmission from these neuromuscular nerve terminals occurs at highly organized structures called active zones (AZs). Within AZs, the relationships between the voltage-gated calcium channels and docked synaptic vesicles govern the probability of acetylcholine release during single action potentials, and the short-term plasticity characteristics during short, high frequency trains of action potentials. Understanding these relationships is important not only for healthy synapses, but also to better understand the pathophysiology of neuromuscular diseases. In particular, we are interested in Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disorder in which neurotransmitter release from the NMJ decreases, leading to severe muscle weakness. In LEMS, the reduced neurotransmission is traditionally thought to be caused by the antibody-mediated removal of presynaptic voltage-gated calcium channels. However, recent experimental data and AZ computer simulations have predicted that a disruption in the normally highly organized active zone structure, and perhaps autoantibodies to other presynaptic proteins, contribute significantly to pathological effects in the active zone and the characteristics of chemical transmitters.

وصف الملف: electronic resource

العلاقة: https://www.mdpi.com/2218-273X/12/6/740Test; https://doaj.org/toc/2218-273XTest

الوصول الحر: https://doaj.org/article/34274bdc074a4bab861fb4333552e71bTest

المؤلفون: Scott P. Ginebaugh, Yomna Badawi, Rozita Laghaei, Glenn Mersky, Caleb J. Wallace, Tyler B. Tarr, Cassandra Kaufhold, Stephen Reddel, Stephen D. Meriney

المصدر: Journal of Neurophysiology. 129:1259-1277

مصطلحات موضوعية: Physiology, General Neuroscience

الوصف: We used a computational model of the active zone (AZ) in the mammalian neuromuscular junction to investigate Lambert-Eaton myasthenic syndrome (LEMS) pathophysiology. This model suggests that disruptions in presynaptic active zone organization and protein content (particularly synaptotagmin), beyond the simple removal of presynaptic calcium channels, play an important role in LEMS pathophysiology.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::7af4c9305ed1891771b91135ad629001Test
https://doi.org/10.1152/jn.00404.2022Test

المؤلفون: Brian M. Wolpin, Jonathan A. Nowak, Andrew J. Aguirre, Alex K. Shalek, William C. Hahn, Aram F. Hezel, Albert C. Koong, Daniel T. Chang, Richard F. Dunne, David C. Linehan, Margaret M. Kozak, Emma R. Hill, Lauren K. Brais, Joseph D. Mancias, Jiping Wang, Thomas E. Clancy, James M. Cleary, Kimberly Perez, Harshabad Singh, Douglas A. Rubinson, Vicente Morales-Oyarvide, Dalia Elganainy, Mai Chan Lau, Kristen E. Lowder, Radha L. Kalekar, Timothy L. Bosse, Annan Yang, Junning Wang, Andrew W. Navia, Chen Yuan, Juha P. Väyrynen, Sara A. Väyrynen, Scott P. Ginebaugh, Kevin S. Kapner, Peter S. Winter, Srivatsan Raghavan, Jinming Zhang, Andressa Dias Costa, Hannah L. Williams

الوصف: Pancreatic ductal adenocarcinoma (PDAC) has been classified into classical and basal-like transcriptional subtypes by bulk RNA measurements. However, recent work has uncovered greater complexity to transcriptional subtypes than was initially appreciated using bulk RNA expression profiling. To provide a deeper understanding of PDAC subtypes, we developed a multiplex immunofluorescence (mIF) pipeline that quantifies protein expression of six PDAC subtype markers (CLDN18.2, TFF1, GATA6, KRT17, KRT5, and S100A2) and permits spatially resolved, single-cell interrogation of pancreatic tumors from resection specimens and core needle biopsies. Both primary and metastatic tumors displayed striking intratumoral subtype heterogeneity that was associated with patient outcomes, existed at the scale of individual glands, and was significantly reduced in patient-derived organoid cultures. Tumor cells co-expressing classical and basal markers were present in > 90% of tumors, existed on a basal-classical polarization continuum, and were enriched in tumors containing a greater admixture of basal and classical cell populations. Cell–cell neighbor analyses within tumor glands further suggested that co-expressor cells may represent an intermediate state between expression subtype poles. The extensive intratumoral heterogeneity identified through this clinically applicable mIF pipeline may inform prognosis and treatment selection for patients with PDAC.Significance:A high-throughput pipeline using multiplex immunofluorescence in pancreatic cancer reveals striking expression subtype intratumoral heterogeneity with implications for therapy selection and identifies co-expressor cells that may serve as intermediates during subtype switching.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::58901a59881a291461b659642ab759fdTest
https://doi.org/10.1158/0008-5472.c.6514341.v1Test

المؤلفون: Brian M. Wolpin, Jonathan A. Nowak, Andrew J. Aguirre, Alex K. Shalek, William C. Hahn, Aram F. Hezel, Albert C. Koong, Daniel T. Chang, Richard F. Dunne, David C. Linehan, Margaret M. Kozak, Emma R. Hill, Lauren K. Brais, Joseph D. Mancias, Jiping Wang, Thomas E. Clancy, James M. Cleary, Kimberly Perez, Harshabad Singh, Douglas A. Rubinson, Vicente Morales-Oyarvide, Dalia Elganainy, Mai Chan Lau, Kristen E. Lowder, Radha L. Kalekar, Timothy L. Bosse, Annan Yang, Junning Wang, Andrew W. Navia, Chen Yuan, Juha P. Väyrynen, Sara A. Väyrynen, Scott P. Ginebaugh, Kevin S. Kapner, Peter S. Winter, Srivatsan Raghavan, Jinming Zhang, Andressa Dias Costa, Hannah L. Williams

الوصف: Supplementary Figures

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::05b8ab2de7cfe7f7ab46e4e9b09fa895Test
https://doi.org/10.1158/0008-5472.22433331.v1Test

المؤلفون: Scott P. Ginebaugh, Matthias Hagner, Anuradha Ray, Serpil C. Erzurum, Suzy A.A. Comhair, Loren C. Denlinger, Nizar N. Jarjour, Mario Castro, Prescott G. Woodruff, Stephanie A. Christenson, Eugene R. Bleecker, Deborah A. Meyers, Annette T. Hastie, Wendy C. Moore, David T. Mauger, Elliot Israel, Bruce D. Levy, Sally E. Wenzel, Matthew J. Camiolo

المصدر: Journal of Allergy and Clinical Immunology.

مصطلحات موضوعية: Immunology, Immunology and Allergy

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::a9d11fa09aa12ba8e0d11a7f34906e2dTest
https://doi.org/10.1016/j.jaci.2023.01.028Test

المؤلفون: Hannah L. Williams, Andressa Dias Costa, Jinming Zhang, Srivatsan Raghavan, Peter S. Winter, Kevin S. Kapner, Scott P. Ginebaugh, Sara A. Väyrynen, Juha P. Väyrynen, Chen Yuan, Andrew W. Navia, Junning Wang, Annan Yang, Timothy L. Bosse, Radha L. Kalekar, Kristen E. Lowder, Mai Chan Lau, Dalia Elganainy, Vicente Morales-Oyarvide, Douglas A. Rubinson, Harshabad Singh, Kimberly Perez, James M. Cleary, Thomas E. Clancy, Jiping Wang, Joseph D. Mancias, Lauren K. Brais, Emma R. Hill, Margaret M. Kozak, David C. Linehan, Richard F. Dunne, Daniel T. Chang, Albert C. Koong, Aram F. Hezel, William C. Hahn, Alex K. Shalek, Andrew J. Aguirre, Jonathan A. Nowak, Brian M. Wolpin

المصدر: Cancer research.

مصطلحات موضوعية: Cancer Research, Oncology

الوصف: Pancreatic ductal adenocarcinoma (PDAC) has been classified into classical and basal-like transcriptional subtypes by bulk RNA measurements. However, recent work has uncovered greater complexity to transcriptional subtypes than was initially appreciated using bulk RNA expression profiling. To provide a deeper understanding of PDAC subtypes, we developed a multiplex immunofluorescence (mIF) pipeline that quantifies protein expression of six PDAC subtype markers (CLDN18.2, TFF1, GATA6, KRT17, KRT5, and S100A2) and permits spatially resolved, single-cell interrogation of pancreatic tumors from resection specimens and core needle biopsies. Both primary and metastatic tumors displayed striking intratumoral subtype heterogeneity that was associated with patient outcomes, existed at the scale of individual glands, and was significantly reduced in patient-derived organoid cultures. Tumor cells co-expressing classical and basal markers were present in > 90% of tumors, existed on a basal-classical polarization continuum, and were enriched in tumors containing a greater admixture of basal and classical cell populations. Cell–cell neighbor analyses within tumor glands further suggested that co-expressor cells may represent an intermediate state between expression subtype poles. The extensive intratumoral heterogeneity identified through this clinically applicable mIF pipeline may inform prognosis and treatment selection for patients with PDAC. Significance: A high-throughput pipeline using multiplex immunofluorescence in pancreatic cancer reveals striking expression subtype intratumoral heterogeneity with implications for therapy selection and identifies co-expressor cells that may serve as intermediates during subtype switching.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::5578179943efe3fd30fd9632eb9d837dTest
https://pubmed.ncbi.nlm.nih.gov/36459568Test

المؤلفون: Eric D. Cyphers, Scott P. Ginebaugh, Stephen D. Meriney, Rozita Laghaei, Viswanath Lanka, Evan W. Miller, Gloria Ortiz

المصدر: J Neurosci

مصطلحات موضوعية: Male, 0301 basic medicine, Time Factors, Neuromuscular Junction, Action Potentials, Motor nerve, Neurotransmission, Neuromuscular junction, Synapse, 03 medical and health sciences, Organ Culture Techniques, 0302 clinical medicine, medicine, Animals, Waveform, Research Articles, Neurotransmitter Agents, Node of Ranvier, Chemistry, General Neuroscience, Rana pipiens, Electrophysiology, 030104 developmental biology, medicine.anatomical_structure, Terminal (electronics), Female, Neuroscience, 030217 neurology & neurosurgery, Forecasting, Sodium Channel Blockers

الوصف: The action potential (AP) waveform controls the opening of voltage-gated calcium channels and contributes to the driving force for calcium ion flux that triggers neurotransmission at presynaptic nerve terminals. Although the frog neuromuscular junction (NMJ) has long been a model synapse for the study of neurotransmission, its presynaptic AP waveform has never been directly studied, and thus the AP waveform shape and propagation through this long presynaptic nerve terminal are unknown. Using a fast voltage-sensitive dye, we have imaged the AP waveform from the presynaptic terminal of male and female frog NMJs and shown that the AP is very brief in duration and actively propagated along the entire length of the terminal. Furthermore, based on measured AP waveforms at different regions along the length of the nerve terminal, we show that the terminal is divided into three distinct electrical regions: A beginning region immediately after the last node of Ranvier where the AP is broadest, a middle region with a relatively consistent AP duration, and an end region near the tip of nerve terminal branches where the AP is briefer. We hypothesize that these measured changes in the AP waveform along the length of the motor nerve terminal may explain the proximal-distal gradient in transmitter release previously reported at the frog NMJ.SIGNIFICANCE STATEMENTThe AP waveform plays an essential role in determining the behavior of neurotransmission at the presynaptic terminal. Although the frog NMJ is a model synapse for the study of synaptic transmission, there are many unknowns centered around the shape and propagation of its presynaptic AP waveform. Here, we demonstrate that the presynaptic terminal of the frog NMJ has a very brief AP waveform and that the motor nerve terminal contains three distinct electrical regions. We propose that the changes in the AP waveform as it propagates along the terminal can explain the proximal-distal gradient in transmitter release seen in electrophysiological studies.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::66ac3dec7373d4d93f1cde3088ae9c0cTest
https://doi.org/10.1523/jneurosci.2415-19.2020Test

المؤلفون: Stephen D. Meriney, Manuel Covarrubias, Gloria Ortiz, Evan W. Miller, Scott P. Ginebaugh, Kristine S. Ojala, Man Wu

المصدر: The Journal of Biological Chemistry

مصطلحات موضوعية: 0301 basic medicine, Male, NMJ, neuromuscular junction, ROI, region of interest, Action Potentials, Gene Expression, voltage-gated calcium (Cav) channel, Biochemistry, Cav, voltage-gated calcium, Tissue Culture Techniques, Mice, Calcium Channels, N-Type, LEMS, Lambert–Eaton myasthenic syndrome, mEPP, miniature endplate potential, neurological disease, Lambert–Eaton myasthenic syndrome (LEMS), neuromuscular junction (NMJ), Voltage-dependent calcium channel, Chemistry, Transmitter, voltage-gated potassium (Kv) channel, medicine.anatomical_structure, Neuromuscular Agents, Shaw Potassium Channels, FWHM, full width at half maximum, Female, Amifampridine, Lambert-Eaton myasthenic syndrome, Research Article, Agonist, BeRST, Berkeley red-based sensor of transmembrane potential, Calcium Channels, L-Type, medicine.drug_class, Neuromuscular Junction, Presynaptic Terminals, chemistry.chemical_element, Calcium, Neuromuscular junction, 03 medical and health sciences, medicine, Potassium Channel Blockers, Animals, Molecular Biology, QC, quantal content, 030102 biochemistry & molecular biology, Dose-Response Relationship, Drug, Rana pipiens, 3,4-DAP, 3,4-diaminopyridine, Antagonist, Cell Biology, medicine.disease, action potential (AP), Acetylcholine, Electrophysiology, 030104 developmental biology, neurotransmitter release, Biophysics, EPP, endplate potential, AP, action potential, Kv, voltage-gated potassium, Microelectrodes, BTX, alpha-bungarotoxin

الوصف: 3,4-Diaminopyridine (3,4-DAP) increases transmitter release from neuromuscular junctions (NMJs), and low doses of 3,4-DAP (estimated to reach ∼1 μM in serum) are the Food and Drug Administration (FDA)-approved treatment for neuromuscular weakness caused by Lambert-Eaton myasthenic syndrome. Canonically, 3,4-DAP is thought to block voltage-gated potassium (Kv) channels, resulting in prolongation of the presynaptic action potential (AP). However, recent reports have shown that low millimolar concentrations of 3,4-DAP have an off-target agonist effect on the Cav1 subtype ("L-type") of voltage-gated calcium (Cav) channels and have speculated that this agonist effect might contribute to 3,4-DAP effects on transmitter release at the NMJ. To address 3,4-DAP's mechanism(s) of action, we first used the patch-clamp electrophysiology to characterize the concentration-dependent block of 3,4-DAP on the predominant presynaptic Kv channel subtypes found at the mammalian NMJ (Kv3.3 and Kv3.4). We identified a previously unreported high-affinity (1-10 μM) partial antagonist effect of 3,4-DAP in addition to the well-known low-affinity (0.1-1 mM) antagonist activity. We also showed that 1.5-μM DAP had no effects on Cav1.2 or Cav2.1 current. Next, we used voltage imaging to show that 1.5- or 100-μM 3,4-DAP broadened the AP waveform in a dose-dependent manner, independent of Cav1 calcium channels. Finally, we demonstrated that 1.5- or 100-μM 3,4-DAP augmented transmitter release in a dose-dependent manner and this effect was also independent of Cav1 channels. From these results, we conclude that low micromolar concentrations of 3,4-DAP act solely on Kv channels to mediate AP broadening and enhance transmitter release at the NMJ.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::ccbc3cf8e9a87415c7993f465b092f47Test
https://pubmed.ncbi.nlm.nih.gov/33465376Test

المؤلفون: Rebecca Boiarsky, Kevin S. Kapner, Daniel Zhao, Stephanie W. Cheng, Ananya D. Jambhale, Jimmy A. Guo, Patrick Z. Yu, Tyler Jacks, François Aguet, Daniel Y. Kim, Harshabad Singh, Hannah I. Hoffman, Brenton R. Paolella, Maryann Zhao, William L. Hwang, Steven I. Wang, Andrew J. Aguirre, John M. Krill-Burger, Westley W. Wu, Kristen E. Lowder, James M. McFarland, Tobiloba E. Oni, Aviv Regev, Gad Getz, Peter Chen, Scott P. Ginebaugh

المصدر: Cancer Research. 81:PR-006

مصطلحات موضوعية: Cancer Research, FOLFIRINOX, Cancer, Biology, Precision medicine, medicine.disease, medicine.disease_cause, Gemcitabine, Oncology, RNA interference, Pancreatic cancer, medicine, Cancer research, CRISPR, KRAS, medicine.drug

الوصف: Targeted therapies for molecularly-defined subtypes have led to immense clinical benefit for many cancer types but have generally not been successful for pancreatic cancer. Given that the mainstay of treatment remains multi-agent chemotherapy with FOLFIRINOX or gemcitabine/nab-paclitaxel, there remains an urgent need to identify novel actionable vulnerabilities for subsets of PDAC patients. Toward this end, we conducted an integrative, genome-scale examination of genetic dependencies and cell surface targets for PDAC by leveraging CRISPR and RNAi screening data from The Cancer Dependency Map Project, genomic data of bulk patient tumors from The Cancer Genome Atlas, and custom single-nucleus RNA-seq of a 43-patient cohort comprised of untreated and treated specimens. Our results re-affirm the prominence of Ras/MAPK signaling and a synthetically-lethal interaction between VPS4A/B, but also reveal recurrent susceptibilities to genes within the fatty acid metabolism, vesicular transport and exocytosis, and nucleobase synthesis pathways that otherwise have minor to moderate depleting effects on the majority of cell lines. Aberrations in frequent tumor suppressor genes and chromosomal arm-level variations appear to modify the strength of dependencies, including that of KRAS, CCND1, and GPX4, and may serve as predictive biomarkers of response. In addition, we leveraged mRNA profiling of bulk primary tumors as well as metastatic organoid models to conduct a genome-wide search for cell surface targets that are highly-expressed in tumors while lowly or not expressed in other toxicity-prone, non-malignant tissues. These putative drug targets do not need to be cancer dependencies and can be compatible with antibody-based therapeutic strategies that leverage alternative modes of cellular toxicity. Our approach identifies MSLN, NECTIN4, TROP2, and other antigens which have previously been shown to be largely tumor-specific but also reveals the expression of multiple putative targets within the CEACAM, claudin, and tetraspanin families. Finally, molecular subtyping efforts over the past decade have yielded classical and basal-like as consensus subtypes with variations therein, but genetic dependencies and cell surface expression patterns unique to either are insufficiently understood. We identified CLDN18, CEACAM5, and CEACAM6 as cell surface antigens for the classical subtype and MSLN, AQP5, and SLC6A14 for basal-like. Dependency on TLK2 and CCND1 is associated with the basal-like and classical subtype, respectively. Taken together, our integrative genomic approach may provide a precision medicine blueprint for stratifying and targeting pancreatic cancer. Citation Format: Jimmy A. Guo, Daniel Zhao, Scott P. Ginebaugh, Steven Wang, Ananya D. Jambhale, Patrick Z. Yu, Westley W. Wu, Peter Chen, Maryann Zhao, Kristen E. Lowder, Kevin S. Kapner, Hannah I. Hoffman, Stephanie W. Cheng, Daniel Y. Kim, Rebecca Boiarsky, Francois Aguet, Brenton Paolella, John M. Krill-Burger, James M. McFarland, Tobiloba Oni, Tyler Jacks, Aviv Regev, Gad Getz, William L. Hwang, Harshabad Singh, Andrew J. Aguirre. Integrative genomic characterization of therapeutic targets for pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-006.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::3bd2b2ab6c72ca27c45082db78ae73ebTest
https://doi.org/10.1158/1538-7445.panca21-pr-006Test