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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 91 records · Page 5
All sourcesNASA NTRSDOE OSTI
DOE OSTI2016

Identification of Novel Host Interactors of Effectors Secreted by Salmonella and Citrobacter

Many pathogenic bacteria of the family Enterobacteriaceae use type III secretion systems to inject virulence proteins, termed “effectors,” into the host cell cytosol. Although host-cellular activities of several effectors have been demonstrated, the function and host-targeted pathways of most of the effectors identified to date are largely undetermined. To gain insight into host proteins targeted by bacterial effectors, we performed coaffinity purification of host proteins from cell lysates using recombinant effectors from the Enterobacteriaceae intracellular pathogens Salmonella enterica serovar Typhimurium and Citrobacter rodentium . We identified 54 high-confidence host interactors for the Salmonella effectors GogA, GtgA, GtgE, SpvC, SrfH, SseL, SspH1, and SssB collectively and 21 interactors for the Citrobacter effectors EspT, NleA, NleG1, and NleK. We biochemically validated the interaction between the SrfH Salmonella protein and the extracellular signal-regulated kinase 2 (ERK2) host protein kinase, which revealed a role for this effector in regulating phosphorylation levels of this enzyme, which plays a central role in signal transduction. IMPORTANCE During infection, pathogenic bacteria face an adverse environment of factors driven by both cellular and humoral defense mechanisms. To help evade the immune response and ultimately proliferate inside the host, many bacteria evolved specialized secretion systems to deliver effector proteins directly into host cells. Translocated effector proteins function to subvert host defense mechanisms. Numerous pathogenic bacteria use a specialized secretion system called type III secretion to deliver effectors into the host cell cytosol. Here, we identified 75 new host targets of Salmonella and Citrobacter effectors, which will help elucidate their mechanisms of action.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

TIR-only protein RBA1 recognizes a pathogen effector to regulate cell death in Arabidopsis

Detection of pathogens by plants is mediated by intracellular nucleotide-binding site leucine-rich repeat (NLR) receptor proteins. NLR proteins are defined by their stereotypical multidomain structure: an N-terminal Toll–interleukin receptor (TIR) or coiled-coil (CC) domain, a central nucleotide-binding (NB) domain, and a C-terminal leucine-rich repeat (LRR). The plant innate immune system contains a limited NLR repertoire that functions to recognize all potential pathogens. We isolated Response to the bacterial type III effector protein HopBA1 (RBA1), a gene that encodes a TIR-only protein lacking all other canonical NLR domains. RBA1 is sufficient to trigger cell death in response to HopBA1. We generated a crystal structure for HopBA1 and found that it has similarity to a class of proteins that includes esterases, the heme-binding protein ChaN, and an uncharacterized domain of Pasteurella multocida toxin. Self-association, coimmunoprecipitation with HopBA1, and function of RBA1 require two previously identified TIR–TIR dimerization interfaces. Although previously described as distinct in other TIR proteins, in RBA1 neither of these interfaces is sufficient when the other is disrupted. These data suggest that oligomerization of RBA1 is required for function. As a result, our identification of RBA1 demonstrates that “truncated” NLRs can function as pathogen sensors, expanding our understanding of both receptor architecture and the mechanism of activation in the plant immune system.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Overexpression of a plasma membrane protein generated broad-spectrum immunity in soybean

Plants fight-off pathogens and pests by manifesting an array of defence responses using their innate immunity mechanisms. Here we report the identification of a novel soybean gene encoding a plasma membrane protein, transcription of which is suppressed following infection with the fungal pathogen, Fusarium virguliforme. Overexpression of the protein led to enhanced resistance against not only against F. virguliforme, but also against spider mites (Tetranychus urticae, Koch), soybean aphids (Aphis glycines, Matsumura) and soybean cyst nematode (Heterodera glycines). We, therefore, name this protein as Glycine max disease resistance 1 (GmDR1; Glyma.10g094800). The homologues of GmDR1 have been detected only in legumes, cocoa, jute and cotton. The deduced GmDR1 protein contains 73 amino acids. GmDR1 is predicted to contain an ecto- and two transmembrane domains. Transient expression of the green fluorescent protein fused GmDR1 protein in soybean leaves showed that it is a plasma membrane protein. We investigated if chitin, a pathogen-associated molecular pattern (PAMP), common to all pathogen and pests considered in this study, can significantly enhance defence pathways among the GmDR1-overexpressed transgenic soybean lines. Chitin induces marker genes of the salicylic- and jasmonic acid-mediated defence pathways, but suppresses the defence pathway regulated by ethylene. Chitin induced SA- and JA-regulated defence pathways may be one of the mechanisms involved in generating broad-spectrum resistance among the GmDR1-overexpressed transgenic soybean lines against two serious pathogens and two pests including spider mites, against which no known resistance genes have been identified in soybean and among the most other crop species.

plasma membrane↗
DOE OSTI2021

Shotgun Immunoproteomic Approach for the Discovery of Linear B-Cell Epitopes in Biothreat Agents Francisella tularensis and Burkholderia pseudomallei

Peptide-based subunit vaccines are coming to the forefront of current vaccine approaches, with safety and cost-effective production among their top advantages. Peptide vaccine formulations consist of multiple synthetic linear epitopes that together trigger desired immune responses that can result in robust immune memory. The advantages of linear compared to conformational epitopes are their simple structure, ease of synthesis, and ability to stimulate immune responses by means that do not require complex 3D conformation. Prediction of linear epitopes through use of computational tools is fast and cost-effective, but typically of low accuracy, necessitating extensive experimentation to verify results. On the other hand, identification of linear epitopes through experimental screening has been an inefficient process that requires thorough characterization of previously identified full-length protein antigens, or laborious techniques involving genetic manipulation of organisms. In this study, we apply a newly developed generalizable screening method that enables efficient identification of B-cell epitopes in the proteomes of pathogenic bacteria. As a test case, we used this method to identify epitopes in the proteome of Francisella tularensis (Ft), a Select Agent with a well-characterized immunoproteome. Our screen identified many peptides that map to known antigens, including verified and predicted outer membrane proteins and extracellular proteins, validating the utility of this approach. We then used the method to identify seroreactive peptides in the less characterized immunoproteome of Select Agent Burkholderia pseudomallei (Bp). This screen revealed known Bp antigens as well as proteins that have not been previously identified as antigens. Although B-cell epitope prediction tools Bepipred 2.0 and iBCE-EL classified many of our seroreactive peptides as epitopes, they did not score them significantly higher than the non-reactive tryptic peptides in our study, nor did they assign higher scores to seroreactive peptides from known Ft or Bp antigens, highlighting the need for experimental data instead of relying on computational epitope predictions alone. The present workflow is easily adaptable to detecting peptide targets relevant to the immune systems of other mammalian species, including humans (depending upon the availability of convalescent sera from patients), and could aid in accelerating the discovery of B-cell epitopes and development of vaccines to counter emerging biological threats.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Defining Lipidomic Responses to Coronavirus Infection

Highly pathogenic human coronavirus infection can cause a severe atypical, rapid onset pneumonia with a mortality rate of up to 10% for severe acute respiratory syndrome coronavirus 1 (SARS-CoV 1), 35% for Middle East respiratory syndrome coronavirus (MERS-CoV), or 1% for severe acute respiratory syndrome coronavirus 2 (SARS-CoV 2 causative agent of COVID 19). While medical countermeasures successfully controlled the worldwide SARS-CoV epidemic, the MERS-CoV epidemic is still ongoing and continues to be a concern for travelers in the Middle East and the multi-year SARS-CoV 2 pandemic underscore the importance of defining biomarkers that are diagnostic and/or predictive of severe disease outcomes for respiratory viruses. Systems biology approaches provide global snapshots of infection induced changes in host cells/tissues and provide extremely rich datasets for understanding host pathogen interactions. Metabolites, especially lipids, are critical for viral replication but less is understood about infection induced changes to lipids due to limits in lipid species detection and identification. To characterize how individual lipid species and proteins with lipid associated functions contribute to highly pathogenic human coronavirus replication and disease severity, existing datasets were probed to determine cell type specific lipid responses to MERS-CoV infection and verification studies were performed to determine if modification of the host lipid signature (by inhibiting enzymatic functions that produce specific lipid species) can perturb CoV replication in human lung cells. MERS-CoV infects human lung epithelial, endothelial and fibroblast cells. All three cell types were infected with MERS-CoV and samples collected to analyze lipids, proteins, metabolites, and transcripts from 12 to 48 hours post infection. Time matched mock-infected cells were collected in parallel for each cell type. Following sample and statistical analysis, functional enrichment and bioinformatic analysis was performed to identify differentially expressed lipids and proteins. Two lipid species were found to be significantly upregulated following MERS-CoV infection, ceramides, and triacylglycerol both of which are indicative of cells undergoing apoptotic cell death. In contrast, sphingomyelins (lipid molecules that can serve as a precursor for one pathway for ceramide synthesis) had significantly decreased expression in MERS-CoV infected cells. An inhibitor of acid sphingomyelinase (that converts sphingomyelin to ceramides and phosphorylcholine) reduced MERS-CoV replication suggesting that production of ceramides is key for successful viral replication and transmission. Acyl-CoA-synthetase 3 (ACSL3), the only protein whose function is lipid associated and had increased differential expression in our dataset, regulates the synthesis of triacylglycerol (increased expression). Inhibitors that directly block ACSL3 (Triacsin C) but not steps later in the triacylglycerol synthesis pathway (Etomoxir) inhibit MERS-CoV replication suggesting that triacylglycerol production and/or ACSL3 activity is also key for viral replication. ACSL3 expression is also upregulated in lung cancer cells and is predicted to promote continued cell viability which would also enhance viral replication. The differentially expressed lipid and lipid-associated protein species identified in our studies suggest that MERS-CoV infection is activating cellular death pathways to limit the number of cells that become infected but also stimulating the production of lipid-associated enzymes that can prolong host cell viability and the amount of time progeny virions can be produced and released. As the inhibitors that worked against MERS-CoV infection were also efficacious against SARS-CoV 2 infection, countermeasures that target these host pathways may provide novel ways to block highly pathogenic human coronavirus infection and/or prevent severe disease outcomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Genomic reconstruction of Bacillus anthracis from complex environmental samples enables high-throughput identification and lineage assignment in Pakistan

Bacillus anthracis, the causative agent of anthrax, is a highly virulent zoonotic pathogen primarily affecting domesticated and wild herbivores. Human exposure to B. anthracis is primarily through contact with infected animals or contaminated animal products. In Pakistan, where livestock vaccines are largely unavailable and infected carcasses are often disposed of improperly, the risk to humans, wildlife and livestock is significant. Currently, the diagnosis of anthrax infections and outbreak tracing necessitates the isolation and culturing of B. anthracis, a process that requires BSL-3 facilities. In this study, we show that positive identification, genome reconstruction and lineage assignment can be accomplished using bioinformatic analysis of DNA extracted directly from environmental samples that would otherwise provide the starting material for isolation and culturing. This approach does not require laboratory target enrichment as is necessary for other pathogens, due in part to the extremely high bacterial load in the bloodstream in the deceased animals. Using these methods, we greatly expand the knowledge of endemic B. anthracis in Pakistan. We provide the first reference B. anthracis genomes from Pakistan since the 1970s and identify A.Br.014 Aust94 as a minor circulating sublineage alongside the dominant A.Br.047 Vollum. Future work will focus on the limits of detection and will determine if this bioinformatic method can be expanded more broadly for B. anthracis or other pathogens to replace typical culture-based methods.

A.Br.047 Vollum↗
DOE OSTI2016

Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing

Francisella tularensis is classified as a Class A bioterrorism agent by the U.S. government due to its high virulence and the ease with which it can be spread as an aerosol. It is a facultative intracellular pathogen and the causative agent of tularemia. Ciprofloxacin (Cipro) is a broad spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Increased Cipro resistance in pathogenic microbes is of serious concern when considering options for medical treatment of bacterial infections. Identification of genes and loci that are associated with Ciprofloxacin resistance will help advance the understanding of resistance mechanisms and may, in the future, provide better treatment options for patients. It may also provide information for development of assays that can rapidly identify Cipro-resistant isolates of this pathogen. In this study, we then selected a large number of F. tularensis live vaccine strain (LVS) isolates that survived in progressively higher Ciprofloxacin concentrations, screened the isolates using a whole genome F. tularensis LVS tiling microarray and Illumina sequencing, and identified both known and novel mutations associated with resistance. For genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Finally, structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRSDate not supplied

Development of Computational Environmental Microbiome Workflows for the Laboratory and the International Space Station

Identification of microorganisms in the spaceflight environment is critical for crew health risk assessment on the International Space Station (ISS). Since 2017, nanopore sequencing technology has been used to support thein situ identification of microbial species during spaceflight. Beginning in 2018, a culture-independent, swab-to-sequencer method was implemented onboard the ISS to provide a more thorough insight of the ISS microbiome. Eliminating microbial culture enables identification of difficult-to-culture organisms, reduces risks associated with potentially pathogenic cultures, and could significantly reduce the time from sample-to-answer. However, this molecular-based approach generates large metagenomic datasets that require substantial computational resources for analysis. To process nanopore-generated sequencing data, the JSC Microbiology Laboratory established a bioinformatics workflow on Amazon EC2 under the security guidance of the NASA Science Managed Cloud Environment (SMCE).This resource allows for the development, testing, and accessing of computational tools for processing large and complex datasets. The work described here will address the downlinking of data from the ISS, the automated pipeline developed to identify targeted bacterial and fungal organisms, and the time from sampling onboard to microbial identification. The pipelines have been enhanced to address high and low biomass samples using optimization based on sample source (air, water, or surface) and type of collection (filter, colony, or swab).The resulting microbiome data can be assessed beyond microbial identifications to gain understanding toward population changes over time, potential selective environmental pressures, and evaluating correlations with a wide range of additional data sets. Metagenome analysis pipelines in development could allow for simultaneous identification of microbial species, gene function, and gene pathways present in the environment. Beyond the ground processing, the developed analysis pipeline is currently deployed onboard the ISS to allow for near real-time assessments of the ISS microbiome. This study serves as a critical foundation for exploration missions, where rapid microbiome analyses will be required.

G. Marie Sharp↗
DOE OSTI2020

A Glycan Array-Based Assay for the Identification and Characterization of Plant Glycosyltransferases

Growing plants with modified cell wall compositions is a promising strategy to improve resistance to pathogens, increase biomass digestibility, and tune other important properties. In order to alter biomass architecture, a detailed knowledge of cell wall structure and biosynthesis is a prerequisite. We report here a glycan array-based assay for the high-throughput identification and characterization of plant cell wall biosynthetic glycosyltransferases (GTs). We demonstrate that different heterologously expressed galactosyl-, fucosyl-, and xylosyltransferases can transfer azido-functionalized sugar nucleotide donors to selected synthetic plant cell wall oligosaccharides on the array and that the transferred monosaccharides can be visualized “on chip” by a 1,3-dipolar cycloaddition reaction with an alkynyl-modified dye. The opportunity to simultaneously screen thousands of combinations of putative GTs, nucleotide sugar donors, and oligosaccharide acceptors will dramatically accelerate plant cell wall biosynthesis research.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2016

Membrane insertion for the detection of lipopolysaccharides: Exploring the dynamics of amphiphile-in-lipid assays

Shiga toxin-producing Escherichia coli is an important cause of foodborne illness, with cases attributable to beef, fresh produce and other sources. Many serotypes of the pathogen cause disease, and differentiating one serotype from another requires specific identification of the O antigen located on the lipopolysaccharide (LPS) molecule. The amphiphilic structure of LPS poses a challenge when using classical detection methods, which do not take into account its lipoglycan biochemistry. Typically, detection of LPS requires heat or chemical treatment of samples and relies on bioactivity assays for the conserved lipid A portion of the molecule. Our goal was to develop assays to facilitate the direct and discriminative detection of the entire LPS molecule and its O antigen in complex matrices using minimal sample processing. To perform serogroup identification of LPS, we used a method called membrane insertion on a waveguide biosensor, and tested three serogroups of LPS. The membrane insertion technique allows for the hydrophobic association of LPS with a lipid bilayer, where the exposed O antigen can be targeted for specific detection. Samples of beef lysate were spiked with LPS to perform O antigen specific detection of LPS from E. coli O157. To validate assay performance, we evaluated the biophysical interactions of LPS with lipid bilayers both in- and outside of a flow cell using fluorescence microscopy and fluorescently doped lipids. Our results indicate that membrane insertion allows for the qualitative and reliable identification of amphiphilic LPS in complex samples like beef homogenates. In addition, we also demonstrated that LPS-induced hole formation does not occur under the conditions of the membrane insertion assays. Together, these findings describe for the first time the serogroup-specific detection of amphiphilic LPS in complex samples using a membrane insertion assay, and highlight the importance of LPS molecular conformations in detection architectures.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2017

Expansion of Microbial Monitoring Capabilities on the International Space Station (ISS)

Microbial monitoring is one of the tools that the National Aeronautics and Space Administration (NASA) uses on the International Space Station (ISS) to help maintain crew health and safety. In combination with regular housekeeping and disinfection when needed, microbial monitoring provides important information to the crew about the quality of the environment. Rotation of astronauts, equipment, and cargo on the ISS can affect the microbial load in the air, surfaces, and water. The current ISS microbial monitoring methods are focused on culture-based enumeration during flight and require a significant amount of crew time as well as long incubation periods of up to 5 days there by proliferating potential pathogens. In addition, the samples require return to Earth for complete identification of the microorganisms cultivated. Although the current approach assess the quality of the ISS environment, molecular technology offers faster turn-around of information particularly beneficial in an off-nominal situation. In 2011, subject matter experts from industry and academia recommended implementation of molecular-based technologies such as quantitative real-time polymerase chain reaction (qPCR) for evaluation to replace current, culture-based technologies. The RAZOR EX (BioFire Defense, Inc, Salt Lake City, UT) a ruggedized, compact, COTS (commercial off the shelf) qPCR instrument was tested, evaluated and selected in the 2 X 2015 JSC rapid flight hardware demonstration initiative as part of the Water Monitoring Suite. RAZOR EX was launched to ISS on SpaceX-9 in July 2016 to evaluate the precision and accuracy of the hardware by testing various concentrations of DNA in microgravity compared to ground controls. Flight testing was completed between September 2016 and March 2017. Data presented will detail the hardware performance of flight testing results compared to ground controls. Future goals include additional operational ground-based testing and assay development to determine if this technology can meet spaceflight microbial monitoring requirements.

Khodadad, Christina L.↗
DOE OSTI2020

Structures of diverse poxin cGAMP nucleases reveal a widespread role for cGAS-STING evasion in host–pathogen conflict

DNA viruses in the family Poxviridae encode poxin enzymes that degrade the immune second messenger 2'3'-cGAMP to inhibit cGAS-STING immunity in mammalian cells. The closest homologs of poxin exist in the genomes of insect viruses suggesting a key mechanism of cGAS-STING evasion may have evolved outside of mammalian biology. Here we use a biochemical and structural approach to discover a broad family of 369 poxins encoded in diverse viral and animal genomes and define a prominent role for 2'3'-cGAMP cleavage in metazoan host-pathogen conflict. Structures of insect poxins reveal unexpected homology to flavivirus proteases and enable identification of functional self-cleaving poxins in RNA-virus polyproteins. Our data suggest widespread 2'3'-cGAMP signaling in insect antiviral immunity and explain how a family of cGAS-STING evasion enzymes evolved from viral proteases through gain of secondary nuclease activity. Poxin acquisition by poxviruses demonstrates the importance of environmental connections in shaping evolution of mammalian pathogens.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Structure of Dirithromycin Bound to the Bacterial Ribosome Suggests New Ways for Rational Improvement of Macrolides

Although macrolides are known as excellent antibacterials, their medical use has been significantly limited due to the spread of bacterial drug resistance. As such, it is necessary to develop new potent macrolides to combat the emergence of drug-resistant pathogens. One of the key steps in rational drug design is the identification of chemical groups that mediate binding of the drug to its target and their subsequent derivatization to strengthen drug-target interactions. In the case of macrolides, a few groups are known to be important for drug binding to the ribosome, such as desosamine. Search for new chemical moieties that improve the interactions of a macrolide with the 70S ribosome might be of crucial importance for the invention of new macrolides. For this purpose, here we studied a classic macrolide, dirithromycin, which has an extended (2-methoxyethoxy)-methyl side chain attached to the C-9/C-11 atoms of the macrolactone ring that can account for strong binding of dirithromycin to the 70S ribosome. By solving the crystal structure of the 70S ribosome in complex with dirithromycin, we found that its side chain interacts with the wall of the nascent peptide exit tunnel in an idiosyncratic fashion: its side chain forms a lone pair-π stacking interaction with the aromatic imidazole ring of the His69 residue in ribosomal protein uL4. To our knowledge, the ability of this side chain to form a contact in the macrolide binding pocket has not been reported previously and potentially can open new avenues for further exploration by medicinal chemists developing next-generation macrolide antibiotics active against resistant pathogens.

60 APPLIED LIFE SCIENCES↗
NASA NTRS1998

Identification of a dual-specificity protein phosphatase that inactivates a MAP kinase from Arabidopsis

Mitogen-activated protein kinases (MAPKs) play a key role in plant responses to stress and pathogens. Activation and inactivation of MAPKs involve phosphorylation and dephosphorylation on both threonine and tyrosine residues in the kinase domain. Here we report the identification of an Arabidopsis gene encoding a dual-specificity protein phosphatase capable of hydrolysing both phosphoserine/threonine and phosphotyrosine in protein substrates. This enzyme, designated AtDsPTP1 (Arabidopsis thaliana dual-specificity protein tyrosine phosphatase), dephosphorylated and inactivated AtMPK4, a MAPK member from the same plant. Replacement of a highly conserved cysteine by serine abolished phosphatase activity of AtDsPTP1, indicating a conserved catalytic mechanism of dual-specificity protein phosphatases from all eukaryotes.

Non-NASA Center↗
DOE OSTI2022

Tracking ebolavirus genomic drift with a resequencing microarray

Filoviruses are emerging pathogens that cause acute fever with high fatality rate and present a global public health threat. During the 2013–2016 Ebola virus outbreak, genome sequencing allowed the study of virus evolution, mutations affecting pathogenicity and infectivity, and tracing the viral spread. In 2018, early sequence identification of the Ebolavirus as EBOV in the Democratic Republic of the Congo supported the use of an Ebola virus vaccine. However, field-deployable sequencing methods are needed to enable a rapid public health response. Resequencing microarrays (RMA) are a targeted method to obtain genomic sequence on clinical specimens rapidly, and sensitively, overcoming the need for extensive bioinformatic analysis. This study presents the design and initial evaluation of an ebolavirus resequencing microarray (Ebolavirus-RMA) system for sequencing the major genomic regions of four Ebolaviruses that cause disease in humans. The design of the Ebolavirus-RMA system is described and evaluated by sequencing repository samples of three Ebolaviruses and two EBOV variants. The ability of the system to identify genetic drift in a replicating virus was achieved by sequencing the ebolavirus glycoprotein gene in a recombinant virus cultured under pressure from a neutralizing antibody. Comparison of the Ebolavirus-RMA results to the Genbank database sequence file with the accession number given for the source RNA and Ebolavirus-RMA results compared to Next Generation Sequence results of the same RNA samples showed up to 99% agreement.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Abiotic and Biotic Damage of Microalgae Generate Different Volatile Organic Compounds (VOCs) for Early Diagnosis of Algal Cultures for Biofuel Production

Open microalgal ponds used in industrial biomass production are susceptible to a number of biotic and abiotic environmental stressors (e.g., grazers, pathogens, pH, temperature, etc.) resulting in pond crashes with high economic costs. Identification of signature chemicals to aid in rapid, non-invasive, and accurate identification of the stressors would facilitate targeted and effective treatment to save the algal crop from a catastrophic crash. Specifically, we were interested in identifying volatile organic compounds (VOCs) that can be used to as an early diagnostic for algal crop damage. Cultures of Microchloropsis gaditana were subjected to two forms of algal crop damage: (1) active grazing by the marine rotifer, Brachionus plicatilis, or (2) repeated freeze–thaw cycles. VOCs emitted above the headspace of these algal cultures were collected using fieldable solid phase microextraction (SPME) fibers. An untargeted analysis and identification of VOCs was conducted using gas chromatography-mass spectrometry (GC-MS). Diagnostic VOCs unique to each algal crop damage mechanism were identified. Active rotifer grazing of M. gaditana was characterized by the appearance of carotenoid degradation products, including β-cyclocitral and various alkenes. Freeze–thaw algae produced a different set of VOCs, including palmitoleic acid. Both rotifer grazing and freeze–thawed algae produced β-ionone as a VOC, possibly suggesting a common stress-induced cellular mechanism. Importantly, these identified VOCs were all absent from healthy algal cultures of M. gaditana. Early detection of biotic or abiotic environmental stressors will facilitate early diagnosis and application of targeted treatments to prevent algal pond crashes. Thus, our work further supports the use of VOCs for monitoring the health of algal ponds to ultimately enhance algal crop yields for production of biofuel.

09 BIOMASS FUELS↗
NASA NTRS2013

RECOVIR Software for Identifying Viruses

Most single-stranded RNA (ssRNA) viruses mutate rapidly to generate a large number of strains with highly divergent capsid sequences. Determining the capsid residues or nucleotides that uniquely characterize these strains is critical in understanding the strain diversity of these viruses. RECOVIR (an acronym for "recognize viruses") software predicts the strains of some ssRNA viruses from their limited sequence data. Novel phylogenetic-tree-based databases of protein or nucleic acid residues that uniquely characterize these virus strains are created. Strains of input virus sequences (partial or complete) are predicted through residue-wise comparisons with the databases. RECOVIR uses unique characterizing residues to identify automatically strains of partial or complete capsid sequences of picorna and caliciviruses, two of the most highly diverse ssRNA virus families. Partition-wise comparisons of the database residues with the corresponding residues of more than 300 complete and partial sequences of these viruses resulted in correct strain identification for all of these sequences. This study shows the feasibility of creating databases of hitherto unknown residues uniquely characterizing the capsid sequences of two of the most highly divergent ssRNA virus families. These databases enable automated strain identification from partial or complete capsid sequences of these human and animal pathogens.

Chakravarty, Sugoto↗
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↗