A novel CSP C-terminal epitope targeted by an antibody with protective activity against Plasmodium f
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Beutler, Nathan↗
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Results for “Active target”
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Fungal Community, Not Substrate Quality, Drives Soil Microbial Function in Northeastern U.S. Temperate Forests
Mycorrhizal fungi can affect soil organic matter cycling through several mechanisms including priming, nutrient competition, and direct enzyme production. Differences in nutrient foraging strategies between ectomycorrhizal (EcM) and arbuscular mycorrhizal (AM) fungi produce divergent belowground dynamics: where EcM can take up organic nitrogen and directly break down soil organic matter (SOM) by producing enzymes, AM fungi are limited to scavenging mineral N. EcM-associated tree species also have leaf litter with relatively higher ratios of carbon to nitrogen (C:N), and belowground saprotrophic communities more dominated by fungi. Consequently, free-living microbes in EcM-dominated soils should experience nitrogen limitation, with subsequent increases in enzyme production and decreased carbon use efficiency (CUE). However, the relative importance of the effects of substrate quality and fungal community composition on enzyme production and CUE are unclear. To assess this distinction, we sampled the organic horizon and 10 cm of the mineral horizon in northern temperate forest soils along a gradient of EcM dominance. We characterized fungal community composition by measuring EcM relative abundances from extracted fungal DNA and the fungal to bacterial (F:B) ratios from phospholipid fatty acid (PLFA) analysis. We assessed soil substrate quality as the soil C:N ratio. Soil microbial functions were measured as potential activities of five hydrolytic and two oxidative enzymes, and microbial CUE. We found that the fungal community, represented by either the F:B ratio, EcM relative abundance, or both, affected CUE and six measured enzyme activities, while the C:N ratio affected only oxidative and chitin-targeting extracellular enzyme activities. Our results highlight the use of EcM relative dominance as a predictor of soil microbial community composition and function independent of substrate quality.
54 ENVIRONMENTAL SCIENCES↗
TRAC based sensing for autonomous rendezvous
The Targeting Reflective Alignment Concept (TRAC) sensor is to be used in an effort to support an Autonomous Rendezvous and Docking (AR&D) flight experiment. The TRAC sensor uses a fixed-focus, fixed-iris CCD camera and a target that is a combination of active and passive components. The system experiment is anticipated to fly in 1994 using two Commercial Experiment Transporters (COMET's). The requirements for the sensor are: bearing error less than or equal to 0.075 deg; bearing error rate less than 0.3 deg/sec; attitude error less than 0.5 deg.; and attitude rate error less than 2.0 deg/sec. The range requirement depends on the range and the range rate of the vehicle. The active component of the target is several 'kilo-bright' LED's that can emit 2500 millicandela with 40 milliwatts of input power. Flashing the lights in a known pattern eliminates background illumination. The system should be able to rendezvous from 300 meters all the way to capture. A question that arose during the presentation: What is the life time of the LED's and their sensitivity to radiation? The LED's should be manufactured to Military Specifications, coated with silicon dioxide, and all other space qualified precautions should be taken. The LED's will not be on all the time so they should easily last the two-year mission.
Everett, Louis J.↗
The development and validation of command schedules for SeaWiFS
An automated method for developing and assessing spacecraft and instrument command schedules is presented for the Sea-viewing Wide Field-of-view Sensor (SeaWiFS) project. SeaWiFS is to be carried on the polar-orbiting SeaStar satellite in 1995. The primary goal of the SeaWiFS mission is to provide global ocean chlorophyll concentrations every four days by employing onboard recorders and a twice-a-day data downlink schedule. Global Area Coverage (GAC) data with about 4.5 km resolution will be used to produce the global coverage. Higher resolution (1.1 km resolution) Local Area Coverage (LAC) data will also be recorded to calibrate the sensor. In addition, LAC will be continuously transmitted from the satellite and received by High Resolution Picture Transmission (HRPT) stations. The methods used to generate commands for SeaWiFS employ numerous hierarchical checks as a means of maximizing coverage of the Earth's surface and fulfilling the LAC data requirements. The software code is modularized and written in Fortran with constructs to mirror the pre-defined mission rules. The overall method is specifically developed for low orbit Earth-observing satellites with finite onboard recording capabilities and regularly scheduled data downlinks. Two software packages using the Interactive Data Language (IDL) for graphically displaying and verifying the resultant command decisions are presented. Displays can be generated which show portions of the Earth viewed by the sensor and spacecraft sub-orbital locations during onboard calibration activities. An IDL-based interactive method of selecting and testing LAC targets and calibration activities for command generation is also discussed.
Woodward, Robert H.↗
Discovery of BBO-8520, a First-In-Class Direct and Covalent Dual Inhibitor of GTP-Bound (ON) and GDP-Bound (OFF) KRASG12C
Abstract Approved inhibitors of KRASG12C prevent oncogenic activation by sequestering the inactive, GDP-bound (OFF) form rather than directly binding and inhibiting the active, GTP-bound (ON) form. This approach provides no direct target coverage of the active protein. Expectedly, adaptive resistance to KRASG12C (OFF)-only inhibitors is observed in association with increased expression and activity of KRASG12C(ON). To provide optimal KRASG12C target coverage, we have developed BBO-8520, a first-in-class, direct dual inhibitor of KRASG12C(ON) and (OFF) forms. BBO-8520 binds in the Switch-II/Helix3 pocket, covalently modifies the target cysteine, and disables effector binding to KRASG12C(ON). BBO-8520 exhibits potent signaling inhibition in growth factor–activated states, in which current (OFF)-only inhibitors demonstrate little measurable activity. In vivo, BBO-8520 demonstrates rapid target engagement and inhibition of signaling, resulting in durable tumor regression in multiple models, including those resistant to KRASG12C(OFF)-only inhibitors. BBO-8520 is in phase 1 clinical trials in patients with KRASG12C non–small cell lung cancer. Significance: BBO-8520 is a first-in-class direct, small molecule covalent dual inhibitor that engages KRASG12C in the active (ON) and inactive (OFF) conformations. BBO-8520 represents a novel mechanism of action that allows for optimal target coverage and delays the emergence of adaptive resistance seen with (OFF)-only inhibitors in the clinic. See related commentary by Zhou and Westover, p. 455
Oncology↗
Broad-spectrum CRISPR-Cas13a enables efficient phage genome editing
Abstract CRISPR-Cas13 proteins are RNA-guided RNA nucleases that defend against incoming RNA and DNA phages by binding to complementary target phage transcripts followed by general, non-specific RNA degradation. Here we analysed the defensive capabilities of LbuCas13a from Leptotrichia buccalis and found it to have robust antiviral activity unaffected by target phage gene essentiality, gene expression timing or target sequence location. Furthermore, we find LbuCas13a antiviral activity to be broadly effective against a wide range of phages by challenging LbuCas13a against nine E. coli phages from diverse phylogenetic groups. Leveraging the versatility and potency enabled by LbuCas13a targeting, we applied LbuCas13a towards broad-spectrum phage editing. Using a two-step phage-editing and enrichment method, we achieved seven markerless genome edits in three diverse phages with 100% efficiency, including edits as large as multi-gene deletions and as small as replacing a single codon. Cas13a can be applied as a generalizable tool for editing the most abundant and diverse biological entities on Earth.
59 BASIC BIOLOGICAL SCIENCES↗
Comprehensive characterization of hepatocyte-derived extracellular vesicles identifies direct miRNA-based regulation of hepatic stellate cells and DAMP-based hepatic macrophage IL-1ß and IL-17 upregulation in alcoholic hepatitis mice.
Extracellular vesicles (EVs) have been growingly recognized as biomarkers and mediators of alcoholic liver disease (ALD) in human and mice. Here we characterized hepatocyte-derived EVs (HC-EVs) and their cargo for their biological functions in a novel murine model that closely resembles liver pathology observed in patients with alcoholic hepatitis (AH), the most severe spectrum of ALD. The number of circulating EVs and HC-EVs were significantly increased by 10-fold in AH mice compared to control mice. The miRNA(miR)-seq analysis detected 20 up-regulated and 4 down-regulated miRNAs (P < 0.001-0.05) in AH-HC-EVs. Treatment of murine primary hepatic stellate cells (HSCs) with AH-HC-EVs induced ?-SMA (P < 0.05) and Col1a1 (P < 0.001). Smad7, Nr1d2, and Rora genes which regulate HSC activation were predicted targets of 17 miRs up-regulated in AH-HC-EVs. Among them were miR-27a and miR-181 which upon transfection in HSCs, repressed Nr1d2, the HSC quiescence marker. AH-HC-EVs were also enriched in organelle proteins and mitochondrial DNA (10-fold, P < 0.05) and upregulated IL-1??and IL-17 production by hepatic macrophages (HMs) from AH mice in a TLR9-dependent manner. These results demonstrate HC-EV release is intensified in AH and suggest that AH-HC-EVs orchestrate liver fibrogenesis by directly targeting HSC quiescence transcripts via a unique set of miRNAs and by amplifying HSC activation via DAMP-based induction of profibrogenic IL-1? and IL-17 by HMs.
AH, hepatocyte-derived EVs, miRNAs, DAMPs, HSC act↗
Utilization of protein film electrochemistry to characterize the mechanisms imparting aerotolerance and bidirectionality in soluble, multimeric [NiFe]-hydrogenases (Final Report for Grant No. DE-SC0008074)
The overall goal of this project was to better understand how structural features control functional aspects of enzymes involved in catalysis in which a substrate is oxidized or reduced. Two types of model enzymes were explored: the soluble [NiFe]-hydrogenase from Pyrococcus furiousus and the thiosulfate reductase from Pyrobaculum aerophilum. Both are models for complex, multielectron transformations such as nitrogen and carbon dioxide reduction, and parts of important biogeochemical cycles. In this project, electrochemical methods were used to define the activities of these enzymes and targeted variants designed to modulate activity. We showed that the catalytic activity of the soluble hydrogenase from Pyrococcus furiosus can be modulated by removing large parts of the enzyme far from the active site. This surprising result suggests that interactions between functional centers far apart within the enzyme may be essential for controlling and tuning activity. We also showed that the catalytic activity of thiosulfate reductase from Pyrobaculum aerophilum can be modulated by changing the metal at the active site to be either molybdenum or tungsten. These results contribute to our understanding of how the protein environment can tune the reactivity of an embedded metal, catalytic center. The results were and are being published in peer-reviewed, scientific publications.
08 HYDROGEN↗
Proximity operations considerations affecting spacecraft design
Proximity operations can be defined as the maneuvering of two or more spacecraft within 1 nautical mile range, with relative velocity less than 10 feet per second. The passive vehicle is nontranslating and should provide for maintenance of the desired approach attitude. It must accommodate the active (translating) vehicle induced structural loads and performance characteristics (mating hardware tolerances), and support sensor compatibility (transponder, visual targets, etc.). The active vehicle must provide adequate sensor systems (relative state information, field-of-view, redundancy), flight control hardware (thruster sizing, minimal cross-coupling, performance margins, redundancy) and software (reconfigurable, attitude/rate modes, translation and rotation fine control authority) characteristic, and adequate non-propulsive consumables such as power. Operational concerns must be considered. These include the following: (1) the desired approach trajectory and relative orientation; (2) the active vehicle thruster plume effects (forces, torques, contamination) on the passive vehicle; and (3) procedures for contingencies such as loss of communications, sensor or propulsion failures, and target vehicle loss of control.
Staas, Steven K.↗
Guidance, Navigation, and Control for Laser-Based Non-Cooperative Target Detumbling
Orbital debris is a well known and growing problem presenting risks to the future of spaceflight. Many pieces of debris are defunct satellites or rocket upper stages. These large objects could generate an exponentially growing cloud of debris should they be involved in a collision, i.e., Kessler Syndrome. Future orbital debris remediation efforts often propose perturbing the orbit of these large targets to mitigate risk. These plans often involve non-cooperative rendezvous. In the absence of active control, the targets are expected to be tumbling which makes rendezvous a dangerous prospect. In this work we describe a guidance, navigation, and control system for a contactless non-cooperative target detumbling methodology which will significantly increase the safety of non-cooperative rendezvous. An onboard laser generates thrust on the target by ablating its surface material. This thrust is used to control target attitude. Our guidance, navigation, and control system is able to estimate relative pose, target the laser, and maintain formation flight during the detumbling process. The system is validated with Monte Carlo simulation in a variety of scenarios.
Guidance↗
Optogenetic control of protein binding using light-switchable nanobodies
A growing number of optogenetic tools have been developed to reversibly control binding between two engineered protein domains. In contrast, relatively few tools confer lightswitchable binding to a generic target protein of interest. Such a capability would offer substantial advantages, enabling photoswitchable binding to endogenous target proteins in cells or light-based protein purification in vitro. Here, we report the development of optonanobodies (OptoNBs), a versatile class of chimeric photoswitchable proteins whose binding to proteins of interest can be enhanced or inhibited upon blue light illumination. We find that OptoNBs are suitable for a range of applications including reversibly binding to endogenous intracellular targets, modulating signaling pathway activity, and controlling binding to purified protein targets in vitro. This work represents a step towards programmable photoswitchable regulation of a wide variety of target proteins.
59 BASIC BIOLOGICAL SCIENCES↗
Recent 238 Pu Production Activities at Idaho National Laboratory
The Plutonium-238 ( 238 Pu) Production program at Idaho National Laboratory (INL) is actively qualifying irradiation targets containing 237 Np for the Advanced Test Reactor (ATR) to produce 238 Pu for future National Aeronautics and Space Administration missions. INL qualified and loaded seven targets in the ATR’s south flux trap for cycle 169A, which occurred in Spring 2021. The irradiation qualification program has expanded to additional ATR irradiation positions after two baseline production targets in three positions validated significant production of 238 Pu. The validation model was followed by the PFS-1 experimental test in the ATR Critical Facility that verified 238 Pu production cross sections. This paper outlines the progress and status of the 238 Pu production program at INL. The qualification effort, safety analysis, hardware status, and future activities for qualification of an updated target design for use in the ATR are discussed.
07 ISOTOPE AND RADIATION SOURCES↗
Nitric oxide inhibits calpain-mediated proteolysis of talin in skeletal muscle cells
We tested the hypothesis that nitric oxide can inhibit cytoskeletal breakdown in skeletal muscle cells by inhibiting calpain cleavage of talin. The nitric oxide donor sodium nitroprusside prevented many of the effects of calcium ionophore on C(2)C(12) muscle cells, including preventing talin proteolysis and release into the cytosol and reducing loss of vinculin, cell detachment, and loss of cellular protein. These results indicate that nitric oxide inhibition of calpain protected the cells from ionophore-induced proteolysis. Calpain inhibitor I and a cell-permeable calpastatin peptide also protected the cells from proteolysis, confirming that ionophore-induced proteolysis was primarily calpain mediated. The activity of m-calpain in a casein zymogram was inhibited by sodium nitroprusside, and this inhibition was reversed by dithiothreitol. Previous incubation with the active site-targeted calpain inhibitor I prevented most of the sodium nitroprusside-induced inhibition of m-calpain activity. These data suggest that nitric oxide inhibited m-calpain activity via S-nitrosylation of the active site cysteine. The results of this study indicate that nitric oxide produced endogenously by skeletal muscle and other cell types has the potential to inhibit m-calpain activity and cytoskeletal proteolysis.
NASA Discipline Musculoskeletal↗
Global Positioning System Synchronized Active Light Autonomous Docking System
A Global Positioning System Synchronized Active Light Autonomous Docking System (GPSSALADS) for automatically docking a chase vehicle with a target vehicle comprises at least one active light emitting target which is operatively attached to the target vehicle. The target includes a three-dimensional array of concomitantly flashing lights which flash at a controlled common frequency. The GPSSALADS further comprises a visual tracking sensor operatively attached to the chase vehicle for detecting and tracking the target vehicle. Its performance is synchronized with the flash frequency of the lights by a synchronization means which is comprised of first and second internal clocks operatively connected to the active light target and visual tracking sensor, respectively, for providing timing control signals thereto, respectively. The synchronization means further includes first and second Global Positioning System receivers operatively connected to the first and second internal clocks, respectively, for repeatedly providing simultaneous synchronization pulses to the internal clocks, respectively. In addition, the GPSSALADS includes a docking process controller means which is operatively attached to the chase vehicle and is responsive to the visual tracking sensor for producing commands for the guidance and propulsion system of the chase vehicle.
Howard, Richard↗
Global Positioning System Synchronized Active Light Autonomous Docking System
A Global Positioning System Synchronized Active Light Autonomous Docking System (GPSSALADS) for automatically docking a chase vehicle with a target vehicle comprising at least one active light emitting target which is operatively attached to the target vehicle. The target includes a three-dimensional array of concomitantly flashing lights which flash at a controlled common frequency. The GPSSALADS further comprises a visual tracking sensor operatively attached to the chase vehicle for detecting and tracking the target vehicle. Its performance is synchronized with the flash frequency of the lights by a synchronization means which is comprised of first and second internal clocks operatively connected to the active light target and visual tracking sensor, respectively, for providing timing control signals thereto, respectively. The synchronization means further includes first and second Global Positioning System receivers operatively connected to the first and second internal clocks, respectively, for repeatedly providing simultaneous synchronization pulses to the internal clocks, respectively. In addition, the GPSSALADS includes a docking process controller means which is operatively attached to the chase vehicle and is responsive to the visual tracking sensor for producing commands for the guidance and propulsion system of the chase vehicle.
Howard, Richard T.↗
Structure-function analysis of the FCRL5–IgG1 Fc complex reveals an unappreciated pathway for B cell modulation by Fc-attenuated IgG
The Fc region of therapeutic IgG antibodies is often engineered to remove or “silence” Fc effector functions, but it remains unclear whether these mutations eliminate all Fc-mediated effector activity. Human Fc receptor-like 5 (FCRL5/FcRH5) is a low-affinity IgG Fc receptor expressed on B cells and is an actively pursued antibody target in multiple myeloma. Here, we show that common Fc function-silencing mutations do not impair FCRL5-mediated activity and therefore attenuate, rather than eliminate, Fc effector function. The crystal structure of the FCRL5-IgG1 Fc complex, solved at 3.4 Å resolution, revealed that FCRL5 binds IgG1 Fc in a 1:1 complex through a binding mode distinct from that of classical Fcγ receptors, explaining why mutations that attenuate Fc effector function spare FCRL5 binding. Fc-engineered antibodies that selectively engage FCRL5 inhibited B cell receptor-induced Ca 2+ flux in FCRL5-expressing B cells. These findings demonstrate that Fc-attenuated therapeutic IgG retains the ability to engage FCRL5, identifying an unappreciated pathway for B cell modulation.
Herpers, Bart M. [Department of Biomedical Enginee↗
Structure of the WYL-domain containing transcription activator, DriD, in complex with ssDNA effector and DNA target site
Transcription regulators play central roles in orchestrating responses to changing environmental conditions. Recently the Caulobacter crescentus transcription activator DriD, which belongs to the newly defined WYL-domain family, was shown to regulate DNA damage responses independent of the canonical SOS pathway. However, the molecular mechanisms by which DriD and other WYL-regulators sense environmental signals and recognize DNA are not well understood. We showed DriD DNA-binding is triggered by its interaction with ssDNA, which is produced during DNA damage. Here we describe the structure of the full-length C. crescentus DriD bound to both target DNA and effector ssDNA. DriD consists of an N-terminal winged-HTH (wHTH) domain, linker region, three-helix bundle, WYL-domain and C-terminal WCX-dimer domain. Strikingly, DriD binds DNA using a novel, asymmetric DNA-binding mechanism that results from different conformations adopted by the linker. Although the linker does not touch DNA, our data show that contacts it makes with the wHTH are key for specific DNA binding. The structure indicates how ssDNA-effector binding to the WYL-domain impacts wHTH DNA binding. In conclusion, we present the first structure of a WYL-activator bound to both effector and target DNA. The structure unveils a unique, asymmetric DNA binding mode that is likely conserved among WYL-activators.
59 BASIC BIOLOGICAL SCIENCES↗
Proteomic and phosphoproteomic measurements enhance ability to predict ex vivo drug response in AML
Acute Myeloid Leukemia (AML) affects 20,000 patients in the US annually with a five-year survival rate of approximately 25%. One reason for the low survival rate is the high prevalence of clonal evolution that gives rise to heterogeneous sub-populations of leukemic cells with diverse mutation spectra, which eventually leads to disease relapse. This genetic heterogeneity drives the activation of complex signaling pathways that is reflected at the protein level. This diversity makes it difficult to treat AML with targeted therapy, requiring custom patient treatment protocols tailored to each individual’s leukemia. Toward this end, the Beat AML research program prospectively collected genomic and transcriptomic data from over 1000 AML patients and carried out ex vivo drug sensitivity assays to identify genomic signatures that could predict patient-specific drug responses. However, there are inherent weaknesses in using only genetic and transcriptomic measurements as surrogates of drug response, particularly the absence of direct information about phosphorylation-mediated signal transduction. As a member of the Clinical Proteomic Tumor Analysis Consortium, we have extended the molecular characterization of this cohort by collecting proteomic and phosphoproteomic measurements from a subset of these patient samples (38 in total) to evaluate the hypothesis that proteomic signatures can improve the ability to predict response to 26 drugs in AML ex vivo samples. In this work we describe our systematic, multi-omic approach to evaluate proteomic signatures of drug response and compare protein levels to other markers of drug response such as mutational patterns. We explore the nuances of this approach using two drugs that target key pathways activated in AML: quizartinib (FLT3) and trametinib (Ras/MEK), and show how patient-derived signatures can be interpreted biologically and validated in cell lines. In conclusion, this pilot study demonstrates strong promise for proteomics-based patient stratification to assess drug sensitivity in AML.
60 APPLIED LIFE SCIENCES↗