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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 199 records · Page 11
All sourcesNASA NTRSDOE OSTI
NASA NTRS2011

Exploring the Fate of Nitrogen Heterocycles in Complex Prebiotic Mixtures

A long standing question in the field of prebiotic chemistry is the origin of the genetic macromolecules DNA and RNA. DNA and RNA have very complex structures with repeating subunits of nucleotides, which are composed of nucleobases (nitrogen heterocycles) connected to sugar-phosphate. Due to the instability of some nucleobases (e.g. cytosine), difficulty of synthesis and instability of D-ribose, and the likely scarcity of polyphosphates necessary for the modern nucleotides, alternative nucleotides have been proposed for constructing the first genetic material. Thus, we have begun to investigate the chemistry of nitrogen heterocycles in plausible, complex prebiotic mixtures in an effort to identify robust reactions and potential alternative nucleotides. We have taken a complex prebiotic mixture produced by a spark discharge acting on a gas mixture of N2, CO2, CH4, and H2, and reacted it with four nitrogen heterocycles: uracil, 5-hydroxymethyluracil, guanine, and isoxanthopterin (2-amino-4,7-dihydroxypteridine). The products of the reaction between the spark mixture and each nitrogen heterocycle were characterized by liquid chromatography coupled to UV spectroscopy and Orbitrap mass spectrometry. We found that the reaction between the spark mixtUl'e and isoxanthopterin formed one major product, which was a cyanide adduct. 5-hydroxymethyluracil also reacted with the spark mixture to form a cyanide adduct, uracil-5-acetonitrile, which has been synthesized previously by reacting HCN with S-hydroxymethyluracil. Unlike isoxanthopterin, the chromatogram of the 5-hydroxymethyluracil reaction was much more complex with multiple products including spark-modified dimers. Additionally, we observed that HMU readily self-polymerizes in solution to a variety of oligomers consistent with those suggested by Cleaves. Guanine and uracil, the biological nucleobases, did not react with the spark mixture, even at high temperature (100 C). This suggests that there are alternative nucleobases which are more reactive under prebiotic conditions and may have been involved in producing precursor nucleotides.

Smith, Karen E.↗
NASA NTRS1998

Transposon tagging and the study of root development in Arabidopsis

The maize Ac-Ds transposable element family has been used as the basis of transposon mutagenesis systems that function in a variety of plants, including Arabidopsis. We have developed modified transposons and methods which simplify the detection, cloning and analysis of insertion mutations. We have identified and are analyzing two plant lines in which genes expressed either in the root cap cells or in the quiescent cells, cortex/endodermal initial cells and columella cells of the root cap have been tagged with a transposon carrying a reporter gene. A gene expressed in root cap cells tagged with an enhancer-trap Ds was isolated and its corresponding EST cDNA was identified. Nucleotide and deduced amino acid sequences of the gene show no significant similarity to other genes in the database. Genetic ablation experiments have been done by fusing a root cap-specific promoter to the diphtheria toxin A-chain gene and introducing the fusion construct into Arabidopsis plants. We find that in addition to eliminating gravitropism, root cap ablation inhibits elongation of roots by lowering root meristematic activities.

NASA Discipline Plant Biology↗
DOE OSTI2025

Protoplast fusion as a strategy to increase ploidy in Rhodotorula toruloides for strain development

Rhodotorula toruloides is a red oleaginous yeast with growing commercial interest because of its hardiness and exceptional lipid production capacity. Because it is a basidiomycete yeast with a complex life cycle, many of the classical breeding methods used with ascomycetes are unavailable for strain improvement. However, we have been able to construct polyploid yeast by fusing protoplasts of parents with the same mating type. Fusing of Y-6985 (A2) and Y-48190 (A2), which had been transformed with complementary antibiotic markers, led to the recovery of two diploids and one triploid. The stability of the fusion yeasts was tested by plating them on non-selective medium after several growth cycles under antibiotics and then testing five colonies per strain for nuclear DNA contents using flow cytometry and standard cell cycle analysis: the triploid and one diploid were stable. Fusants inherited their mitochondria from a single parent, which was demonstrated using restriction fragment length polymorphism (RFLP) of mitochondrial DNA. The phenotypic properties of the parents and fusants were compared in glucose fed-batch bioreactor studies and cellulosic sugar batch cultures. The final lipid titers for the fed-batch cultures were 24.9–39.7 g/L with Y-6985 and the diploid and triploid performing the best and worst, respectively. The fusants demonstrated intermediate hardiness for growth on hydrolysate prepared with dilute-acid pretreated switchgrass and were outperformed by Y-48190. Unlike one of the haploid parents, the fusants grew in 70% v/v concentrated hydrolysate. Furthermore, they did not grow as fast as the other haploid. In this study, a modernized protoplast fusion method is resurrected a useful tool for strain development in this yeast, which is complementary with other available methods.

Lignocellulose↗
NASA NTRS2002

The Arabidopsis SKU5 gene encodes an extracellular glycosyl phosphatidylinositol-anchored glycoprotein involved in directional root growth

To investigate how roots respond to directional cues, we characterized a T-DNA-tagged Arabidopsis mutant named sku5 in which the roots skewed and looped away from the normal downward direction of growth on inclined agar surfaces. sku5 roots and etiolated hypocotyls were slightly shorter than normal and exhibited a counterclockwise (left-handed) axial rotation bias. The surface-dependent skewing phenotype disappeared when the roots penetrated the agar surface, but the axial rotation defect persisted, revealing that these two directional growth processes are separable. The SKU5 gene belongs to a 19-member gene family designated SKS (SKU5 Similar) that is related structurally to the multiple-copper oxidases ascorbate oxidase and laccase. However, the SKS proteins lack several of the conserved copper binding motifs characteristic of copper oxidases, and no enzymatic function could be assigned to the SKU5 protein. Analysis of plants expressing SKU5 reporter constructs and protein gel blot analysis showed that SKU5 was expressed most strongly in expanding tissues. SKU5 was glycosylated and modified by glycosyl phosphatidylinositol and localized to both the plasma membrane and the cell wall. Our observations suggest that SKU5 affects two directional growth processes, possibly by participating in cell wall expansion.

NASA Discipline Plant Biology↗
DOE OSTI2023

Manipulating the 3D organization of the largest synthetic yeast chromosome

Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Structural and compositional complexities of hierarchical self-assembly: A hypergraph approach

Programmable self-assembly enables the construction of complex molecular, supramolecular, and crystalline architectures from well-designed building blocks. In this work, we introduce a hypergraph-based formalism, Blocks & Bonds (B&B), which generalizes classical chemical graph theory by incorporating directed and multicolored interactions, internal symmetries, and hierarchical organization. Within this framework, we develop the Structure Code (SC), a compact and versatile language for describing self-assembled architectures. We define a Kolmogorov-style structural complexity as the total information content of SC, obtained through its tokenization and Shannon information assignment. Complementing this encoding-based measure, we introduce a much simpler quantity, the compositional complexity, which depends only on the number and cumulative usage of block and bond types in the construction set. A central result of this work is a strong empirical correlation between the token-based structural complexity and the compositional complexity across all examined systems. Owing to this agreement, the compositional complexity emerges as the most practical and broadly applicable measure: it is easy to compute, requires no explicit encoding, and yet closely tracks the actual information content of structurally diverse architectures. Applications to molecular systems (ethylene glycol and glucose), DNA-origami lattices, and crystalline assemblies show that B&B hypergraphs provide a unified, scalable, and information-efficient representation of structural organization, naturally capturing symmetry, modularity, and stereochemistry. This framework establishes a quantitative foundation for complexity-aware classification and inverse design of programmable matter.

36 MATERIALS SCIENCE↗
NASA NTRS1995

Regulation of insulin-like growth factor I transcription by cyclic adenosine 3',5'-monophosphate (cAMP) in fetal rat bone cells through an element within exon 1: protein kinase A-dependent control without a consensus AMP response element

Insulin-like growth factor I (IGF-I) is a locally synthesized anabolic growth factor for bone. IGF-I synthesis by primary fetal rat osteoblasts (Ob) is stimulated by agents that increase the intracellular cAMP concentration, including prostaglandin E2 (PGE2). Previous studies with Ob cultures demonstrated that PGE2 enhanced IGF-I transcription through selective use of IGF-I promoter 1, with little effect on IGF-I messenger RNA half-life. Transient transfection of Ob cultures with an array of promoter 1-luciferase reporter fusion constructs has now allowed localization of a potential cis-acting promoter element(s) responsible for cAMP-stimulated gene expression to the 5'-untranslated region (5'-UTR) of IGF-I exon 1, within a segment lacking a consensus cAMP response element. Our evidence derives from three principal observations: 1) a transfection construct containing only 122 nucleotides (nt) of promoter 1 and 328 nt of the 5'-UTR retained full PGE2-stimulated reporter expression; 2) maximal PGE2-driven reporter expression required the presence of nt 196 to 328 of exon 1 when tested within the context of IGF-I promoter 1; 3) cotransfection of IGF-I promoter-luciferase-reporter constructs with a plasmid encoding the alpha-isoform of the catalytic subunit of murine cAMP-dependent protein kinase (PKA) produced results comparable to those seen with PGE2 treatment, whereas cotransfection with a plasmid encoding a mutant regulatory subunit of PKA that cannot bind cAMP blocked PGE2-induced reporter expression. Deoxyribonuclease I footprinting of the 5'-UTR of exon 1 demonstrated protected sequences at HS3A, HS3B, and HS3D, three of six DNA-protein binding sites previously characterized with rat liver nuclear extracts. Of these three regions, only the HS3D binding site is located within the functionally identified hormonally responsive segment of IGF-I exon 1. These results directly implicate PKA in the control of IGF-I gene transcription by PGE2 and identify a segment of IGF-I exon 1 as being essential for this hormonal regulation.

Non-NASA Center↗
DOE OSTI2022

Data-Driven and Multiscale Modeling of DNA-Templated Dye Aggregates

Dye aggregates are of interest for excitonic applications, including biomedical imaging, organic photovoltaics, and quantum information systems. Dyes with large transition dipole moments (μ) are necessary to optimize coupling within dye aggregates. Extinction coefficients (ε) can be used to determine the μ of dyes, and so dyes with a large ε (>150,000 M –1 cm –1 ) should be engineered or identified. However, dye properties leading to a large ε are not fully understood, and low-throughput methods of dye screening, such as experimental measurements or density functional theory (DFT) calculations, can be time-consuming. In order to screen large datasets of molecules for desirable properties (i.e., large ε and μ), a computational workflow was established using machine learning (ML), DFT, time-dependent (TD-) DFT, and molecular dynamics (MD). ML models were developed through training and validation on a dataset of 8802 dyes using structural features. A Classifier was developed with an accuracy of 97% and a Regressor was constructed with an R2 of above 0.9, comparing between experiment and ML prediction. Using the Regressor, the ε values of over 18,000 dyes were predicted. The top 100 dyes were further screened using DFT and TD-DFT to identify 15 dyes with a μ relative to a reference dye, pentamethine indocyanine dye Cy5. Two benchmark MD simulations were performed on Cy5 and Cy5.5 dimers, and it was found that MD could accurately capture experimental results. The results of this study exhibit that our computational workflow for identifying dyes with a large μ for excitonic applications is effective and can be used as a tool to develop new dyes for excitonic applications.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
NASA NTRS2017

Beyond Nanopore Sequencing in Space: Identifying the Unknown

Astronaut Kate Rubins sequenced DNA on the International Space Station (ISS) for the first time in August 2016 (Figure 1A). A 2D sequencing library containing an equal mixture of lambda bacteriophage, Escherichia coli, and Mus musculus was prepared on the ground with a SQK_MAP006 kit and sent to the ISS frozen and loaded into R7.3 flow cells. After a total of 9 on-orbit sequencing runs over 6 months, it was determined that there was no decrease in sequencing performance on-orbit compared to ground controls (1). A total of ~280,000 and ~130,000 reads generated on-orbit and on the ground, respectively, identified 90% of reads that were attributed to 30% lambda bacteriophage, 30% Escherichia coli, and 30% M. musculus (Figure 1B). Extensive bioinformatics analysis determined comparable 2D and 1D read accuracies between flight and ground runs (Figure 1C), and data collected from the ISS were able to construct directed assemblies of E.coli and lambda genomes at 100% and M. musculus mitochondrial genome at 96.7%. These findings validate sequencing as a viable option for potential on-orbit applications such as environmental microbial monitoring and disease diagnosis. Current microbial monitoring of the ISS applies culture-based techniques that provide colony forming unit (CFU) data for air, water, and surface samples. The identity of the cultured microorganisms in unknown until sample return and ground-based analysis, a process that can take up to 60 days. For sequencing to benefit ISS applications, spaceflight-compatible sample preparation techniques are required. Subsequent to the testing of the MinION on-orbit, a sample-to-sequence method was developed using miniPCR™ and basic pipetting, which was only recently proven to be effective in microgravity. The work presented here details the in- flight sample preparation process and the first application of DNA sequencing on the ISS to identify unknown ISS-derived microorganisms.

Stahl, Sarah E.↗
DOE OSTI2022

Geochemistry and Multiomics Data Differentiate Streams in Pennsylvania Based on Unconventional Oil and Gas Activity

Unconventional oil and gas (UOG) extraction is increasing exponentially around the world, as new technological advances have provided cost-effective methods to extract hard-to-reach hydrocarbons. While UOG has increased the energy output of some countries, past research indicates potential impacts in nearby stream ecosystems as measured by geochemical and microbial markers. Here, we utilized a robust data set that combines 16S rRNA gene amplicon sequencing (DNA), metatranscriptomics (RNA), geochemistry, and trace element analyses to establish the impact of UOG activity in 21 sites in northern Pennsylvania. These data were also used to design predictive machine learning models to determine the UOG impact on streams. We identified multiple biomarkers of UOG activity and contributors of antimicrobial resistance within the order Burkholderiales. Furthermore, we identified expressed antimicrobial resistance genes, land coverage, geochemistry, and specific microbes as strong predictors of UOG status. Of the predictive models constructed (n = 30), 15 had accuracies higher than expected by chance and area under the curve values above 0.70. The supervised random forest models with the highest accuracy were constructed with 16S rRNA gene profiles, metatranscriptomics active microbial composition, metatranscriptomics active antimicrobial resistance genes, land coverage, and geochemistry (n = 23). The models identified the most important features within those data sets for classifying UOG status. These findings identified specific shifts in gene presence and expression, as well as geochemical measures, that can be used to build robust models to identify impacts of UOG development.

16S rRNA↗
NASA NTRS2003

Constant Applied Force Stimulates Osteoblast Proliferation Via Matrix-Integrin-Signaling Pathways

Reduced weight-bearing caused by immobilization, bed-rest or microgravity leads to atrophy in mechanosensitive tissue such as muscle and bone. We hypothesize that bone tissue requires earth s gravity (1-g) for the maintenance of extracellular matrix, integrin, and kinase-mediated cell growth and survival pathways. We investigate the role of matrix-integrin signaling in bone cells using cell culture centrifugation to provide different levels of hypergravity mechanostimulation. The 10-50-g range we use also mimics physiological intermedullary pressure (1.2 - 5 kPa). 24 hours at 50-g increased primary rat osteoblast proliferation on collagen Type I and fibronectin, but not laminin or uncoated plastic. BrdU incorporation in primary osteoblasts over 24 h showed hypergravity increased the number of cells actively synthesizing DNA from about 60% at 1-g to over 90% at 25-g. Primary rat fibroblasts grown at 50-g (24 h) showed no proliferation increase, suggesting this is a tissue-specific phenomenon. These results suggest that the betal and alpha4 integrins may be involved. To further test this, we used osteocytic-like MLO-Y4 cells that showed increased proliferation at 1-g with stable expression of a betal integrin cytoplasmic tail and transmembrane domain construct. At 50-g, MLO-Y4/betal cells showed greater MAPK activation than MLO-Y4 vector controls, suggesting that betal integrin is involved in transducing mitogenic signals in response to hypergravity. Preliminary results also show that interfering with the alpha4 integrin in primary osteoblasts grown on fibronectin blocked the proliferation response. These results indicate that cells from mechanosensitive bone tissue can respond to gravity-generated forces, and this response involves specific matrix and integrin-dependent signaling pathways.

Vercoutere, W.↗
NASA NTRS2002

Plant adaptation to low atmospheric pressures: potential molecular responses

There is an increasing realization that it may be impossible to attain Earth normal atmospheric pressures in orbital, lunar, or Martian greenhouses, simply because the construction materials do not exist to meet the extraordinary constraints imposed by balancing high engineering requirements against high lift costs. This equation essentially dictates that NASA have in place the capability to grow plants at reduced atmospheric pressure. Yet current understanding of plant growth at low pressures is limited to just a few experiments and relatively rudimentary assessments of plant vigor and growth. The tools now exist, however, to make rapid progress toward understanding the fundamental nature of plant responses and adaptations to low pressures, and to develop strategies for mitigating detrimental effects by engineering the growth conditions or by engineering the plants themselves. The genomes of rice and the model plant Arabidopsis thaliana have recently been sequenced in their entirety, and public sector and commercial DNA chips are becoming available such that thousands of genes can be assayed at once. A fundamental understanding of plant responses and adaptation to low pressures can now be approached and translated into procedures and engineering considerations to enhance plant growth at low atmospheric pressures. In anticipation of such studies, we present here the background arguments supporting these contentions, as well as informed speculation about the kinds of molecular physiological responses that might be expected of plants in low-pressure environments.

NASA Discipline Plant Biology↗
DOE OSTI2019

Improved Stress Tolerance of Saccharomyces cerevisiae by CRISPR-Cas-Mediated Genome Evolution

In bioprocesses, a microorganism with high tolerance to various stresses would be advantageous for efficient bio-based chemical production. Yeast Saccharomyces cerevisiae has long been used in the food industry because of its safety and convenience, and genetically engineered S. cerevisiae strains have been constructed and used for the production of various bio-based chemicals. In this study, we developed a novel genome shuffling method for S. cerevisiae using CRISPR-Cas. By using this, the thermotolerant mutant strain T8-292, which can grow well at 39 °C, was successfully created. The strain also showed higher cell viability in low pH and high ethanol concentration. In addition, the differences in genome structure between mutant and parent strains were suggested by random amplified polymorphic DNA PCR method. Our genome shuffling method could be a promising strategy for improvement of various stress tolerance in S. cerevisiae.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Hidden Markov Model: a shortest unique representative approach to detect the protein toxins, virulence factors and antibiotic resistance genes

Objective: Currently, next generation sequencing (NGS) is widely used to decode potential novel or variant pathogens both in emergent outbreaks and in routine clinical practice. However, the efficient identification of novel or diverged pathogenomic compositions remains a big challenge. It is especially true for short DNA sequence fragments from NGS, since sequence similarity searching is vulnerable to false negatives or false positives, as is mismatching or matching with unrelated proteins. Therefore, this study aimed to establish a bioinformatics approach that can generate unique motif sequences for profiling searching, resulting in high specificity and sensitivity. Results: In this study, we introduced a Shortest Unique Representative Hidden Markov Model (HMM) approach to identify bacterial toxin, virulence factor (VF), and antimicrobial resistance (AR) in short sequence reads. We first construct unique representative domain sequences of toxin genes, VFs, and ARs to avoid potential false positives, and then to use HMM models to accurately identify potential toxin, VF, and AR fragments. The benchmark shows this approach can achieve relatively high specificity and sensitivity if the appropriate cutoff value is applied. Our approach can be used to recognize the protein sequences of known toxins and pathogens, identifies their common characteristics and then searches for similar sequences in other organisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Exploiting regulatory heterogeneity to systematically identify enhancers with high accuracy

Identifying functional enhancer elements in metazoan systems is a major challenge. Large-scale validation of enhancers predicted by ENCODE reveal false-positive rates of at least 70%. We used the pregrastrula-patterning network of Drosophila melanogaster to demonstrate that loss in accuracy in held-out data results from heterogeneity of functional signatures in enhancer elements. We show that at least two classes of enhancers are active during early Drosophila embryogenesis and that by focusing on a single, relatively homogeneous class of elements, greater than 98% prediction accuracy can be achieved in a balanced, completely held-out test set. The class of well-predicted elements is composed predominantly of enhancers driving multistage segmentation patterns, which we designate segmentation driving enhancers (SDE). Prediction is driven by the DNA occupancy of early developmental transcription factors, with almost no additional power derived from histone modifications. We further show that improved accuracy is not a property of a particular prediction method: after conditioning on the SDE set, naïve Bayes and logistic regression perform as well as more sophisticated tools. Applying this method to a genome-wide scan, we predict 1,640 SDEs that cover 1.6% of the genome. An analysis of 32 SDEs using whole-mount embryonic imaging of stably integrated reporter constructs chosen throughout our prediction rank-list showed >90% drove expression patterns. We achieved 86.7% precision on a genome-wide scan, with an estimated recall of at least 98%, indicating high accuracy and completeness in annotating this class of functional elements.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Expanded encyclopaedias of DNA elements in the human and mouse genomes

The human and mouse genomes contain instructions that specify RNAs and proteins and govern the timing, magnitude, and cellular context of their production. To better delineate these elements, phase III of the Encyclopedia of DNA Elements (ENCODE) Project has expanded analysis of the cell and tissue repertoires of RNA transcription, chromatin structure and modification, DNA methylation, chromatin looping, and occupancy by transcription factors and RNA-binding proteins. Here we summarize these efforts, which have produced 5,992 new experimental datasets, including systematic determinations across mouse fetal development. All data are available through the ENCODE data portal (https://www.encodeproject.org), including phase II ENCODE1 and Roadmap Epigenomics2 data. We have developed a registry of 926,535 human and 339,815 mouse candidate cis-regulatory elements, covering 7.9 and 3.4% of their respective genomes, by integrating selected datatypes associated with gene regulation, and constructed a web-based server (SCREEN; http://screen.encodeproject.org) to provide flexible, user-defined access to this resource. Collectively, the ENCODE data and registry provide an expansive resource for the scientific community to build a better understanding of the organization and function of the human and mouse genomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Expanding the genetic toolkit: adenine and cytosine base editors for gene disruption in Aspergillus niger

Despite revolutionizing fungal genetic engineering, conventional CRISPR/Cas9-mediated knockouts rely on DNA double-strand breaks (DSBs), which can cause unwanted insertions and deletions, chromosomal abnormalities, and cytotoxicity. Base editors such as adenine base editors (ABEs), which convert A‧T to G‧C, and cytosine base editors (CBEs), which convert C‧G to T‧A, offer a safer alternative by enabling predictable, target-specific single-nucleotide changes without introducing DSBs. To overcome the limitations of traditional genome editing in filamentous fungi, we developed efficient base-editing systems in Aspergillus niger . For the first time, we constructed an ABE in A. niger , achieving up to 80% editing efficiency and inducing predictable A-to-G mutations at the intended intron sites, disrupting gene function through mRNA mis-splicing. We also developed a highly efficient CBE system, capable of introducing premature stop codons with 50–100% efficiency. To broaden the editing scope, we implemented a Cas9-NG variant recognizing a relaxed PAM sequence requiring only a single guanine (G), enabling editing at start codons and splice sites. Leveraging this expanded scope, we established gene disruption approaches by targeting start codons via ABE-mediated A-to-G conversions (ATG-to-GTG and ATG-to-ACG) and CBE-mediated C-to-T conversion (ATG-to-ATA). Additionally, our base-editing systems enable multiplex gRNA delivery and marker-free editing of multiple genes. Collectively, the scope-expanding strategies increase the number of genes targetable for disruption by base-editing in A. niger by 26.3% and enable near-complete coverage of 96% of the coding genes. Overall, this work demonstrates the potential of ABE and CBE systems as versatile, efficient, and safer alternatives to DSBs-based gene disruption in filamentous fungi.

Aspergillus↗
NASA NTRS1997

17beta-estradiol potently suppresses cAMP-induced insulin-like growth factor-I gene activation in primary rat osteoblast cultures

Insulin-like growth factor-I (IGF-I) is a key factor in bone remodeling. In osteoblasts, IGF-I synthesis is enhanced by parathyroid hormone and prostaglandin E2 (PGE2) through cAMP-activated protein kinase. In rats, estrogen loss after ovariectomy leads to a rise in serum IGF-I and an increase in bone remodeling, both of which are reversed by estrogen treatment. To examine estrogen-dependent regulation of IGF-I expression at the molecular level, primary fetal rat osteoblasts were co-transfected with the estrogen receptor (hER, to ensure active ER expression), and luciferase reporter plasmids controlled by promoter 1 of the rat IGF-I gene (IGF-I P1), used exclusively in these cells. As reported, 1 microM PGE2 increased IGF-I P1 activity by 5-fold. 17beta-Estradiol alone had no effect, but dose-dependently suppressed the stimulatory effect of PGE2 by up to 90% (ED50 approximately 0.1 nM). This occurred within 3 h, persisted for at least 16 h, required ER, and appeared specific, since 17alpha-estradiol was 100-300-fold less effective. By contrast, 17beta-estradiol stimulated estrogen response element (ERE)-dependent reporter expression by up to 10-fold. 17beta-Estradiol also suppressed an IGF-I P1 construct retaining only minimal promoter sequence required for cAMP-dependent gene activation, but did not affect the 60-fold increase in cAMP induced by PGE2. There is no consensus ERE in rat IGF-I P1, suggesting novel downstream interactions in the cAMP pathway that normally enhances IGF-I expression in skeletal cells. To explore this, nuclear extract from osteoblasts expressing hER were examined by electrophoretic mobility shift assay using the atypical cAMP response element in IGF-I P1. Estrogen alone did not cause DNA-protein binding, while PGE2 induced a characteristic gel shift complex. Co-treatment with both hormones caused a gel shift greatly diminished in intensity, consistent with their combined effects on IGF-I promoter activity. Nonetheless, hER did not bind IGF-I cAMP response element or any adjacent sequences. These results provide new molecular evidence that estrogen may temper the biological effects of hormones acting through cAMP to regulate skeletal IGF-I expression and activity.

Non-NASA Center↗