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At least 523 records · Page 29

Targeted protein degradation: from small molecules to complex organelles—a Keystone Symposia report

Targeted protein degradation is critical for proper cellular function and development. Protein degradation pathways, such as the ubiquitin proteasomes system, autophagy, and endosome–lysosome pathway, must be tightly regulated to ensure proper elimination of misfolded and aggregated proteins and regulate changing protein levels during cellular differentiation, while ensuring that normal proteins remain unscathed. Protein degradation pathways have also garnered interest as a means to selectively eliminate target proteins that may be difficult to inhibit via other mechanisms. Additionally, on June 7 and 8, 2021, several experts in protein degradation pathways met virtually for the Keystone eSymposium “Targeting protein degradation: from small molecules to complex organelles.” The event brought together researchers working in different protein degradation pathways in an effort to begin to develop a holistic, integrated vision of protein degradation that incorporates all the major pathways to understand how changes in them can lead to disease pathology and, alternatively, how they can be leveraged for novel therapeutics.

59 BASIC BIOLOGICAL SCIENCES↗

Metabolic and transcriptomic study of pennycress natural variation identifies targets for oil improvement

Pennycress (Thlaspi arvense L.), a member of the Brassicaceae family, produces seed oil high in erucic acid, suitable for biodiesel and aviation fuel. Although pennycress, a winter annual, could be grown as a dedicated bioenergy crop, an increase in its seed oil content is required to improve its economic competitiveness. The success of crop improvement relies upon finding the right combination of biomarkers and targets, and the best genetic engineering and/or breeding strategies. In this work, we combined biomass composition with metabolomic and transcriptomic studies of developing embryos from 22 pennycress natural variants to identify targets for oil improvement. The selected accession collection presented diverse levels of fatty acids at maturity ranging from 29% to 41%. Pearson correlation analyses, weighted gene co‐expression network analysis and biomarker identifications were used as complementary approaches to detect associations between metabolite level or gene expression and oil content at maturity. The results indicated that improving seed oil content can lead to a concomitant increase in the proportion of erucic acid without affecting the weight of embryos. Processes, such as carbon partitioning towards the chloroplast, lipid metabolism, photosynthesis, and a tight control of nitrogen availability, were found to be key for oil improvement in pennycress. Besides identifying specific targets, our results also provide guidance regarding the best timing for their modification, early or middle maturation. Thus, this work lays out promising strategies, specific for pennycress, to accelerate the successful development of lines with increased seed oil content for biofuel applications.

59 BASIC BIOLOGICAL SCIENCES↗

On-target uniformity of the OMEGA 60-beam inertial confinement fusion laser

Successful direct-drive inertial confinement fusion (ICF) experiments require excellent on-target laser 9 irradiance uniformity maintained over the duration of the pulse. Achieving this symmetry relies on maintaining an 10 energy, power, and fluence balance among all beams in a multibeam laser system. As a result of the improvements 11 described in this paper, the OMEGA 60-beam laser performance has been assessed at approximately 2% on-target 12 irradiance nonuniformity using an updated performance assessment metric that accounts for the laser diagnostic noise 13 floor. The performance is measurement-limited, prompting the need for an improved diagnostic suite. This manuscript 14 provides a comprehensive first-order assessment of extant status of on-target energy, power, and fluence uniformity 15 and explores avenues for improvements.

Diagnostics↗

Signal sequences target enzymes and structural proteins to bacterial microcompartments and are critical for microcompartment formation

ABSTRACT Spatial organization of pathway enzymes has emerged as a promising tool to address several challenges in metabolic engineering, such as flux imbalances and off-target product formation. Bacterial microcompartments (MCPs) are a spatial organization strategy used natively by many bacteria to encapsulate metabolic pathways that produce toxic, volatile intermediates. Several recent studies have focused on engineering MCPs to encapsulate heterologous pathways of interest, but how this engineering affects MCP assembly and function is poorly understood. In this study, we investigated the role of signal sequences, short domains that target proteins to the MCP core, in the assembly of 1,2-propanediol utilization (Pdu) MCPs. We characterized two novel Pdu signal sequences on the structural proteins PduM and PduB, which constitute the first report of metabolosome signal sequences on structural proteins rather than enzymes. We then explored the role of enzymatic and structural Pdu signal sequences on MCP assembly by deleting their encoding sequences from the genome alone and in combination. Deleting enzymatic signal sequences decreased the MCP formation, but this defect could be recovered in some cases by overexpressing genes encoding the knocked-out signal sequence fused to a heterologous protein. By contrast, deleting structural signal sequences caused similar defects to knocking out the genes encoding the full-length PduM and PduB proteins. Our results contribute to a growing understanding of how MCPs form and function in bacteria and provide strategies to mitigate assembly disruption when encapsulating heterologous pathways in MCPs. IMPORTANCE Spatially organizing biosynthetic pathway enzymes is a promising strategy to increase pathway throughput and yield. Bacterial microcompartments (MCPs) are proteinaceous organelles that many bacteria natively use as a spatial organization strategy to encapsulate niche metabolic pathways, providing significant metabolic benefits. Encapsulating heterologous pathways of interest in MCPs could confer these benefits to industrially relevant pathways. Here, we investigate the role of signal sequences, short domains that target proteins for encapsulation in MCPs, in the assembly of 1,2-propanediol utilization (Pdu) MCPs. We characterize two novel signal sequences on structural proteins, constituting the first Pdu signal sequences found on structural proteins rather than enzymes, and perform knockout studies to compare the impacts of enzymatic and structural signal sequences on MCP assembly. Our results demonstrate that enzymatic and structural signal sequences play critical but distinct roles in Pdu MCP assembly and provide design rules for engineering MCPs while minimizing disruption to MCP assembly.

Johnson, Elizabeth R. (ORCID:0000000179236881)↗

Actinide targets for the synthesis of superheavy nuclei

The use of heavy actinide targets, including 243 Am, 240,242,244 Pu, 245,248 Cm, 249 Bk, and 249 Cf, irradiated by intense heavy ion beams of 48 Ca has resulted in a significant expansion of the periodic table since 2000, including the discovery of five new heaviest elements and more than 50 new isotopes. These actinide materials can only be produced by intense neutron irradiation in very high flux reactors followed by chemical processing and purification in specialized hot cell facilities available in only a few locations worldwide. This paper reviews the reactor production of heavy actinides, the recovery and chemical separation of actinide materials, and the preparation of actinide targets for superheavy element experiments. The focus is on 248 Cm, 249 Bk, mixed 249–251 Cf, and 254 Es, including current availabilities and new production processes. The impacts of new facilities, including the Superheavy Element Factory at Dubna, accelerator and separator upgrades at RIKEN, and proposed upgrades to the High Flux Isotope Reactor at Oak Ridge are also described. Examples of recent superheavy element research are discussed as well as future opportunities for superheavy research using actinide targets.

72 PHYSICS OF ELEMENTARY PARTICLES AND FIELDS↗

Kinematics reconstruction in solenoidal spectrometers operated in active target mode

Here, we discuss the reconstruction of low-energy nuclear reaction kinematics from charged-particle tracks in solenoidal spectrometers working in Active Target Time Projection Chamber mode. In this operation mode, reaction products are tracked within the active gas medium of the Active Target with a three dimensional space point cloud. We have inferred the reaction kinematics from the point cloud using an algorithm based on a linear quadratic estimator (Kalman filter). The performance of this algorithm has been evaluated using experimental data from nuclear reactions measured with the Active Target Time Projection Chamber (AT-TPC) detector.

73 NUCLEAR PHYSICS AND RADIATION PHYSICS↗

Experiments towards a neutron target for measurements in inverse kinematics

Neutron-induced reactions play an important role in fundamental nuclear physics, nuclear astrophysics, and applications. In the case of reactions on rare isotopes, there are limited options for direct experimental measurements. The Neutron Target Demonstrator project at Los Alamos National Laboratory seeks to test the feasibility of moderating spallation neutrons within a 1 m 3 graphite cube to create a standing neutron target for neutron-induced reaction measurements in inverse kinematics. This paper presents the results of experimental neutron flux distribution tests using neutron sources (ranging from 1 keV to 50 MeV) created by accelerators at the University of Notre Dame and Texas A&M University. Measurements were made with both the full graphite cube as well as a ”half cube” setup in which half of the graphite cube was removed. The measured distributions agree with simulated distributions in the case of the full cube moderator, although there remain discrepancies in certain cases for the half cube moderator. The results shown here will provide useful information for an upcoming experimental campaign to test the neutron target proof-of-principle.

73 NUCLEAR PHYSICS AND RADIATION PHYSICS↗

Polarized target nuclear magnetic resonance measurements with deep neural networks

Continuous-wave Nuclear Magnetic Resonance (CW-NMR) operated in constant-current mode has served as a foundational technique for polarization measurement in solid-state dynamically polarized targets within nuclear and high-energy physics experiments for several decades, and it remains an essential tool. Conventional Q-meter-based phase-sensitive detection is critical for precise real-time determination of target polarization during scattering runs. However, the accuracy and reliability of these measurements are frequently compromised by elevated noise levels, baseline drift, and systematic uncertainties arising from signal isolation and fitting, ultimately degrading the overall experimental figure of merit. In this work, we report the first successful application of neural network architectures to continuous-wave NMR polarization metrology. By leveraging advanced machine learning techniques for signal extraction and denoising, we achieve a substantial reduction of fitting uncertainties under a variety of realistic simulated and experimental conditions. These improvements translate directly into more robust real-time (online) polarization monitoring and higher precision in subsequent offline analysis. By reducing analysis-induced uncertainty, the resulting methodology can improve the effective figure of merit for scattering experiments employing dynamically polarized targets and provides a new toolset for NMR-based polarimetry in high-energy and nuclear physics.

Metrology↗

Treatment of Prostate Cancer with CD46-targeted 225 Ac Alpha Particle Radioimmunotherapy

Radiopharmaceutical therapy is changing the standard of care in prostate cancer and other malignancies. We previously reported high CD46 expression in prostate cancer and developed an antibody–drug conjugate and immunoPET agent based on the YS5 antibody, which targets a tumor-selective CD46 epitope. Here, we present the preparation, preclinical efficacy, and toxicity evaluation of [ 225 Ac]DOTA-YS5, a radioimmunotherapy agent based on the YS5 antibody. [ 225 Ac]DOTA-YS5 was developed, and its therapeutic efficiency was tested on cell-derived (22Rv1, DU145), and patient-derived (LTL-545, LTL484) prostate cancer xenograft models. Biodistribution studies were carried out on 22Rv1 tumor xenograft models to confirm the targeting efficacy. Toxicity analysis of the [ 225 Ac]DOTA-YS5 was carried out on nu/nu mice to study short-term (acute) and long-term (chronic) toxicity. Biodistribution study shows that [ 225 Ac]DOTA-YS5 agent delivers high levels of radiation to the tumor tissue (11.64% ± 1.37%ID/g, 28.58% ± 10.88%ID/g, 29.35% ± 7.76%ID/g, and 31.78% ± 5.89%ID/g at 24, 96, 168, and 408 hours, respectively), compared with the healthy organs. [ 225 Ac]DOTA-YS5 suppressed tumor size and prolonged survival in cell line–derived and patient-derived xenograft models. Toxicity analysis revealed that the 0.5 μCi activity levels showed toxicity to the kidneys, likely due to redistribution of daughter isotope 213 Bi. [ 225 Ac]DOTA-YS5 suppressed the growth of cell-derived and patient-derived xenografts, including prostate-specific membrane antigen–positive and prostate-specific membrane antigen–deficient models. Overall, this preclinical study confirms that [ 225 Ac]DOTA-YS5 is a highly effective treatment and suggests feasibility for clinical translation of CD46-targeted radioligand therapy in prostate cancer.

62 RADIOLOGY AND NUCLEAR MEDICINE↗

Targeting CCL2/CCR2 Signaling Overcomes MEK Inhibitor Resistance in Acute Myeloid Leukemia

Emerging evidence underscores the critical role of extrinsic factors within the microenvironment in protecting leukemia cells from therapeutic interventions, driving disease progression, and promoting drug resistance in acute myeloid leukemia (AML). This finding emphasizes the need for the identification of targeted therapies that inhibit intrinsic and extrinsic signaling to overcome drug resistance in AML. We performed a comprehensive analysis utilizing a cohort of ~300 AML patient samples. This analysis encompassed the evaluation of secreted cytokines/growth factors, gene expression, and ex vivo drug sensitivity to small molecules. Our investigation pinpointed a notable association between elevated levels of CCL2 and diminished sensitivity to the MEK inhibitors (MEKi). We validated this association through loss-of-function and pharmacologic inhibition studies. Further, we deployed global phosphoproteomics and CRISPR/Cas9 screening to identify the mechanism of CCR2-mediated MEKi resistance in AML. Our multifaceted analysis unveiled that CCL2 activates multiple prosurvival pathways, including MAPK and cell-cycle regulation in MEKi-resistant cells. Employing combination strategies to simultaneously target these pathways heightened growth inhibition in AML cells. Both genetic and pharmacologic inhibition of CCR2 sensitized AML cells to trametinib, suppressing proliferation while enhancing apoptosis. These findings underscore a new role for CCL2 in MEKi resistance, offering combination therapies as an avenue to circumvent this resistance. Our study demonstrates a compelling rationale for translating CCL2/CCR2 axis inhibitors in combination with MEK pathway-targeting therapies, as a potent strategy for combating drug resistance in AML. This approach has the potential to enhance the efficacy of treatments to improve AML patient outcomes.

60 APPLIED LIFE SCIENCES↗

Establishing Pb-203 production from electrodeposited Tl targets at Brookhaven National Laboratory

Background: Promising developments in Pb-212 radiopharmaceutical therapies have increased demand for Pb-203 diagnostic agents. Building on previous work from various isotope production facilities, this study optimized Pb-203 production from electrodeposited Tl targets at Brookhaven National Laboratory (BNL). The additional supply of Pb-203 may help meet growing preclinical and clinical demands. Results: Two Tl targets were irradiated at the Brookhaven Linac Isotope Producer facility with 30 ± 1 MeV protons, measured using previously published cross section data. Distribution coefficients for Pb Resin in acetate media were investigated for both Na + and K + cations, where potassium acetate was ~ 4 times more effective at stripping Pb from the Pb Resin. The Tl electrodeposition was optimized to deposit 350 mg of Tl (~ 60 mg/cm 2 ) on Au backing in under 6 h. The proposed separation process was completed in < 1.5 h and achieved > 98% and 92 ± 3% recovery of Tl and Pb, respectively, with an overall Tl-Pb separation factor of 6 × 10 5 . The experimentally measured half-life of Pb-203 was 52.4 ± 0.7 h, agreeing with 51.93 ± 0.02 h reported by the National Nuclear Data Center. The radioisotopic purity of the Pb fraction at 24 h post end of bombardment (EOB) from a 24 h irradiation was 66% Pb-203, 28% Pb-201, and 6% Pb-200. Following chemical separation, the Pb-203 produced in this work (21 MBq Pb-203 EOB) achieved apparent molar activities of 10 ± 5 and 0.9 ± 0.5 GBq/µmol for [ 203 Pb]Pb-DOTAM and [ 203 Pb]Pb-DO3A, respectively, decay corrected to EOB. Data derived from this work suggests BNL can produce > 10’s GBq Pb-203 with > 99% radiochemical and radioisotopic purity from Tl-205 for worldwide distribution. Conclusions: The production and separation of Pb-203 from natural Tl target material was successfully demonstrated at BNL. Existing methods were adapted and optimized for the facilities at BNL. Results from this work will guide future large-scale Pb-203 production opportunities at BNL for clinical applications.

38 RADIATION CHEMISTRY, RADIOCHEMISTRY, AND NUCLEA↗

A microCT-based platform to quantify drug targeting

Abstract Background Heterotopic ossification (HO) is a frequent and debilitating complication of traumatic musculoskeletal injuries and orthopedic procedures. Prophylactic dosing of botulinum toxin type A (BTxA) holds potential as a novel treatment option if accurately distributed throughout soft-tissue volumes where protection is clinically desired. We developed a high-resolution, microcomputed tomography (microCT)-based imaging strategy to assess drug distribution and validated this platform by quantifying distribution achieved via a prototype delivery system versus a single-bolus injection. Methods We injected an iodine-containing contrast agent (iodixanol 320 mg I/mL) into dissected rabbit musculature followed by microCT imaging and analysis. To contrast the performance of distributed versus bolus injections, a three-dimensional (3D) 64-cm 3 -printed soft-tissue holder was developed. A centered 2-cm 3 volume of interest (VOI) was targeted with a single-bolus injection or an equal volume distributed injection delivered via a 3D-printed prototype. VOI drug coverage was quantified as a percentage of the VOI volume that was < 1.0 mm from the injected fluid. Results The microCT-based approach enabled high-resolution quantification of injection distribution within soft tissue. The distributed dosing prototype provided significantly greater tissue coverage of the targeted VOI (72 ± 3%, mean ± standard deviation) when compared to an equal volume bolus dose (43 ± 5%, p = 0.031) while also enhancing the precision of injection targeting. Conclusions A microCT-based imaging technique precisely quantifies drug distribution within a soft-tissue VOI, providing a path to overcome a barrier for clinical translation of prophylactic inhibition of HO by BTxA. Relevance statement This platform will facilitate rapid optimization of injection parameters for clinical devices used to effectively and safely inhibit the formation of heterotopic ossification. Key points • MicroCT provides high-resolution quantification of soft-tissue drug distribution. • Distributed dosing is required to maximize soft-tissue drug coverage. • Imaging platform will enable rapid screening of 3D-printed drug distribution prototypes. Graphical Abstract

Ausk, Brandon J. (ORCID:0000000335338303)↗

SMAD4 target genes are part of a transcriptional network that integrates the response to BMP and SHH signaling during early limb bud patterning

SMAD4 regulates gene expression in response to BMP and TGFβ signal transduction, and is required for diverse morphogenetic processes, but its target genes have remained largely elusive. Here, we identify the SMAD4 target genes in mouse limb buds using an epitope-tagged Smad4 allele for ChIP-seq analysis in combination with transcription profiling. This analysis shows that SMAD4 predominantly mediates BMP signal transduction during early limb bud development. Unexpectedly, the expression of cholesterol biosynthesis enzymes is precociously downregulated and intracellular cholesterol levels are reduced in Smad4-deficient limb bud mesenchymal progenitors. Most importantly, our analysis reveals a predominant function of SMAD4 in upregulating target genes in the anterior limb bud mesenchyme. Analysis of differentially expressed genes shared between Smad4- and Shh-deficient limb buds corroborates this function of SMAD4 and also reveals the repressive effect of SMAD4 on posterior genes that are upregulated in response to SHH signaling. This analysis uncovers opposing trans-regulatory inputs from SHH- and SMAD4-mediated BMP signal transduction on anterior and posterior gene expression during the digit patterning and outgrowth in early limb buds.

59 BASIC BIOLOGICAL SCIENCES↗

Data for Comparison of Genotyping Assays for Detection of Targeted CRISPR/Cas Mutagenesis in Highly Polyploid Sugarcane

Sugarcane ( Saccharum spp.) is an important biofuel feedstock and a leading source of global table sugar. Saccharum hybrid cultivars are highly polyploid (2n = 100–130), containing large numbers of functionally redundant hom(e)ologs in their genomes. Genome editing with sequence-specific nucleases holds tremendous promise for sugarcane breeding. However, identification of plants with the desired level of co-editing within a pool of primary transformants can be difficult. While DNA sequencing provides direct evidence of targeted mutagenesis, it is cost-prohibitive as a primary screening method in sugarcane and most other methods of identifying mutant lines have not been optimized for use in highly polyploid species. In this study, non-sequencing methods of mutant screening, including capillary electrophoresis (CE), Cas9 RNP assay, and high-resolution melt analysis (HRMA), were compared to assess their potential for CRISPR/Cas9-mediated mutant screening in sugarcane. These assays were used to analyze sugarcane lines containing mutations at one or more of six sgRNA target sites. All three methods distinguished edited lines from wild type, with co-mutation frequencies ranging from 2% to 100%. Cas9 RNP assays were able to identify mutant sugarcane lines with as low as 3.2% co-mutation frequency, and samples could be scored based on undigested band intensity. CE was highlighted as the most comprehensive assay, delivering precise information on both mutagenesis frequency and indel size to a 1 bp resolution across all six targets. This represents an economical and comprehensive alternative to sequencing-based genotyping methods which could be applied in other polyploid species.

Genomics↗

Joule-class THz pulses from microchannel targets

Inference of joule-class THz radiation sources from microchannel targets driven with hundreds of joule, picosecond lasers is reported. Here, THz sources of this magnitude are useful for nonlinear pumping of matter and for charged-particle acceleration and manipulation. Microchannel targets demonstrate increased laser–THz conversion efficiency compared to planar foil targets, with laser energy to THz energy conversion up to ~0.9% in the best cases.

47 OTHER INSTRUMENTATION↗

Directed evolution of a stem-helix–targeting antibody enables MERS-CoV cross-neutralization through enhanced binding affinity

Broadly neutralizing antibodies (bnAbs) targeting conserved regions of the betacoronavirus spike are important for pan-betacoronavirus protection and pandemic preparedness. Here, we report the isolation of a human monoclonal antibody, CC65.1, from a SARS-CoV-2 convalescent donor that targets the conserved S2 stem helix region. CC65.1 neutralizes various sarbecoviruses, including SARS-CoV-2, and binds to the MERS-CoV spike but lacks MERS-CoV-neutralizing activity due to insufficient binding affinity. We utilized directed evolution to enhance the binding affinity of CC65.1 for the MERS-CoV S2 stem helix, yielding engineered antibody variants with newly acquired MERS-CoV-neutralizing activity. High-resolution structural analysis reveals key paratope mutations that enhance binding and stabilize epitope engagement. Our findings demonstrate the potential of in vitro affinity maturation to expand the neutralization breadth of stem-helix-targeting antibodies across divergent betacoronaviruses. This work supports the development of engineered bnAbs for broadly protective betacoronavirus countermeasures and provides a strategy for achieving cross-lineage neutralization.

Zhou, Panpan↗

Evolution of the high-power spallation neutron mercury target at the SNS

The Spallation Neutron Source (SNS) began operation in 2006 and first operated at its full 1.4 MW power in 2013. Targets, which receive the pulsed proton beam, were a limiting factor for reliable full power operation for several years. Reaching reliable target operation at 1.4 MW required not only changes to the target design but also support and coordination across the entire SNS enterprise. The history and some key lessons learned are presented.

Winder, Drew E↗

Target Optimization Study: Tolerance Sensitivity

A Work Package was initiated to investigate the potential for target optimization. The current design is made up of 82 disks 0.5 mm thick spaced 0.25 mm apart for helium coolant flow. This disk thickness and gap width are a result of continual increases is beam power and hence volumetric heating rate. Thickening the disks would mean higher disk temperatures, but this would be weighed against the prospect of fewer, thicker disks and wider coolant gap spaces, and the relaxed tolerances that would result. Fabrication of the target holder would be easier and less expensive, and target assembly would also be easier.

43 PARTICLE ACCELERATORS↗