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At least 109 records · Page 6

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NASA NTRS2017

Identification of Novel Desiccation-Tolerant S. cerevisiae Strains for Deep Space Biosensors

NASA's BioSentinel mission, a secondary payload that will fly on the Space Launch System's first Exploration Mission (EM-1), utilizes the budding yeast S. cerevisiae to study the biological response to the deep space radiation environment. Yeast samples are desiccated prior to launch to suspend growth and metabolism while the spacecraft travels to its target heliocentric orbit beyond Low Earth Orbit. Each sample is then rehydrated at the desired time points to reactivate the cells. A major risk in this mission is the loss of cell viability that occurs in the recovery period following the desiccation and rehydration process. Cell survival is essential for the detection of the biological response to features in the deep space environment, including ionizing radiation. The aim of this study is to mitigate viable cell loss in future biosensors by identifying mutations and genes that confer tolerance to desiccation stress in rad51, a radiation-sensitive yeast strain. We initiated a screen for desiccation-tolerance after rehydrating cells that were desiccated for three years, and selected various clones exhibiting robust growth. To verify retention of radiation sensitivity in the isolated clones - a crucial feature for a successful biosensor - we exposed them to ionizing radiation. Finally, to elucidate the genetic and molecular bases for observed desiccation-tolerance, we will perform whole-genome sequencing of those rad51 clones that exhibit both robust growth and radiation sensitivity following desiccation. The identification and characterization of desiccation-tolerant strains will allow us to engineer a biological model that will be resilient in face of the challenges of the deep space environment, and will thus ensure the experimental success of future biosensor missions.

S. cerevisiae↗

NASA NTRS2017

Identification of Novel Desiccation-Tolerant S. cerevisiae Strains for Deep Space Biosensors

NASA's BioSentinel mission, a secondary payload that will fly on the Space Launch Systems first Exploration Mission (EM-1), utilizes the budding yeast S. cerevisiae to study the biological response to the deep space radiation environment. Yeast samples are desiccated prior to launch to suspend growth and metabolism while the spacecraft travels to its target heliocentric orbit beyond Low Earth Orbit. Each sample is then rehydrated at the desired time points to reactivate the cells. A major risk in this mission is the loss of cell viability that occurs in the recovery period following the desiccation and rehydration process. Cell survival is essential for the detection of the biological response to features in the deep space environment, including ionizing radiation.The aim of this study is to mitigate viable cell loss in future biosensors by identifying mutations and genes that confer tolerance to desiccation stress in rad51, a radiation-sensitive yeast strain. We initiated a screen for desiccation-tolerance after rehydrating cells that were desiccated for three years, and selected various clones exhibiting robust growth. To verify retention of radiation sensitivity in the isolated clonesa crucial feature for a successful biosensorwe exposed them to ionizing radiation. Finally, to elucidate the genetic and molecular bases for observed desiccation-tolerance, we will perform whole-genome sequencing of those rad51 clones that exhibit both robust growth and radiation sensitivity following desiccation. The identification and characterization of desiccation-tolerant strains will allow us to engineer a biological model that will be resilient in face of the challenges of the deep space environment, and will thus ensure the experimental success of future biosensor missions.

S. cerevisiae↗

NASA NTRS2024

Space Algae-2 Ground and Lunar Analog Studies in Preparation for Long-Duration Propagation of Cyanobacteria in Spaceflight

There are numerous applications for microalgae in spaceflight missions and on Earth, such as oxygen production, carbon dioxide removal, nutrition, wastewater processing, and biofuel production. Space Algae-2 aims to test the genetic stability of Arthrospira platensis, commonly known as spirulina, during six-months of continuous culture on the International Space Station. Long-duration exposure to ionizing radiation and microgravity may impact growth, nutrient composition, and genetic stability. The high protein, vitamin, antioxidant content, and radiation resistance make spirulina a promising candidate for bioregenerative life support systems. A concept of operations was developed to grow and harvest algal biomass in space. Preflight testing experiments optimized conditions for an extended growth period in a gas permeable bioreactor bag. Preflight and post-harvest storage methods were developed in addition to a novel cryopreservation method. After sample return, multi-omics analysis will be conducted to determine the mutation rate, gene expression, and the protein and metabolite profile. The concept of operations for Space Algae-2 was tested during a lunar mission simulation within a semi-controlled environment. During a six-day lunar analog mission at the Hawai’i Space Exploration Analog and Simulation (HI-SEAS) A. platensis was successfully grown using flight-like hardware. The cyanobacteria were harvested and used to supplement bread as an example of spirulina biomass utilization. Overall, the data collected from Space Algae-2 will inform potential bioengineering of spirulina for space and terrestrial applications.

Algae↗

NASA NTRSDate not supplied

Space Algae-2 Ground and Lunar Analog Studies in Preparation for Long-Duration Propagation of Cyanobacteria in Spaceflight

There are numerous applications for microalgae in spaceflight missions and on Earth, such as, oxygen production, carbon dioxide removal, nutrition, wastewater processing, and biofuel production. Space Algae-2 aims to test the genetic stability of Arthrospira platensis, commonly known as spirulina, during six-months of continuous culture in spaceflight on the International Space Station. Long-duration exposure to ionizing radiation and microgravity may impact growth, nutrient composition, and genetic stability. The high protein, vitamin, antioxidant content, and radiation resistance make spirulina a promising candidate for bioregenerative life support systems during long-duration missions. A concept of operations was developed to grow and harvest algal biomass in space. Preflight testing experiments were conducted to optimize conditions for an extended growth period in a gas permeable bioreactor bag. Preflight and post-harvest storage methods were developed in addition to a novel cryopreservation method. After sample return, multi-omics analysis will be conducted to determine the mutation rate, gene expression, and protein and metabolite profile. The concept of operations for Space Algae-2 was tested at HI-SEAS (Hawai’i Space Exploration Analog and Simulation) during a six-day lunar analog mission (EMMIHS23, EuroMoonMars, International MoonBase Alliance, HI-SEAS, 2023). A. platensis was grown in the semi-controlled environment using flight-like hardware and solar powered LED lights. Then, the biomass was harvested and used to supplement bread as an example of A. platensis utilization. Overall, the data collected from Space Algae-2 will inform potential bioengineering of spirulina for space and terrestrial applications.

Algae↗

NASA NTRSDate not supplied

Space Algae-2 Ground and Lunar Analog Studies in Preparation for Long-Duration Propagation of Cyanobacteria in Spaceflight

Algae↗

DOE OSTI2023

Mutations in the Second Alternative Oxidase Gene: A New Approach to Group Aspergillus niger Strains

Alternative oxidase is a terminal oxidase in the branched mitochondrial electron transport chain of most fungi including Aspergillus niger (subgenus Circumdati, section Nigri). A second, paralogous aox gene (aoxB) is extant in some A. niger isolates but also present in two divergent species of the subgenus Nidulantes—A. calidoustus and A. implicatus—as well as in Penicillium swiecickii. Black aspergilli are cosmopolitan opportunistic fungi that can cause diverse mycoses and acute aspergillosis in immunocompromised individuals. Amongst the approximately 75 genome-sequenced A. niger strains, aoxB features considerable sequence variation. Five mutations were identified that rationally affect transcription or function or terminally modify the gene product. One mutant allele that occurs in CBS 513.88 and A. niger neotype strain CBS 554.65 involves a chromosomal deletion that removes exon 1 and intron 1 from aoxB. Another aoxB allele results from retrotransposon integration. Three other alleles result from point mutations: a missense mutation of the start codon, a frameshift, and a nonsense mutation. A. niger strain ATCC 1015 has a full-length aoxB gene. The A. niger sensu stricto complex can thus be subdivided into six taxa according to extant aoxB allele, which may facilitate rapid and accurate identification of individual species

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Targeted mutagenesis with sequence–specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects

Many of the world's most important crops are polyploid. The presence of more than two sets of chromosomes within their nuclei and frequently aberrant reproductive biology in polyploids present obstacles to conventional breeding. The presence of a larger number of homoeologous copies of each gene makes random mutation breeding a daunting task for polyploids. Genome editing has revolutionized improvement of polyploid crops as multiple gene copies and/or alleles can be edited simultaneously while preserving the key attributes of elite cultivars. Most genome–editing platforms employ sequence–specific nucleases (SSNs) to generate DNA double–stranded breaks at their target gene. Such DNA breaks are typically repaired via the error–prone nonhomologous end–joining process, which often leads to frame shift mutations, causing loss of gene function. Genome editing has enhanced the disease resistance, yield components, and end–use quality of polyploid crops. However, identification of candidate targets, genotyping, and requirement of high mutagenesis efficiency remain bottlenecks for targeted mutagenesis in polyploids. In this review, we will survey the tremendous progress of SSN–mediated targeted mutagenesis in polyploid crop improvement, discuss its challenges, and identify optimizations needed to sustain further progress.

60 APPLIED LIFE SCIENCES↗

NASA NTRS1998

The Toll pathway is required in the epidermis for muscle development in the Drosophila embryo

The Toll signaling pathway functions in several Drosophila processes, including dorsal-ventral pattern formation and the immune response. Here, we demonstrate that this pathway is required in the epidermis for proper muscle development. Previously, we showed that the zygotic Toll protein is necessary for normal muscle development; in the absence of zygotic Toll, close to 50% of hemisegments have muscle patterning defects consisting of missing, duplicated and misinserted muscle fibers (Halfon, M.S., Hashimoto, C., and Keshishian, H., Dev. Biol. 169, 151-167, 1995). We have now also analyzed the requirements for easter, spatzle, tube, and pelle, all of which function in the Toll-mediated dorsal-ventral patterning pathway. We find that spatzle, tube, and pelle, but not easter, are necessary for muscle development. Mutations in these genes give a phenotype identical to that seen in Toll mutants, suggesting that elements of the same pathway used for Toll signaling in dorsal-ventral development are used during muscle development. By expressing the Toll cDNA under the control of distinct Toll enhancer elements in Toll mutant flies, we have examined the spatial requirements for Toll expression during muscle development. Expression of Toll in a subset of epidermal cells that includes the epidermal muscle attachment cells, but not Toll expression in the musculature, is necessary for proper muscle development. Our results suggest that signals received by the epidermis early during muscle development are an important part of the muscle patterning process.

Non-NASA Center↗

DOE OSTI2026

Synthetic overlapping genes stabilize genetic systems

Overlapping genes—wherein two different proteins are translated from alternative reading frames of the same DNA sequence—provide a means to stabilize an engineered gene by directly linking its evolutionary fate with that of an overlapping gene. However, creating overlapping gene pairs is challenging, as it requires redesigning both protein products to accommodate overlap constraints. Here, we present a new “overlapping, alternate-frame insertion” (OAFI) method for creating synthetic overlapping genes by inserting an “inner” gene, encoded in an alternate frame, into a flexible region of an “outer” gene. Using OAFI, we create new overlapping gene pairs of genetic reporters and bacterial toxins within an antibiotic resistance gene. We show that both the inner and outer genes retain function despite redesign, with translation of the inner gene influenced by its overlap position in the outer gene. Importantly, we show that, despite these inner gene sequences not contributing to outer gene function, selection for the outer gene alters the permitted inactivating mutations in the inner gene, and that overlapping toxins can restrict horizontal gene transfer of the antibiotic resistance gene. Overall, OAFI offers a versatile tool for synthetic biology, expanding the applications of overlapping genes in gene stabilization and biocontainment.

Biological and medical sciences↗

DOE OSTI2022

Knockout of ABC transporter gene ABCA2 confers resistance to Bt toxin Cry2Ab in Helicoverpa zea

Evolution of pest resistance reduces the benefits of widely cultivated genetically engineered crops that produce insecticidal proteins derived from Bacillus thuringiensis (Bt). Better understanding of the genetic basis of pest resistance to Bt crops is needed to monitor, manage, and counter resistance. Previous work shows that in several lepidopterans, resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2. The results here show that mutations introduced by CRISPR/Cas9 gene editing in the Helicoverpa zea (corn earworm or bollworm) gene encoding ABCA2 (HzABCA2) can cause resistance to Cry2Ab. Disruptive mutations in HzABCA2 facilitated the creation of two Cry2Ab-resistant strains. A multiple concentration bioassay with one of these strains revealed it had > 200-fold resistance to Cry2Ab relative to its parental susceptible strain. All Cry2Ab-resistant individuals tested had disruptive mutations in HzABCA2. We identified five disruptive mutations in HzABCA2 gDNA. The most common mutation was a 4-bp deletion in the expected Cas9 guide RNA target site. The results here indicate that HzABCA2 is a leading candidate for monitoring Cry2Ab resistance in field populations of H. zea.

60 APPLIED LIFE SCIENCES↗

NASA NTRS2001

Loss of chromosomal integrity in human mammary epithelial cells subsequent to escape from senescence

The genomic changes that foster cancer can be either genetic or epigenetic in nature. Early studies focused on genetic changes and how mutational events contribute to changes in gene expression. These point mutations, deletions and amplifications are known to activate oncogenes and inactivate tumor suppressor genes. More recently, multiple epigenetic changes that can have a profound effect on carcinogenesis have been identified. These epigenetic events, such as the methylation of promoter sequences in genes, are under active investigation. In this review we will describe a methylation event that occurs during the propagation of human mammary epithelial cells (HMEC) in culture and detail the accompanying genetic alterations that have been observed.

Review↗

DOE OSTI2021

The Sorghum (Sorghum bicolor) Brown Midrib 30 Gene Encodes a Chalcone Isomerase Required for Cell Wall Lignification

In sorghum ( Sorghum bicolor ) and other C 4 grasses, brown midrib ( bmr ) mutants have long been associated with plants impaired in their ability to synthesize lignin. The brown midrib 30 ( Bmr30 ) gene, identified using a bulk segregant analysis and next-generation sequencing, was determined to encode a chalcone isomerase (CHI). Two independent mutations within this gene confirmed that loss of its function was responsible for the brown leaf midrib phenotype and reduced lignin concentration. Loss of the Bmr30 gene function, as shown by histochemical staining of leaf midrib and stalk sections, resulted in altered cell wall composition. In the bmr30 mutants, CHI activity was drastically reduced, and the accumulation of total flavonoids and total anthocyanins was impaired, which is consistent with its function in flavonoid biosynthesis. The level of the flavone lignin monomer tricin was reduced 20-fold in the stem relative to wild type, and to undetectable levels in the leaf tissue of the mutants. The bmr30 mutant, therefore, harbors a mutation in a phenylpropanoid biosynthetic gene that is key to the interconnection between flavonoids and monolignols, both of which are utilized for lignin synthesis in the grasses.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS2002

Mutations in the dopamine beta-hydroxylase gene are associated with human norepinephrine deficiency

Norepinephrine (NE), a key neurotransmitter of the central and peripheral nervous systems, is synthesized by dopamine beta-hydroxylase (DBH) that catalyzes oxidation of dopamine (DA) to NE. NE deficiency is a congenital disorder of unknown etiology, in which affected patients suffer profound autonomic failure. Biochemical features of the syndrome include undetectable tissue and circulating levels of NE and epinephrine, elevated levels of DA, and undetectable levels of DBH. Here, we report identification of seven novel variants including four potentially pathogenic mutations in the human DBH gene (OMIM 223360) from analysis of two unrelated patients and their families. Both patients are compound heterozygotes for variants affecting expression of DBH protein. Each carries one copy of a T-->C transversion in the splice donor site of DBH intron 1, creating a premature stop codon. In patient 1, there is a missense mutation in DBH exon 2. Patient 2 carries missense mutations in exons 1 and 6 residing in cis. We propose that NE deficiency is an autosomal recessive disorder resulting from heterogeneous molecular lesions at DBH. Copyright 2002 Wiley-Liss, Inc.

Case Reports↗

DOE OSTI2020

Mutation of the d-hordein gene by RNA-guided Cas9 targeted editing reducing the grain size and changing grain compositions in barley

Not Available

Yang, Qiang↗

DOE OSTI2021

Disease modeling for Mucopolysaccharidosis type IIIB using patient derived induced pluripotent stem cells

Highlights: • Establishment of a patient-specific neuronal model for Mucopolysaccharidosis type IIIB using iPSCs. • Lysosomes enlarged and secondary lipid accumulated in Mucopolysaccharidosis type IIIB iPSC-derived neuronal cells. • Recombinant NAGLU, δ-tocopherol, and HPBCD reduced the disease phenotypes in MPS IIIB iPSC-derived neuronal cells. Mucopolysaccharidosis type IIIB (MPS IIIB) is a lysosomal disease caused by mutations in the NAGLU gene encoding α-N-acetylglucosaminidase (NAGLU) which degrades heparan sulfate in lysosomes. Deficiency in NAGLU results in lysosomal accumulation of glycosaminoglycans (GAGs) and neurological symptoms. Currently, there is no effective treatment or cure for this disease. In this study, induced pluripotent stem cell lines were established from two MPS IIIB patient fibroblast lines and differentiated into neural stem cells and neurons. MPS IIIB neural stem cells exhibited NAGLU deficiency accompanied with GAG accumulation, as well as lysosomal enlargement and secondary lipid accumulation. Treatments with recombinant NAGLU, δ-tocopherol, and 2-hydroxypropyl-b-cyclodextrin significantly reduced the disease phenotypes in these cells. These results indicate the MPS IIIB neural stem cells and neurons have the disease relevant phenotype and can be used as a cell-based disease model system for evaluation of drug efficacy and compound screening for drug development.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2022

Optimizing neural networks

A system and method design and optimize neural networks. The system and method include a data store that stores a plurality of gene vectors that represent diverse and distinct neural networks and an evaluation queue stored with the plurality of gene vectors. Secondary nodes construct, train, and evaluate the neural network and automatically render a plurality of fitness values asynchronously. A primary node executes a gene amplification on a select plurality of gene vectors, a crossing-over of the amplified gene vectors, and a mutation of the crossing-over gene vectors automatically and asynchronously, which are then transmitted to the evaluation queue. The process continuously repeats itself by processing the gene vectors inserted into the evaluation queue until a fitness level is reached, a network's accuracy level plateaus, a processing time period expires, or when some stopping condition or performance metric is met or exceeded.

97 MATHEMATICS AND COMPUTING↗

NASA NTRS1971

Comparisons of the polypeptide chains of globins.

Discussion of the amino acid differences and minimum base differences per codon due to mutations which took place during divergent evolution of vertebrates from a common ancestral gene. The ?random mutation model' of evolution is examined by comparing a carp alpha Hb chain and six mammalian alpha Hb chains in terms of the genetic code. The occurrence of recognizable three-base changes is analyzed and a summary of the distribution of changes in the hemoglobin and myoglobin chains is given for 148 sites.

Jukes, T. H.↗

DOE OSTI2021

Simple, efficient and open-source CRISPR/Cas9 strategy for multi-site genome editing in Populus tremula × alba

Although the CRISPR/Cas9 system has been successfully used for crop breeding, its application remains limited in forest trees. Here, we describe an efficient gene editing strategy for hybrid poplar, (Populus tremula × alba INRA clone 717-1B4) based on the Golden Gate MoClo cloning. To test the system efficiency for generating single gene mutants, two single guide RNAs (sgRNAs) were designed and incorporated into the MoClo Tool Kit level 2 binary vector with the Cas9 expression cassette to mutate the SHORT ROOT (SHR) gene. Moreover, we also tested its efficiency for introducing mutations in two genes simultaneously by expressing one sgRNA targeting a single site of the YUC4 gene and the other sgRNA targeting the PLT1 gene. For a robust evaluation of the approach, we repeated the strategy to target the LBD12 and LBD4 genes simultaneously, using an independent construct. We generated hairy roots by Agrobacterium rhizogenes-mediated leaf transformation. Sequencing results confirmed the CRISPR/Cas9-mediated mutation in the targeted sites of PtaSHR. Biallelic and homozygous knockout mutations were detected. A deletion spanning both target sites and small insertions/deletions were the most common mutations. Out of the 22 SHR alleles sequenced, 21 were mutated. The phenotype’s characterization showed that transgenic roots with biallelic mutations for the SHR gene lacked a defined endodermal single cell layer, suggesting a conserved gene function similar to its homolog in Arabidopsis Arabidopsis thaliana (L.) Heynh. Sequencing results also revealed the high efficiency of the system for generating double mutants. Biallelic mutations for both genes in the yuc4/plt1 and lbd12/lbd4 roots were detected in three (yuc4/plt1) and two (lbd12/lbd4) out of four transgenic roots evaluated. A small deletion or a single nucleotide insertion at the single target site was the most common mutations. This CRISPR/Cas9 strategy arises as a rapid, simple and standardized gene-editing tool to evaluate the gene role in essential developmental programs such as radial cell differentiation of poplar roots.

59 BASIC BIOLOGICAL SCIENCES↗