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At least 793 records · Page 44
All sourcesNASA NTRSDOE OSTI
DOE OSTI2014

The Microbial Detection Array for Detection of Emerging Viruses in Clinical Samples - A Useful Panmicrobial Diagnostic Tool

Emerging viruses are usually endemic to tropical and sub-tropical regions of the world, but increased global travel, climate change and changes in lifestyle are believed to contribute to the spread of these viruses into new regions. Many of these viruses cause similar disease symptoms as other emerging viruses or common infections, making these unexpected pathogens difficult to diagnose. Broad-spectrum pathogen detection microarrays containing probes for all sequenced viruses and bacteria can provide rapid identification of viruses, guiding decisions about treatment and appropriate case management. We report a modified Whole Transcriptome Amplification (WTA) method that increases unbiased amplification, particular of RNA viruses. Using this modified WTA method, we tested the specificity and sensitivity of the Lawrence Livermore Microbial Detection Array (LLMDA) against a wide range of emerging viruses present in both nonclinical and clinical samples using two different microarray data analysis methods.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRSDate not supplied

My Journey to NASA and role as a (Molecular) Biologist…

My journey to NASA started in undergrad, where I performed microbiology research and became hooked in the STEM fields. From there I earned a Ph.D. in Biochemistry and Molecular Biology from Mayo Graduate school where I performed Pancreatic Cancer research and then went on to perform postdoctoral research in Developmental Biology at the University of Miami. Trained as a biologist, I landed my first job at NASA as a mission scientist on the Rodent Research project. As a mission scientist, I led science activities associated with development, flight, and post-flight analysis of rodent research missions on the ISS in consultation with project engineers, operational specialists, mission PI(s), and mission Implementation Partner(s). I also assessed the feasibility of scientific experiments as a technical and scientific expert in rodent research on the ISS. Eager to learn more about the molecular changes that occur as a result of spaceflight exposure, I transitioned to the NASA GeneLab project where I eventually became the data processing lead. NASA’s GeneLab helps scientists understand how the fundamental building blocks of life – DNA, RNA, proteins, and metabolites – change from exposure to the space environment including microgravity and cosmic radiation exposure. GeneLab does so by providing fully coordinated epigenomics, genomics, transcriptomics, proteomics, and metabolomics data (collectively known as omics data) alongside essential metadata describing each spaceflight and space-relevant experiment. To maximize the intelligibility of these data, particularly for users with limited bioinformatics knowledge, GeneLab has started processing and analyzing these datasets to generate differential gene expression data and identify biological and physiological pathways that are dysregulated as a result of spaceflight. The user interface was designed to be accessible to a broad variety of users, including high school and college students who can use it to learn about omics data analysis and space biology. During the CA Space Grant Women in STEM Panel event, I will provide an overview of my journey to NASA including my work at NASA on both the Rodent Research and GeneLab projects. I will conclude by providing resources for opportunities to work with GeneLab and NASA at large followed by links to programs designed specifically to engage women in STEM fields.

Amanda M Saravia-Butler↗
NASA NTRSDate not supplied

My Journey to NASA and Role as a (Molecular) Biologist…

My journey to NASA started in undergrad, where I performed microbiology research and became hooked in the STEM fields. From there I earned a Ph.D. in Biochemistry and Molecular Biology from Mayo Graduate school where I performed Pancreatic Cancer research and then went on to perform postdoctoral research in Developmental Biology at the University of Miami. Trained as a biologist, I landed my first job at NASA as a mission scientist on the Rodent Research project. As a mission scientist, I led science activities associated with development, flight, and post-flight analysis of rodent research missions on the ISS in consultation with project engineers, operational specialists, mission PI(s), and mission Implementation Partner(s). I also assessed the feasibility of scientific experiments as a technical and scientific expert in rodent research on the ISS. Eager to learn more about the molecular changes that occur as a result of spaceflight exposure, I transitioned to the NASA GeneLab project where I eventually became the data processing lead. NASA’s GeneLab helps scientists understand how the fundamental building blocks of life – DNA, RNA, proteins, and metabolites – change from exposure to the space environment including microgravity and cosmic radiation exposure. GeneLab does so by providing fully coordinated epigenomics, genomics, transcriptomics, proteomics, and metabolomics data (collectively known as omics data) alongside essential metadata describing each spaceflight and space-relevant experiment. To maximize the intelligibility of these data, particularly for users with limited bioinformatics knowledge, GeneLab has started processing and analyzing these datasets to generate differential gene expression data and identify biological and physiological pathways that are dysregulated as a result of spaceflight. The user interface was designed to be accessible to a broad variety of users, including high school and college students who can use it to learn about omics data analysis and space biology. During the NASA Ames ECN at Lincoln HS Virtual Panel event, I will provide an overview of my journey to NASA including my work at NASA on both the Rodent Research and GeneLab projects. I will conclude by providing resources for opportunities to work with GeneLab and NASA at large followed by links to programs designed specifically to engage women in STEM fields.

Amanda M Saravia-Butler↗
NASA NTRSDate not supplied

Working at NASA as a (Molecular) Biologist...

Trained as a biologist, I landed my first job at NASA as a mission scientist on the Rodent Research project. As a mission scientist, I led science activities associated with development, flight, and post-flight analysis of rodent research missions on the ISS in consultation with project engineers, operational specialists, mission PI(s), and mission Implementation Partner(s). I also assessed the feasibility of scientific experiments as a technical and scientific expert in rodent research on the ISS. Eager to learn more about the molecular changes that occur as a result of spaceflight exposure, I transitioned to the NASA GeneLab project where I eventually became the data processing lead. NASA’s GeneLab helps scientists understand how the fundamental building blocks of life – DNA, RNA, proteins, and metabolites – change from exposure to the space environment including microgravity and cosmic radiation exposure. GeneLab does so by providing fully coordinated epigenomics, genomics, transcriptomics, proteomics, and metabolomics data (collectively known as omics data) alongside essential metadata describing each spaceflight and space-relevant experiment. In order to maximize the intelligibility of these data, particularly for users with limited bioinformatics knowledge, GeneLab has started processing and analyzing these datasets to generate differential gene expression data and identify biological and physiological pathways that are dysregulated as a result of spaceflight. The user interface was designed to be accessible to a broad variety of users, including high school and college students who can use it to learn about omics data analysis and space biology. During the CA Space Grant NASA Panel event, I will provide an overview of my work at NASA on both the Rodent Research and GeneLab projects and will conclude by providing resources for opportunities to work with GeneLab and NASA at large.

Amanda M Saravia-Butler↗
DOE OSTI2017

Gene expression analysis of whole blood RNA from pigs infected with low and high pathogenic African swine fever viruses

African swine fever virus (ASFV) is a macrophage-tropic virus responsible for ASF, a transboundary disease that threatens swine production world-wide. Since there are no vaccines available to control ASF after an outbreak, obtaining an understanding of the virus-host interaction is important for developing new intervention strategies. In this study, a whole transcriptomic RNA-Seq method was used to characterize differentially expressed genes in pigs infected with a low pathogenic ASFV isolate, OUR T88/3 (OURT), or the highly pathogenic Georgia 2007/1 (GRG). After infection, pigs infected with OURT showed no or few clinical signs; whereas, GRG produced clinical signs consistent with acute ASF. RNA-Seq detected the expression of ASFV genes from the whole blood of the GRG, but not the OURT pigs, consistent with the pathotypes of these strains and the replication of GRG in circulating monocytes. Even though GRG and OURT possess different pathogenic properties, there was significant overlap in the most upregulated host genes. A small number of differentially expressed microRNAs were also detected in GRG and OURT pigs. These data confirm previous studies describing the response of macrophages and lymphocytes to ASFV infection, as well as reveal unique gene pathways upregulated in response to infection with GRG.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Identification of a novel nematotoxic protein by challenging the model mushroom Coprinopsis cinerea with a fungivorous nematode

The dung of herbivores, the natural habitat of the model mushroom Coprinopsis cinerea, is a nutrient-rich but also very competitive environment for a saprophytic fungus. Previously we showed that C. cinerea expresses constitutive, tissue-specific armories against antagonists such as animal predators and bacterial competitors. In order to dissect the inducible armories against such antagonists, we sequenced the poly(A)-positive transcriptome of C. cinerea vegetative mycelium upon challenge with fungivorous and bacterivorous nematodes, Gram-negative and Gram-positive bacteria and mechanical damage. As a response to the fungivorous nematode Aphelenchus avenae, C. cinerea was found to specifically induce the transcription of several genes encoding previously characterized nematotoxic lectins. In addition, a previously not characterized gene encoding a cytoplasmic protein with several predicted Ricin B-fold domains, was found to be strongly up-regulated under this condition. Functional analysis of the recombinant protein revealed a high toxicity towards the bacterivorous nematode Caenorhabditis elegans. Challenge of the mycelium with A. avenae also lead to the induction of several genes encoding putative antibacterial proteins. Some of these genes were also induced upon challenge of the mycelium with the bacteria Escherichia coli and Bacillus subtilis. Lastly, these results suggest that fungi have the ability to induce specific innate defense responses similar to plants and animals.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Identification and molecular cloning of novel transcripts of the human kallikrein-related peptidase 10 (KLK10) gene using next-generation sequencing

Tissue kallikrein and kallikrein-related peptidases (KLKs) form the largest group of serine proteases in the human genome, sharing many structural and functional characteristics. Multiple alternative transcripts have been reported for the most human KLK genes, while many of them are aberrantly expressed in various malignancies, thus possessing significant prognostic and/or diagnostic value. Alternative splicing of cancer-related genes is a common cellular mechanism accounting for cancer cell transcriptome complexity, as it affects cell cycle control, proliferation, apoptosis, invasion, and metastasis. In this study, we describe the identification and molecular cloning of eight novel transcripts of the human KLK10 gene using 3′ rapid amplification of cDNA ends (3′ RACE) and next-generation sequencing (NGS), as well as their expression analysis in a wide panel of cell lines, originating from several distinct cancerous and normal tissues. Bioinformatic analysis revealed that the novel KLK10 transcripts contain new alternative splicing events between already annotated exons as well as novel exons. In addition, investigation of their expression profile in a wide panel of cell lines was performed with nested RT-PCR using variant-specific pairs of primers. Since many KLK mRNA transcripts possess clinical value, these newly discovered alternatively spliced KLK10 transcripts appear as new potential biomarkers for diagnostic and/or prognostic purposes or as targets for therapeutic strategies. - Highlights: • NGS was used to identify novel transcripts of the human KLK10 gene. • 8 novel KLK10 transcripts were identified. • A novel 3′UTR was detected and characterized. • The expression profiles of all 8 novel KLK10 transcripts were identified.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2012

Soybean Knowledge Base (SoyKB): a Web Resource for Soybean Translational Genomics

Background: Soybean Knowledge Base (SoyKB) is a comprehensive all-inclusive web resource for soybean translational genomics. SoyKB is designed to handle the management and integration of soybean genomics, transcriptomics, proteomics and metabolomics data along with annotation of gene function and biological pathway. It contains information on four entities, namely genes, microRNAs, metabolites and single nucleotide polymorphisms (SNPs). Methods: SoyKB has many useful tools such as Affymetrix probe ID search, gene family search, multiple gene/ metabolite search supporting co-expression analysis, and protein 3D structure viewer as well as download and upload capacity for experimental data and annotations. It has four tiers of registration, which control different levels of access to public and private data. It allows users of certain levels to share their expertise by adding comments to the data. It has a user-friendly web interface together with genome browser and pathway viewer, which display data in an intuitive manner to the soybean researchers, producers and consumers. Conclusions: SoyKB addresses the increasing need of the soybean research community to have a one-stop-shop functional and translational omics web resource for information retrieval and analysis in a user-friendly way. SoyKB can be publicly accessed at http://soykb.org/.

Environmental Molecular Sciences Laboratory↗
DOE OSTI2023

Guided construction of single cell reference for human and mouse lung

Accurate cell type identification is a key and rate-limiting step in single-cell data analysis. Single-cell references with comprehensive cell types, reproducible and functionally validated cell identities, and common nomenclatures are much needed by the research community for automated cell type annotation, data integration, and data sharing. Here, we develop a computational pipeline utilizing the LungMAP CellCards as a dictionary to consolidate single-cell transcriptomic datasets of 104 human lungs and 17 mouse lung samples to construct LungMAP single-cell reference (CellRef) for both normal human and mouse lungs. CellRefs define 48 human and 40 mouse lung cell types catalogued from diverse anatomic locations and developmental time points. We demonstrate the accuracy and stability of LungMAP CellRefs and their utility for automated cell type annotation of both normal and diseased lungs using multiple independent methods and testing data. We develop user-friendly web interfaces for easy access and maximal utilization of the LungMAP CellRefs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Provitamin A biofortification of cassava enhances shelf life but reduces dry matter content of storage roots due to altered carbon partitioning into starch

Storage roots of cassava (Manihot esculenta Crantz), a major subsistence crop of sub-Saharan Africa, are calorie rich but deficient in essential micronutrients, including provitamin A bcarotene. In this study, b-carotene concentrations in cassava storage roots were enhanced by coexpression of transgenes for deoxy-D-xylulose-5-phosphate synthase (DXS) and bacterial phytoene synthase (crtB), mediated by the patatin-type 1 promoter. Storage roots harvested from field-grown plants accumulated carotenoids to ≤50 lg/g DW, 15- to 20-fold increases relative to roots from nontransgenic plants. Approximately 85%–90% of these carotenoids accumulated as all-trans-b-carotene, the most nutritionally efficacious carotenoid. b-Caroteneaccumulating storage roots displayed delayed onset of postharvest physiological deterioration, a major constraint limiting utilization of cassava products. Large metabolite changes were detected in b-carotene-enhanced storage roots. Most significantly, an inverse correlation was observed between b-carotene and dry matter content, with reductions of 50%–60% of dry matter content in the highest carotenoid-accumulating storage roots of different cultivars. Further analysis confirmed a concomitant reduction in starch content and increased levels of total fatty acids, triacylglycerols, soluble sugars and abscisic acid. Potato engineered to co-express DXS and crtB displayed a similar correlation between b-carotene accumulation, reduced dry matter and starch content and elevated oil and soluble sugars in tubers. Transcriptome analyses revealed a reduced expression of genes involved in starch biosynthesis including ADP-glucose pyrophosphorylase genes in transgenic, carotene-accumulating cassava roots relative to nontransgenic roots. These findings highlight unintended metabolic consequences of provitamin A biofortification of starch-rich organs and point to strategies for redirecting metabolic flux to restore starch production.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2014

Functional Genomics with a Comprehensive Library of Transposon Mutants for the Sulfate-Reducing Bacterium Desulfovibrio alaskensis G20

The genomes of sulfate-reducing bacteria remain poorly characterized, largely due to a paucity of experimental data and genetic tools. To meet this challenge, we generated an archived library of 15,477 mapped transposon insertion mutants in the sulfate-reducing bacterium Desulfovibrio alaskensis G20. To demonstrate the utility of the individual mutants, we profiled gene expression in mutants of six regulatory genes and used these data, together with 1,313 high-confidence transcription start sites identified by tiling microarrays and transcriptome sequencing (5' RNA-Seq), to update the regulons of Fur and Rex and to confirm the predicted regulons of LysX, PhnF, PerR, and Dde_3000, a histidine kinase. In addition to enabling single mutant investigations, the D. alaskensis G20 transposon mutants also contain DNA bar codes, which enables the pooling and analysis of mutant fitness for thousands of strains simultaneously. Using two pools of mutants that represent insertions in 2,369 unique protein-coding genes, we demonstrate that the hypothetical gene Dde_3007 is required for methionine biosynthesis. Using comparative genomics, we propose that Dde_3007 performs a missing step in methionine biosynthesis by transferring a sulfur group to O-phosphohomoserine to form homocysteine. Additionally, we show that the entire choline utilization cluster is important for fitness in choline sulfate medium, which confirms that a functional microcompartment is required for choline oxidation. Finally, we demonstrate that Dde_3291, a MerR-like transcription factor, is a choline-dependent activator of the choline utilization cluster. Taken together, our data set and genetic resources provide a foundation for systems-level investigation of a poorly studied group of bacteria of environmental and industrial importance.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2011

Unexpected Diversity of Chloroplast Noncoding RNAs as Revealed by Deep Sequencing of the Arabidopsis Transcriptome

Noncoding RNAs (ncRNA) are widely expressed in both prokaryotes and eukaryotes. Eukaryotic ncRNAs are commonly micro- and small-interfering RNAs (18–25 nt) involved in posttranscriptional gene silencing, whereas prokaryotic ncRNAs vary in size and are involved in various aspects of gene regulation. Given the prokaryotic origin of organelles, the presence of ncRNAs might be expected; however, the full spectrum of organellar ncRNAs has not been determined systematically. Here, strand-specific RNA-Seq analysis was used to identify 107 candidate ncRNAs from Arabidopsis thaliana chloroplasts, primarily encoded opposite protein-coding and tRNA genes. Forty-eight ncRNAs were shown to accumulate by RNA gel blot as discrete transcripts in wild-type (WT) plants and/or the pnp1-1 mutant, which lacks the chloroplast ribonuclease polynucleotide phosphorylase (cpPNPase). Ninety-eight percent of the ncRNAs detected by RNA gel blot had different transcript patterns between WT and pnp1-1, suggesting cpPNPase has a significant role in chloroplast ncRNA biogenesis and accumulation. Analysis of materials deficient for other major chloroplast ribonucleases, RNase R, RNase E, and RNase J, showed differential effects on ncRNA accumulation and/or form, suggesting specificity in RNase-ncRNA interactions. 59 end mapping demonstrates that some ncRNAs are transcribed from dedicated promoters, whereas others result from transcriptional read-through. Finally, correlations between accumulation of some ncRNAs and the symmetrically transcribed sense RNA are consistent with a role in RNA stability. Overall, our data suggest that this extensive population of ncRNAs has the potential to underpin a previously underappreciated regulatory mode in the chloroplast.

transcription↗
DOE OSTI2024

Building a FAIR data ecosystem for incorporating single-cell transcriptomics data into agricultural genome to phenome research

Introduction The agriculture genomics community has numerous data submission standards available, but the standards for describing and storing single-cell (SC, e.g., scRNA- seq) data are comparatively underdeveloped. Methods To bridge this gap, we leveraged recent advancements in human genomics infrastructure, such as the integration of the Human Cell Atlas Data Portal with Terra, a secure, scalable, open-source platform for biomedical researchers to access data, run analysis tools, and collaborate. In parallel, the Single Cell Expression Atlas at EMBL-EBI offers a comprehensive data ingestion portal for high-throughput sequencing datasets, including plants, protists, and animals (including humans). Developing data tools connecting these resources would offer significant advantages to the agricultural genomics community. The FAANG data portal at EMBL-EBI emphasizes delivering rich metadata and highly accurate and reliable annotation of farmed animals but is not computationally linked to either of these resources. Results Herein, we describe a pilot-scale project that determines whether the current FAANG metadata standards for livestock can be used to ingest scRNA-seq datasets into Terra in a manner consistent with HCA Data Portal standards. Importantly, rich scRNA-seq metadata can now be brokered through the FAANG data portal using a semi-automated process, thereby avoiding the need for substantial expert curation. We have further extended the functionality of this tool so that validated and ingested SC files within the HCA Data Portal are transferred to Terra for further analysis. In addition, we verified data ingestion into Terra, hosted on Azure, and demonstrated the use of a workflow to analyze the first ingested porcine scRNA-seq dataset. Additionally, we have also developed prototype tools to visualize the output of scRNA-seq analyses on genome browsers to compare gene expression patterns across tissues and cell populations. This JBrowse tool now features distinct tracks, showcasing PBMC scRNA-seq alongside two bulk RNA-seq experiments. Discussion We intend to further build upon these existing tools to construct a scientist-friendly data resource and analytical ecosystem based on Findable, Accessible, Interoperable, and Reusable (FAIR) SC principles to facilitate SC-level genomic analysis through data ingestion, storage, retrieval, re-use, visualization, and comparative annotation across agricultural species.

Genetics & Heredity↗
DOE OSTI2014

Community-integrated omics links dominance of a microbial generalist to fine-tuned resource usage

Microbial communities are complex and dynamic systems that are primarily structured according to their members’ ecological niches. To investigate how niche breadth (generalist versus specialist lifestyle strategies) relates to ecological success, we develop and apply an integrative workflow for the multi-omic analysis of oleaginous mixed microbial communities from a biological wastewater treatment plant. Time- and space-resolved coupled metabolomic and taxonomic analyses demonstrate that the community-wide lipid accumulation phenotype is associated with the dominance of the generalist bacterium Candidatus Microthrix spp. By integrating population-level genomic reconstructions (reflecting fundamental niches) with transcriptomic and proteomic data (realised niches), we identify finely tuned gene expression governing resource usage by Candidatus Microthrix parvicella over time. Moreover, our results indicate that the fluctuating environmental conditions constrain the accumulation of genetic variation in Candidatus Microthrix parvicella likely due to fitness trade-offs. Based on our observations, niche breadth has to be considered as an important factor for understanding the evolutionary processes governing (microbial) population sizes and structures in situ.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRSDate not supplied

Space Algae-2: Preflight Testing for A Long-Duration, Multi-Omics Analysis of Arthrospira Platensis

The cyanobacteria Arthrospira platensis NIES-39, commonly known as spirulina, could provide a fresh supply of nutrients for crew on long-duration spaceflight missions. Spirulina is a readily digestible food that is high in protein with all essential amino acids as well as significant levels of B vitamins, antioxidants, and anti-inflammatory metabolites. Spaceflight has multiple abiotic stressors such as increased ionizing radiation and microgravity, which causes a lack of convective mixing. These environmental conditions may impact productivity, nutritional composition, and in long-duration propagation, spaceflight stress may impact the genetic stability of spirulina cultures. We are developing an International Space Station experiment to continuously culture A. platensis for six months. Multi-omics profiling will be used to monitor for changes in the genome, transcriptome, proteome, and metabolome to determine if A. platensis is a suitable nutritional supplement on long-duration missions. During preflight testing we developed a protocol for inoculated liquid cultures to survive a 10-week storage period prior to photo-incubation. The bioreactor bag, temperature, and lighting conditions that support a 14-day growth cycle between passages were also determined. Media testing identified minimal salts supporting robust growth that can be stored in liquid or dry form. A simple filtration method was developed to dewater cultures and harvest biomass for frozen sample return. We optimized a cryopreservation method to enable return of live cells for isolation of individual A. platensis clones. The concept of operations for Space Algae-2 developed from these test results as well as progress on multi-omics analysis methods will be presented.

Algae↗
NASA NTRSDate not supplied

Space Algae-2: Preflight Testing for A Long-Duration, Multi-Omics Analysis of Arthrospira Platensis

The cyanobacteria Arthrospira platensis NIES-39, commonly known as spirulina, could provide a fresh supply of nutrients for crew on long-duration spaceflight missions. Spirulina is a readily digestible food that is high in protein with all essential amino acids as well as significant levels of B vitamins, antioxidants, and anti-inflammatory metabolites. Spaceflight has multiple abiotic stressors such as increased ionizing radiation and microgravity, which causes a lack of convective mixing. These environmental conditions may impact productivity, nutritional composition, and in long-duration propagation, spaceflight stress may impact the genetic stability of spirulina cultures. We are developing an International Space Station experiment to continuously culture A. platensis for six months. Multi-omics profiling will be used to monitor for changes in the genome, transcriptome, proteome, and metabolome to determine if A. platensis is a suitable nutritional supplement on long-duration missions. During preflight testing we developed a protocol for inoculated liquid cultures to survive a 10-week storage period prior to photo-incubation. The bioreactor bag, temperature, and lighting conditions that support a 14-day growth cycle between passages were also determined. Media testing identified minimal salts supporting robust growth that can be stored in liquid or dry form. A simple filtration method was developed to dewater cultures and harvest biomass for frozen sample return. We optimized a cryopreservation method to enable return of live cells for isolation of individual A. platensis clones. The concept of operations for Space Algae-2 developed from these test results as well as progress on multi-omics analysis methods will be presented.

Algae↗