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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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At least 685 records · Page 38
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Supramolecular assembly of polycation/mRNA nanoparticles and in vivo monocyte programming

Size-dependent phagocytosis is a well-characterized phenomenon in monocytes and macrophages. However, this size effect for preferential gene delivery to these important cell targets has not been fully exploited because commonly adopted stabilization methods for electrostatically complexed nucleic acid nanoparticles, such as PEGylation and charge repulsion, typically arrest the vehicle size below 200 nm. Here, we bridge the technical gap in scalable synthesis of larger submicron gene delivery vehicles by electrostatic self-assembly of charged nanoparticles, facilitated by a polymer structurally designed to modulate internanoparticle Coulombic and van der Waals forces. Specifically, our strategy permits controlled assembly of small poly(β-amino ester)/messenger ribonucleic acid (mRNA) nanoparticles into particles with a size that is kinetically tunable between 200 and 1,000 nm with high colloidal stability in physiological media. We found that assembled particles with an average size of 400 nm safely and most efficiently transfect monocytes following intravenous administration and mediate their differentiation into macrophages in the periphery. When a CpG adjuvant is co-loaded into the particles with an antigen mRNA, the monocytes differentiate into inflammatory dendritic cells and prime adaptive anticancer immunity in the tumor-draining lymph node. This platform technology offers a unique ligand-independent, particle-size-mediated strategy for preferential mRNA delivery and enables therapeutic paradigmsviamonocyte programming.

Science & Technology - Other Topics↗
DOE OSTI2025

Cryosectioning-enhanced super-resolution microscopy for single-protein imaging across cells and tissues

DNA-points accumulation for imaging in nanoscale topography (DNA-PAINT) enables nanoscale imaging with virtually unlimited multiplexing and molecular counting. Here, we address challenges, such as variable imaging performance and target accessibility, that can limit its broader applicability. Specifically, we enhance its capacity for robust single-protein imaging and molecular counting by optimizing the integration of total internal reflection fluorescence microscopy with physical sectioning, in particular, Tokuyasu cryosectioning. Our method, tomographic and kinetically enhanced DNA-PAINT (tkPAINT), achieves 3 nm localization precision across diverse samples, enhanced imager binding, and improved cellular integrity. tkPAINT can facilitate molecular counting with DNA-PAINT inside the nucleus, as demonstrated through its quantification of the in situ abundance of RNA Polymerase II in both HeLa cells as well as mouse tissues. Anticipating that tkPAINT could become a versatile tool for the exploration of biomolecular organization and interactions across cells and tissues, we also demonstrate its capacity to support multiplexing, multimodal targeting of proteins and nucleic acids, and three-dimensional (3D) imaging.

Science & Technology - Other Topics↗
DOE OSTI2020

Csx3 is a cyclic oligonucleotide phosphodiesterase associated with type III CRISPR–Cas that degrades the second messenger cA 4

Cas10 is the signature gene for type III CRISPR–Cas surveillance complexes. Unlike type I and type II systems, type III systems do not require a protospacer adjacent motif and target nascent RNA associated with transcriptionally active DNA. Further, target RNA recognition activates the cyclase domain of Cas10, resulting in the synthesis of cyclic oligoadenylate second messengers. These second messengers are recognized by ancillary Cas proteins harboring CRISPR-associated Rossmann fold (CARF) domains and regulate the activities of these proteins in response to invading nucleic acid. Csx3 is a distant member of the CARF domain superfamily previously characterized as a Mn 2+ -dependent deadenylation exoribonuclease. However, its specific role in CRISPR–Cas defense remains to be determined. Here we show that Csx3 is strongly associated with type III systems and that Csx3 binds cyclic tetra-adenylate (cA 4 ) second messenger with high affinity. Further, Csx3 harbors cyclic oligonucleotide phosphodiesterase activity that quickly degrades this cA 4 signal. In addition, structural analysis identifies core elements that define the CARF domain fold, and the mechanistic basis for ring nuclease activity is discussed. Overall, the work suggests that Csx3 functions within CRISPR–Cas as a counterbalance to Cas10 to regulate the duration and amplitude of the cA 4 signal, providing an off ramp from the programmed cell death pathway in cells that successfully cure viral infection.

36 MATERIALS SCIENCE↗
DOE OSTI2021

LAMP Diagnostics at the Point-of-Care: Emerging Trends and Perspectives for the Developer Community

Over the past decade, loop-mediated isothermal amplification (LAMP) technology has played an important role in molecular diagnostics. Amongst numerous nucleic acid amplification assays, LAMP stands out in terms of sample-to-answer time, sensitivity, specificity, cost, robustness, and accessibility, making it ideal for field-deployable diagnostics in resource-limited regions. In this review, we outline the front-end LAMP design practices for point-of-care (POC) applications, including sample handling and various signal readout methodologies. Next, we explore existing LAMP technologies that have been validated with clinical samples in the field. We summarize recent work that utilizes reverse transcription (RT) LAMP to rapidly detect SARS-CoV-2 as an alternative to standard PCR protocols. Finally, we describe challenges in translating LAMP from the benchtop to the field and opportunities for future LAMP assay development and performance reporting. Despite the popularity of LAMP in the academic research community and a recent surge in interest in LAMP due to the COVID-19 pandemic, there are numerous areas for improvement in the fundamental understanding of LAMP, which are needed to elevate the field of LAMP assay development and characterization.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Synthesis of two fluorescent GTP$\gamma$S molecules and their biological relevance

Fluorescent GTP analogues are utilized for an assortment of nucleic acid and protein characterization studies. Non-hydrolysable analogues such as GTPγS offer the advantage of keeping proteins in a GTP-bound conformation due to their resistance to hydrolysis into GDP. Two novel fluorescent GTPγS molecules were developed by linking fluorescein and tetramethylrhodamine to the γ-thiophosphate of GTPγS. Chemical and biological analysis of these two compounds revealed their successful synthesis and ability to bind to the nucleotide-binding site of tubulin. These two new fluorescent non-hydrolysable nucleotides offer new possibilities for biophysical and biochemical characterization of GTP-binding proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Differential effects of divalent cations on elk prion protein fibril formation and stability

Misfolding of the normally folded prion protein of mammals (PrP C ) into infectious fibrils causes a variety of diseases, from scrapie in sheep to chronic wasting disease (CWD) in cervids. The misfolded form of PrP C , termed PrP Sc , or in this case PrP CWD , interacts with PrP C to create more PrP CWD . This process is not clearly defined but is affected by the presence and interactions of biotic and abiotic cofactors. These include nucleic acids, lipids, glycosylation, pH, and ionic character. PrP C has been shown to act as a copper-binding protein in vivo, though it also binds to other divalents as well. The significance of this action has not been conclusively elucidated. Previous reports have shown that metal binding sites occur throughout the N-terminal region of PrP C . Other cations like manganese have also been shown to affect PrP C abundance in a transcript-independent fashion. Here, we examined the ability of different divalent cations to influence the stability and in vitro conversion of two variants of PrP from elk (L/M132, 26–234). We find that copper and zinc destabilize PrP. We also find that PrP M132 exhibits a greater degree of divalent cation induced destabilization than L132. This supports findings that leucine at position 132 confers resistance to CWD, while M132 is susceptible. However, in vitro conversion of PrP is equally suppressed by either copper or zinc, in both L132 and M132 backgrounds. This report demonstrates the complex importance of ionic character on the PrP C folding pathway selection on the route to PrP Sc formation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Catalytically inactive, purified RNase H1: A specific and sensitive probe for RNA–DNA hybrid imaging

R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA–DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic because it readily binds to double-stranded RNA (dsRNA) in vitro and in vivo, giving rise to nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged with GFP (GFP-dRNH1) is a more specific reagent for imaging RNA–DNA hybrids. GFP-dRNH1 binds strongly to RNA–DNA hybrids but not to dsRNA oligonucleotides in fixed human cells and is not susceptible to binding endogenous RNA. Furthermore, we demonstrate that purified GFP-dRNH1 can be applied to fixed cells to detect hybrids after their induction, thereby bypassing the need for cell line engineering. GFP-dRNH1 therefore promises to be a versatile tool for imaging and quantifying RNA–DNA hybrids under a wide range of conditions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Metagenomic Methods for Addressing NASA's Planetary Protection Policy Requirements on Future Missions: A Workshop Report

Molecular biology methods and technologies have advanced substantially over the past decade. These new molecular methods should be incorporated among the standard tools of planetary protection (PP) and could be validated for incorporation by 2026. To address the feasibility of applying modern molecular techniques to such an application, NASA conducted a technology workshop with private industry partners, academics, and government agency stakeholders, along with NASA staff and contractors. The technical discussions and presentations of the Multi-Mission Metagenomics Technology Development Workshop focused on modernizing and supplementing the current PP assays. The goals of the workshop were to assess the state of metagenomics and other advanced molecular techniques in the context of providing a validated framework to supplement the bacterial endospore-based NASA Standard Assay and to identify knowledge and technology gaps. In particular, workshop participants were tasked with discussing metagenomics as a stand-alone technology to provide rapid and comprehensive analysis of total nucleic acids and viable microorganisms on spacecraft surfaces, thereby allowing for the development of tailored and cost-effective microbial reduction plans for each hardware item on a spacecraft. Workshop participants recommended metagenomics approaches as the only data source that can adequately feed into quantitative microbial risk assessment models for evaluating the risk of forward (exploring extraterrestrial planet) and back (Earth harmful biological) contamination. Participants were unanimous that a metagenomics workflow, in tandem with rapid targeted quantitative (digital) PCR, represents a revolutionary advance over existing methods for the assessment of microbial bioburden on spacecraft surfaces. The workshop highlighted low biomass sampling, reagent contamination, and inconsistent bioinformatics data analysis as key areas for technology development. Finally, it was concluded that implementing metagenomics as an additional workflow for addressing concerns of NASA's robotic mission will represent a dramatic improvement in technology advancement for PP and will benefit future missions where mission success is affected by backward and forward contamination.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Virus-Like Particle: Evolving Meanings in Different Disciplines

Virus-like particle (VLP) is a term that has been in use for about 80 years. Usually, VLP has meant a particle that is like a virus, generally by appearance, but without either proven or actual virus functionality. Initially VLP referred to particles seen in electron microscope images of tissues. More recently, VLP has come to mean other things to other researchers. A key divergence has been use of VLP in association with vaccine and biotechnology applications versus use of VLP in enumeration of viruses in environmental samples. To these viral ecologists, a VLP is a particle that is virus sized, has nucleic acid, and could be a functional virus. But to vaccine developers and biotechnology researchers a VLP instead is a viral structure that intentionally lacks a viral genome. Here, we look at the history of use of VLP, following changes in meaning as the technology to study VLPs changed.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Strategies for community-sourced biocuration in bioinformatics: a case study on MIBiG 4.0

Biocuration is essential to transform molecular sequence data into standardized, machine-readable resources. Such curated datasets enable comparative analysis, predictive modeling, and data integration across bioinformatics platforms. While professional biocuration is resource-intensive and usually limited to institutional settings, community-driven approaches can mobilize large-scale annotation of specialized datasets and are more resilient to disruptions in scientific funding. Here, we present a model for community-powered curation applied to the Minimum Information about a Biosynthetic Gene Cluster (MIBiG) repository. Through a framework of workflows for metadata capture, annotation validation, and contributor coordination, the MIBiG 4.0 initiative recruited 267 scientists across 178 institutions from 33 countries, volunteering an estimated 4000 h of work. These efforts expanded the MIBiG repository by 22% and enhanced its usability in downstream molecular data analyses in comparative genomic analyses, natural product discovery, and machine learning applications. We provide strategies and actionable lessons for adopting this model, supporting the sustainability of curated bioinformatics resources central to nucleic acid research and related fields.

biocuration↗
DOE OSTI2021

Draft genome sequences of strains CBS6241 and CBS6242 of the basidiomycetous yeast Filobasidium floriforme

The Tremellomycetes are a species-rich group within the basidiomycete fungi; however, most analyses of this group to date have focused on pathogenic Cryptococcus species within the order Tremellales. Recent genome-assisted studies of other Tremellomycetes have identified interesting features with respect to biotechnological applications as well as the evolution of genes involved in mating and sexual development. Here, we report genome sequences of two strains of Filobasidium floriforme, a species from the order Filobasidiales, which branches basally to the Tremellales, Trichosporonales, and Holtermanniales. The assembled genomes of strains CBS6241 and CBS6242 are 27.4 Mb and 26.4 Mb in size, respectively, with 8314 and 7695 predicted protein-coding genes. Overall sequence identity at nucleic acid level between the strains is 97%. Among the predicted genes are pheromone precursor and pheromone receptor genes as well as two genes encoding homedomain (HD) transcription factors, which are predicted to be part of the mating type (MAT) locus. Sequence analysis indicates that CBS6241 and CBS6242 carry different alleles for both the pheromone/receptor genes as well as the HD transcription factors. Orthology inference identified 1482 orthogroups exclusively found in F. floriforme, some of which were involved in carbohydrate transport and metabolism. Subsequent CAZyme repertoire characterization identified 267 and 247 enzymes for CBS6241 and CBS6242, respectively, the second highest number of CAZymes among the analyzed Tremellomycete species. In addition, F. floriforme contains five CAZymes absent in other species and several plant-cell-wall degrading CAZymes with the highest copy number in Tremellomycota, indicating the biotechnological potential of this species.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Engineered dsRNA–protein nanoparticles for effective systemic gene silencing in plants

Long-distance transport or systemic silencing effects of exogenous biologically active RNA molecules in higher plants have not been reported. Here, we report that cationized bovine serum albumin (cBSA) avidly binds double-stranded beta-glucuronidase RNA (dsGUS RNA) to form nucleic acid–protein nanocomplexes. In our experiments with tobacco and poplar plants, we have successfully demonstrated systemic gene silencing effects of cBSA/dsGUS RNA nanocomplexes when we locally applied the nanocomplexes from the basal ends of leaf petioles or shoots. We have further demonstrated that the cBSA/dsGUS RNA nanocomplexes are highly effective in silencing both the conditionally inducible DR5-GUS gene and the constitutively active 35S-GUS gene in leaf, shoot, and shoot meristem tissues. This cBSA/dsRNA delivery technology may provide a convenient, fast, and inexpensive tool for characterizing gene functions in plants and potentially for in planta gene editing.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

CasPEDIA Database: a functional classification system for class 2 CRISPR-Cas enzymes

Abstract CRISPR-Cas enzymes enable RNA-guided bacterial immunity and are widely used for biotechnological applications including genome editing. In particular, the Class 2 CRISPR-associated enzymes (Cas9, Cas12 and Cas13 families), have been deployed for numerous research, clinical and agricultural applications. However, the immense genetic and biochemical diversity of these proteins in the public domain poses a barrier for researchers seeking to leverage their activities. We present CasPEDIA (http://caspedia.org), the Cas Protein Effector Database of Information and Assessment, a curated encyclopedia that integrates enzymatic classification for hundreds of different Cas enzymes across 27 phylogenetic groups spanning the Cas9, Cas12 and Cas13 families, as well as evolutionarily related IscB and TnpB proteins. All enzymes in CasPEDIA were annotated with a standard workflow based on their primary nuclease activity, target requirements and guide-RNA design constraints. Our functional classification scheme, CasID, is described alongside current phylogenetic classification, allowing users to search related orthologs by enzymatic function and sequence similarity. CasPEDIA is a comprehensive data portal that summarizes and contextualizes enzymatic properties of widely used Cas enzymes, equipping users with valuable resources to foster biotechnological development. CasPEDIA complements phylogenetic Cas nomenclature and enables researchers to leverage the multi-faceted nucleic-acid targeting rules of diverse Class 2 Cas enzymes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

The Human Phenotype Ontology in 2017

Deep phenotyping has been defined as the precise and comprehensive analysis of phenotypic abnormalities in which the individual components of the phenotype are observed and described. The three components of the Human PhenotypeOntology (HPO; www.human-phenotype-ontology.org) project are the phenotype vocabulary, disease-phenotype annotations and the algorithms that operate on these. These components are being used for computational deep phenotyping and precision medicine as well as integration of clinical data into translational research. The HPO is being increasingly adopted as a standard for phenotypic abnormalities by diverse groups such as international rare disease organizations, registries, clinical labs, biomedical resources, and clinical software tools and will thereby contribute toward nascent efforts at global data exchange for identifying disease etiologies. This update article reviews the progress of the HPO project since the debut Nucleic Acids Research database article in 2014, including specific areas of expansion such as common (complex) disease, new algorithms for phenotype driven genomic discovery and diagnostics, integration of cross-species mapping efforts with the Mammalian Phenotype Ontology, an improved quality control pipeline, and the addition of patient-friendly terminology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

DNA microstructure influences selective binding of small molecules designed to target mixed-site DNA sequences

Specific targeting of protein–nucleic acid interactions is an area of current interest, for example, in the regulation of gene-expression. Most transcription factor proteins bind in the DNA major groove; however, we are interested in an approach using small molecules to target the minor groove to control expression by an allosteric mechanism. In an effort to broaden sequence recognition of DNA-targeted-small-molecules to include both A·T and G·C base pairs, we recently discovered that the heterocyclic diamidine, DB2277, forms a strong monomer complex with a DNA sequence containing 5'-AAAGTTT-3'. Competition mass spectrometry and surface plasmon resonance identified new monomer complexes, as well as unexpected binding of two DB2277 with certain sequences. Inherent microstructural differences within the experimental DNAs were identified through computational analyses to understand the molecular basis for recognition. These findings emphasize the critical nature of the DNA minor groove microstructure for sequence-specific recognition and offer new avenues to design synthetic small molecules for effective regulation of gene-expression.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

5-Formylcytosine mediated DNA–protein cross-links block DNA replication and induce mutations in human cells

Nucleic Acids Research, 46(13):6455–6469, https://doi.org/10.1093/nar/gky444. There was a typographical error on page 6458, column 1, line 12 and on page 6459, column 2, line 53. In both instances, dNTP concentrations should be listed as ‘5, 10, 25, 50, 100, 150, 250, 500 μM’. The same error has been corrected in the labels of Supplementary Figure S2. These typos do not affect the kinetics parameters listed in Table 2 and do not influence the results and conclusions of the article. The article and supplementary file have been updated.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Structural organization of a Type III-A CRISPR effector subcomplex determined by X-ray crystallography and cryo-EM

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins provide an immune-like response in many prokaryotes against extraneous nucleic acids. CRISPR–Cas systems are classified into different classes and types. Class 1 CRISPR–Cas systems form multi-protein effector complexes that includes a guide RNA (crRNA) used to identify the target for destruction. Here we present crystal structures of Staphylococcus epidermidis Type III-A CRISPR subunits Csm2 and Csm3 and a 5.2 Å resolution single-particle cryo-electron microscopy (cryo-EM) reconstruction of an in vivo assembled effector subcomplex including the crRNA. The structures help to clarify the quaternary architecture of Type III-A effector complexes, and provide details on crRNA binding, target RNA binding and cleavage, and intermolecular interactions essential for effector complex assembly. The structures allow a better understanding of the organization of Type III-A CRISPR effector complexes as well as highlighting the overall similarities and differences with other Class 1 effector complexes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

CasCollect: targeted assembly of CRISPR-associated operons from high-throughput sequencing data

Abstract CRISPR arrays and CRISPR-associated (Cas) proteins comprise a widespread adaptive immune system in bacteria and archaea. These systems function as a defense against exogenous parasitic mobile genetic elements that include bacteriophages, plasmids and foreign nucleic acids. With the continuous spread of antibiotic resistance, knowledge of pathogen susceptibility to bacteriophage therapy is becoming more critical. Additionally, gene-editing applications would benefit from the discovery of new cas genes with favorable properties. While next-generation sequencing has produced staggering quantities of data, transitioning from raw sequencing reads to the identification of CRISPR/Cas systems has remained challenging. This is especially true for metagenomic data, which has the highest potential for identifying novel cas genes. We report a comprehensive computational pipeline, CasCollect, for the targeted assembly and annotation of cas genes and CRISPR arrays—even isolated arrays—from raw sequencing reads. Benchmarking our targeted assembly pipeline demonstrates significantly improved timing by almost two orders of magnitude compared with conventional assembly and annotation, while retaining the ability to detect CRISPR arrays and cas genes. CasCollect is a highly versatile pipeline and can be used for targeted assembly of any specialty gene set, reconfigurable for user provided Hidden Markov Models and/or reference nucleotide sequences.

Podlevsky, Joshua D.↗