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Results for “sequence analysis”

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 667 records · Page 37
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Improved high-throughput screening technique to rapidly isolate Chlamydomonas transformants expressing recombinant proteins

Abstract The single-celled eukaryotic green alga Chlamydomonas reinhardtii has long been a model system for developing genetic tools for algae, and is also considered a potential platform for the production of high-value recombinant proteins. Identifying transformants with high levels of recombinant protein expression has been a challenge in this organism, as random integration of transgenes into the nuclear genome leads to low frequency of cell lines with high gene expression. Here, we describe the design of an optimized vector for the expression of recombinant proteins in Chlamydomonas , that when transformed and screened using a dual antibiotic selection, followed by screening using fluorescence activated cell sorting (FACS), permits rapid identification and isolation of microalgal transformants with high expression of a recombinant protein. This process greatly reduces the time required for the screening process, and can produce large populations of recombinant algae transformants with between 60 and 100% of cells producing the recombinant protein of interest, in as little as 3 weeks, that can then be used for whole population sequencing or individual clone analysis. Utilizing this new vector and high-throughput screening (HTS) process resulted in an order of magnitude improvement over existing methods, which normally produced under 1% of algae transformants expressing the protein of interest. This process can be applied to other algal strains and recombinant proteins to enhance screening efficiency, thereby speeding up the discovery and development of algal-derived recombinant protein products. Key points • A protein expression vector using double-antibiotic resistance genes was designed • Double antibiotic selection causes fewer colonies with more positive for phenotype • Coupling the new vector with FACS improves microalgal screening efficiency > 60%

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2013

In search of genome annotation consistency: solid gene clusters and how to use them

Maintaining consistency in genome annotations is important for supporting many computational tasks, particularly metabolic modeling. The SEED project has implemented a process that improves annotation consistencies across microbial genomes for proteins with conserved sequences and genomic context. In this research report, we describe this process and show how this effort has resulted in improvements to microbial genome annotations in the SEED. We also compare SEED annotation consistencies with other commonly used resources such as IMG (the Joint Genome Institute’s Integrated Microbial Genomes system), RefSeq (the National Center for Biotechnology Information’s Reference Sequence Database), Swiss-Prot (the annotated protein sequence database of the Swiss Institute of Bioinformatics, European Molecular Biology Laboratory and the European Bioinformatics Institute) and TrEMBL (Translated European Molecular Biology Laboratory nucleotide sequence data Library). Our analysis indicates that manual and computational efforts are paying off for the databases where consistency is a major goal.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Extracting data from the muck: deriving biological insight from complex microbial communities and non-model organisms with next generation sequencing

It is becoming increasingly clear that microbes within microbial communities, for which cultured isolates have not yet been obtained, have an immense, untapped reservoir of enzymes that could help address grand challenges in human health, energy, and sustainability. Despite the obstacles associated with culturing these microbes, recent advances in next-generation sequencing (NGS) have made it possible to explore complex microbial communities in their native context for the first time. Key to extracting meaning from rapidly growing NGS datasets are bioinformatics tools that assemble the sequence data, annotate homologous sequences and interrogate it to reveal regulatory patterns. Complementing this are advances in proteomics that can link NGS data to biological function. Finally, this combination of next generation sequencing, proteomics and bioinformatic analysis forms a powerful tool to study non-model microbes, which will transform what we know about these dynamic systems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Chain elongators, friends, and foes

Bioproduction of medium chain carboxylic acids has recently emerged as an alternative strategy to valorize low-value organic waste and side-streams. Key to this route is chain elongation, an anaerobic microbial process driven by ethanol, lactic acid, or carbohydrates. Additionally, because these technologies use wastes as feedstocks, mixed microbial communities are often considered as biocatalysts. Understanding and steering these microbiomes is key to optimize bioprocess performance. From a meta-analysis of publicly available sequencing data, we (i) explore how the current collection of isolated chain elongators compares to microbiome members, (ii) discuss the main beneficial and antagonistic interactions with community partners, and (iii) identify the key research gaps and needs to help understand chain elongation microbiomes, and design/steer these novel bioproduction processes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2012

Identification of an S-adenosylmethionine (SAM) dependent arsenic methyltransferase in Danio rerio

Arsenic methylation is an important cellular metabolic process that modulates arsenic toxicity and carcinogenicity. Biomethylation of arsenic produces a series of mono-, di- and tri-methylated arsenic metabolites that can be detected in tissues and excretions. Here we report that zebrafish exposed to arsenite (As{sup III}) produces organic arsenicals, including MMA{sup III}, MMA{sup V} and DMA{sup V} with characteristic tissue ratios, demonstrating that an arsenic methylation pathway exists in zebrafish. In mammals, cellular inorganic arsenic is methylated by a SAM-dependent arsenic methyltransferase, AS3MT. A zebrafish arsenic methyltransferase homolog, As3mt, was identified by sequence alignment. Western blotting analysis showed that As3mt was universally expressed in zebrafish tissues. Prominent expression in liver and intestine correlated with methylated arsenic metabolites detected in those tissues. As3mt was expressed in and purified from Escherichia coli for in vitro functional studies. Our results demonstrated that As3mt methylated As{sup III} to DMA{sup V} as an end product and produced MMA{sup III} and MMA{sup V} as intermediates. The activity of As3mt was inhibited by elevated concentrations of the substrate As{sup III} as well as the metalloid selenite, which is a well-known antagonistic micronutrient of arsenic toxicity. The activity As3mt was abolished by substitution of either Cys160 or Cys210, which corresponds to conserved cysteine residues in AS3MT homologs, suggesting that they are involved in catalysis. Expression in zebrafish of an enzyme that has a similar function to human and rodent orthologs in catalyzing intracellular arsenic biomethylation validates the applicability of zebrafish as a valuable vertebrate model for understanding arsenic-associated diseases in humans. -- Highlights: ► Zebrafish methylated As{sup III} to MMA{sup III}, MMA{sup V} and DMA{sup V}. ► A zebrafish arsenic methyltransferase (As3mt) was purified in E. coli. ► As3mt catalyzed biomethylation of As{sup III} to DMA{sup V} and produced toxic intermediates. ► As3mt activity is inhibited by elevated substrate concentrations and selenite. ► C160 and C165 are predicted as As{sup III} binding sites.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

Thiol dependent NF-κB suppression and inhibition of T-cell mediated adaptive immune responses by a naturally occurring steroidal lactone Withaferin A

Withaferin A (WA), a steroidal lactone isolated from ayurvedic medicinal plant Withania somnifera, was shown to inhibit tumor growth by inducing oxidative stress and suppressing NF-κB pathway. However, its effect on T-cell mediated adaptive immune responses and the underlying mechanism has not been investigated. Since both T-cell responses and NF-κB pathway are known to be redox sensitive, the present study was undertaken to elucidate the effect of WA on adaptive immune responses in vitro and in vivo. WA inhibited mitogen induced T-cell and B-cell proliferation in vitro without inducing any cell death. It inhibited upregulation of T-cell (CD25, CD69, CD71 and CD54) and B-cell (CD80, CD86 and MHC-II) activation markers and secretion of Th1 and Th2 cytokines. WA induced oxidative stress by increasing the basal ROS levels and the immunosuppressive effects of WA were abrogated only by thiol anti-oxidants. The redox modulatory effects of WA in T-cells were attributed to its ability to directly interact with free thiols. WA inhibited NF-κB nuclear translocation in lymphocytes and prevented the direct binding of nuclear NF-κB to its consensus sequence. MALDI-TOF analysis using a synthetic NF-κB-p50 peptide containing Cys-62 residue suggested that WA can modify the cysteine residue of NF-κB. The pharmacokinetic studies for WA were also carried out and in vivo efficacy of WA was studied using mouse model of Graft-versus-host disease. In conclusion, WA is a potent inhibitor of T-cell responses and acts via a novel thiol dependent mechanism and inhibition of NF-κB pathway. - Highlights:: • Withaferin A (WA) inhibited T-cell and B-cell mediated immune responses. • WA increased basal ROS levels in lymphocytes. • WA directly interacted with GSH as studied using spectrophotometry and HPLC. • WA inhibited NF-κB nuclear translocation and binding of nuclear NF-κB to DNA. • WA inhibited induction of the graft-versus-host disease in mice.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Characterization of Coffee ringspot virus-Lavras: A model for an emerging threat to coffee production and quality

The emergence of viruses in Coffee (Coffea arabica and Coffea canephora), the most widely traded agricultural commodity in the world, is of critical concern. The RNA1 (6552 nt) of Coffee ringspot virus is organized into five open reading frames (ORFs) capable of encoding the viral nucleocapsid (ORF1p), phosphoprotein (ORF2p), putative cell-to-cell movement protein (ORF3p), matrix protein (ORF4p) and glycoprotein (ORF5p). Each ORF is separated by a conserved intergenic junction. RNA2 (5945 nt), which completes the bipartite genome, encodes a single protein (ORF6p) with homology to RNA-dependent RNA polymerases. Phylogenetic analysis of L protein sequences firmly establishes CoRSV as a member of the recently proposed Dichorhavirus genus. Predictive algorithms, in planta protein expression, and a yeast-based nuclear import assay were used to determine the nucleophillic character of five CoRSV proteins. Finally, the temperature-dependent ability of CoRSV to establish systemic infections in an initially local lesion host was quantified. - Highlights: • We report genome sequence determination for Coffee ringspot virus (CoRSV). • CoRSV should be considered a member of the proposed Dichorhavirus genus. • We report temperature-dependent systemic infection of an initially local lesion host. • We report in planta protein and localization data for five CoRSV proteins. • In silico predictions of the CoRSV proteins were validated using in vivo assays.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2024

Pressure–Temperature–Magnetic Field Phase Diagram of Multiferroic (NH 4 ) 2 FeCl 5 ·H 2 O

We combined synchrotron-based infrared absorbance and Raman scattering spectroscopies with diamond anvil cell techniques and a symmetry analysis to explore the properties of multiferroic (NH 4 ) 2 FeCl 5 ·H 2 O under extreme pressure–temperature conditions. Compression-induced splitting of the Fe–Cl stretching, Cl–Fe–Cl and Cl–Fe–O bending, and NH 4 + librational modes defines two structural phase transitions, and a group–subgroup analysis reveals space group sequences that vary depending upon proximity to the unexpectedly wide order–disorder transition. Here, we bring these findings together with prior high-field work to develop the pressure–temperature–magnetic field phase diagram uncovering competing polar, chiral, and magnetic phases in this system.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2018

Primary and Higher Order Structure of the Reaction Center from the Purple Phototrophic Bacterium Blastochloris viridis : A Test for Native Mass Spectrometry

The reaction center (RC) from the phototrophic bacterium Blastochloris viridis was the first integral membrane protein complex to have its structure determined by X-ray crystallography and has been studied extensively since then. It is composed of four protein subunits, H, M, L, and C, as well as cofactors, including bacteriopheophytin (BPh), bacteriochlorophyll (BCh), menaquinone, ubiquinone, heme, carotenoid, and Fe. In this study, we utilized mass spectrometry-based proteomics to study this protein complex via bottom-up sequencing, intact protein mass analysis, and native MS ligand-binding analysis. Its primary structure shows a series of mutations, including an unusual alteration and extension on the C-terminus of the M-subunit. In terms of quaternary structure, proteins such as this containing many cofactors serve to test the ability to introduce native-state protein assemblies into the gas phase because the cofactors will not be retained if the quaternary structure is seriously perturbed. Furthermore, this specific RC, under native MS, exhibits a strong ability not only to bind the special pair but also to preserve the two peripheral BCh’s.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

A diminutive perinate European Enantiornithes reveals an asynchronous ossification pattern in early birds

Fossils of juvenile Mesozoic birds provide insight into the early evolution of avian development, however such fossils are rare. The analysis of the ossification sequence in these early-branching birds has the potential to address important questions about their comparative developmental biology and to help understand their morphological evolution and ecological differentiation. Here we report on an early juvenile enantiornithine specimen from the Early Cretaceous of Europe, which sheds new light on the osteogenesis in this most species-rich clade of Mesozoic birds. Consisting of a nearly complete skeleton, it is amongst the smallest known Mesozoic avian fossils representing post-hatching stages of development. Finally, comparisons between this new specimen and other known early juvenile enantiornithines support a clade-wide asynchronous pattern of osteogenesis in the sternum and the vertebral column, and strongly indicate that the hatchlings of these phylogenetically basal birds varied greatly in size and tempo of skeletal maturation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Probing the active fraction of soil microbiomes using BONCAT-FACS

The ability to link soil microbial diversity to soil processes requires technologies that differentiate active microbes from extracellular DNA and dormant cells. Here, we use BONCAT (bioorthogonal non-canonical amino acid tagging) to measure translationally active cells in soils. We compare the active population of two soil depths from Oak Ridge (Tennessee, USA) and find that a maximum of 25-70% of the extractable cells are active. Analysis of 16S rRNA sequences from BONCAT-positive cells recovered by fluorescence-activated cell sorting (FACS) reveals that the phylogenetic composition of the active fraction is distinct from the total population of extractable cells. Some members of the community are found to be active at both depths independently of their abundance rank, suggesting that the incubation conditions favor the activity of similar organisms. We conclude that BONCAT-FACS is effective for interrogating the active fraction of soil microbiomes in situ and provides a new approach for uncovering the links between soil processes and specific microbial groups.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Deciphering triterpenoid saponin biosynthesis by leveraging transcriptome response to methyl jasmonate elicitation in Saponaria vaccaria

Abstract Methyl jasmonate (MeJA) is a known elicitor of plant specialized metabolism, including triterpenoid saponins. Saponaria vaccaria is an annual herb used in traditional Chinese medicine, containing large quantities of oleanane-type triterpenoid saponins with anticancer properties and structural similarities to the vaccine adjuvant QS-21. Leveraging the MeJA-elicited saponin biosynthesis, we identify multiple enzymes catalyzing the oxidation and glycosylation of triterpenoids in S. vaccaria . This exploration is aided by Pacbio full-length transcriptome sequencing and gene expression analysis. A cellulose synthase-like enzyme can not only glucuronidate triterpenoid aglycones but also alter the product profile of a cytochrome P450 monooxygenase via preference for the aldehyde intermediate. Furthermore, the discovery of a UDP-glucose 4,6-dehydratase and a UDP-4-keto-6-deoxy-glucose reductase reveals the biosynthetic pathway for the rare nucleotide sugar UDP- d -fucose, a likely sugar donor for fucosylation of plant natural products. Our work enables the production and optimization of high-value saponins in microorganisms and plants through synthetic biology approaches.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Multimodal framework for the joint analysis of single-cell RNA and T cell receptor sequencing data predicts T cell response to cancer immunotherapy

T cell states are prognostic in different cancer types. Recent technologies enable joint profiling of T cell RNA and T cell receptor (TCR) sequences at single-cell resolution. Here we present the TCR-RNA Integrating Model (TRIM), a multi-modal variational autoencoder framework that integrates RNA-TCR data and predicts T cell clonality and transcriptional states. TRIM learns a shared representation of the data conditioned on patient, tissue source, and treatment timepoint. We applied TRIM to three independent datasets that included T cells collected before and after checkpoint inhibitor treatment, sourced either from blood and tumor biopsies in patients with head and neck squamous cell carcinoma and colorectal cancer, or from tumor and adjacent tissue in a pan-cancer dataset. In all settings, TRIM accurately predicted intra-tumor T cell clonal expansion and transcriptional status based on T cells from blood or normal tissue before treatment, demonstrating its utility in modeling multimodal T cell data and predicting T cell response to treatment and disease progression.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Genome expansion and lineage-specific genetic innovations in the forest pathogenic fungi Armillaria

Armillaria species are both devastating forest pathogens and some of the largest terrestrial organisms on Earth. They forage for hosts and achieve immense colony sizes via rhizomorphs, root-like multicellular structures of clonal dispersal. Here, we sequenced and analysed the genomes of four Armillaria species and performed RNA sequencing and quantitative proteomic analysis on the invasive and reproductive developmental stages of A. ostoyae. Comparison with 22 related fungi revealed a significant genome expansion in Armillaria, affecting several pathogenicity-related genes, lignocellulose-degrading enzymes and lineage-specific genes expressed during rhizomorph development. Rhizomorphs express an evolutionarily young transcriptome that shares features with the transcriptomes of both fruiting bodies and vegetative mycelia. Several genes show concomitant upregulation in rhizomorphs and fruiting bodies and share cis-regulatory signatures in their promoters, providing genetic and regulatory insights into complex multicellularity in fungi. Our results suggest that the evolution of the unique dispersal and pathogenicity mechanisms of Armillaria might have drawn upon ancestral genetic toolkits for wood-decay, morphogenesis and complex multicellularity.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Structure determination of the HgcAB complex using metagenome sequence data: insights into microbial mercury methylation

Bacteria and archaea possessing the hgcAB gene pair methylate inorganic mercury (Hg) to form highly toxic methylmercury. HgcA consists of a corrinoid binding domain and a transmembrane domain, and HgcB is a dicluster ferredoxin. However, their detailed structure and function have not been thoroughly characterized. We modeled the HgcAB complex by combining metagenome sequence data mining, coevolution analysis, and Rosetta structure calculations. In addition, we overexpressed HgcA and HgcB in Escherichia coli, confirmed spectroscopically that they bind cobalamin and [4Fe-4S] clusters, respectively, and incorporated these cofactors into the structural model. Surprisingly, the two domains of HgcA do not interact with each other, but HgcB forms extensive contacts with both domains. The model suggests that conserved cysteines in HgcB are involved in shuttling HgII, methylmercury, or both. These findings refine our understanding of the mechanism of Hg methylation and expand the known repertoire of corrinoid methyltransferases in nature.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Metallomics of two microorganisms relevant to heavy metal bioremediation reveal fundamental differences in metal assimilation and utilization

Although as many as half of all proteins are thought to require a metal cofactor, the metalloproteomes of microorganisms remain relatively unexplored. Microorganisms from different environments are likely to vary greatly in the metals that they assimilate, not just among the metals with well-characterized roles but also those lacking any known function. Herein we investigated the metal utilization of two microorganisms that were isolated from very similar environments and are of interest because of potential roles in the immobilization of heavy metals, such as uranium and chromium. The metals assimilated and their concentrations in the cytoplasm of Desulfovibrio vulgaris strain Hildenborough (DvH) and Enterobacter cloacae strain Hanford (EcH) varied dramatically, with a larger number of metals present in Enterobacter. For example, a total of 9 and 19 metals were assimilated into their cytoplasmic fractions, respectively, and DvH did not assimilate significant amounts of zinc or copper whereas EcH assimilated both. However, bioinformatic analysis of their genome sequences revealed a comparable number of predicted metalloproteins, 813 in DvH and 953 in EcH. These allowed some rationalization of the types of metal assimilated in some cases (Fe, Cu, Mo, W, V) but not in others (Zn, Nd, Ce, Pr, Dy, Hf and Th). It was also shown that U binds an unknown soluble protein in EcH but this incorporation was the result of extracellular U binding to cytoplasmic components after cell lysis.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2019

The impact of tomato fruits containing multi-walled carbon nanotube residues on human intestinal epithelial cell barrier function and intestinal microbiome composition

Carbon nanomaterials (CNMs) can positively regulate seed germination and enhance plant growth. However, clarification of the impact of plant organs containing absorbed CNMs on animal and human health is a critical step of risk assessment for new nano–agro-technology. In this study, we have taken a comprehensive approach to studying the effect tomato fruits derived from plants exposed to multi-walled carbon nanotubes (CNTs) have on gastrointestinal epithelial barrier integrity and their impact on the human commensal intestinal microbiota using an in vitro cell culture and batch human fecal suspension models. The effects of CNTs on selected pure cultures of Salmonella enterica Typhimurium and Lactobacillus acidophilus were also evaluated. This study demonstrated that CNT-containing fruits or the corresponding residual level of pure CNTs (0.001 μg ml –1 ) was not sufficient to initiate a significant change in transepithelial resistance and on gene expression of the model T-84 human intestinal epithelial cells. However, at 10 μg ml –1 concentration CNTs were able to penetrate the cell membrane and change the gene expression profile of exposed cells. Furthermore, extracts from CNT-containing fruits had minimal to no effect on human intestinal microbiota as revealed by culture-based analysis and 16S rRNA sequencing.

60 APPLIED LIFE SCIENCES↗