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All sources NASA NTRS DOE OSTI

DOE OSTI2024

Correlating transcription and protein expression profiles of immune biomarkers following lipopolysaccharide exposure in lung epithelial cells

Universal and early recognition of pathogens occurs through recognition of evolutionarily conserved pathogen associated molecular patterns (PAMPs) by innate immune receptors and the consequent secretion of cytokines and chemokines. The intrinsic complexity of innate immune signaling and associated signal transduction challenges our ability to obtain physiologically relevant, reproducible and accurate data from experimental systems. One of the reasons for the discrepancy in observed data is the choice of measurement strategy. Immune signaling is regulated by the interplay between pathogen-derived molecules with host cells resulting in cellular expression changes. However, these cellular processes are often studied by the independent assessment of either the transcriptome or the proteome. Correlation between transcription and protein analysis is lacking in a variety of studies. In order to methodically evaluate the correlation between transcription and protein expression profiles associated with innate immune signaling, we measured cytokine and chemokine levels following exposure of human cells to the PAMP lipopolysaccharide (LPS) from the Gram-negative pathogen Pseudomonas aeruginosa . Expression of 84 messenger RNA (mRNA) transcripts and 69 proteins, including 35 overlapping targets, were measured in human lung epithelial cells. We evaluated 50 biological replicates to determine reproducibility of outcomes. Following pairwise normalization, 16 mRNA transcripts and 6 proteins were significantly upregulated following LPS exposure, while only five (CCL2, CSF3, CXCL5, CXCL8/IL8, and IL6) were upregulated in both transcriptomic and proteomic analysis. This lack of correlation between transcription and protein expression data may contribute to the discrepancy in the immune profiles reported in various studies. The use of multiomic assessments to achieve a systems-level understanding of immune signaling processes can result in the identification of host biomarker profiles for a variety of infectious diseases and facilitate countermeasure design and development.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1992

Survival of epiphytic bacteria from seed stored on the Long Duration Exposure Facility (LDEF)

Microbial contamination in American spacecraft has previously been documented, however, potential risks to plants and humans in future space based controlled ecological life support systems (CELSS) have yet to be addressed directly. The current study was designed to determine the survival of microorganisms exposed to the relatively harsh conditions found in low Earth orbit (LEO). Total mean dosage for flight and ground control seeds were 210.2 and 0.9 rads, respectively. Bacteria were isolated by plating samples of seedwashings onto dilute tryptic soy agar. Pure isolates of morphologically distinct bacteria were obtained by standard microbiological procedures. Bacteria were grouped according to colony type and preliminary identification was completed using a fatty acid analysis system. Bacillus spp. were the primary microorganisms that survived on seed during the experiment. Results support the hypothesis that terrestrial microorganisms can survive long periods of time in relatively harsh LEO environments.

Schuerger, Andrew C.↗

NASA NTRS2014

NASA's Asteroid Redirect Mission: A Robotic Boulder Capture Option for Science, Human Exploration, Resource Utilization, and Planetary Defense

NASA is examining two options for the Asteroid Redirect Mission (ARM), which will return asteroid material to a Lunar Distant Retrograde Orbit (LDRO) using a robotic solar electric propulsion spacecraft, called the Asteroid Redirect Vehicle (ARV). Once the ARV places the asteroid material into the LDRO, a piloted mission will rendezvous and dock with the ARV. After docking, astronauts will conduct two extravehicular activities (EVAs) to inspect and sample the asteroid material before returning to Earth. One option involves capturing an entire small (~4 - 10 m diameter) near-Earth asteroid (NEA) inside a large inflatable bag. However, NASA is also examining another option that entails retrieving a boulder (~1 - 5 m) via robotic manipulators from the surface of a larger (~100+ m) pre-characterized NEA. The Robotic Boulder Capture (RBC) option can leverage robotic mission data to help ensure success by targeting previously (or soon to be) well- characterized NEAs. For example, the data from the Japan Aerospace Exploration Agency's (JAXA) Hayabusa mission has been utilized to develop detailed mission designs that assess options and risks associated with proximity and surface operations. Hayabusa's target NEA, Itokawa, has been identified as a valid target and is known to possess hundreds of appropriately sized boulders on its surface. Further robotic characterization of additional NEAs (e.g., Bennu and 1999 JU3) by NASA's OSIRIS REx and JAXA's Hayabusa 2 missions is planned to begin in 2018. This ARM option reduces mission risk and provides increased benefits for science, human exploration, resource utilization, and planetary defense. Science: The RBC option is an extremely large sample-return mission with the prospect of bringing back many tons of well-characterized asteroid material to the Earth-Moon system. The candidate boulder from the target NEA can be selected based on inputs from the world-wide science community, ensuring that the most scientifically interesting boulder be returned for subsequent sampling. In addition, the material surrounding the boulder can be collected from the surface, thus providing geological contextual information and additional samples of NEA regolith. The robotic manipulators used for capturing the boulder will ensure some of the surface remains undisturbed and that the boulder will retain its structural integrity, which will preserve the context of any samples collected by the astronauts and ensure a high level of science return. Human Exploration: Due to the coherent nature of the boulder that will be collected, entire encapsulation of the asteroid material is not required. This facilitates exploration and sample collection of the boulder by astronauts in a variety of ways. The total time for EVA during the crew portion of the mission is very limited. Current estimates are that each of the two EVAs will only last four hours. The RBC option will allow crew members to have good situational awareness of the work site and quickly identify sample sites of interest. In addition, the samples to be collected can be readily accessed without having to deal with removal of an encapsulation system, which adds extra complexity and risk for the astronauts during EVA. Resource Utilization: One of the most crucial aspects for resource utilization is the identification and collection of appropriate materials (e.g., volatiles, organics, metals, etc.) that contain components of interest. Prior characterization of NEAs is required in order to increase the likelihood that appropriate materials will be returned. Ground-based observations of small (<10 m) NEAs are challenging, but characterization efforts of larger targets have demonstrated that NEAs with volatiles and organics have been identified. Two potential targets for the RBC option (Bennu and 1999 JU3) have been previously identified as potentially rich in resources, and both are already targets of currently planned robotic missions that will characterize their physical properties in great detail. Planetary Defense: The RBC option involves interaction with a well- characterized potentially hazardoussized NEA that would enable NASA to conduct one or more planetary defense demonstrations. The primary method would use the collected boulder to augment the mass of the ARV and perform an Enhanced Gravity Tractor (EGT) demonstration on the NEA. Additionally, other approaches could be demonstrated during the mission, such as Ion Beam Deflection (IBD) and/or observation of a Kinetic Impactor (KI). The relative effectiveness of a slow push-pull method such as the EGT or IBD could be directly compared and contrasted with the results of the more energetic KI method on the target NEA. Conclusions: This boulder option for NASA's ARM can leverage knowledge of previously characterized NEAs from prior robotic missions, which provides more certainty of the target NEA's physical characteristics and reduces mission risk. This increases the return on investment for NASA's future activities with respect to human exploration, resource utilization, and planetary defense.

Abell, P.↗

DOE OSTI2023

TopPICR: A Companion R Package for Top-Down Proteomics Data Analysis

Top-down proteomics is the analysis of proteins in their intact form without proteolysis, thus preserving valuable information about post-translational modifications, isoforms, and proteolytic processing. However, it is still a developing field due to limitations in the instrumentation, difficulties with interpretation of complex mass spectra, and a lack of well-established quantification approaches. TopPIC is one of the popular tools for proteoform identification. Here we extended its capabilities into label-free proteoform quantification by developing a companion R package (TopPICR). Key steps in the TopPICR pipeline include filtering identifications, inferring a minimal set of protein accessions explaining the observed sequences, aligning retention times, recalibrating measured masses, clustering features across datasets, and finally compiling feature intensities using the match-between-runs approach. The output of the pipeline is an MSnSet object which makes downstream data analysis seamlessly compatible with packages from the Bioconductor project. It also provides the capability for visualizing proteoforms within the context of the parent protein sequence. The functionality of TopPICR is demonstrated on top-down LC-MS/MS datasets of 10 human-in-mouse xenografts of luminal and basal breast tumor samples.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

The Campylobacter jejuni CiaD effector co-opts the host cell protein IQGAP1 to promote cell entry

Campylobacter jejuni is a foodborne pathogen that binds to and invades the epithelial cells lining the human intestinal tract. Maximal invasion of host cells by C. jejuni requires cell binding as well as delivery of the Cia proteins (Campylobacter invasion antigens) to the host cell cytosol via the flagellum. Here, we show that CiaD binds to the host cell protein IQGAP1 (a Ras GTPase-activating-like protein), thus displacing RacGAP1 from the IQGAP1 complex. This, in turn, leads to the unconstrained activity of the small GTPase Rac1, which is known to have roles in actin reorganization and internalization of C. jejuni. Our results represent the identification of a host cell protein targeted by a flagellar secreted effector protein and demonstrate that C. jejuni-stimulated Rac signaling is dependent on IQGAP1.

59 BASIC BIOLOGICAL SCIENCES↗

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↗

NASA NTRS2011

Musculoskeletal Modeling Component of the NASA Digital Astronaut Project

The NASA Digital Astronaut Project s (DAP) objective is to provide computational tools that support research of the physiological response to low gravity environments and analyses of how changes cause health and safety risks to the astronauts and to the success of the mission. The spaceflight risk associated with muscle atrophy is impaired performance due to reduced muscle mass, strength and endurance. Risks of early onset of osteoporosis and bone fracture are among the spaceflight risks associated with loss of bone mineral density. METHODS: Tools under development include a neuromuscular model, a biomechanical model and a bone remodeling model. The neuromuscular model will include models of neuromuscular drive, muscle atrophy, fiber morphology and metabolic processes as a function of time in space. Human movement will be modeled with the biomechanical model, using muscle and bone model parameters at various states. The bone remodeling model will allow analysis of bone turnover, loss and adaptation. A comprehensive trade study was completed to identify the current state of the art in musculoskeletal modeling. The DAP musculoskeletal models will be developed using a combination of existing commercial software and academic research codes identified in the study, which will be modified for use in human spaceflight research. These individual models are highly dependent upon each other and will be integrated together once they reach sufficient levels of maturity. ANALYSES: The analyses performed with these models will include comparison of different countermeasure exercises for optimizing effectiveness and comparison of task requirements and the state of strength and endurance of a crew member at a particular time in a mission. DISCUSSION: The DAP musculoskeletal model has the potential to complement research conducted on spaceflight induced changes to the musculoskeletal system. It can help with hypothesis formation, identification of causative mechanisms and supplementing small data samples.

Lewandowski, B. E.↗

NASA NTRS2003

Truly incomplete and complex exchanges in prematurely condensed chromosomes of human fibroblasts exposed in vitro to energetic heavy ions

Confluent human fibroblast cells (AG1522) were irradiated with gamma rays, 490 MeV/nucleon silicon ions, or iron ions at either 200 or 500 MeV/nucleon. The cells were allowed to repair at 37 degrees C for 24 h after exposure, and a chemically induced premature chromosome condensation (PCC) technique was used to condense chromosomes in the G2 phase of the cell cycle. Incomplete and complex exchanges were analyzed in the irradiated samples. To verify that chromosomal breaks were truly unrejoined, chromosome aberrations were analyzed using a combination of whole-chromosome specific probes and probes specific for the telomere region of the chromosome. Results showed that the frequency of unrejoined chromosome breaks was higher after irradiation with the heavy ions of high LET, and consequently the ratio of incomplete to complete exchanges increased steadily with LET up to 440 keV/microm, the highest LET included in the present study. For samples exposed to 200 MeV/nucleon iron ions, chromosome aberrations were analyzed using the multicolor FISH (mFISH) technique, which allows identification of both complex and truly incomplete exchanges. Results of the mFISH study showed that 0.7 and 3 Gy iron ions produced similar ratios of complex to simple exchanges and incomplete to complete exchanges; these ratios were higher than those obtained after exposure to 6 Gy gamma rays. After 0.7 Gy of iron ions, most complex aberrations were found to involve three or four chromosomes, which is a likely indication of the maximum number of chromosome domains traversed by a single iron-ion track.

NASA Discipline Radiation Health↗

DOE OSTI2022

Cryo-electron Microscopy of Adeno-associated Virus

Adeno-associated virus (AAV) has a single-stranded DNA genome encapsidated in a small icosahedrally symmetric protein shell with 60 subunits. AAV is the leading delivery vector in emerging gene therapy treatments for inherited disorders, so its structure and molecular interactions with human hosts are of intense interest. A wide array of electron microscopic approaches have been used to visualize the virus and its complexes, depending on the scientific question, technology available, and amenability of the sample. Approaches range from subvolume tomographic analyses of complexes with large and flexible host proteins to detailed analysis of atomic interactions within the virus and with small ligands at resolutions as high as 1.6 Å. Analyses have led to the reclassification of glycan receptors as attachment factors, to structures with a new-found receptor protein, to identification of the epitopes of antibodies, and a new understanding of possible neutralization mechanisms. AAV is now well-enough characterized that it has also become a model system for EM methods development. Heralding a new era, cryo-EM is now also being deployed as an analytic tool in the process development and production quality control of high value pharmaceutical biologics, namely AAV vectors.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1982

The identification of the variation of atherosclerosis plaques by invasive and non-invasive methods

Computer-enhanced visualization of coronary arteries and lesions within them is discussed, comparing invasive and noninvasive methods. Trial design factors in computer lesions assessment are briefly discussed, and the use of the computer edge-tracking technique in that assessment is described. The results of a small pilot study conducted on serial cineangiograms of men with premature atherosclerosis are presented. A canine study to determine the feasibility of quantifying atherosclerosis from intravenous carotid angiograms is discussed. Comparative error for arterial and venous injection in the canines is determined, and the mode of processing the films to achieve better visualization is described. The application of the computer edge-tracking technique to an ultrasound image of the human carotid artery is also shown and briefly discussed.

Selzer, R. H.↗

DOE OSTI2022

Comparing variability in diagnosis of upper respiratory tract infections in patients using syndromic, next generation sequencing, and PCR-based methods

Early and accurate diagnosis of respiratory pathogens and associated outbreaks can allow for the control of spread, epidemiological modeling, targeted treatment, and decision making–as is evident with the current COVID-19 pandemic. Many respiratory infections share common symptoms, making them difficult to diagnose using only syndromic presentation. Yet, with delays in getting reference laboratory tests and limited availability and poor sensitivity of point-of-care tests, syndromic diagnosis is the most-relied upon method in clinical practice today. Here, we examine the variability in diagnostic identification of respiratory infections during the annual infection cycle in northern New Mexico, by comparing syndromic diagnostics with polymerase chain reaction (PCR) and sequencing-based methods, with the goal of assessing gaps in our current ability to identify respiratory pathogens. Of 97 individuals that presented with symptoms of respiratory infection, only 23 were positive for at least one RNA virus, as confirmed by sequencing. Whereas influenza virus (n = 7) was expected during this infection cycle, we also observed coronavirus (n = 7), respiratory syncytial virus (n = 8), parainfluenza virus (n = 4), and human metapneumovirus (n = 1) in individuals with respiratory infection symptoms. Four patients were coinfected with two viruses. In 21 individuals that tested positive using PCR, RNA sequencing completely matched in only 12 (57%) of these individuals. Few individuals (37.1%) were diagnosed to have an upper respiratory tract infection or viral syndrome by syndromic diagnostics, and the type of virus could only be distinguished in one patient. Thus, current syndromic diagnostic approaches fail to accurately identify respiratory pathogens associated with infection and are not suited to capture emerging threats in an accurate fashion. We conclude there is a critical and urgent need for layered agnostic diagnostics to track known and unknown pathogens at the point of care to control future outbreaks.

60 APPLIED LIFE SCIENCES↗

NASA NTRS2015

In-Flight Microbial Monitor

Previous research has shown that potential human pathogens have been detected on the International Space Station (ISS). New microorganisms are introduced with every exchange of crew and cargo. Microorganisms introduced to the ISS are readily transferred between crew and subsystems (i.e., ECLSS, environmental control and life support systems). Current microbial characterization methods require a culture-based enrichment of microorganisms and at least a 48-hour incubation time. This increases the microbial load while detecting only a limited number of microorganisms. The culture-based method detects approximately 1-10% of the total organisms present and provides no identification. To identify and enumerate ISS samples requires that the microbes be returned to Earth for complete analysis. Therefore, a more expedient, low-cost, inflight method of microbial detection, identification, and enumeration is needed. The RAZOR EX, a ruggedized, commercial off the shelf, real-time PCR field instrument was tested for its ability to detect microorganisms at low concentrations within one hour. Escherichia coli, Salmonella enterica Typhimurium, and Pseudomonas aeruginosa were detected at low levels using real-time DNA amplification. Total heterotrophic counts could also be detected using a 16S gene marker that can identify up to 98% of all bacteria. To reflect viable cells found in the samples, RNA was also detectable using a modified, single-step reverse transcription reaction.

Technology Portfolio System↗

DOE OSTI2021

The Crown Pearl: a draft genome assembly of the European freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758)

Abstract Since historical times, the inherent human fascination with pearls turned the freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758) into a highly valuable cultural and economic resource. Although pearl harvesting in M. margaritifera is nowadays residual, other human threats have aggravated the species conservation status, especially in Europe. This mussel presents a myriad of rare biological features, e.g. high longevity coupled with low senescence and Doubly Uniparental Inheritance of mitochondrial DNA, for which the underlying molecular mechanisms are poorly known. Here, the first draft genome assembly of M. margaritifera was produced using a combination of Illumina Paired-end and Mate-pair approaches. The genome assembly was 2.4 Gb long, possessing 105,185 scaffolds and a scaffold N50 length of 288,726 bp. The ab initio gene prediction allowed the identification of 35,119 protein-coding genes. This genome represents an essential resource for studying this species’ unique biological and evolutionary features and ultimately will help to develop new tools to promote its conservation.

Gomes-dos-Santos, André (ORCID:0000000199734861)↗

NASA NTRSDate not supplied

Machine Learning Based Crater Detection for Terrain Relative Navigation

As Lunar exploration continues to become more commonplace, reliable methods of precise Terrain Relative Navigation (TRN) are needed. While there are many TRN techniques available, one that has received increased interest in the past few years is that of crater based navigation. Crater based navigation has numerous benefits, including being a human recognizable feature (important for crewed missions), as well as the fact that craters are often possible hazards that need to be detected and avoided. The use of crater based navigation has been limited however. This has been due to the difficulty of running such algorithms on board a spacecraft, as well as the difficulty in procuring large amounts of the required training data. This paper presents a new rendering tool for generating large amounts of high quality training data. It then looks at two recently developed machine learning techniques for crater detection and crater identification in real-time on near-future space hardware.

computer vision↗

DOE OSTI2022

A non-traditional crystal-based compound screening method targeting the ATP binding site of Plasmodium falciparum GRP78 for identification of novel nucleoside analogues

Drug resistance to front-line malarial treatments represents an ongoing threat to control malaria, a vector borne infectious disease. The malarial parasite, Plasmodium falciparum has developed genetic variants, conferring resistance to the current standard therapeutic artemisinin and its derivatives commonly referred to as artemisinin-combination therapies (ACTs). Emergence of multi-drug resistance parasite genotypes is a warning of potential treatment failure, reaffirming the urgent and critical need to find and validate alternate drug targets to prevent the spread of disease. An attractive and novel drug target includes glucose-regulated protein 78 kDa (GRP78, or BiP), an essential molecular chaperone protein involved in the unfolded protein response that is upregulated in ACT treated P. falciparum parasites. We have shown that both sequence and structure are closely related to human GRP78 (hGRP78), a chaperone belonging to the HSP70 class of ATPase proteins, which is often upregulated in cellular stress responses and cancer. By screening a library of nucleoside analogues, we identified eight ‘hit’ compounds binding at the active site of the ATP binding domain of P. falciparum GRP78 using a high-throughput ligand soaking screen using x-ray crystallography. These compounds were further evaluated using protein thermal shift assays to assess target binding activity. The nucleoside analogues identified from our screen provide a starting point for the development of more potent and selective antimalarial inhibitors. In addition, we have established a well-defined, high-throughput crystal-based screening approach that can be applied to many crystallizable P. falciparum proteins for generating anti-Plasmodium specific compounds.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1998

Identification of phosphoproteins coupled to initiation of motility in live epididymal mouse sperm

A method for collecting live immotile cauda epididymal mouse sperm that initiate motility by dilution into an activation buffer is described. Sperm in collection buffer showed low percent motility (MOT) and population progression (PRG) that increased 10-fold and 9-fold, respectively, during the first 2 min after dilution into activation buffer. Western phosphoserine (pS), phosphothreonine (pT), and phosphotyrosine (pY) analysis revealed a 120 kDa protein that markedly increased in pT content during initiation of motility and may be related to FP130, the motility-coupled axonemal protein of sea urchin sperm. A prominent 82 kDa protein that was pS and pT-phosphorylated in immotile and motile sperm is likely the fibrous sheath component AKAP82 that is phosphorylated during spermatogenesis. Analysis of live human sperm also identified a prominent 120 kDa pT protein. Thus it appears that phosphorylation of FP130 and related 120 kDa proteins in mouse, and perhaps human sperm, represent common targets during motility initiation in sperm. Copyright 1998 Academic Press.

Non-NASA Center↗

NASA NTRS2015

Inflight Microbial Monitoring- An Alternative Method to Culture Based Detection Currently Used on the International Space Station

Previous research has shown that potentially destructive microorganisms and human pathogens have been detected on the International Space Station (ISS). The likelihood of introducing new microorganisms occurs with every exchange of crew or addition of equipment or supplies. Microorganisms introduced to the ISS are readily transferred between crew and subsystems (i.e. ECLSS, environmental control and life support systems). Current microbial characterization methods require enrichment of microorganisms and at least a 48-hour incubation time. This increases the microbial load while detecting only a limited number of the total microorganisms. The culture based method detects approximately 1-10% of the total organisms present and provides no identification. To identify and enumerate ISS microbes requires that samples be returned to Earth for complete analysis. Therefore, a more expedient, low-cost, in-flight method of microbial detection, identification, and enumeration is warranted. The RAZOR EX, a ruggedized, commercial off the shelf, real-time PCR field instrument was tested for its ability to detect microorganisms at low concentrations within one hour. Escherichia coli, Salmonella enterica Typhimurium, and Pseudomonas aeruginosa were detected at low levels using real-time DNA amplification. Total heterotrophic counts could also be detected using a 16S gene marker that can identify up to 98% of all bacteria. To reflect viable cells found in the samples, RNA was also detectable using a modified, single-step reverse transcription reaction.

Inflight Microbial↗

DOE OSTI2025

Discovering methylated DNA motifs in bacterial nanopore sequencing data with MIJAMP

Abstract Bacterial DNA methylation is involved in diverse cellular functions, including modulation of gene expression, DNA repair, and restriction–modification systems for defense against viruses and other foreign DNA. Restriction systems hinder efforts to engineer organisms to produce fuels and chemicals from waste and renewable feedstocks by degrading DNA during transformation. Methylome analysis allows identification of motifs within a bacterial chromosome that may be targeted by native restriction enzymes. Further expression of the corresponding methyltransferases in Escherichia coli allows plasmid DNA to be protected from restriction in the target organism, thereby drastically enhancing transformation efficiency. Nanopore sequencing can detect methylated bases, but software is needed to transform modified base coordinates into methylated motifs. Here, we develop MIJAMP (MIJAMP Is Just A MethylBED Parser), a software package that was developed to discover methylated motifs from the output of ONT’s Modkit or other data in the methylBED format. MIJAMP employs a human-driven refinement strategy that empirically validates all motifs against genome-wide methylation data, thus eliminating incorrect motifs. MIJAMP also reports methylation data on specific, user-defined motifs. Using MIJAMP, we determined the methylated motifs both in a control strain (wild-type E. coli) and in Synecococcus sp. strain PCC7002, laying the foundation for improved transformation in this organism. MIJAMP is available at https://code.ornl.gov/alexander-public/mijamp/. One Sentence Summary: Here we describe software written to discover DNA methylation motifs from nanopore sequencing data.

59 BASIC BIOLOGICAL SCIENCES↗