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NASA NTRS2006

Integrated human-robotic missions to the Moon and Mars : mission operations design implications

For most of the history of space exploration, human and robotic programs have been independent, and have responded to distinct requirements. The NASA Vision for Space Exploration calls for the return of humans to the Moon, and the eventual human exploration of Mars; the complexity of this range of missions will require an unprecedented use of automation and robotics in support of human crews. The challenges of human Mars missions, including roundtrip communications time delays of 6 to 40 minutes, interplanetary transit times of many months, and the need to manage lifecycle costs, will require the evolution of a new mission operations paradigm far less dependent on real-time monitoring and response by an Earthbound operations team. Robotic systems and automation will augment human capability, increase human safety by providing means to perform many tasks without requiring immediate human presence, and enable the transfer of traditional mission control tasks from the ground to crews. Developing and validating the new paradigm and its associated infrastructure may place requirements on operations design for nearer-term lunar missions. The authors, representing both the human and robotic mission operations communities, assess human lunar and Mars mission challenges, and consider how human-robot operations may be integrated to enable efficient joint operations, with the eventual emergence of a unified exploration operations culture.

Lee, Young↗

NASA NTRSDate not supplied

Updated Human Mars Ascent Vehicle Concept in Support of NASA’s Strategic Analysis Cycle 2021

The NASA Artemis program has brought significant change to the agency’s human exploration strategy over the last several years. The human exploration of Mars remains an ultimate objective of the overall human exploration strategy. However, how the agency intends to execute initial human exploration of Mars has shifted to better align with current policy. These changes find their way into the design of key architecture elements, such as the Mars Ascent Vehicle (MAV). Of the numerous changes in the conceptual human Mars architecture, two in particular have had significant impact on the design of the human MAV: the desire for minimal surface infrastructure for initial human Mars missions, and technology investment timelines in support of initial human missions to Mars. The first leads to a surface mission architecture targeting a 30-day surface duration with two crewmembers. The second drives out in-situ resource utilization (ISRU) from the initial sortie. As a result, the reference MAV design for initial human Mars missions in the Human Exploration and Operations Mission Directorate’s Strategic Analysis Cycle 2021 (SAC21) is based around supporting the return of 2 crew from the surface of Mars, without surface ISRU. This has led to several design changes since 2019, when details of a reference human MAV were last published. The MAV concept for SAC21 consists of a two-stage vehicle capable of supporting two crew for 84 hours. This duration is derived from a baseline ascent trajectory targeting an apoapsis rendezvous with the habitation element in a nominal 5-sol Mars aggregation orbit. With the goal of minimizing technology investments for the initial surface sortie removing ISRU from the trade space, the MAV must now be landed either fully loaded with propellant, or with some portion of the propellant off-loaded. Landing a partially-fueled MAV will require additional surface assets to perform robotic propellant transfer on the surface of Mars from a pre-emplaced propellant depot. Furthermore, cryogenic fluid management and storage for extended duration with no losses represents additional technology investment that would have to be made to support cryogenic-based propellant concepts that have been the baseline in past architectures. As a result, the SAC21 architecture utilizes a storable propellant-based MAV propulsion system to minimize these potential technology investments incurred by cryogenic-based propulsion systems. Recent efforts also focused on evaluating the sensitivity of key design parameters, such as landing site latitude, elevation, and local atmospheric conditions. All play a large role in determining the predicted propellant requirements of the MAV. Understanding their potential impacts is important because of the rippling effects of changes to the MAV design on other architecture elements, such as entry, descent, landing, and transportation systems. Results indicate up to +2% to -4% wet mass variation due to landing site latitude, with higher latitudes greater than 70 degrees North resulting in greater than 6% wet mass increases. The MAV saw reduced impact due to atmospheric design parameters, which only accounting for -0.6% to +0.4% wet mass variation.

Douglas J. Trent↗

NASA NTRSDate not supplied

Can Resilience Assessments Inform Early Design Human Factors Decision-making?

There is a growing call among researchers for tighter coupling between human factors and human reliability assessments. In this research, we explore if early design stage resilience assessments can help bridge some of the gaps between human factors and human reliability assessments. Resilience in systems is their ability to recover reasonably and operate within acceptable bounds during failures and unexpected events. The fmdtools toolkit allows designers to assess the resilience of a system by modeling the human error and machine-related failure propagation in both nominal and faulty scenarios during the early design stages. As a result, the fmdtools toolkit has a low-fidelity dynamic human reliability assessment component built into it. In this paper, we study if the results from the fmdtools simulations can help inform and prioritize human factors design decision-making, resulting in tighter coupling between human factors and human reliability assessments during the design process. Specifically, we explore the results from a rover design example to understand the types of information that can help guide human factor-related decision-making.

Resilience-based Design↗

NASA NTRSDate not supplied

Identifying Human Errors and Error Mechanisms From Accident Reports Using Large Language Models

Emerging operational concepts for aviation hinge on novel paradigms for human machine interaction. Critical to their safe operation is early consideration of human error into the design process. Existing methods for consideration of human error require significant expert input, which is challenging both in early design and in novel systems for which there is little existing safety expertise. In this research, we propose a methodology for identifying human error, error producing factors, and mechanisms in early design from historical incident reports. Additionally, we hypothesize that cross-domain sharing of lessons learned can aid with early design human considerations in circumstances where data is not relevant or incomplete. This is addressed by identifying causes of human error in aviation and railway domains through applying state-of-the art natural language processing techniques to historical incident reports. Using this method, it is possible to extract extensive reports on human error from past incidents. Using the proposed approach, we identify nine human errors from railway reports and fourteen from aviation reports, with three errors common to both domains. There is at least one error producing conditions for each human error while a majority of the errors have more than one error mechanism. We also found that a majority of the human errors, error producing factors, and error mechanisms (even if they are not common between the domains) can be used to inform safe operations across domains as long as the errors are not domain specific and are interpreted and contextualized using engineering judgement.

Human Errors↗

DOE OSTI2017

The overview in safety review of human factors engineering and control room design in chinese AP1000 nuclear power plant - 174

The first AP1000 nuclear power plant is constructed in Sanmen county in Zhejiang Province in China, which has creative and distinctive design characteristics. Human factors engineering disciplines are applied to the design of the AP1000. In addition to the elements of the program review model(including human factors engineering program management, operating experience review, functional requirements analysis and allocation, task analysis, staffing, integration of human reliability analysis with human factors engineering, human system interface design, procedure development, training program development, human system interface verification and validation, design implementation, human performance monitoring),the minimum inventory of controls, displays, and alarms present in the main control room and at the remote shutdown workstation. These contents mentioned above are reviewed according to standards, rules and regulations. This article introduces review process, and several important issues during the reviewing are described in detail. These issues include many elements, such as operating experience review, task analysis, human system interface design, verification and validation,and so on. This paper emphasizes on the main control room design(including environment and layout). Background noise in the actual main control room may exceed too much to design value. Another important issue is about main control room habitability systems(VES) changes to satisfy post-actuation performance requirements in AP1000 design change proposal. All the wall panel displays will be closed in this design change proposal. Review process and proposal are described in the paper. Since the AP1000 unit in China is the first constructed all over the world, verification and validation are especially important and necessary. The plan of verification and validation, the results summary report of verification and validation, the report of human engineering discrepancies are focus attention. Verification and validation in human factors engineering include HSI task support verification, HFE design verification and integrated system validation. This paper introduces the review process of verification and validation, the issues found in verification and validation. (authors)

21 SPECIFIC NUCLEAR REACTORS AND ASSOCIATED PLANTS↗

DOE OSTI2017

Determination of a Generic Human Error Probability Distribution, Part 2: A Dynamic SPAR-H Example Application

Humans are an integral part of a nuclear power plant (NPP) from its construction, to its everyday maintenance and operation. In the nuclear industry there are many approaches to quantifying component reliability, which is called probability risk assessment (PRA). In PRA there are two considerations given to the quantification, static and dynamic. Static is quantified before or after an operation; and dynamic is time dependent, in that the probability changes as real time data are provided. In PRA, the importance of the components is calculated, but what of the importance of the human component? There are several methods for human reliability analysis (HRA), which include but are not limited to: THERP, ATHEANA, CREAM, SPAR-H, ASEP, and SLIM. Each of these methods aims to provide a more accurate and objective human error probability (HEP) quantification. Of the methods listed above, ASEP, THERP, and SPAR-H are all inextricably linked. The Standardized Plant Analysis Risk-Human Reliability Analysis (SPAR-H) method was generated to better generalize the Technique for Human Error Rate Prediction (THERP) and Accident Sequence Precursor (ASEP). THERP is mapped to specific scenario templates, and ASEP is a simplified screening method, while SPAR-H employs generalizations to describe the spectrum of human behavior while maintaining the same theoretical underpinnings. SPAR-H is a well-known and accepted HRA method that uses performance shaping factors (PSFs) to classify inputs to the human component. PSFs capture a wide variety of input data, including plant status, crew dynamics, task description, and psychological aspects of the human operator. Current efforts have been focused to further generalize SPAR-H in to a dynamic framework. These methods can fit into dynamic NPP simulations such as RAVEN / RELAP-7. In order to do this, a dynamic methodology is applied to model the human as would be applied to the components. This method is applied in part one in the accompanying paper.

22 GENERAL STUDIES OF NUCLEAR REACTORS↗

DOE OSTI2015

Challenges in leveraging existing human performance data for quantifying the IDHEAS HRA method

Our article documents an exploratory study for collecting and using human performance data to inform human error probability (HEP) estimates for a new human reliability analysis (HRA) method, the IntegrateD Human Event Analysis System (IDHEAS). The method was based on cognitive models and mechanisms underlying human behaviour and employs a framework of 14 crew failure modes (CFMs) to represent human failures typical for human performance in nuclear power plant (NPP) internal, at-power events [1]. A decision tree (DT) was constructed for each CFM to assess the probability of the CFM occurring in different contexts. Data needs for IDHEAS quantification are discussed. Then, the data collection framework and process is described and how the collected data were used to inform HEP estimation is illustrated with two examples. Next, five major technical challenges are identified for leveraging human performance data for IDHEAS quantification. Furthermore, these challenges reflect the data needs specific to IDHEAS. More importantly, they also represent the general issues with current human performance data and can provide insight for a path forward to support HRA data collection, use, and exchange for HRA method development, implementation, and validation.

22 GENERAL STUDIES OF NUCLEAR REACTORS↗

DOE OSTI2016

Isolation and characterization of human islet stellate cells

Background and aims: We have previously demonstrated that islet stellate cells (ISCs) exhibiting a similar phenotype to classical pancreatic stellate cells (PSCs) could be isolated from rat islets, where they may contribute to islet fibrosis in type 2 diabetes mellitus (T2DM). This study was designed to determine whether human islets also contain ISC. Materials and methods: Using standard explants techniques, human ISCs were enriched from freshly isolated human islets. Immunofluorescence visualization of markers for PSCs(α-smooth muscle actin;α-SMA), desmin, vimentin, glial fibrillary acidic protein (GFAP) was used to characterize the human ISC. Cell counting kit-8 (CCK-8) was used to assess the proliferation of ISC. The wound-healing assay and the transwell migration were used to assess the migration capacity of ISC. Immunofluorescence against collagen typesI (col-I), collagen typesIII (col-III) and fibronectin (FN) was performed to identify extracellular matrix (ECM) component synthesized by ISC. Adipogenic and osteogenic differentiation were tried to detected stem cell potential. Results: In culture, ISC with triangular shape grow out from human islets. The passaged ISC expressed α-SMA, desmin, vimentin, GFAP and was positive for col-I, col-III and FN. The proliferation and migration ability of ISC was significantly slower than those of PSC. And both the human PSC and ISC were able to differentiate in vitro into adipocyte- and osteoblast-like cells. Conclusion: Similar to our previous rat experiment, the current study shows that human islets also contain ISC which is phenotypically similar but not identical to human PSC. - Highlights: • ISCs grow out from human islets. ISC expressed α-SMA, desmin, vimentin, GFAP and was positive for col-I, col-III and FN. The proliferation and migration ability of human islets contain ISC. • ISC is phenotypically similar but not identical to PSC. • ISC may play an important role in the islet fibrosis in T2DM.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2020

An Integrated Energy Systems Prototype Human-System Interface for a Steam Extraction Loop System to Support Joint Electricity-Hydrogen Flexible Operations

Due to increasing economic competition from renewables and combined-cycle natural gas plants, nuclear power plants are looking toward flexible operations to enhance their cost competitiveness. The Integrated Energy Systems project under the Light Water Reactor Sustainability program of the U.S. Department of Energy focuses on joint electricity-hydrogen flexible operations. Joint electricity-hydrogen flexible operations entail the nuclear power plant diverting thermal energy via main steam to a hydrogen production plant located nearby. The steam serves to enhance the efficiency of the hydrogen production. Furthermore, high temperature electrolysis requires a large amount of electricity, which the plant can also provide. The plant provides steam and electricity to the hydrogen plant throughout the day, but during peak demand hours the nuclear power plant returns to solely providing electricity to meet the high demand. Through this flexible concept of operations, the plant can optimize the thermal energy it produces without having to maneuver the power of the plant. This report documents the human factors process to design and develop a prototype human-system interface for the steam extraction loop that serves as the conduit between the nuclear power plant and the adjacent hydrogen plant. The design process followed the human factors guidelines set by NUREG-0711, Human Factors Engineering Program Review Model (O’Hara, Higgins, & Fleger, 2012), and expanded upon by the Guideline for Operational Nuclear Usability and Knowledge Elicitation (GONUKE; Boring, Ulrich, Joe, & Lew, 2015; Boring, Lew, & Ulrich, 2016). The design process entailed operator interviews to determine the concept of operations for the steam extraction loop, a review and adaptation of digital interface design concepts developed by the team in prior projects, an iterative design process, and reviews conducted by both operators and human factors experts. Several versions of the prototype human-system interface were developed. Operators were interviewed to determine what design features they found useful and would like to see in the interface. The design underwent a review by human factors experts against NUREG-0700, Human Interface Design Review Guidelines (U.S. Nuclear Regulatory Commission, 2019), to ensure compliance with the latest human factors standards for digital interfaces in nuclear applications. The design was then prototyped as a functional windows-based application integrated with the Generic Pressurized Water Reactor simulator modified to include the steam extraction loop. The simulation is supported by the Human Systems Simulation Laboratory at Idaho National Laboratory, which supports operator-in-the-loop testing. This is an ongoing project and the next phase of the project entails performing an operator-in-the-loop usability study to evaluate the interface and examine the proposed concept of operations to extraction steam from the nuclear power plant for delivery to the coupled hydrogen production plant.

99 GENERAL AND MISCELLANEOUS↗

DOE OSTI2025

Implications of Safety and Operational Features of Small, Advanced Reactors for the Evaluation of Important Human Actions

The design and operational characteristics of non-light water reactors are likely to change the role of human actions in safety function management and the types of human actions that are deemed important. The objectives of this report are to: • Identify the implications of small, advanced reactor design characteristics on human performance and the changing role of human actions in the management of safety functions. • Identify the methods that may be used to identify important human actions. • Identify how HFE safety reviewers can help ensure that the methods adequately model human actions to identify those that are important to safety. We identified the implications of small, advanced reactor characteristics on the role of personnel in safety function management. Then we addressed how designers can identify which human actions are important to safety using both probabilistic risk assessment (PRA) and deterministic analyses. PRA identifies important human actions using risk-importance criteria. Deterministically identified important human actions include those identified by analyses of situations such as transients and accidents and defense in depth. In all cases, the acceptability of the analyses is dependent on the modeling, quantification, and criterion selection to determine which human actions are important. How well the designers address these processes determines the acceptability of their methodology.

22 GENERAL STUDIES OF NUCLEAR REACTORS↗

NASA NTRS2017

What Machines Need to Learn to Support Human Problem-Solving

In the development of intelligent systems that interact with humans, there is often confusion between how the system functions with respect to the humans it interacts with and how it interfaces with those humans. The former is a much deeper challenge than the latter it requires a system-level understanding of evolving human roles as well as an understanding of what humans need to know (and when) in order to perform their tasks. This talk will focus on some of the challenges in getting this right as well as on the type of research and development that results in successful human-autonomy teaming. Brief Bio: Dr. Alonso Vera is Chief of the Human Systems Integration Division at NASA Ames Research Center. His expertise is in human-computer interaction, information systems, artificial intelligence, and computational human performance modeling. He has led the design, development and deployment of mission software systems across NASA robotic and human space flight missions, including Mars Exploration Rovers, Phoenix Mars Lander, ISS, Constellation, and Exploration Systems. Dr. Vera received a Bachelor of Science with First Class Honors from McGill University in 1985 and a Ph.D. from Cornell University in 1991. He went on to a Post-Doctoral Fellowship in the School of Computer Science at Carnegie Mellon University from 1990-93.

intelligent systems↗

NASA NTRSDate not supplied

Logistics Rates and Assumptions for Future Human Spaceflight Missions Beyond LEO

As NASA prepares for future human spaceflight missions with extended crew duration in destinations beyond low Earth orbit (LEO), the Agency has focused itself on understanding the drivers to sustainably support human life beyond Earth’s atmosphere. Future missions to deep space, the lunar surface, and eventually the Martian surface pose new challenges in ensuring the crew is sufficiently supplied with all necessary materials. To mitigate the risk of not delivering sufficient consumables and logistics for human spaceflight missions, NASA has examined past human space mission data and developed metabolic modeling to determine estimates for the crew consumption rates of fluids, solid consumables, and additional equipment needed. This paper is a compilation of guidelines, rates, and assumptions necessary to evaluate the logistics needs for future human exploration conceptual missions beyond LEO, providing a starting point and resource of information regarding usage rates and overall logistics supply planning for crewed exploration missions. Logistics represent all equipment and supplies not installed as part of the vehicle that are needed to support mission activities. Logistics can be further divided into specific categories, including consumables, maintenance items, spares, utilization, outfitting, as well as any packaging required. This paper will also provide use case examples of logistics needs to support human missions in deep space, including a conceptual lunar surface mission. The paper provides information necessary to calculate the mass and volume of known logistics for conceptual future human exploration missions beyond LEO. The assumptions in the paper are updated versions of previous assumptions made by the Agency and were derived from a number of sources, including International Space Station (ISS) historical usage and resupply rates, the Life Support Baseline Values and Assumptions Document (BVAD) 2022, Human Integration Design Handbook (HIDH) 2014, and data gathered from NASA human spaceflight programs and projects. The primary goal of the paper is to establish a set of consistent reference rates that multiple teams and groups can utilize to conduct logistics analysis and compare cases. This methodology is for initial estimates of conceptual human missions and does not take the place of detailed analysis for programs, nor does it provide requirements for programs.

Human Spaceflight↗

NASA NTRSDate not supplied

Logistics Rates and Assumptions for Future Human Spaceflight Missions Beyond LEO

Human Spaceflight↗

DOE OSTI2024

An Approach to Dynamic Human Reliability Analysis and Its Data Collection Framework

Human reliability analysis (HRA) is a method for evaluating human errors in a variety of complex systems such as nuclear power plants, military systems, aircraft, and chemical plants. Most HRA methods currently used by regulatory institutes or utilities are called static HRA and are carried out by simple worksheets or simple calculators. To date, there are many unsolved or intrinsic challenges in static HRA. For example, existing static HRA does not realistically model and evaluate human actions as they would be performed at actual systems. There is no method with HRA to objectively estimate the time required for human actions despite being essential to HRA processes. In addition, many HRA methods still rely on a dataset generated prior to the 1980s, from unrelated industry experience or simply from expert judgment. Accordingly, this study attempted to research how to overcome the challenges of existing HRA via dynamic risk assessment (a.k.a., simulation-based or computation-based risk assessment) techniques. First, this study developed a dynamic HRA method, named as PRocedure-based Investigation Method of EMRALD Risk Assessment – HRA (PRIMERA-HRA). The PRIMERA-HRA mainly concentrates on providing HRA analysts with specific guidelines on how to reasonably model human actions, assign human reliability data and evaluate output of simulation within a dynamic probabilistic risk assessment tool, called as Event Modeling Risk Assessment using Linked Diagrams (EMRALD). Second, this study also developed a module for performance shaping factors (i.e., the key concept in HRA quantification) applicable to dynamic HRA, then implemented it based on PRIMERA-HRA within the EMRALD tool. Third, this study developed an HRA data collection framework to support dynamic HRA, called as Simplified Human Error Experimental Program (SHEEP). Originally, the SHEEP study aimed to support static HRA and its data collection, but recently extended the scope to the new technologies such as dynamic HRA or HRA for advanced reactors. SHEEP focuses on the use of data collected from simplified simulators to complement—but not replace—data collection studies using full-scope simulators and actual operators. To date, many experiments were conducted under the SHEEP framework. Multiple analyses, such as human performance analysis, human error analysis, task complexity analysis, learning effect analysis and time distribution analysis, were also carried out using the collected data. Then, based on the major insights, an approach to inferring full-scope data based on simplified simulator data was proposed. The PRIMERA-HRA and SHEEP research are expected to evaluate human actions more realistically than existing static HRA, provide an opportunity to collect more HRA data with reasonable cost and labor, then contribute to enhance the quality of HRA.

99 - GENERAL AND MISCELLANEOUS↗

NASA NTRS2002

On the nature and evolution of the neural bases of human language

The traditional theory equating the brain bases of language with Broca's and Wernicke's neocortical areas is wrong. Neural circuits linking activity in anatomically segregated populations of neurons in subcortical structures and the neocortex throughout the human brain regulate complex behaviors such as walking, talking, and comprehending the meaning of sentences. When we hear or read a word, neural structures involved in the perception or real-world associations of the word are activated as well as posterior cortical regions adjacent to Wernicke's area. Many areas of the neocortex and subcortical structures support the cortical-striatal-cortical circuits that confer complex syntactic ability, speech production, and a large vocabulary. However, many of these structures also form part of the neural circuits regulating other aspects of behavior. For example, the basal ganglia, which regulate motor control, are also crucial elements in the circuits that confer human linguistic ability and abstract reasoning. The cerebellum, traditionally associated with motor control, is active in motor learning. The basal ganglia are also key elements in reward-based learning. Data from studies of Broca's aphasia, Parkinson's disease, hypoxia, focal brain damage, and a genetically transmitted brain anomaly (the putative "language gene," family KE), and from comparative studies of the brains and behavior of other species, demonstrate that the basal ganglia sequence the discrete elements that constitute a complete motor act, syntactic process, or thought process. Imaging studies of intact human subjects and electrophysiologic and tracer studies of the brains and behavior of other species confirm these findings. As Dobzansky put it, "Nothing in biology makes sense except in the light of evolution" (cited in Mayr, 1982). That applies with as much force to the human brain and the neural bases of language as it does to the human foot or jaw. The converse follows: the mark of evolution on the brains of human beings and other species provides insight into the evolution of the brain bases of human language. The neural substrate that regulated motor control in the common ancestor of apes and humans most likely was modified to enhance cognitive and linguistic ability. Speech communication played a central role in this process. However, the process that ultimately resulted in the human brain may have started when our earliest hominid ancestors began to walk.

Review, Tutorial↗

DOE OSTI2016

Differential sensitivity of Chironomus and human hemoglobin to gamma radiation

Chironomus ramosus is known to tolerate high doses of gamma radiation exposure. Larvae of this insect possess more than 95% of hemoglobin (Hb) in its circulatory hemolymph. This is a comparative study to see effect of gamma radiation on Hb of Chironomus and humans, two evolutionarily diverse organisms one having extracellular and the other intracellular Hb respectively. Stability and integrity of Chironomus and human Hb to gamma radiation was compared using biophysical techniques like Dynamic Light Scattering (DLS), UV-visible spectroscopy, fluorescence spectrometry and CD spectroscopy after exposure of whole larvae, larval hemolymph, human peripheral blood, purified Chironomus and human Hb. Sequence- and structure-based bioinformatics methods were used to analyze the sequence and structural similarities or differences in the heme pockets of respective Hbs. Resistivity of Chironomus Hb to gamma radiation is remarkably higher than human Hb. Human Hb exhibited loss of heme iron at a relatively low dose of gamma radiation exposure as compared to Chironomus Hb. Unlike human Hb, the heme pocket of Chironomus Hb is rich in aromatic amino acids. Higher hydophobicity around heme pocket confers stability of Chironomus Hb compared to human Hb. Previously reported gamma radiation tolerance of Chironomus can be largely attributed to its evolutionarily ancient form of extracellular Hb as evident from the present study. -- Highlights: •Comparison of radiation tolerant Chironomus Hb and radiation sensitive Human Hb. •Amino acid composition of midge and human heme confer differential hydrophobicity. •Heme pocket of evolutionarily ancient midge Hb provide gamma radiation resistivity.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2015

Using populations of human and microbial genomes for organism detection in metagenomes

Identifying causative disease agents in human patients from shotgun metagenomic sequencing (SMS) presents a powerful tool to apply when other targeted diagnostics fail. Numerous technical challenges remain, however, before SMS can move beyond the role of research tool. Accurately separating the known and unknown organism content remains difficult, particularly when SMS is applied as a last resort. The true amount of human DNA that remains in a sample after screening against the human reference genome and filtering nonbiological components left from library preparation has previously been underreported. In this study, we create the most comprehensive collection of microbial and reference-free human genetic variation available in a database optimized for efficient metagenomic search by extracting sequences from GenBank and the 1000 Genomes Project. The results reveal new human sequences found in individual Human Microbiome Project (HMP) samples. Individual samples contain up to 95% human sequence, and 4% of the individual HMP samples contain 10% or more human reads. In conclusion, left unidentified, human reads can complicate and slow down further analysis and lead to inaccurately labeled microbial taxa and ultimately lead to privacy concerns as more human genome data is collected.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Characterization of a monoclonal antibody specific to California serogroup orthobunyaviruses and development as a chimeric immunoglobulin M-positive control in human diagnostics

ABSTRACT California serogroup viruses (CSGVs) of medical importance in the United States include La Crosse virus, Jamestown Canyon virus (JCV), California encephalitis virus, and snowshoe hare virus. Current diagnosis of CSGVs relies heavily on serologic techniques for detecting immunoglobulin M (IgM), an indication of a recent CSGV infection. However, human-positive control sera reactive to viruses in the serogroup are scarce because detection of recent infections is rare. Here, we describe the development of new murine monoclonal antibodies (MAbs) reactive to CSGVs and the engineering of a human-murine chimeric antibody by combining the variable regions of the broadly CSGV cross-reactive murine MAb, 3-3B6/2-3B2 and the constant region of the human IgM. MAb 3-3B6/2-3B2 recognizes a tertiary epitope on the Gn/Gc heterodimer, and epitopes important in JCV neutralization were mapped to the Gc glycoprotein. This engineered human IgM constitutively expressed in a HEK-293 stable cell line can replace human-positive control sera in diagnostic serological techniques such as IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA). Compared to the parent murine MAbs, the human-chimeric IgM antibody had identical serological activity to CSGVs in ELISA and demonstrated equivalent reactivity compared to human immune sera in the MAC-ELISA. Importance Orthobunyaviruses in the California serogroup cause severe neurological disease in children and adults. While these viruses are known to circulate widely in North America, their occurrence is rare. Serological testing for CSGVs is hindered by the limited availability and volumes of human-positive specimens needed as controls in serologic assays. Here, we described the development of a murine monoclonal antibody cross-reactive to CSGVs engineered to contain the variable regions of the murine antibody on the backbone of human IgM. The chimeric IgM produced from the stably expressing HEK293 cell line was evaluated for use as a surrogate human-positive control in a serologic diagnostic test.

Microbiology↗