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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 127 records · Page 7
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Analysis of ionizing radiation induced DNA damage response in human adult stem cells and differentiated neurons

Findings of neurodegenerative features associated with human radiosensitive syndromes such as Ataxia telangiectasia suggest that DNA repair efficiency is crucial for maintaining the functional integrity of central nervous system. To gain a better understanding of ionizing radiation (IR) induced DNA damage response in undifferentiated and differentiated neural cell types and to evaluate the role of ATM in DNA double strand break (DSB) repair, an in vitro human neural cell differentiation model system was utilized in this study. As compared to adult stem cells, differentiated neurons displayed an attenuated DSB repair response (as judged by the persistence of 53BP1 foci) after IR exposure and the attenuation was even more pronounced in stem cells and neurons after suppression of ATM (Ataxia Telangiectasia Mutated) gene product suggesting the importance of ATM for an optimal DSB repair efficiency in human neural cell types. In corroboration with an attenuated DNA damage response, a sharp decline in the expression levels of several DSB repair genes was observed in neurons. Furthermore, our results suggest that cellular differentiation modulates the expression of several genes thereby compromising the DSB repair fidelity in post mitotic neurons. Further studies are required to verify whether or not ATM mediated exacerbation of DNA repair deficiency in differentiated neurons leads to neurodegeneration.

53 binding protein 1↗
DOE OSTI2021

In vivo evolution of lactic acid hyper-tolerant Clostridium thermocellum

Lactic acid (LA) has several applications in the food, cosmetics and pharmaceutical industries, as well as in the production of biodegradable plastic polymers, namely polylactides. Industrial production of LA is essentially based on microbial fermentation. Recent reports have shown the potential of the cellulolytic bacterium Clostridium thermocellum for direct LA production from inexpensive lignocellulosic biomass. However, C. thermocellum is highly sensitive to acids and does not grow at pH < 6.0. Improvement of LA tolerance of this microorganism is pivotal for its application in cost-efficient production of LA. In the present study, the LA tolerance of C. thermocellum strains LL345 (wild-type fermentation profile) and LL1111 (high LA yield) was increased by adaptive laboratory evolution. At large inoculum size (10 %), the maximum tolerated LA concentration of strain LL1111 was more than doubled, from 15 g/L to 35 g/L, while subcultures evolved from LL345 showed 50–85 % faster growth in medium containing 45 g/L LA. Gene mutations (pyruvate phosphate dikinase, histidine protein kinase/phosphorylase) possibly affecting carbohydrate and/or phosphate metabolism have been detected in most LA-adapted populations. Although improvement of LA tolerance may sometimes also enable higher LA production in microorganisms, C. thermocellum LA-adapted cultures showed a yield of LA, and generally of other organic acids, similar to or lower than parental strains. Based on its improved LA tolerance and LA titer similar to its parent strain (LL1111), mixed adapted culture LL1630 showed the highest performing phenotype and could serve as a framework for improving LA production by further metabolic engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Highly efficient and simple SSPER and rrPCR approaches for the accurate site-directed mutagenesis of large and small plasmids

Advances are needed in the site-directed mutagenesis of large plasmids for protein structure-function studies, as current methods are often inefficient, complicated and time-consuming. Here two new methods are reported that overcome these difficulties, namely the single primer extension reaction (SSPER) strategy that reaches 100% efficiency and the reduce recycle PCR (rrPCR) method that is advantageous in generating single and pairwise combinations of mutations. Both methods are distinguished from current technologies by the addition of a step that easily removes the oligonucleotide primer(s) after the first reaction, thus allowing for the addition of a second reaction in chronological sequence to generate and isolate the appropriate DNA product with the site-directed mutation(s). High efficiency of the methods is demonstrated by generating single and paired combinations of the 11 site-directed mutations targeted on 5 different plasmid DNA templates ranging from 10 to 12 kb and 57–60% GC-content at a rate of 50–100%. Overall, the methods are demonstrated to be (i) highly accurate, allowing for screening of plasmids by DNA sequencing, (ii) streamlined to generate the mutations within a single day, (iii) cost-effective in requiring only two primers and two enzymes (DpnI and a proofreading DNA polymerase), (iv) straightforward in primer design, (v) applicable for both large and small plasmids, and (vi) easily implemented by entry level researchers.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Drug discovery efforts at George Mason University

With over 39,000 students, and research expenditures in excess of $200 million, George Mason University (GMU) is the largest R1 (Carnegie Classification of very high research activity) university in Virginia. Mason scientists have been involved in the discovery and development of novel diagnostics and therapeutics in areas as diverse as infectious diseases and cancer. Below are highlights of the efforts being led by Mason researchers in the drug discovery arena. To enable targeted cellular delivery, and non-biomedical applications, Veneziano and colleagues have developed a synthesis strategy that enables the design of self-assembling DNA nanoparticles (DNA origami) with prescribed shape and size in the 10 to 100 nm range. The nanoparticles can be loaded with molecules of interest such as drugs, proteins and peptides, and are a promising new addition to the drug delivery platforms currently in use. The investigators also recently used the DNA origami nanoparticles to fine tune the spatial presentation of immunogens to study the impact on B cell activation. These studies are an important step towards the rational design of vaccines for a variety of infectious agents. To elucidate the parameters for optimizing the delivery efficiency of lipid nanoparticles (LNPs), Buschmann, Paige and colleagues have devised methods for predicting and experimentally validating the pKa of LNPs based on the structure of the ionizable lipids used to formulate the LNPs. These studies may pave the way for the development of new LNP delivery vehicles that have reduced systemic distribution and improved endosomal release of their cargo post administration. To better understand protein-protein interactions and identify potential drug targets that disrupt such interactions, Luchini and colleagues have developed a methodology that identifies contact points between proteins using small molecule dyes. The dye molecules noncovalently bind to the accessible surfaces of a protein complex with very high affinity, but are excluded from contact regions. When the complex is denatured and digested with trypsin, the exposed regions covered by the dye do not get cleaved by the enzyme, whereas the contact points are digested. The resulting fragments can then be identified using mass spectrometry. The data generated can serve as the basis for designing small molecules and peptides that can disrupt the formation of protein complexes involved in disease processes. For example, using peptides based on the interleukin 1 receptor accessory protein (IL-1RAcP), Luchini, Liotta, Paige and colleagues disrupted the formation of IL-1/IL-R/IL-1RAcP complex and demonstrated that the inhibition of complex formation reduced the inflammatory response to IL-1B. Working on the discovery of novel antimicrobial agents, Bishop, van Hoek and colleagues have discovered a number of antimicrobial peptides from reptiles and other species. DRGN-1, is a synthetic peptide based on a histone H1-derived peptide that they had identified from Komodo Dragon plasma. DRGN-1 was shown to disrupt bacterial biofilms and promote wound healing in an animal model. The peptide, along with others, is being developed and tested in preclinical studies. Other research by van Hoek and colleagues focuses on in silico antimicrobial peptide discovery, screening of small molecules for antibacterial properties, as well as assessment of diffusible signal factors (DFS) as future therapeutics. The above examples provide insight into the cutting-edge studies undertaken by GMU scientists to develop novel methodologies and platform technologies important to drug discovery.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A FAIR-compliant parts catalogue for genome engineering and expression control in Saccharomyces cerevisiae

The synthetic biology toolkit for baker's yeast, Saccharomyces cerevisiae, includes extensive genome engineering toolkits and parts repositories. However, with the increasing complexity of engineering tasks and versatile applications of this model eukaryote, there is a continued interest to expand and diversify the rational engineering capabilities in this chassis by FAIR (findable, accessible, interoperable, and reproducible) compliance. In this study, we designed and characterised 41 synthetic guide RNA sequences to expand the CRISPR-based genome engineering capabilities for easy and efficient replacement of genomically encoded elements. Moreover, we characterize in high temporal resolution 20 native promoters and 18 terminators using fluorescein and LUDOX CL-X as references for GFP expression and OD600 measurements, respectively. Additionally, all data and reported analysis is provided in a publicly accessible jupyter notebook providing a tool for researchers with low-coding skills to further explore the generated data as well as a template for researchers to write their own scripts. We expect the data, parts, and databases associated with this study to support a FAIR-compliant resource for further advancing the engineering of yeasts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Directed Evolution of CRISPR/Cas Systems for Precise Gene Editing

CRISPR technology is a universal tool for genome engineering that has revolutionized biotechnology. Recently identified unique CRISPR/Cas systems, as well as re-engineered Cas proteins, have rapidly expanded the functions and applications of CRISPR/Cas systems. The structures of Cas proteins are complex, containing multiple functional domains. These protein domains are evolutionarily conserved polypeptide units that generally show independent structural or functional properties. Here, we propose using protein domains as a new way to classify protein engineering strategies for these proteins and discuss common ways to engineer key domains to modify the functions of CRISPR/Cas systems.

59 BASIC BIOLOGICAL SCIENCES↗