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Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

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All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

AGTR1 blocker attenuates activation of Tenon's capsule fibroblasts after glaucoma filtration surgery via the NF-κB signaling pathway

Activation of Tenon's capsule fibroblasts limits the success rate of glaucoma filtration surgery (GFS), the most efficacious therapy for patients with glaucoma. Angiotensin type 1 receptor (AGTR1) is involved in tissues remodeling and fibrogenesis. However, whether AGTR1 is involved in the progress of fibrogenesis after GFS is not fully elucidated. The aim of this study was to investigate the role of an AGTR1 in scar formation after GFS and the potential anti-fibrosis effect of AGTR1 blocker. AGTR1 expression level was increased in subconjunctival tissues in a rat model of GFS and transforming growth factor-beta 2 (TGF-β2)-induced human Tenon's capsule fibroblasts (HTFs). AGTR1 blocker treatment suppressed TGF-β2-induced HTF migration and α-smooth muscle actin (α-SMA) and fibronectin (FN) expression. AGTR1 blocker treatment also attenuated collagen deposition and α-SMA and FN expression in subconjunctival tissues of the rat model after GFS. Moreover, AGTR1 blocker decreased TGF-β2-induced P65 phosphorylation, P65 nuclear translocation, and nuclear factor kappa B (NF-κB) luciferase activity. Additionally, BAY 11–7082 (an NF-κB inhibitor) significantly suppressed HTF fibrosis. In conclusion, our results indicate that AGTR1 is involved in scar formation after GFS. The AGTR1 blocker attenuates subconjunctival fibrosis after GFS by inhibiting the NF-κB signaling pathway. These findings indicate that targeting AGTR1 is a potential approach to attenuate fibrosis after GFS.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

ESR1 mediated circ{sub 0}004018 suppresses angiogenesis in hepatocellular carcinoma via recruiting FUS and stabilizing TIMP2 expression

Angiogenesis has been certified to account for tumor pathobiology. Circular RNAs (circRNAs) have been demonstrated to be involved in angiogenesis-related diseases, including hepatocellular carcinoma (HCC). Nevertheless, the regulatory roles of most circRNAs remain obscure. This study aims to uncover the function of hsa{sub c}irc{sub 0}004018 on angiogenesis in HCC. Firstly, quantitative real-time RT-PCR (RT-qPCR) analyzed that circ{sub 0}004018 was definitely down-regulated in HCC. Western blot analysis was conducted to detect the protein level of fused protein in sarcoma (FUS) and TIMP metallopeptidase inhibitor 2 (TIMP2). Functional assays were carried out to assess the impacts of circ{sub 0}004018 on HCC. From the experimental results, we found that overexpression of circ{sub 0}004018 significantly inhibited angiogenesis in HCC. The regulatory mechanism of circ{sub 0}004018 in HCC was determined by chromatin immunoprecipitation (ChIP), luciferase reporter assays and RNA immunoprecipitation (RIP) assay. Therefore, we proved that estrogen receptor 1 (ESR1) mediated circ{sub 0}004018 regulated TIMP2 by recruiting FUS. A series of rescue assays verified that circ{sub 0}004018 participated in angiogenesis in HCC via modulating TIMP2. In summary, this paper disclosed that ESR1 activated circ{sub 0}004018 inhibited angiogenesis in HCC via binding to FUS and stabilizing TIMP2 expression.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

MiR-125b enhances autophagic flux to improve septic cardiomyopathy via targeting STAT3/HMGB1

We explore the role of miR-125b in septic cardiomyopathy, focusing on miR-125b/STAT3/HMGB1 axis. CLP mouse model and LPS-stimulated primary rat cardiomyocytes (CMs) and H9C2 cell were used as in vivo and in vitro models of septic cardiomyopathy, respectively. qRT-PCR and western blot were performed to measure expression levels of miR-125b, STAT3, HMGB1, and autophagy-related proteins. MTT assay was employed to examine LPS toxicity. Dual luciferase activity assay and CHIP were performed to validate interactions of miR-125b/STAT3 and STAT3/HMGB1 promoter. Immunostaining was used to assess the level of autophagic flux. ROS level was measured by fluorescence assay. Heart functions were examined via intracoronary Doppler ultrasound. miR-125b was diminished while STAT3 and HMGB1 were elevated in the heart tissue following CLP surgery and in LPS-treated H9C2 cells. LPS treatment up-regulated ROS generation and suppressed autophagic flux. Overexpression of miR-125b mimics or knockdown of STAT3 or HMGB1 alleviated LPS-induced hindrance of autophagic flux and ROS production. miR-125b directly targeted STAT3 mRNA and STAT3 bound with HMGB1 promoter. Overexpression of miR-125b mitigated myocardial dysfunction induced by CLP in vivo. Hyperactivation of STAT3/HMGB1 caused by reduced miR-125b contributes to ROS generation and the hindrance of autophagic flux during septic cardiomyopathy, leading to myocardial dysfunction.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

LncRNA HOTTIP from synovial fibroblast-derived exosomes: A novel molecular target for rheumatoid arthritis through the miR-1908–5p/STAT3 axis

Rheumatoid arthritis (RA) is a chronic inflammation mediated by autoimmune responses. HOTTIP, a long noncoding RNA (lncRNA), participates in cell proliferation and invasion. However, the correlation between HOTTIP and RA remains unclear. Therefore, this study aimed to clarify how HOTTIP works in RA and to investigate its role in the development of RA. Flow cytometry was used to analyze cell cycle progression. Binding between HOTTIP, signal transducer and activator of transcription 3 (STAT3) and miR-1908–5p was demonstrated by dual-luciferase assays. Quantitative real-time polymerase chain reaction (qRT–PCR) was used to measure the expression of T cell differentiation-related proteins. We found that HOTTIP was upregulated in rheumatoid arthritis synovial fibroblasts (RASFs). HOTTIP directly bound to miR-1908–5p and negatively modulated miR-1908–5p expression while positively regulating STAT3. The effects of HOTTIP overexpression on regulating the balance of the Th17/Treg cell ratio were partly reversed by miR-1908–5p overexpression. In addition, in vivo experiments demonstrated that overexpression of HOTTIP aggravated inflammation in RA mice, which was demonstrated by hematoxylin and eosin (HE) staining and the increased expression levels of CD4{sup +} interleukin (IL)-17{sup +}, forkhead Box P3 (FOXP3) and retinoid-related orphan receptor gamma-t (RORγt). In summary, our study suggests that HOTTIP plays a damaging role in RA by promoting inflammation, which may be related to the regulation of miR-1908–5p expression and the STAT3 signaling pathway. These results suggest that the regulation of HOTTIP may be a promising therapeutic strategy for RA.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Triggering cell death in cancers using self-illuminating nanocomposites

Bioinspired photocatalysis has resulted in efficient solutions for many areas of science and technology spanning from solar cells to medicine. Here we show a new bioinspired semiconductor nanocomposite (nanoTiO 2 DOPA-luciferase, TiDoL) capable of converting light energy within cancerous tissues into chemical species that are highly disruptive to cell metabolism and lead to cell death. This localized activity of semiconductor nanocomposites is triggered by cancer-generated activators. Adenosine triphosphate (ATP) is produced in excess in cancer tissues only and activates nearby immobilized TiDoL composites, thereby eliminating its off-target toxicity. The interaction of TiDoL with cancerous cells was probed in situand in real-time to establish a detailed mechanism of nanoparticle activation, triggering of the apoptotic signaling cascade, and finally, cancer cell death. Activation of TiDoL with non-cancerous cells did not result in cell toxicity. Exploring the activation of antibody-targeted semiconductor conjugates using ATP is a step toward a universal approach to single-cell-targeted medical therapies with more precision, efficacy, and potentially fewer side effects.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2026

A Fluorescence‐Based Transient Expression Assay for the Analysis of Upstream Open Reading Frames in Plants

Upstream open reading frames (uORFs) are regulatory elements present in the 5′ leaders of mRNA that can significantly impact downstream gene expression in eukaryotes. In crop engineering, editing of uORFs can provide an avenue to upregulate expression of native genes without the need to add persistent transgenic copies. Even with genome-wide methods to identify translated uORFs such as ribosome profiling, their functional characterization depends on validation through reporter gene assays and mutagenesis studies. Current screening methods for plants use luciferases or protoplasts to measure differential gene expression between wild-type and mutated transcript leaders, which requires tissue processing and/or substrate addition. Here, we present a time- and cost-efficient alternative to investigate transcript leaders by co-expression of two fluorescent proteins in Nicotiana benthamiana leaf tissue and test our assay on genes involved in photoprotection, editing of which could provide a pathway to increase CO 2 assimilation during sun–shade transitions.

Nicotiana benthamiana↗
DOE OSTI2023

An extensible vector toolkit and parts library for advanced engineering of plant genomes

Abstract Plant biotechnology is rife with new advances in transformation and genome engineering techniques. A common requirement for delivery and coordinated expression in plant cells, however, places the design and assembly of transformation constructs at a crucial juncture as desired reagent suites grow more complex. Modular cloning principles have simplified some aspects of vector design, yet many important components remain unavailable or poorly adapted for rapid implementation in biotechnology research. Here, we describe a universal Golden Gate cloning toolkit for vector construction. The toolkit chassis is compatible with the widely accepted Phytobrick standard for genetic parts, and supports assembly of arbitrarily complex T‐DNAs through improved capacity, positional flexibility, and extensibility in comparison to extant kits. We also provision a substantial library of newly adapted Phytobricks, including regulatory elements for monocot and dicot gene expression, and coding sequences for genes of interest such as reporters, developmental regulators, and site‐specific recombinases. Finally, we use a series of dual‐luciferase assays to measure contributions to expression from promoters, terminators, and from cross‐cassette interactions attributable to enhancer elements in certain promoters. Taken together, these publicly available cloning resources can greatly accelerate the testing and deployment of new tools for plant engineering.

42 ENGINEERING↗
DOE OSTI2024

Omics-driven onboarding of the carotenoid producing red yeast Xanthophyllomyces dendrorhous CBS 6938

Transcriptomics is a powerful approach for functional genomics and systems biology, yet it can also be used for genetic part discovery. Here, we derive constitutive and light-regulated promoters directly from transcriptomics data of the basidiomycete red yeast Xanthophyllomyces dendrorhous CBS 6938 (anamorph Phaffia rhodozyma) and use these promoters with other genetic elements to create a modular synthetic biology parts collection for this organism. X. dendrorhous is currently the sole biotechnologically relevant yeast in the Tremellomycete class-it produces large amounts of astaxanthin, especially under oxidative stress and exposure to light. Thus, we performed transcriptomics on X. dendrorhous under different wavelengths of light (red, green, blue, and ultraviolet) and oxidative stress. Differential gene expression analysis (DGE) revealed that terpenoid biosynthesis was primarily upregulated by light through crtI, while oxidative stress upregulated several genes in the pathway. Further gene ontology (GO) analysis revealed a complex survival response to ultraviolet (UV) where X. dendrorhous upregulates aromatic amino acid and tetraterpenoid biosynthesis and downregulates central carbon metabolism and respiration. The DGE data was also used to identify 26 constitutive and regulated genes, and then, putative promoters for each of the 26 genes were derived from the genome. Simultaneously, a modular cloning system for X. dendrorhous was developed, including integration sites, terminators, selection markers, and reporters. Each of the 26 putative promoters were integrated into the genome and characterized by luciferase assay in the dark and under UV light. The putative constitutive promoters were constitutive in the synthetic genetic context, but so were many of the putative regulated promoters. Notably, one putative promoter, derived from a hypothetical gene, showed ninefold activation upon UV exposure. Thus, this study reveals metabolic pathway regulation and develops a genetic parts collection for X. dendrorhous from transcriptomic data. Therefore, this study demonstrates that combining systems biology and synthetic biology into an omics-to-parts workflow can simultaneously provide useful biological insight and genetic tools for nonconventional microbes, particularly those without a related model organism. This approach can enhance current efforts to engineer diverse microbes.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Constructing the Mechanism of Dinoflagellate Luciferin Bioluminescence Using Computation

Dinoflagellate luciferin bioluminescence is unique since it does not rely on decarboxylation but is poorly understood compared to that of firefly, bacteria, and coelenterata luciferins. Here, in this work, we computationally investigate possible protonation states, stereoisomers, a chemical mechanism, and the dynamics of the bioluminescence intermediate that is responsible for chemiexcitation. Using semiempirical dynamics, time-dependent density functional theory static calculations, and a correlation diagram, we find that the intermediate’s functional group that is likely responsible for chemiexcitation is a 4-member ring, a dioxetanol, that undergoes [2π + 2π] cycloreversion and the biolumiphore is the cleaved structure. The simulated emission spectra and luciferase-dependent absorbance spectra agree with the experimental data, giving support to our proposed mechanism and biolumiphore. We also compute circular dichroism spectra of the intermediate’s four stereoisomers to guide future experiments in differentiating them.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2022

Omicron mutations enhance infectivity and reduce antibody neutralization of SARS-CoV-2 virus-like particles

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant contains extensive sequence changes relative to the earlier-arising B.1, B.1.1, and Delta SARS-CoV-2 variants that have unknown effects on viral infectivity and response to existing vaccines. Using SARS-CoV-2 virus-like particles (VLPs), we examined mutations in all four structural proteins and found that Omicron and Delta showed 4.6-fold higher luciferase delivery overall relative to the ancestral B.1 lineage, a property conferred mostly by enhancements in the S and N proteins, while mutations in M and E were mostly detrimental to assembly. Thirty-eight antisera samples from individuals vaccinated with Pfizer/BioNTech, Moderna, or Johnson & Johnson vaccines and convalescent sera from unvaccinated COVID-19 survivors had 15-fold lower efficacy to prevent cell transduction by VLPs containing the Omicron mutations relative to the ancestral B.1 spike protein. A third dose of Pfizer vaccine elicited substantially higher neutralization titers against Omicron, resulting in detectable neutralizing antibodies in eight out of eight subjects compared to one out of eight preboosting. Furthermore, the monoclonal antibody therapeutics casirivimab and imdevimab had robust neutralization activity against B.1 and Delta VLPs but no detectable neutralization of Omicron VLPs, while newly authorized bebtelovimab maintained robust neutralization across variants. Our results suggest that Omicron has similar assembly efficiency and cell entry compared to Delta and that its rapid spread is due mostly to reduced neutralization in sera from previously vaccinated subjects. In addition, most currently available monoclonal antibodies will not be useful in treating Omicron-infected patients with the exception of bebtelovimab.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Leaky ribosomal scanning enables tunable translation of bicistronic ORFs in green algae

Advances in sequencing technology have unveiled examples of nucleus-encoded polycistrons, once considered rare. Exclusively polycistronic transcripts are prevalent in green algae, although the mechanism by which multiple polypeptides are translated from a single transcript is unknown. Here, we used bioinformatic and in vivo mutational analyses to evaluate competing mechanistic models for translation of bicistronic mRNAs in green algae. High-confidence manually curated datasets of bicistronic loci from two divergent green algae, Chlamydomonas reinhardtii and Auxenochlorella protothecoides, revealed a preference for weak Kozak-like sequences for ORF 1 and an underrepresentation of potential initiation codons before the ORF 2 start codon, which are suitable conditions for leaky ribosome scanning to allow ORF 2 translation. We used mutational analysis in A. protothecoides to test the mechanism. In vivo manipulation of the ORF 1 Kozak-like sequence and start codon altered reporter expression at ORF 2, with a weaker Kozak-like sequence enhancing expression and a stronger one diminishing it. A synthetic bicistronic dual reporter demonstrated inversely adjustable activity of green fluorescent protein expressed from ORF 1 and luciferase from ORF 2, depending on the strength of the ORF 1 Kozak-like sequence. Our findings demonstrate that translation of multiple ORFs in green algal bicistronic transcripts is consistent with episodic leaky scanning of ORF 1 to allow translation at ORF 2. This work has implications for the potential functionality of upstream open reading frames (uORFs) found across eukaryotic genomes and for transgene expression in synthetic biology applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Fluorescent and Bioluminescent Reporter Mouse-Adapted Ebola Viruses Maintain Pathogenicity and Can Be Visualized in Vivo

Abstract Ebola virus (EBOV) causes lethal disease in humans but not in mice. Here, we generated recombinant mouse-adapted (MA) EBOVs, including 1 based on the previously reported serially adapted strain (rMA-EBOV), along with single-reporter rMA-EBOVs expressing either fluorescent (ZsGreen1 [ZsG]) or bioluminescent (nano-luciferase [nLuc]) reporters, and dual-reporter rMA-EBOVs expressing both ZsG and nLuc. No detriment to viral growth in vitro was seen with inclusion of MA-associated mutations or reporter proteins. In CD-1 mice, infection with MA-EBOV, rMA-EBOV, and single-reporter rMA-EBOVs conferred 100% lethality; infection with dual-reporter rMA-EBOV resulted in 73% lethality. Bioluminescent signal from rMA-EBOV expressing nLuc was detected in vivo and ex vivo using the IVIS Spectrum CT. Fluorescent signal from rMA-EBOV expressing ZsG was detected in situ using handheld blue-light transillumination and ex vivo through epi-illumination with the IVIS Spectrum CT. These data support the use of reporter MA-EBOV for studies of Ebola virus in animal disease models.

Immunology↗
DOE OSTI2021

GTP binding by Arabidopsis extra-large G protein 2 is not essential for its functions

The extra-large guanosine-5'-triphosphate (GTP)-binding protein 2, XLG2, is an unconventional Gα subunit of the Arabidopsis (Arabidopsis thaliana) heterotrimeric GTP-binding protein complex with a major role in plant defense. In vitro biochemical analyses and molecular dynamic simulations show that affinity of XLG2 for GTP is two orders of magnitude lower than that of the conventional Gα, AtGPA1. Here we tested the physiological relevance of GTP binding by XLG2. We generated an XLG2(T476N) variant with abolished GTP binding, as confirmed by in vitro GTPγS binding assay. Yeast three-hybrid, bimolecular fluorescence complementation, and split firefly-luciferase complementation assays revealed that the nucleotide-depleted XLG2(T476N) retained wild-type XLG2-like interactions with the Gβγ dimer and defense-related receptor-like kinases. Both wild-type and nucleotide-depleted XLG2(T476N) restored the defense responses against Fusarium oxysporum and Pseudomonas syringae compromised in the xlg2 xlg3 double mutant. Additionally, XLG2(T476N) was fully functional restoring stomatal density, root growth, and sensitivity to NaCl, but failed to complement impaired germination and vernalization-induced flowering. We conclude that XLG2 is able to function in a GTP-independent manner and discuss its possible mechanisms of action.

Plant Sciences↗
DOE OSTI2020

Hsa_circ_0000515 is a novel circular RNA implicated in the development of breast cancer through its regulation of the microRNA‐296‐5p/CXCL10 axis

Cancer metastasis is a major cause of death among women afflicted with breast cancer (BC) and understanding the molecular processes involved is a major focus in BC research. Circular RNAs (circRNAs) have emerged as genomic regulatory molecules in carcinogenesis and metastasis; however, their role in BC is unclear. We characterized a novel circRNA, hsa_circ_0000515, in context of BC. We collected 340 cancerous tissues surgically resected from BC patients and found hsa_circ_0000515 was upregulated in BC tissues and associated with poor prognosis of BC. Silencing of hsa_circ_0000515 impaired cell cycle progression, cell proliferation, and invasion, attenuated inflammatory response, and reduced the proangiogenetic potential of BC cells. RNA pull‐down and dual‐luciferase reporter gene assays showed that hsa_circ_0000515 binds miR‐296‐5p, preventing it from repressing CXCL10 expression. We also observed that miR‐296‐5p inhibition or CXCL10 overexpression promoted cell cycle progression, restored proliferative, invasive and proangiogenetic abilities, and increased inflammatory response in MCF‐7 cells in the absence of hsa_circ_0000515. In vivo analyses showed that partial loss of hsa_circ_0000515 reduced the tumor growth of MCF‐7 cells in nude mice. The key findings from this study revealed that targeting hsa_circ_0000515 might be an effective strategy to combat BC.

Cai, Fenglin↗
DOE OSTI2022

GhBZR3 suppresses cotton fiber elongation by inhibiting very‐long‐chain fatty acid biosynthesis

SUMMARY The BRASSINAZOLE‐RESISTANT (BZR) transcription factor is a core component of brassinosteroid (BR) signaling and is involved in the development of many plant species. BR is essential for the initiation and elongation of cotton fibers. However, the mechanism of BR‐regulating fiber development and the function of BZR is poorly understood in Gossypium hirsutum L. (cotton). Here, we identified a BZR family transcription factor protein referred to as GhBZR3 in cotton. Overexpression of GhBZR3 in Arabidopsis caused shorter root hair length, hypocotyl length, and hypocotyl cell length, indicating that GhBZR3 negatively regulates cell elongation. Pathway enrichment analysis from VIGS ‐GhBZR3 cotton plants found that fatty acid metabolism and degradation might be the regulatory pathway that is primarily controlled by GhBZR3. Silencing GhBZR3 expression in cotton resulted in taller plant height as well as longer fibers. The very‐long‐chain fatty acid (VLCFA) content was also significantly increased in silenced GhBZR3 plants compared with the wild type. The GhKCS13 promoter, a key gene for VLCFA biosynthesis, contains two GhBZR3 binding sites. The results of yeast one‐hybrid, electrophoretic mobility shift, and luciferase assays revealed that GhBZR3 directly interacted with the GhKCS13 promoter to suppress gene expression. Taken together, these results indicate that GhBZR3 negatively regulates cotton fiber development by reducing VLCFA biosynthesis. This study not only deepens our understanding of GhBZR3 function in cotton fiber development, but also highlights the potential of improving cotton fiber length and plant growth using GhBZR3 and its related genes in future cotton breeding programs.

Shi, Zemin↗
DOE OSTI2022

Pleiotropic Effects of Influenza H1, H3, and B Baloxavir-Resistant Substitutions on Replication, Sensitivity to Baloxavir, and Interferon Expression

Baloxavir is an anti-influenza endonuclease inhibitor that targets the polymerase acidic (PA) protein of influenza A and B viruses. Our knowledge regarding the pleiotropic effects of baloxavir resistance-associated substitutions is limited. We generated recombinant A/California/04/09 (H1N1)-, A/Hong Kong/218849/2006 (H3N2)-, and B/Victoria/504/2000-like viruses that contained PA substitutions identified in baloxavir clinical trials and surveillance that could potentially be associated with baloxavir resistance. We characterized their susceptibility to baloxavir, impact on polymerase activity, viral growth, and ability to induce interferon (IFN) and IFN-stimulated genes expression in vitro. Four PA substitutions, H1N1 I38L/T, E199D, and B G199R, significantly reduced the sensitivity of the recombinant viruses to baloxavir (14.1-fold). We confirmed our findings by using the luciferase-based ribonucleoprotein minigenome assay and by using virus yield reduction assay in Calu-3 and normal human bronchial epithelial (NHBE) cells. We observed that I38L and E199D resulted in decreased viral replication of the H1N1 wild-type virus (1.4-fold) but the H1N1 I38T and B G199R substitutions did not significantly alter replication capacity in Calu-3 cells. In addition, H1N1 variants with PA I38L/T and E199D induced significantly higher levels of IFNB1 gene expression compared to the wild-type virus (4.2-fold). In contrast, the B variant, G199R, triggered the lowest levels of IFN genes in Calu-3 cells (1.6-fold). Because baloxavir is a novel anti-influenza therapeutic agent, identifying and characterizing substitutions associated with reduced sensitivity to baloxavir, as well as the impact of these substitutions on viral fitness, is paramount to the strategic implementation of this novel countermeasure.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2024

CD46-Targeted Theranostics for PET and 225 Ac-Radiopharmaceutical Therapy of Multiple Myeloma

Multiple myeloma is a plasma cell malignancy with an unmet clinical need for improved imaging methods and therapeutics. Recently, we identified CD46 as an overexpressed therapeutic target in multiple myeloma and developed the antibody YS5, which targets a cancer-specific epitope on this protein. We further developed the CD46-targeting PET probe [ 89 Zr]Zr-DFO-YS5 for imaging and [ 225 Ac]Ac-DOTA-YS5 for radiopharmaceutical therapy of prostate cancer. These prior studies suggested the feasibility of the CD46 antigen as a theranostic target in multiple myeloma. Herein, we validate [ 89 Zr]Zr-DFO-YS5 for immunoPET imaging and [ 225 Ac]Ac-DOTA-YS5 for radiopharmaceutical therapy of multiple myeloma in murine models. In vitro saturation binding was performed using the CD46 expressing MM.1S multiple myeloma cell line. ImmunoPET imaging using [ 89 Zr]Zr-DFO-YS5 was performed in immunodeficient (NSG) mice bearing subcutaneous and systemic multiple myeloma xenografts. For radioligand therapy, [ 225 Ac]Ac-DOTA-YS5 was prepared, and both dose escalation and fractionated dose treatment studies were performed in mice bearing MM1.S-Luc systemic xenografts. Tumor burden was analyzed using BLI, and body weight and overall survival were recorded to assess antitumor effect and toxicity. [ 89 Zr]Zr-DFO-YS5 demonstrated high affinity for CD46 expressing MM.1S multiple myeloma cells (K d = 16.3 nmol/L). In vitro assays in multiple myeloma cell lines demonstrated high binding, and bioinformatics analysis of human multiple myeloma samples revealed high CD46 expression. [ 89 Zr]Zr-DFO-YS5 PET/CT specifically detected multiple myeloma lesions in a variety of models, with low uptake in controls, including CD46 knockout (KO) mice or multiple myeloma mice using a nontargeted antibody. In the MM.1S systemic model, localization of uptake on PET imaging correlated well with the luciferase expression from tumor cells. A treatment study using [ 225 Ac]Ac-DOTA-YS5 in the MM.1S systemic model demonstrated a clear tumor volume and survival benefit in the treated groups. Our study showed that the CD46-targeted probe [ 89 Zr]Zr-DFO-YS5 can successfully image CD46-expressing multiple myeloma xenografts in murine models, and [ 225 Ac]Ac-DOTA-YS5 can effectively inhibit the growth of multiple myeloma. These results demonstrate that CD46 is a promising theranostic target for multiple myeloma, with the potential for clinical translation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

In vitro activity and in vivo efficacy of omadacycline against Plasmodium species

Abstract Background Doxycycline is currently the only tetracycline-class antibiotic recommended for malaria prophylaxis. Omadacycline, a semisynthetic aminomethylcycline approved for treatment of adults with community-acquired bacterial pneumonia and acute bacterial skin and skin structure infections, has a well-established safety profile. This study evaluated the in vitro activity of omadacycline againstPlasmodium falciparumandPlasmodium cynomolgiand its in vivo efficacy againstPlasmodium bergheiin experimental malaria models to assess its potential as an antimalarial drug. Methods Fluorescence-based assays were used to assess the in vitro blood and liver stage activity of omadacycline and doxycycline againstP. falciparumandP. cynomolgilaboratory clones. In vivo liver and early-stage blood stage efficacy were evaluated in a murine model ofP. bergheiinfection, utilizing in vivo imaging of luciferase-expressingP. berghei(ANKA strain) sporozoites in female albino C57Bl/6 mice. Parasitaemia was monitored by flow cytometry for up to 30 days post-infection. Results Omadacycline demonstrated comparable in vitro activity to doxycycline against both drug-sensitive and drug-resistantP. falciparumclones, while doxycycline showed reduced activity against two drug-resistant clones. Notably, omadacycline exhibited superior anti-schizont activity in theP. cynomolgiliver stage assay. In theP. bergheimurine model, omadacycline was efficacious in both liver and early blood stages compared to the untreated control group, and demonstrated improved survival compared to doxycycline. Conclusions Omadacycline demonstrated enhanced antimalarial efficacy over doxycycline in vitro in liver stage activity and in overcoming resistance in the blood stage, and in survival in an in vivo model ofP. bergheiinfection. These findings support further investigation of omadacycline as a potential candidate for malaria prophylaxis and treatment.

Infectious Diseases↗