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At least 253 records · Page 14
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Pooled PPIseq: Screening the SARS-CoV-2 and human interface with a scalable multiplexed protein-protein interaction assay platform

Protein-Protein Interactions (PPIs) are a key interface between virus and host, and these interactions are important to both viral reprogramming of the host and to host restriction of viral infection. In particular, viral-host PPI networks can be used to further our understanding of the molecular mechanisms of tissue specificity, host range, and virulence. At higher scales, viral-host PPI screening could also be used to screen for small-molecule antivirals that interfere with essential viral-host interactions, or to explore how the PPI networks between interacting viral and host genomes co-evolve. Current high-throughput PPI assays have screened entire viral-host PPI networks. However, these studies are time consuming, often require specialized equipment, and are difficult to further scale. Here, we develop methods that make larger-scale viral-host PPI screening more accessible. This approach combines the mDHFR split-tag reporter with the iSeq2 interaction-barcoding system to permit massively-multiplexed PPI quantification by simple pooled engineering of barcoded constructs, integration of these constructs into budding yeast, and fitness measurements by pooled cell competitions and barcode-sequencing. We applied this method to screen for PPIs between SARS-CoV-2 proteins and human proteins, screening in triplicate >180,000 ORF-ORF combinations represented by >1,000,000 barcoded lineages. Our results complement previous screens by identifying 74 putative PPIs, including interactions between ORF7A with the taste receptors TAS2R41 and TAS2R7, and between NSP4 with the transmembrane KDELR2 and KDELR3. We show that this PPI screening method is highly scalable, enabling larger studies aimed at generating a broad understanding of how viral effector proteins converge on cellular targets to effect replication.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

TRACKING LIGNOCELLULOSIC BREAKDOWN BY ANAEROBIC FUNGI AND FUNGAL CELLULOSOMES

Anaerobic fungi degrade plant biomass through invasive, filamentous growth, and the secretion of multi-protein biomass-degrading complexes called fungal cellulosomes. This project developed new tools for anaerobic, non-destructive, real-time imaging of cellulosomes across spatial and temporal scales. Novel nanobody tools were synthesized and deployed to image native fungal cellulosomes. Antibodies raised against key fungal cellulosome components were also used to define the localization patterns of cellulosomes in mature fungal mats vs. fungal zoospores, and revealed direct connections between cellular life stage progression and the regulation of cellulosome production. New procedures were developed to purify native cellulosomes and study their morphology and sub-structures, and genetic engineering tools were also developed and applied to anaerobic fungi to advance in vivo labeling capabilities. A cryoEM structure of a native fungal cellulosome was achieved, as well as a proof of concept for transformation of fungi with flavin-based anaerobic reporter proteins for in vivo labeling of cellulosome components.

09 BIOMASS FUELS↗
DOE OSTI2025

Feeding from the sun—Successes and prospects in bioengineering photosynthesis for food security

There is an urgent need for increased crop productivity to reduce food insecurity and improve sustainability. Photosynthesis converts sunlight energy into carbohydrates, providing the source of nearly all of humanity’s food. Photosynthesis is a key target for improvement, owing to inherent inefficiencies in the biochemical process. Over the last decade of advancements in bioengineering, strategies to increase the efficiency of photosynthesis were tested with proven enhancements to crop yields in field trials. Simple strategies like increasing the content of photosynthetic proteins have reliably increased photosynthesis and productivity in crops, as have more complex strategies such as bypassing photorespiration. While insertion of carbon-concentrating mechanisms into C3 plants remains an engineering challenge, modeling suggests that achieving that would have the greatest gain for crop improvement. This review discusses the many successes in improving photosynthesis achieved over the past decade and quantifies the potential for future engineering targets to increase crop productivity.

Long, Stephen P. [University of Illinois, Urbana, ↗
NASA NTRS2000

Protein Crystals Grow Purer in Space: Physics of Phenomena

This presentation will summarize the quantitative experimental and theoretical results obtained by B.R. Thomas, P.G. Vekilov, D.C. Carter, A.M. Holmes, W.K. Widierow and the Author, the team with expertise in physics, biochemistry, crystallography and engineering. Impurities inhomogeneously trapped by a growing crystal - e.g., producing sectorial structure and/or striations - may induce macroscopic internal stress in it if an impurity molecule has slightly (less than 10%) different shape or volume than the regular one(s) they replace. We tested for the first time plasticity and measured Young modulus E of the triclinic, not cross-linked lysozyme by triple point bending technique. Triclinic lysozyme crystals are purely elastic with E similar or equal to 1/5 (raised dot) 10 (exp 9) partial derivative yn/sq cm. The strength limit, sigma (sub c) similar or equal to 10 (exp -3)E similar or equal to Epsilon (sub c), where sigma (sub c) and epsilon (sub c) are critical stress and strain, respectively. Scaling E and sigma (sub c) with the lattice spacing suggests similar binding stiffness in inorganic and biomolecular crystals. The inhomogeneous internal stress may be resolved in these brittle crystals either by cracking or by creation of misoriented mosaic blocks during, not after growth. If each impurity molecule induces in the lattice elementary strain epsilon (sub 0) similar or equal to 3 (raised dot) 10 (exp -2) (this is maximal elementary strain that can arise at the supersaturation DELTA mu/kT similar or equal to 2 and macroscopic molecular concentration difference between subsequent macrolayers or growth sectors is partial derivativeC similar or equal to 5 (raised dot) 10 (exp -3), the internal strain epsilon similar or equal to epsilon (sub 0) partial derivative C similar or equal to 10 (exp -4). Mosaic misorientation resolving such strain is approximately 30 arcsec. Tenfold increase of impurity concentration may cause cracking. Estimates of stress in an isometric sectorial crystal show that lysozyme crystals can tolerate the stress till the size of 0.5mm. Dissolving mosaic lysozyme crystal shows that the mosaicity, indeed, is absent below that size.

Chernov, Alex A.↗
DOE OSTI2025

A small number of point mutations confer formate tolerance in Shewanella oneidensis

ABSTRACT Microbial electrosynthesis (MES) is a sustainable approach to chemical production from CO 2 and clean electricity. However, limitations in electron transfer efficiency and gaps in understanding of electron transfer pathways in MES systems prevent full realization of this technology. Shewanella oneidensis could serve as an MES biocatalyst because it has a well-studied, efficient transmembrane electron transfer pathway. A key first step in MES in this organism could be CO 2 reduction to formate. However, we report that wild-type S. oneidensis does not tolerate high levels of formate. In this work, we created and characterized formate-tolerant strains of S. oneidensis for further engineering and future use in MES systems through adaptive laboratory evolution. Two different point mutations in a gene encoding a predicted sodium-dependent bicarbonate transporter and a DUF2721-containing protein separately confer formate tolerance to S. oneidensis . The mutations were further evaluated to understand their role in improving formate tolerance. We also show that the wild-type and mutant versions of the putative sodium-dependent bicarbonate transporter improve formate tolerance of Zymomonas mobilis , indicating the potential of transferring this formate tolerance phenotype to other organisms. IMPORTANCE Shewanella oneidensis is a bacterium with a well-studied, efficient extracellular electron transfer pathway. This capability could make this organism a suitable host for microbial electrosynthesis using CO 2 or formate as feedstocks. However, we report here that formate is toxic to S. oneidensis , limiting the potential for its use in these systems. In this work, we evolve several strains of S. oneidensis that have improved formate tolerance, and we investigate some mutations that confer this phenotype. The phenotype is confirmed to be attributed to several single point mutations by transferring the wild-type and mutant versions of each gene to the wild-type strain. Finally, the formate tolerance mechanism of one variant is studied using structural modeling and expression in another host. This study, therefore, presents a simple method for conferring formate tolerance to bacterial hosts.

Cross, Megan C. Gruenberg (ORCID:0000000291589900)↗
DOE OSTI2025

Nitrogen limitation causes a seismic shift in redox state and phosphorylation of proteins implicated in carbon flux and lipidome remodeling in Rhodotorula toruloides

Background: Oleaginous yeast are prodigious producers of oleochemicals, offering alternative and secure sources for applications in foodstuff, skincare, biofuels, and bioplastics. Nitrogen starvation is the primary strategy used to induce oil accumulation in oleaginous yeast as part of a global stress response. While research has demonstrated that post-translational modifications (PTMs), including phosphorylation and protein cysteine thiol oxidation (redox PTMs), are involved in signaling pathways that regulate stress responses in metazoa and algae, their role in oleaginous yeast remain understudied and unexplored. Results: Towards linking the yeast oleaginous phenotype to protein function, we integrated lipidomics, redox proteomics, and phosphoproteomics to investigate Rhodotorula toruloides under nitrogen-rich and starved conditions over time. Our lipidomics results unearthed interactions involving sphingolipids and cardiolipins with ER stress and mitophagy. Our redox and phosphoproteomics data highlighted the roles of the AMPK, TOR, and calcium signaling pathways in regulation of lipogenesis, autophagy, and oxidative stress response. As a first, we also demonstrated that lipogenic enzymes including fatty acid synthase are modified as a consequence of shifts in cellular redox states due to nutrient availability. Conclusions: We conclude that lipid accumulation is largely a consequence of carbon rerouting and autophagy governed by changes to PTMs, and not increases in the abundance of enzymes involved in central carbon metabolism and fatty acid biosynthesis. Our systems-level approach sets the stage for acquiring multidimensional data sets for protein structural modeling and predicting the functional relevance of PTMs using Artificial Intelligence/Machine Learning (AI/ML). Coupled to those bioinformatics approaches, the putative PTM switches that we delineate will enable advanced metabolic engineering strategies to decouple lipid accumulation from nitrogen limitation.

Lipid Signalling↗
DOE OSTI2024

CRISPR-Cas9/Cas12a systems for efficient genome editing and large genomic fragment deletions in Aspergillus niger

CRISPR technology has revolutionized fungal genetic engineering by accelerating the pace and expanding the feasible scope of experiments in this field. Among various CRISPR-Cas systems, Cas9 and Cas12a are widely used in genetic and metabolic engineering. In filamentous fungi, both Cas9 and Cas12a have been utilized as CRISPR nucleases. In this work we first compared efficacies and types of genetic edits for CRISPR-Cas9 and -Cas12a systems at the polyketide synthase (albA) gene locus in Aspergillus niger. By employing a tRNA-based gRNA polycistronic cassette, both Cas9 and Cas12a have demonstrated equally remarkable editing efficacy. Cas12a showed potential superiority over Cas9 protein when one gRNA was used for targeting, achieving an editing efficiency of 86.5% compared to 31.7% for Cas9. Moreover, when employing two gRNAs for targeting, both systems achieved up to 100% editing efficiency for single gene editing. In addition, the CRISPR-Cas9 system has been reported to induce large genomic deletions in various species. However, its use for engineering large chromosomal segments deletions in filamentous fungi still requires optimization. Here, we engineered Cas9 and -Cas12a-induced large genomic fragment deletions by targeting various genomic regions of A. niger ranging from 3.5 kb to 40 kb. Our findings demonstrate that targeted engineering of large chromosomal segments can be achieved, with deletions of up to 69.1% efficiency. Furthermore, by targeting a secondary metabolite gene cluster, we show that fragments over 100 kb can be efficiently and specifically deleted using the CRISPR-Cas9 or -Cas12a system. Overall, in this paper, we present an efficient multi-gRNA genome editing system utilizing Cas9 or Cas12a that enables highly efficient targeted editing of genes and large chromosomal regions in A. niger.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Activation Domain Hunter (ADhunter) v2.0

ADhunter is a software program that enables accurate identification and quantification of transcriptional activation domains. Unlike previous software, ADhunter uses protein representations from a pre-trained protein language model, model ensembling, and a training dataset from a diverse sampling of protein sequence space for state-of-the-art performance. These advantages enable improved perception of transcriptional activation domains across sequence space that can be used for mapping natural genetic circuits and engineering synthetic genetic circuits. In particular, ADhunter enables fine-tuned control of gene expression through synthetic transcription factors that can be used for complex control of cellular programs.

Waldburger, Lucas [Lawrence Berkeley National Labo↗
NASA NTRS2005

Electroporation System for Sterilizing Water

A prototype of an electroporation system for sterilizing wastewater or drinking water has been developed. In electroporation, applied electric fields cause transient and/or permanent changes in the porosities of living cells. Electroporation at lower field strengths can be exploited to increase the efficiency of chemical disinfection (as in chlorination). Electroporation at higher field strengths is capable of inactivating and even killing bacteria and other pathogens, without use of chemicals. Hence, electroporation is at least a partial alternative to chlorination. The transient changes that occur in micro-organisms at lower electric-field strengths include significantly increased uptake of ions and molecules. Such increased uptake makes it possible to achieve disinfection at lower doses of chemicals (e.g., chlorine or ozone) than would otherwise be needed. Lower doses translate to lower costs and reduced concentrations of such carcinogenic chemical byproducts as trichloromethane. Higher electric fields cause cell membranes to lose semipermeability and thereby become unable to function as selective osmotic barriers between the cells and the environment. This loss of function is the cause of the cell death at higher electric-field intensities. Experimental evidence does not indicate cell lysis but, rather, combined leaking of cell proteins out of the cells as well as invasion of foreign chemical compounds into the cells. The concept of electroporation is not new: it has been applied in molecular biology and genetic engineering for decades. However, the laboratory-scale electroporators used heretofore have been built around small (400-microliter) cuvettes, partly because the smallness facilitates the generation of electric fields of sufficient magnitude to cause electroporation. Moreover, most laboratory- scale electroporators have been designed for testing static water. In contrast, the treatment cell in the present system is much larger and features a flow-through geometry, such that electric fields strong enough to effect 99.9- percent disinfection can be applied to water flowing in a pipe.

Schlager, Kenneth J.↗
DOE OSTI2025

Engineered IL-18 variants with half-life extension and improved stability for cancer immunotherapy

Background The pro-inflammatory cytokine, interleukin-18 (IL-18), plays an instrumental role in bolstering anti-tumor immunity. However, the therapeutic application of IL-18 has been limited due to its susceptibility to neutralization by IL-18 binding protein (IL-18BP), short in vivo half-life, and unfavorable physicochemical properties. Methods In order to overcome the poor drug-like properties of IL-18, we installed an artificial disulfide bond, removed the native, unpaired cysteines, and fused the stabilized cytokine to an IgG Fc domain. The stability, potency, pharmacokinetic and pharmacodynamic properties as well as efficacy of disulfide-stabilized IL-18 Fc-fusion (dsIL-18-Fc) were assessed via in vitro and in vivo studies. Results The stability and mammalian host cell production yields of dsIL-18-Fc were improved, compared to the wild-type (WT) cytokine, while maintaining its biological potency and interactions with IL-18 receptor α (IL-18Rα) and IL-18BP. Recombinant fusion of the cytokine to an IgG Fc domain provided extended half-life. Notably, despite maintaining sensitivity to IL-18BP, dsIL-18-Fc was effective at activating both T and natural killer (NK) cells, and elicited a strong anti-tumor response, either as a single agent, or in conjunction with anti-programmed cell death-ligand 1 (anti-PD-L1) therapy. Conclusions We engineered IL-18 for reinforced stability, extended half-life, and improved manufacturability. The therapeutic benefit of dsIL-18-Fc, coupled with a more favorable manufacturability profile and enhanced drug-like properties, underscores the potential utility of this engineered cytokine in cancer immunotherapy.

Immunology↗
DOE OSTI2025

Structural analysis of the NifL-NifA complex reveals the molecular basis of anti-activation of nitrogen fixation gene expression in Azotobacter vinelandii

Understanding the molecular basis of regulated nitrogen (N 2 ) fixation is essential for engineering N 2 -fixing bacteria that fulfill the demand of crop plants for fixed nitrogen, reducing our reliance on synthetic nitrogen fertilizers. In Azotobacter vinelandii and many other members of Proteobacteria, the two-component system comprising the anti-activator protein (NifL) and the Nif-specific transcriptional activator (NifA)controls the expression of nif genes, encoding the nitrogen fixation machinery. The NifL-NifA system evolved the ability to integrate several environmental cues, such as oxygen, nitrogen, and carbon availability. The nitrogen fixation machinery is thereby only activated under strictly favorable conditions, enabling diazotrophs to thrive in competitive environments. While genetic and biochemical studies have enlightened our understanding of how NifL represses NifA, the molecular basis of NifA sequestration by NifL depends on structural information on their interaction. Here, we present mechanistic insights into how nitrogen fixation is regulated by combining biochemical and genetic approaches with a low-resolution cryo-electron microscopy (cryo-EM) map of the oxidized NifL-NifA complex. Our findings define the interaction surface between NifL and NifA and reveal how this interaction can be manipulated to generate bacterial strains with increased nitrogen fixation rates able to secrete surplus nitrogen outside the cell, a crucial step in engineering improved nitrogen delivery to crop plants.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2000

International Space Station: Expedition 2000

Live footage of the International Space Station (ISS) presents an inside look at the groundwork and assembly of the ISS. Footage includes both animation and live shots of a Space Shuttle liftoff. Phil West, Engineer; Dr. Catherine Clark, Chief Scientist ISS; and Joe Edwards, Astronaut, narrate the video. The first topic of discussion is People and Communications. Good communication is a key component in our ISS endeavor. Dr. Catherine Clark uses two soup cans attached by a string to demonstrate communication. Bill Nye the Science Guy talks briefly about science aboard the ISS. Charlie Spencer, Manager of Space Station Simulators, talks about communication aboard the ISS. The second topic of discussion is Engineering. Bonnie Dunbar, Astronaut at Johnson Space Flight Center, gives a tour of the Japanese Experiment Module (JEM). She takes us inside Node 2 and the U.S. Lab Destiny. She also shows where protein crystal growth experiments are performed. Audio terminal units are used for communication in the JEM. A demonstration of solar arrays and how they are tested is shown. Alan Bell, Project Manager MRMDF (Mobile Remote Manipulator Development Facility), describes the robot arm that is used on the ISS and how it maneuvers the Space Station. The third topic of discussion is Science and Technology. Dr. Catherine Clark, using a balloon attached to a weight, drops the apparatus to the ground to demonstrate Microgravity. The bursting of the balloon is observed. Sherri Dunnette, Imaging Technologist, describes the various cameras that are used in space. The types of still cameras used are: 1) 35 mm, 2) medium format cameras, 3) large format cameras, 4) video cameras, and 5) the DV camera. Kumar Krishen, Chief Technologist ISS, explains inframetrics, infrared vision cameras and how they perform. The Short Arm Centrifuge is shown by Dr. Millard Reske, Senior Life Scientist, to subject astronauts to forces greater than 1-g. Reske is interested in the physiological effects of the eyes and the muscular system after their exposure to forces greater than 1-g.

Source record↗
NASA NTRS1999

Space travel directly induces skeletal muscle atrophy

Space travel causes rapid and pronounced skeletal muscle wasting in humans that reduces their long-term flight capabilities. To develop effective countermeasures, the basis of this atrophy needs to be better understood. Space travel may cause muscle atrophy indirectly by altering circulating levels of factors such as growth hormone, glucocorticoids, and anabolic steroids and/or by a direct effect on the muscle fibers themselves. To determine whether skeletal muscle cells are directly affected by space travel, tissue-cultured avian skeletal muscle cells were tissue engineered into bioartificial muscles and flown in perfusion bioreactors for 9 to 10 days aboard the Space Transportation System (STS, i.e., Space Shuttle). Significant muscle fiber atrophy occurred due to a decrease in protein synthesis rates without alterations in protein degradation. Return of the muscle cells to Earth stimulated protein synthesis rates of both muscle-specific and extracellular matrix proteins relative to ground controls. These results show for the first time that skeletal muscle fibers are directly responsive to space travel and should be a target for countermeasure development.

NASA Discipline Musculoskeletal↗
NASA NTRS2022

Neurosymbolic Hybrid Approach to Driver Collision Warning

There are two main algorithmic approaches to autonomous driving systems: (1) An end-to-end system in which a single deep neural network learns to map sensory input directly into appropriate warning and driving responses. (2) A mediated hybrid recognition system in which a system is created by combining independent modules that detect each semantic feature. While some researchers believe that deep learning can solve any problem, others believe that a more engineered and symbolic approach is needed to cope with complex environments with less data. Deep learning alone has achieved state-of-the-art results in many areas, from complex gameplay to predicting protein structures. In particular, in image classification and recognition, deep learning models have achieved accuracies as high as humans. But sometimes it can be very difficult to debug if the deep learning model doesn't work. Deep learning models can be vulnerable and are very sensitive to changes in data distribution. Generalization can be problematic. It's usually hard to prove why it works or doesn't. Deep learning models can also be vulnerable to adversarial attacks. Here, we combine deep learning-based object recognition and tracking with an adaptive neurosymbolic network agent, called the Non-Axiomatic Reasoning System (NARS), that can adapt to its environment by building concepts based on perceptual sequences. We achieved an improved intersection-over-union (IOU) object recognition performance of 0.65 in the adaptive retraining model compared to IOU 0.31 in the COCO data pre-trained model. We improved the object detection limits using RADAR sensors in a simulated environment, and demonstrated the weaving car detection capability by combining deep learning-based object detection and tracking with a neurosymbolic model.

Wang, Pei↗
DOE OSTI2026

Data for Expression of a Bacterial Trehalose 6-Phosphate Synthase Gene otsA in Camelina sativa Seeds Promotes the Channelling of Carbon Towards Oil Accumulation

Improving seed oil yield is essential for developing Camelina sativa as a sustainable biofuel crop. Fatty acid synthesis depends on the production of acetyl-CoA from photosynthetically derived sugars. Trehalose 6-phosphate (T6P), a proxy for sucrose availability, can link sugar status to plant growth and development. Synthesised by trehalose 6-phosphate synthase (TPS) from UDP-glucose and glucose-6-phosphate, T6P plays a regulatory role in metabolism. Our previous studies on Arabidopsis transgenic lines constitutively expressing the E. coli otsA (encoding TPS) showed increased T6P levels and seed triacylglycerol, along with stunted growth. In the present study we express otsA in camelina under the control of a seed-specific Phaseolin promoter. Seeds of the resulting transgenic lines accumulated high levels of T6P, and a 15%–20% increase in total fatty acids and triacylglycerol compared to wild-type. Molecular analysis showed the transgenic seeds had reduced SnRK1 activity, elevated WRI1 protein levels, and increased the levels of WRI1 and its target genes, along with enhanced rates of fatty acid synthesis that increased seed weights relative to wild type. Notably, the increase in oil did not affect seed protein levels but did reduce the soluble metabolite fraction. Crucially, seed-specific expression of otsA mitigated the growth defects associated with constitutive otsA expression, and the transgenic lines showed normal seed development and germination. These findings demonstrate that targeted T6P modulation via seed-specific otsA expression is an effective metabolic engineering strategy to boost oil production in camelina and potentially in other oilseed crops and bioenergy crops such as energycane, sorghum and miscanthus.

Lipids↗
DOE OSTI2025

Viral delivery of recombinases activates heritable genetic switches in plants

Viral vectors provide an increasingly versatile platform for transformation-free reagent delivery to plants. RNA viral vectors can be used to induce gene silencing, overexpress proteins, or introduce gene editing reagents; however, they are often constrained by carrying capacity or restricted tropism in germline cells. Site-specific recombinases that catalyze precise genetic rearrangements are powerful tools for genome engineering that vary in size and, potentially, efficacy in plants. In this work, we show that viral vectors based on tobacco rattle virus (TRV) deliver and stably express four recombinases ranging in size from ~0.6kb to ~1.5kb and achieve simultaneous marker removal and reporter activation through targeted excision in transgenic Nicotiana benthamiana lines. TRV vectors with Cre, FLP, CinH, and Integrase13 efficiently mediated recombination in infected somatic tissue and led to heritable modifications at high frequency. Here, an excision-activated Ruby reporter enabled simple and high-resolution tracing of infected cell lineages without the need for molecular genotyping. Together, our experiments broaden the scope of viral recombinase delivery and offer insights into infection dynamics that may be useful in developing future viral vectors.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Data for "Viral Delivery of Recombinases Activates Heritable Genetic Switches in Plants"

Viral vectors provide an increasingly versatile platform for transformation-free reagent delivery to plants. RNA viral vectors can be used to induce gene silencing, overexpress proteins, or introduce gene editing reagents; however, they are often constrained by carrying capacity or restricted tropism in germline cells. Site-specific recombinases that catalyze precise genetic rearrangements are powerful tools for genome engineering that vary in size and, potentially, efficacy in plants. In this work, we show that viral vectors based on tobacco rattle virus (TRV) deliver and stably express four recombinases ranging in size from ∼0.6 to ∼1.5 kb and achieve simultaneous marker removal and reporter activation through targeted excision in transgenic Nicotiana benthamiana lines. TRV vectors with Cre, FLP, CinH, and Integrase13 efficiently mediated recombination in infected somatic tissue and led to heritable modifications at high frequency. An excision-activated Ruby reporter enabled simple and high-resolution tracing of infected cell lineages without the need for molecular genotyping. Together, our experiments broaden the scope of viral recombinase delivery and offer insights into infection dynamics that may be useful in developing future viral vectors.

gene editing↗
DOE OSTI2025

Nanoparticle-mediated antagonism of sustained endosomal signaling of the calcitonin receptor-like receptor provides enhanced and persistent relief of oral cancer pain

By improving the delivery and tumor retention of chemotherapeutics, nanomedicines hold potential for cancer treatment. The usefulness of nanoparticle (NP)-encapsulated analgesics for the cancer pain treatment is comparatively unexplored. We investigated whether NPs encapsulating olcegepant (OCP), an antagonist of the calcitonin receptor-like receptor (CLR) for the calcitonin gene-related peptide (CGRP), effectively relieved oral cancer pain in mice. Because persistent endosomal CLR signaling in Schwann cells mediates craniofacial pain, we reasoned that the predisposition of NPs to accumulate in endosomes could be leveraged to effectively relieve oral cancer pain. By expressing biosensors for activated CLR, Gα proteins and β-arrestins in HEK293T and Schwann cells, we found that CGRP activates CLR signaling first at the plasma membrane and then in early, late and recycling endosomes and the cis- and trans-Golgi apparatus. We synthesized biocompatible NPs encapsulating OCP and fluorophores by integrating hydrophobic ion pairing nanoformulation with Flash NanoPrecipitation. NPs slowly released OCP and accumulated in early endosomes, leading to sustained inhibition of endosomal CLR signaling in HEK293T and Schwann cells. Oral cancers were established in mice, which led to heightened pain-like responses. After intra-tumoral injection, NPs were retained in tumors for at least one week. OCP-loaded NPs almost completely reversed allodynia and hyperalgesia for a prolonged period, whereas unencapsulated OCP had small and transient effects. The NP accumulation in endosomal sites of pain signaling, the sustained release of antagonist, and the retention of NPs in tumors explain their beneficial actions. Thus, NP-encapsulation holds promise for the relief of painful cancers that are inadequately treated by opioids.

Calcitonin gene-related peptide↗