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Results for “In vivo assays”

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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At least 73 records · Page 4
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Library Screening, In Vivo Confirmation, and Structural and Bioinformatic Analysis of Pentapeptide Sequences as Substrates for Protein Farnesyltransferase

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This process often causes proteins to associate with the membrane and participate in signal transduction pathways. The most common substrates of FTase are proteins that have C-terminal tetrapeptide CaaX box sequences where the cysteine is the site of modification. However, recent work has shown that five amino acid sequences can also be recognized, including the pentapeptides CMIIM and CSLMQ. In this work, peptide libraries were initially used to systematically vary the residues in those two parental sequences using an assay based on Matrix Assisted Laser Desorption Ionization–Mass Spectrometry (MALDI-MS). In addition, 192 pentapeptide sequences from the human proteome were screened using that assay to discover additional extended CaaaX-box motifs. Selected hits from that screening effort were rescreened using an in vivo yeast reporter protein assay. The X-ray crystal structure of CMIIM bound to FTase was also solved, showing that the C-terminal tripeptide of that sequence interacted with the enzyme in a similar manner as the C-terminal tripeptide of CVVM, suggesting that the tripeptide comprises a common structural element for substrate recognition in both tetrapeptide and pentapeptide sequences. Molecular dynamics simulation of CMIIM bound to FTase further shed light on the molecular interactions involved, showing that a putative catalytically competent Zn(II)-thiolate species was able to form. Bioinformatic predictions of tetrapeptide (CaaX-box) reactivity correlated well with the reactivity of pentapeptides obtained from in vivo analysis, reinforcing the importance of the C-terminal tripeptide motif. This analysis provides a structural framework for understanding the reactivity of extended CaaaX-box motifs and a method that may be useful for predicting the reactivity of additional FTase substrates bearing CaaaX-box sequences.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Synthase-Selective Exploration of a Tunicate Microbiome by Activity-Guided Single-Cell Genomics

While thousands of environmental metagenomes have been mined for the presence of novel biosynthetic gene clusters, such computational predictions do not provide evidence of their in vivo biosynthetic functionality. Using fluorescent in situ enzyme assay targeting carrier proteins common to polyketide (PKS) and nonribosomal peptide synthetases (NRPS), we applied fluorescence-activated cell sorting to tunicate microbiome to enrich for microbes with active secondary metabolic capabilities. Single-cell genomics uncovered the genetic basis for a wide biosynthetic diversity in the enzyme-active cells and revealed a member of marine Oceanospirillales harboring a novel NRPS gene cluster with high similarity to phylogenetically distant marine and terrestrial bacteria. Interestingly, this synthase belongs to a larger class of siderophore biosynthetic gene clusters commonly associated with pestilence and disease. Furthermore, this demonstrates activity-guided single-cell genomics as a tool to guide novel biosynthetic discovery.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2020

Sclerostin inhibits Wnt signaling through tandem interaction with two LRP6 ectodomains

Low-density lipoprotein receptor-related protein 6 (LRP6) is a coreceptor of the β-catenin-dependent Wnt signaling pathway. The LRP6 ectodomain binds Wnt proteins, as well as Wnt inhibitors such as sclerostin (SOST), which negatively regulates Wnt signaling in osteocytes. Although LRP6 ectodomain 1 (E1) is known to interact with SOST, several unresolved questions remain, such as the reason why SOST binds to LRP6 E1E2 with higher affinity than to the E1 domain alone. Here, we present the crystal structure of the LRP6 E1E2–SOST complex with two interaction sites in tandem. The unexpected additional binding site was identified between the C-terminus of SOST and the LRP6 E2 domain. This interaction was confirmed by in vitro binding and cell-based signaling assays. Its functional significance was further demonstrated in vivo using Xenopus laevis embryos. Our results provide insights into the inhibitory mechanism of SOST on Wnt signaling.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Zam Is a Redox-Regulated Member of the RNB-Family Required for Optimal Photosynthesis in Cyanobacteria

The zam gene mediating resistance to acetazolamide in cyanobacteria was discovered thirty years ago during a drug tolerance screen. We use phylogenetics to show that Zam proteins are distributed across cyanobacteria and that they form their own unique clade of the ribonuclease II/R (RNB) family. Despite being RNB family members, multiple sequence alignments reveal that Zam proteins lack conservation and exhibit extreme degeneracy in the canonical active site—raising questions about their cellular function(s). Several known phenotypes arise from the deletion of zam, including drug resistance, slower growth, and altered pigmentation. Using room-temperature and low-temperature fluorescence and absorption spectroscopy, we show that deletion of zam results in decreased phycocyanin synthesis rates, altered PSI:PSII ratios, and an increase in coupling between the phycobilisome and PSII. Conserved cysteines within Zam are identified and assayed for function using in vitro and in vivo methods. We show that these cysteines are essential for Zam function, with mutation of either residue to serine causing phenotypes identical to the deletion of Zam. Redox regulation of Zam activity based on the reversible oxidation-reduction of a disulfide bond involving these cysteine residues could provide a mechanism to integrate the ‘central dogma’ with photosynthesis in cyanobacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Compositions for therapeutics, targeted PET imaging and methods of their use

Described herein is a chelator for radiolabels (e.g., 89Zr) for targeted PET imaging that is an alternative to DFO. In certain embodiments, the chelator for 89Zr is the ligand, 3,4,3-(LI-1,2-HOPO) (“HOPO”), which exhibits equal or superior stability compared to DFO in chemical and biological assays across a period of several days in vivo. As shown in FIG. 1, the HOPO is an octadentate chelator that stabilizes chelation of radiolabels (e.g., 89Zr). A bifunctional ligand comprising p-SCN-Bn-HOPO is shown in FIG. 4 and FIG. 5. Such a bifunctional ligand can eliminate (e.g., 89Zr) loss from the chelate in vivo and reduce uptake in bone and non-target tissue. Therefore, the bifunctional HOPO ligand can facilitate safer and improved PET imaging with radiolabeled antibodies.

Lewis, Jason S.↗
DOE OSTI2022

Activating alternative transport modes in a multidrug resistance efflux pump to confer chemical susceptibility

Small multidrug resistance (SMR) transporters contribute to antibiotic resistance through proton-coupled efflux of toxic compounds. Previous biophysical studies of the E. coli SMR transporter EmrE suggest that it should also be able to perform proton/toxin symport or uniport, leading to toxin susceptibility rather than resistance in vivo. Here we show EmrE does confer susceptibility to several previously uncharacterized small-molecule substrates in E. coli, including harmane. In vitro electrophysiology assays demonstrate that harmane binding triggers uncoupled proton flux through EmrE. Assays in E. coli are consistent with EmrE-mediated dissipation of the transmembrane pH gradient as the mechanism underlying the in vivo phenotype of harmane susceptibility. Furthermore, checkerboard assays show this alternative EmrE transport mode can synergize with some existing antibiotics, such as kanamycin. These results demonstrate that it is possible to not just inhibit multidrug efflux, but to activate alternative transport modes detrimental to bacteria, suggesting a strategy to address antibiotic resistance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Streptococcus pneumoniae HtrA is a dynamic and monomeric virulence factor capable of forming larger oligomeric complexes

Abstract High‐temperature requirement A (HtrA) proteases are a conserved family of serine proteases central to protein quality control and bacterial virulence. While Gram‐negative and human HtrAs are structurally well studied, Gram‐positive homologs remain essentially uncharacterized. Here, we present the first integrated structural and mechanistic analysis of a Gram‐positive HtrA, from Streptococcus pneumoniae , a virulence factor essential for adhesion and infection in vivo. Proteomic profiling of an htrA knockout and cleavage assays demonstrate that S. pneumoniae HtrA is required for protein quality control, with the PDZ domain mediating substrate recognition. Biochemically, S. pneumoniae HtrA exists exclusively as a monomer in solution, a striking divergence from canonical trimeric HtrAs that we show is shared with other Gram‐positive homologs. NMR analyses reveal that the monomer dynamically samples open and closed conformations, while cryo‐EM of a catalytic mutant identifies a hexamer stabilized by a unique LoopA–PDZ interaction. Together, these findings define S. pneumoniae HtrA as a dynamic monomer with interdomain coupling between its protease and PDZ domains, establishing Gram‐positive HtrAs as a mechanistically divergent subgroup within the HtrA family.

Lee, Eunjeong [Department of Biochemistry and Mole↗
DOE OSTI2020

A Cytoplasmic Receptor-like Kinase Contributes to Salinity Tolerance

Receptor-like cytoplasmic kinases (RLCKs) are receptor kinases that lack extracellular ligand-binding domains and have emerged as a major class of signaling proteins that regulate plant cellular activities in response to biotic/abiotic stresses and endogenous extracellular signaling molecules. We have identified a rice RLCK (OsRLCK311) that was significantly higher in transgenic pSARK-IPT rice (Oryza sativa) that exhibited enhanced growth under saline conditions. Overexpression of OsRLCK311 full-length protein (RLCK311FL) and the C-terminus of OsRLCK311 (ΔN) in Arabidopsis confirmed its role in salinity tolerance, both in seedlings and mature plants. Protein interaction assays indicated that OsRLCK311 and ΔN interacted in-vivo with the plasma membrane AQP AtPIP2;1. The RLCK311-PIP2;1 binding led to alterations in the stomata response to ABA, which was characterized by more open stomata of transgenic plants. Moreover, OsRLCK311-ΔN effect in mediating enhanced plant growth under saline conditions was also observed in the perennial grass Brachypodium sylvaticum, confirming its role in both dicots and monocots species. Lastly, OsRLCK311 interacted with the rice OsPIP2;1. We suggest that the rice OsRLCK311 play a role in regulating the plant growth response under saline conditions via the regulation of the stomata response to stress. This role seems to be independent of the RLCK311 kinase activity, since the overexpression of the RLCK311 C-terminus (ΔN), which lacks the kinase full domain, has a similar phenotype to RLCK311FL.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Evaluation of an in vitro three-dimensional HepaRG spheroid model for genotoxicity testing using the high-throughput CometChip platform

Three-dimensional (3D) culture systems are increasingly being used for genotoxicity studies due to improved cell-to-cell interactions and tissue-like structures that are limited or lacking in 2D cultures. The present study optimized a 3D culture system using metabolically competent HepaRG cells for in vitro genotoxicity testing. 3D HepaRG spheroids, formed in 96- or 384-well ultra-low attachment plates, were exposed to various concentrations of 34 test articles, including 8 direct-acting and 11 indirect-acting genotoxicants/carcinogens as well as 15 compounds that show different genotoxic responses in vitro and in vivo. DNA damage was evaluated using the high-throughput CometChip assay with concurrent cytotoxicity assessment by the ATP assay in both 2D and 3D cultures. 3D HepaRG spheroids maintained a stable phenotype for up to 30 days with higher levels of albumin secretion, cytochrome P450 gene expression, and enzyme activities compared to 2D cultures. 3D spheroids also demonstrated a higher sensitivity than 2D cultures for detecting both direct- and indirect-acting genotoxicants/carcinogens, indicating a better prediction of in vivo genotoxicity responses. When DNA damage dose-response data were quantified using PROAST software, 3D spheroids generally had lower or similar benchmark dose values compared to 2D HepaRG cells and were more comparable with primary human hepatocytes. These results demonstrate that 3D models can be adapted to the CometChip technology for high-throughput genotoxicity testing and that 3D HepaRG spheroids may be used as a reliable and pragmatic in vitro approach to better support the hazard identification and risk assessment of potential human genotoxic carcinogens.

60 APPLIED LIFE 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↗
DOE OSTI2025

A split ribozyme system for in vivo plant RNA imaging and genetic engineering

RNA plays a central role in plants, governing various cellular and physiological processes. Monitoring its dynamic abundance provides a discerning understanding of molecular mechanisms underlying plant responses to internal (developmental) and external (environmental) stimuli, paving the way for advances in plant biotechnology to engineer crops with improved resilience, quality and productivity. In general, traditional methods for analysis of RNA abundance in plants require destructive, labour-intensive and time-consuming assays. To overcome these limitations, we developed a transformative innovation for in vivo RNA imaging in plants. Specifically, we established a synthetic split ribozyme system that converts various RNA signals to orthogonal protein outputs, enabling in vivo visualisation of various RNA signals in plants. We demonstrated the utility of this system in transient expression experiments (i.e., leaf infiltration in Nicotiana benthamiana ) to detect RNAs derived from transgenes and tobacco rattle virus, respectively. Also, we successfully engineered a split ribozyme-based biosensor in Arabidopsis thaliana for in vivo visualisation of endogenous gene expression at the cellular level, demonstrating the feasibility of multi-scale (e.g., cellular and tissue level) RNA imaging in plants. Furthermore, we developed a platform for easy incorporation of different protein outputs, allowing for flexible choice of reporters to optimise the detection of target RNAs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Characterization of Mammalian In Vivo Enhancers Using Mouse Transgenesis and CRISPR Genome Editing [Book Chapter]

Embryonic morphogenesis is strictly dependent on tight spatiotemporal control of developmental gene expression, which is typically achieved through the concerted activity of multiple enhancers driving cell type-specific expression of a target gene. Mammalian genomes are organized in topologically associated domains, providing a preferred environment and framework for interactions between transcriptional enhancers and gene promoters. While epigenomic profiling and three-dimensional chromatin conformation capture have significantly increased the accuracy of identifying enhancers, assessment of subregional enhancer activities via transgenic reporter assays in mice remains the gold standard for assigning enhancer activity in vivo. Once this activity is defined, the ideal method to explore the functional necessity of a transcriptional enhancer and its contribution to target gene dosage and morphological or physiological processes is deletion of the enhancer sequence from the mouse genome. Here we present detailed protocols for efficient introduction of enhancer-reporter transgenes and CRISPR-mediated genomic deletions into the mouse genome, including a step-by-step guide for pronuclear microinjection of fertilized mouse eggs. We provide instructions for the assembly and genomic integration of enhancer-reporter cassettes that have been used for validation of thousands of putative enhancer sequences accessible through the VISTA enhancer browser, including a recently published method for robust site-directed transgenesis at the H11 safe-harbor locus. Together, these methods enable rapid and large-scale assessment of enhancer activities and sequence variants in mice, which is essential to understand mammalian genome function and genetic diseases.

cis-regulatory elements↗
DOE OSTI2022

Detection of innate immune response modulating impurities (IIRMI) in therapeutic peptides and proteins: Impact of excipients

Unintended immunogenicity can affect the safety and efficacy of therapeutic proteins and peptides, so accurate assessments of immunogenicity risk can aid in the selection, development, and regulation of biologics. Product- and process- related impurities can act as adjuvants that activate the local or systemic innate immune response increasing the likelihood of product immunogenicity. Thus, assessing whether products have innate immune response modulating impurities (IIRMI) is a key component of immunogenicity risk assessments. Identifying trace levels of individual IIRMI can be difficult and testing individually for all potential impurities is not feasible. Therefore, to mitigate the risk, cell-based assays that use human blood cells or monocyte-macrophage reporter cell lines are being developed to detect minute quantities of impurities capable of eliciting innate immune activation. As these are cell-based assays, there is concern that excipients could blunt the cell responses, masking the presence of immunogenic IIRMI. Here, we explore the impact of frequently used excipients (non-ionic detergents, sugars, amino acids, bulking agents) on the sensitivity of reporter cell lines (THP-1- and RAW-Blue cells) and fresh human blood cells to detect purified TLR agonists as model IIRMI. We show that while excipients do not modulate the innate immune response elicited by TLR agonists in vivo, they can impact on the sensitivity of cell-based IIRMI assays. Reduced sensitivity to detect LPS, FSL-1, and other model IIRMI was also evident when testing 3 different recombinant drug products, product A (a representative mAb), B (a representative growth factor), C (a representative peptide), and their corresponding formulations. These results indicate that product formulations need to be considered when developing and validating cell-based assays for assessing clinically relevant levels of IIRMI in therapeutic proteins. Optimization of reporter cells, culture conditions and drug product concentration appear to be critical to minimize the impact of excipients and attain sensitive and reproducible assays.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Application of Cell Painting for chemical hazard evaluation in support of screening-level chemical assessments

‘Cell Painting’ is an imaging-based high-throughput phenotypic profiling (HTPP) method in which cultured cells are fluorescently labeled to visualize subcellular structures (i.e., nucleus, nucleoli, endoplasmic reticulum, cytoskeleton, Golgi apparatus / plasma membrane and mitochondria) and to quantify morphological changes in response to chemicals or other perturbagens. HTPP is a high-throughput and cost-effective bioactivity screening method that detects effects associated with many different molecular mechanisms in an untargeted manner, enabling rapid in vitro hazard assessment for thousands of chemicals. Here, 1201 chemicals from the ToxCast library were screened in concentration-response up to ~100 μM in human U-2 OS cells using HTPP. A phenotype altering concentration (PAC) was estimated for chemicals active in the tested range. PACs tended to be higher than lower bound potency values estimated from a broad collection of targeted high-throughput assays, but lower than the threshold for cytotoxicity. In vitro to in vivo extrapolation (IVIVE) was used to estimate administered equivalent doses (AEDs) based on PACs for comparison to human exposure predictions. AEDs for 18/412 chemicals overlapped with predicted human exposures. Phenotypic profile information was also leveraged to identify putative mechanisms of action and group chemicals. Of 58 known nuclear receptor modulators, only glucocorticoids and retinoids produced characteristic profiles; and both receptor types are expressed in U-2 OS cells. Thirteen chemicals with profile similarity to glucocorticoids were tested in a secondary screen and one chemical, pyrene, was confirmed by an orthogonal gene expression assay as a novel putative GR modulating chemical. Most active chemicals demonstrated profiles not associated with a known mechanism-of-action. However, many structurally related chemicals produced similar profiles, with exceptions such as diniconazole, whose profile differed from other active conazoles. Overall, the present study demonstrates how HTPP can be applied in screening-level chemical assessments through a series of examples and brief case studies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Enzyme Complexes of Ptr4CL and PtrHCT Modulate Co-enzyme A Ligation of Hydroxycinnamic Acids for Monolignol Biosynthesis in Populus trichocarpa

Co-enzyme A (CoA) ligation of hydroxycinnamic acids by 4-coumaric acid:CoA ligase (4CL) is a critical step in the biosynthesis of monolignols. Perturbation of 4CL activity significantly impacts the lignin content of diverse plant species. In Populus trichocarpa, two well-studied xylem-specific Ptr4CLs (Ptr4CL3 and Ptr4CL5) catalyze the CoA ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. Subsequently, two 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) mediate the conversion of 4-coumaroyl-CoA to caffeoyl-CoA. Here, we show that the CoA ligation of 4-coumaric and caffeic acids is modulated by Ptr4CL/PtrHCT protein complexes. Downregulation of PtrHCTs reduced Ptr4CL activities in the stem-differentiating xylem (SDX) of transgenic P. trichocarpa. The Ptr4CL/PtrHCT interactions were then validated in vivo using biomolecular fluorescence complementation (BiFC) and protein pull-down assays in P. trichocarpa SDX extracts. Enzyme activity assays using recombinant proteins of Ptr4CL and PtrHCT showed elevated CoA ligation activity for Ptr4CL when supplemented with PtrHCT. Numerical analyses based on an evolutionary computation of the CoA ligation activity estimated the stoichiometry of the protein complex to consist of one Ptr4CL and two PtrHCTs, which was experimentally confirmed by chemical cross-linking using SDX plant protein extracts and recombinant proteins. Based on these results, we propose that Ptr4CL/PtrHCT complexes modulate the metabolic flux of CoA ligation for monolignol biosynthesis during wood formation in P. trichocarpa.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

AFF4 facilitates melanoma cell progression by regulating c-Jun activity

Melanoma is characterized by high mortality and poor prognosis due to metastasis. AFF4 (AF4/FMR2 family member 4), as a scaffold protein, is a component of the super elongation complex (SEC), and is involved in the progression of tumors, e.g., leukemia, head and neck squamous cell carcinoma (HNSCC). However, few studies on AFF4 have focused on melanoma. Here, AFF4 expression levels and clinicopathological features were evaluated in melanoma tissue samples. Then, we performed cell proliferation, migration and invasion assays in A375 and A2058 cells lines in vitro to evaluate the role of AFF4 in melanoma. The effects of AFF4 knockdown in vivo were characterized via a xenograft mouse model. Finally, the correlation between c-Jun and AFF4 protein levels in melanoma was analyzed by rescue assay and immunohistochemistry (IHC). We found that AFF4 expression was upregulated in melanoma tumor tissues and that AFF4 protein expression was also closely related to the prognosis of patients with cutaneous melanoma. Moreover, AFF4 could promote the invasion and migration of melanoma cells by mediating epithelial to mesenchymal transition (EMT). AFF4 might regulate c-Jun activity to promote the invasion and migration of melanoma cells. Importantly, c-Jun was regulated by the AFF4 promoted melanoma tumorigenesis in vivo. Taken together, AFF4 may be a novel oncogene that promotes melanoma progression through regulation of c-Jun activity.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

The Preclinical Validation of 405 nm Light Parasiticidal Efficacy on Leishmania donovani in Ex Vivo Platelets in a Rag2 -/- Mouse Model

Violet–blue light of 405 nm in the visible spectrum at a dose of 270 J/cm 2 alone has been shown to be an effective microbicidal tool for inactivating several bacteria, HIV-1, and Trypanosoma cruzi in ex vivo plasma and platelets. Unlike chemical- and ultraviolet (UV)-based pathogen inactivation methods for plasma and platelet safety, 405 nm light is shown to be less toxic to host cells at light doses that are microbicidal. In this report, we evaluated the parasiticidal activity of a 405 nm light treatment on platelets spiked with the Leishmania donovani parasite. Following the light treatment, parasite viability was observed to be near zero in both low- and high-titer-spiked platelets relative to controls. Furthermore, to test the residual infectivity after inactivation in vivo, the light-treated low-titer L. donovani-spiked platelets were evaluated in an immunodeficient Rag2 -/- mouse model and monitored for 9 weeks. The parasiticidal efficacy of 405 nm light was evident from the lack of a presence of parasites in the mice spleens. Parasiticidal activity was confirmed to be mediated through 405 nm light-induced reactive oxygen species (ROS), as quantitatively measured by a 2',7'-Dichlorodihydrofluorescein diacetate (H 2 DCFDA)-based assay. Overall, these results confirm the complete inactivation of L. donovani spiked in ex vivo platelets by 405 nm light treatment and exemplify the utility of the Rag2 -/- mouse infection model for the preclinical validation of the parasiticidal efficacy of 405 nm light and this light-based technology as a potential PRT for ex vivo platelets.

405 nm light↗
DOE OSTI2020

Evaluating 225 Ac and 177 Lu Radioimmunoconjugates against Antibody–Drug Conjugates for Small-Cell Lung Cancer

Interest in the use of 225 Ac for targeted alpha therapies has increased dramatically over the past few years, resulting in a multitude of new isotope production and translational research efforts. However, 225 Ac radioimmunoconjugate (RIC) research is still in its infancy, with most prior experience in hematologic malignancies and only one reported preclinical solid tumor study using 225 Ac RICs. In an effort to compare 225 Ac RICs to other current antibody conjugates, a variety of RICs are tested against intractable small-cell lung cancer (SCLC). Here we directly compare, in vitro and in vivo , two promising candidates of each α or β - category, 225 Ac and 177 Lu, versus pyrrolobenzodiazepine (PBD) nonradioactive benchmarks. The monoclonal antibody constructs are targeted to either delta like 3 protein (DLL3), a recently discovered SCLC target, or CD46 as a positive control. An immunocompromised maximum tolerated dose assay is performed on NOD SCID mice, along with tumor efficacy proof-of-concept studies in vivo . We overview the conjugation techniques required to create serum-stable RICs and characterize and compare in vitro cell killing with RICs conjugated to nonspecific antibodies (huIgG1) with either native or site-specific thiol loci against tumor antigen DLL3-expressing and nonexpressing cell lines. Using patient-derived xenografts of SCLC onto NOD SCID mice, solid tumor growth was controlled throughout 3 weeks before growth appeared, in comparison to PBD conjugate controls. NOD SCID mice showed lengthened survival using 225 Ac compared to 177 Lu RICs, and PBD dimers showed full tumor suppression with nine out of ten mice. The exploration of RICs on a variety of antibody-antigen systems is necessary to direct efforts in cancer research toward promising candidates. However, the anti-DLL3-RIC system with 225 Ac and 177 Lu appears to be not as effective as the anti-DLL3-PBD counterpart in SCLC therapy with matched antibodies and portrays the challenges in both SCLC therapy as well as the specialized utility of RICs in cancer treatment.

60 APPLIED LIFE SCIENCES↗