Actinin BioID reveals sarcomere crosstalk with oxidative metabolism through interactions with IGF2BP2.

Actinins are strain-sensing actin cross-linkers that are ubiquitously expressed and harbor mutations in human diseases. We utilize CRISPR, pluripotent stem cells, and BioID to study actinin interactomes in human cardiomyocytes. We identify 324 actinin proximity partners, including those that are dependent on sarcomere assembly. We confirm 19 known interactors and identify a network of RNA-binding proteins, including those with RNA localization functions. In vivo and biochemical interaction studies support that IGF2BP2 localizes electron transport chain transcripts to actinin neighborhoods through interactions between its K homology (KH) domain and actinin\u27s rod domain. We combine alanine scanning mutagenesis and metabolic assays to disrupt and functionally interrogate actinin-IGF2BP2 interactions, which reveal an essential role in metabolic responses to pathological sarcomere activation using a hypertrophic cardiomyopathy model. This study expands our functional knowledge of actinin, uncovers sarcomere interaction partners, and reveals sarcomere crosstalk with IGF2BP2 for metabolic adaptation relevant to human disease

Reading Frame Repair of TTN Truncation Variants Restores Titin Quantity and Functions

BACKGROUND: Titin truncation variants (TTNtvs) are the most common inheritable risk factor for dilated cardiomyopathy (DCM), a disease with high morbidity and mortality. The pathogenicity of TTNtvs has been associated with structural localization as A-band variants overlapping myosin heavy chain-binding domains are more pathogenic than I-band variants by incompletely understood mechanisms. Demonstrating why A-band variants are highly pathogenic for DCM could reveal new insights into DCM pathogenesis, titin (TTN) functions, and therapeutic targets. METHODS: We constructed human cardiomyocyte models harboring DCM-associated TTNtvs within A-band and I-band structural domains using induced pluripotent stem cell and CRISPR technologies. We characterized normal TTN isoforms and variant-specific truncation peptides by their expression levels and cardiomyocyte localization using TTN protein gel electrophoresis and immunofluorescence, respectively. Using CRISPR to ablate A-band variant-specific truncation peptides through introduction of a proximal I-band TTNtv, we studied genetic mechanisms in single cardiomyocyte and 3-dimensional, biomimetic cardiac microtissue functional assays. Last, we engineered a full-length TTN protein reporter assay and used next-generation sequencing assays to develop a CRISPR therapeutic for somatic cell genome editing TTNtvs. RESULTS: An A-band TTNtv dose-dependently impaired cardiac microtissue twitch force, reduced full-length TTN levels, and produced abundant TTN truncation peptides. TTN truncation peptides integrated into nascent myofibril-like structures and impaired myofibrillogenesis. CRISPR ablation of TTN truncation peptides using a proximal I-band TTNtv partially restored cardiac microtissue twitch force deficits. Cardiomyocyte genome editing using SpCas9 and a TTNtv-specific guide RNA restored the TTN protein reading frame, which increased full-length TTN protein levels, reduced TTN truncation peptides, and increased sarcomere function in cardiac microtissue assays. CONCLUSIONS: An A-band TTNtv diminished sarcomere function greater than an I-band TTNtv in proportion to estimated DCM pathogenicity. Although both TTNtvs resulted in full-length TTN haploinsufficiency, only the A-band TTNtv produced TTN truncation peptides that impaired myofibrillogenesis and sarcomere function. CRISPR-mediated reading frame repair of the A-band TTNtv restored functional deficits, and could be adapted as a one-and-done genome editing strategy to target ≈30% of DCM-associated TTNtvs

Reading Frame Repair of TTN Truncation Variants Restores Titin Quantity and Functions.

BACKGROUND: Titin truncation variants (TTNtvs) are the most common inheritable risk factor for dilated cardiomyopathy (DCM), a disease with high morbidity and mortality. The pathogenicity of TTNtvs has been associated with structural localization as A-band variants overlapping myosin heavy chain-binding domains are more pathogenic than I-band variants by incompletely understood mechanisms. Demonstrating why A-band variants are highly pathogenic for DCM could reveal new insights into DCM pathogenesis, titin (TTN) functions, and therapeutic targets. METHODS: We constructed human cardiomyocyte models harboring DCM-associated TTNtvs within A-band and I-band structural domains using induced pluripotent stem cell and CRISPR technologies. We characterized normal TTN isoforms and variant-specific truncation peptides by their expression levels and cardiomyocyte localization using TTN protein gel electrophoresis and immunofluorescence, respectively. Using CRISPR to ablate A-band variant-specific truncation peptides through introduction of a proximal I-band TTNtv, we studied genetic mechanisms in single cardiomyocyte and 3-dimensional, biomimetic cardiac microtissue functional assays. Last, we engineered a full-length TTN protein reporter assay and used next-generation sequencing assays to develop a CRISPR therapeutic for somatic cell genome editing TTNtvs. RESULTS: An A-band TTNtv dose-dependently impaired cardiac microtissue twitch force, reduced full-length TTN levels, and produced abundant TTN truncation peptides. TTN truncation peptides integrated into nascent myofibril-like structures and impaired myofibrillogenesis. CRISPR ablation of TTN truncation peptides using a proximal I-band TTNtv partially restored cardiac microtissue twitch force deficits. Cardiomyocyte genome editing using SpCas9 and a TTNtv-specific guide RNA restored the TTN protein reading frame, which increased full-length TTN protein levels, reduced TTN truncation peptides, and increased sarcomere function in cardiac microtissue assays. CONCLUSIONS: An A-band TTNtv diminished sarcomere function greater than an I-band TTNtv in proportion to estimated DCM pathogenicity. Although both TTNtvs resulted in full-length TTN haploinsufficiency, only the A-band TTNtv produced TTN truncation peptides that impaired myofibrillogenesis and sarcomere function. CRISPR-mediated reading frame repair of the A-band TTNtv restored functional deficits, and could be adapted as a one-and-done genome editing strategy to target ≈30% of DCM-associated TTNtvs

TRPM2 enhances ischemic excitotoxicity by associating with PKCγ

Summary: N-methyl-D-aspartate receptor (NMDAR)-mediated glutamate excitotoxicity significantly contributes to ischemic neuronal death and post-recanalization infarction expansion. Despite tremendous efforts, targeting NMDARs has proven unsuccessful in clinical trials for mitigating brain injury. Here, we show the discovery of an interaction motif for transient receptor potential melastatin 2 (TRPM2) and protein kinase Cγ (PKCγ) association and demonstrate that TRPM2-PKCγ uncoupling is an effective therapeutic strategy for attenuating NMDAR-mediated excitotoxicity in ischemic stroke. We demonstrate that the TRPM2-PKCγ interaction allows TRPM2-mediated Ca2+ influx to promote PKCγ activation, which subsequently enhances TRPM2-induced potentiation of extrasynaptic NMDAR (esNMDAR) activity. By identifying the PKCγ binding motif on TRPM2 (M2PBM), which directly associates with the C2 domain of PKCγ, an interfering peptide (TAT-M2PBM) is developed to disrupt TRPM2-PKCγ interaction without compromising PKCγ function. M2PBM deletion or TRPM2-PKCγ dissociation abolishes both TRPM2-PKCγ and TRPM2-esNMDAR couplings, resulting in reduced excitotoxic neuronal death and attenuated ischemic brain injury

Design and Integration of a High-Powered Model Rocket – III

The paper describes a project to design, assemble, and test propulsion, stage separation, and recovery systems for a high-powered model rocket. A first-principles model was used to evaluate the pressurization produced by the CO2 separation system. A prototype system was fabricated and tested. Circuit and coil design for the electromagnetic booster separation system was used to minimize the capacitance, reducing vehicle mass. Results of this analysis are presented. Two models were used to evaluate rocket motor performance, one to estimate the thrust and specific impulse, and the other the heat transfer rates in the motor. Descriptions of these models are presented. Finally, an autorotation recovery system was designed based on turbine, helicopter blade, and blade element momentum theories

Development of a Cardiac Sarcomere Functional Genomics Platform to Enable Scalable Interrogation of Human

BACKGROUND: Pathogenic METHODS: We created a toolkit of human induced pluripotent stem cell models and functional assays using CRISPR/Cas9 to study RESULTS: Hypertrophic cardiomyopathy-associated CONCLUSIONS: Our study found that hypertrophic cardiomyopathy-associate

Flight System Technologies Enabling the Twin-CubeSat FIREBIRD-II Scientific Mission

Recent technological developments have enabled a CubeSat-based targeted science investigation to unravel a mysterious process that results in the Earth being bombarded by relativistic electrons. The Focused Investigations of Relativistic Electron Burst Intensity, Range, and Dynamics (FIREBIRD) mission is an-NSF funded collaboration carried out by Montana State University, the University of New Hampshire, The Aerospace Corporation and Los Alamos National Laboratory. Four satellites were placed into low Earth orbit in pairs on December 6, 2013 (FIREBIRD-I) and January 31, 2015 (FIREBIRD-II) as auxiliary payloads under NASA’s CubeSat Launch Initiative. Enabling technologies carried on the twin FIREBIRD-II CubeSats include Vanguard Space Technologies, Inc. high-efficiency body-mounted solar panels affixed to the four 10x15 cm sidewalls of each 1.5U CubeSat. These solar panels provide energy to a custom MSU-designed-and-built electrical power system that includes two 2600mAh Li-Ion cells with integrated battery protection circuitry. Each spacecraft carried GPS receivers enabling precise timing and position information necessary for science operations. These technologies together with Montana State’s custom avionics and operations software (built upon L-3’s In-Control package) enabled exciting, unique, and insightful measurements of the near-Earth radiation environment to unravel the spatio-temporal ambiguities of relativistic electron bursts previously observed only by single spacecraft. Without these technologies the mission would not have been possible utilizing CubeSat-class spacecraft measuring merely 15x10x10 cm

Synthesis, Characterization, and Topoisomerase Studies with a Novel Cobalt(III) Complex Coordinated by an Aromatic Bidentate Ligand and N-(3,5-bis(trifluoromethyl)phenyl)pyridine-2-thiocarboxamide

The need for novel antibiotic drugs is urgent. Tuberculosis (TB) is one of the top ten leading causes of death worldwide, with 1.8 million deaths per year, and the number one killer of people who are HIV-positive. The emergence of multidrug-resistant TB (roughly 20% of new cases in 2015) and even extensively drug-resistant TB is a major cause for concern. As the war against bacterial pathogens continues, finding novel drugs and drug targets is essential. Topoisomerase IA is a novel and attractive drug target because it has never yet been targeted by antibiotics used clinically. Since the clinical success of cisplatin as an anticancer drug in the early 19th century, there has been substantial effort to discover novel metal-based therapeutics. In our contribution to the fight against bacterial infection, [[Co(phen)2(PCA-CF3)2)](PF6)2•1.25H2O 1 (where phen = 1,10-phenanthroline and PCA-(CF3)2 = N-(3,5-bis(trifluoromethyl)phenyl)pyridine-2-thiocarboxamide) was synthesized with PCA-(CF3)2 as a mixed ligand. Elemental analysis, FTIR spectroscopy, 1H, 13C, and 59Co NMR spectroscopy, and high resolution electrospray ionization mass spectroscopy were used to determine the structure of complex 1. Complex 1 was tested for inhibition of bacterial topoisomerase I as well as antibacterial activities. Complex 1 had an MtbTopI relaxation inhibition IC50 value of 55.5 µM when compared to an IC50 value of 0.8 µM for [Co(phen)2(MeATSC)](NO3)3·2.5H2O·C2H5OH 2 (where MeATSC = 9-anthraldehyde-N(4)-methylthiosemicarbazone). Complex 1 was able to prevent the growth of M. smegmatis, with an MIC value of 0.89 µM when compared to an MIC value of 6.25 µM for complex 2

CCDC 1956421: Experimental Crystal Structure Determination

Related Article: Michael J. Celestine, Mark A.W. Lawrence, Nicholas K. Evaristo, Benjamin W. Legere, James K. Knarr, Olivier Schott, Vincent Picard, Jimmie L. Bullock, Garry S. Hanan, Colin D. McMillen, Craig A. Bayse, Alvin A. Holder|2020|Inorg.Chim.Acta|510|119726|doi:10.1016/j.ica.2020.11972