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At least 199 records · Page 11

Considering Cell Proliferation to Optimize Detection of Radiation-induced 53BP1+ Foci in 15 Mouse Strains ex vivo

Due to high metabolic activity, proliferating cells continuously generate free radicals, which induce DNA double strand breaks (DSB). Fluorescently tagged nuclear foci of DNA repair protein 53 binding protein-1(53BP1) are used as a standard metric for measuring DSB formation at baseline and in response to environmental insults such as radiation. Here we demonstrate that the background level of spontaneous 53BP1+ foci formation can be modeled mathematically as a function of cell confluence, which is a metric of proliferation rate. This model was validated using spontaneous 53BP1+ foci data from 68 cultures of primary skin fibroblasts derived from 15 different strains of mice, showing a ~10 fold decrease from low to full confluence that is independent of mouse strain. We developed an online open access tool to correct for the impact of cell confluence on the detection of radiation-induced 53BP1+ foci (RIF). This tool provides guidelines for the number of cells required to reach statistical significance for the detection of excess foci induced by low doses of ionizing radiation as a function of confluence and time post-irradiation. We hope that this quantification tool will help the radiation biology community in the design of future experiments that utilize 53BP1+ foci-based quantification of radiation responses in vitro. Our “tool for enhanced results of RIF in cells” (terRIFic) can be found at: https://radbiolab.shinyapps.io/terrific/

Radiation, DNA damage, confluence↗

Atmosphere stabilization and element recycle in an experimental mouse-algal system

Life support systems based on bioregeneration rely on the control and manipulation of organisms. Experiments conducted with a gas-closed mouse-algal system designed to investigate principles of photosynthetic gas exchange focus primarily on observing gas exchange phenomena under varying algal environmental conditions and secondarily on studying element cycling through compartments of the experimental system. Inherent instabilities exit between the uptake and release of carbon dioxide CO2 and oxygen O2 by the mouse and algae. Variations in light intensity and cell density alter the photosynthetic rate of the algae and enable maintenance of physiologic concentrations of CO2 and O2. Different nitrogen sources (urea and nitrate) result in different algal assimilatory quotients (AQ). Combinations of photosynthetic rate and AQ ratio manipulations have been examined for their potential in stabilizing atmospheric gas concentrations in the gas-closed algal-mouse system. Elemental mass balances through the experimental systems compartments are being studied with the concurrent development of a mathematical simulation model. Element cycling experiments include quantification of elemental flows through system compartments and wet oxidation of system waste materials for use as an algal nutrient source. Oxidized waste products demonstrate inhibitory properties although dilution has been shown to allow normal growth.

Smernoff, David T.↗

Mouse Behavior on ISS: The Emergence of a Distinctive, Organized Group Circling Behavior Unique to Spaceflight

As interest in long duration effects of space habitation increases, understanding the behavior of model organisms living within the habitats engineered to fly them is vital for designing, validating, and interpreting future spaceflight studies. Only a handful of papers have previously reported behavior of mice and rats in the weightless environment of space (Andreev-Andrievskiy, et al., 2013; Cancedda et al., 2012; Ronca et al., 2008). The Rodent Research Hardware and Operations Validation Mission (Rodent Research-1; RR1) utilized the Rodent Habitat (RH) developed at NASA Ames Research Center to fly mice on the ISS. Ten adult (16-week-old) female C57BL6J mice were launched on September 21st, 2014 in an unmanned Dragon Capsule, and spent 37 days in flight. Here we report group behavioral phenotypes of the RR1 Flight (FLT) and environment-matched Ground Control (GC) mice in the RH during this long duration flight. Video was recorded for 34 days on the ISS, permitting daily assessments of overall health and well being of the mice, and providing a valuable repository for detailed behavioral analysis. As compared to GC mice, RR1 FLT mice exhibited the same range of behaviors, including eating, drinking, exploration, self- and allogrooming, and social interactions at similar or greater levels of occurrence. Overall activity was greater in FLT as compared to GC mice, with spontaneous ambulatory behavior, including organized circling or race-tracking behavior that emerged within the first few days of flight following a common developmental sequence, comprising the primary dark cycle activity of FLT mice. Circling participation by individual mice persisted throughout the mission. Analysis of group behavior over mission days revealed recruitment of mice into the group phenotype, coupled with decreasing numbers of collisions between circling mice. This analysis provides insights into the behavior of mice in microgravity, and clear evidence for the emergence of a distinctive, organized group behavior unique to the weightless space environment. Supported by the NASA Rodent Research Project, Space Biology Program, and Space Life Sciences Training Program.

spaceflight↗

Anticipatory Eye Movements in Interleaving Templates of Human Behavior

Performance modeling has been made easier by architectures which package psychological theory for reuse at useful levels of abstraction. CPM-GOMS uses templates of behavior to package at a task level (e.g., mouse move-click, typing) predictions of lower-level cognitive, perceptual, and motor resource use. CPM-GOMS also has a theory for interleaving resource use between templates. One example of interleaving is anticipatory eye movements. This paper describes the use of ACT-Stitch, a framework for translating CPM-GOMS templates and interleaving theory into ACT-R, to model anticipatory eye movements in skilled behavior. The anticipatory eye movements explain performance in a well-practiced perceptual/motor task, and the interleaving theory is supported with results from an eye-tracking experiment.

Matessa, Michael↗

Evidence for a cytokine-sensitive network of iron-associated genes that protects pancreatic islets against ferroptosis

Background/Objectives: The micronutrient iron is closely connected to inflammation and is among the complex factors contributing to beta-cell failure in diabetes. High levels of dietary iron increase the risk of developing type 2 diabetes, and excessive iron uptake by beta-cells can cause oxidative stress and inhibit function. Elevated levels of proinflammatory cytokines in obese individuals, such as interleukin (IL)-1beta and IL-6, increase the risk of developing type 2 diabetes, and there is evidence that these low levels of circulating cytokines can lead to islet dysfunction. Methods: In this study, gene microarray and other data were analyzed for expression differences in islets treated for 48 h with 10 pg/mL IL-1beta + 20 pg/mL IL-6 as a model of low-grade inflammation versus untreated. Results: Three iron-associated genes were among the most cytokine-sensitive in the mouse genome: Hamp, Steap4, and Lcn2. These proteins are all involved with increasing/retaining cellular iron. We hypothesized that increased cellular iron would lead to increased susceptibility to ferroptosis. Surprisingly, 24 h pre-exposure to low-grade inflammation, which upregulates this iron-gene network, prevented subsequent erastin-induced ferroptosis. We also found that Steap4 overexpression reduced islet dysfunction caused by high-dose proinflammatory cytokines (10× low-dose), suggesting an overall protective effect. Steap4 overexpression also upregulated Hamp and Lcn2, suggesting Steap4 regulates these cytokine-sensitive iron genes.; in contrast, ferritin and ferroportin gene expression, which are not sensitive to cytokines, were unchanged. Conclusions: These data suggest an inflammation-induced network of genes involved in cellular iron uptake and retention plays a protective role in islets against oxidative stress and ferroptosis.

IL-1β↗

A walk through the planned CS building

Using the architectural plan views of our future computer science building as test objects, we have completed the first stage of a Building walkthrough system. The inputs to our system are AutoCAD files. An AutoCAD converter translates the geometrical information in these files into a format suitable for 3D rendering. Major model errors, such as incorrect polygon intersections and random face orientations, are detected and fixed automatically. Interactive viewing and editing tools are provided to view the results, to modify and clean the model and to change surface attributes. Our display system provides a simple-to-use user interface for interactive exploration of buildings. Using only the mouse buttons, the user can move inside and outside the building and change floors. Several viewing and rendering options are provided, such as restricting the viewing frustum, avoiding wall collisions, and selecting different rendering algorithms. A plan view of the current floor, with the position of the eye point and viewing direction on it, is displayed at all times. The scene illumination can be manipulated, by interactively controlling intensity values for 5 light sources.

Khorramabadi, Delnaz↗

Proceedings of the 1972 Lyndon B. Johnson Space Center Endocrine Program Conference

Subjects covered during the Endocrine Program Conference include the following: (1) endocrine/metabolic studies on the Apollo 16 crewmen; (2) changes in glucose, insulin, and growth hormone levels associated with bed rest; (3) circadian rhythms of heart rate and body temperature during 56 days of bed rest; (4) stress-induced changes in corticosteroid metabolism in man; (5) present status of physiological studies on parathyroid hormone and vitamin D; (6) antagonistic effect of lithium on antidiuretic hormone action; (7) proposed Skylab body-fluid volumes study; (8) daily rhythmic changes in serotonin content in areas of the mouse brain and norepinephrine content in areas of the hamster brain; (9) studies of sodium homeostasis during simulated weightlessness; and (10) application of the water immersion model to man.

Source record↗

Biological Research and Space Health Enabled by Machine Learning to Support Deep Space Missions

A key science goal of the NASA “Moon to Mars” campaign is to understand how biology responds to the Lunar, Martian, and deep space environments in order to advance fundamental knowledge, reduce risk, and support safe, productive human space missions. Through the powerful emerging computer science approaches of artificial intelligence (AI) and machine learning (ML), a paradigm shift has begun in biomedical science and engineered astronaut health systems, to enable Earth-independence and autonomy of mission operations. We present a decadal view of AI/ML architecture to support deep space mission goals, developed in concert with leaders in the field. We describe current AI/ML methods to support 1) fundamental biology, 2) in situ analytics, 3) high performance computing hardware, 4) automated science, 5) self-driving labs, 6) remote data management, 7) integrated real-time mission biomonitoring, and 8) a Precision Space Health system. Cutting-edge AI/ML approaches that can be integrated to support these domains include active learning, explainable AI, adaptive learning, causal inference, knowledge graphs, federated learning, transfer learning, and large language models. Finally, we present results from several current ML projects that are underway in the field to address key challenges of small sample n, high feature count, heterogeneity, and sparse data. These include 1) connecting omics data to phenotypic data using an ensemble model to infer causality of spaceflight rodent liver health disruption, 2) usage of explainable ML to interrogate the muscular underpinnings of spaceflight muscle atrophy, 3) ML models analyzing and determining directed acyclic graphs of human space health risk leveraging rodent bone datasets, 4) usage of large pre-trained models connecting biomedical knowledgebases with small spaceflight datasets to understand gene-to-gene interaction networks, and 5) a suite of benchmarked open science datasets (spaceflight mouse liver; radiation DNA damage) enabling programmers to identify the best ML algorithms to answer space biological science questions.

space biology↗

Biological Research and Space Health Enabled by Machine Learning to Support Deep Space Missions

A key science goal of the NASA “Moon to Mars” campaign is to understand how biology responds to the Lunar, Martian, and deep space environments in order to advance fundamental knowledge, reduce risk, and support safe, productive human space missions. Through the powerful emerging computer science approaches of artificial intelligence (AI) and machine learning (ML), a paradigm shift has begun in biomedical science and engineered astronaut health systems, to enable Earth-independence and autonomy of mission operations. We present a decadal view of AI/ML architecture to support deep space mission goals, developed in concert with leaders in the field. We describe current AI/ML methods to support 1) fundamental biology, 2) in situ analytics, 3) high performance computing hardware, 4) automated science, 5) self-driving labs, 6) remote data management, 7) integrated real-time mission biomonitoring, and 8) a Precision Space Health system. Cutting-edge AI/ML approaches that can be integrated to support these domains include active learning, explainable AI, adaptive learning, causal inference, knowledge graphs, federated learning, transfer learning, and large language models. Finally, we present results from several current ML projects that are underway in the field to address key challenges of small sample n, high feature count, heterogeneity, and sparse data. These include 1) connecting omics data to phenotypic data using an ensemble model to infer causality of spaceflight rodent liver health disruption, 2) usage of explainable ML to interrogate the muscular underpinnings of spaceflight muscle atrophy, 3) ML models analyzing and determining directed acyclic graphs of human space health risk leveraging rodent bone datasets, 4) usage of large pre-trained models connecting biomedical knowledgebases with small spaceflight datasets to understand gene-to-gene interaction networks, and 5) a suite of benchmarked open science datasets (spaceflight mouse liver; radiation DNA damage) enabling programmers to identify the best ML algorithms to answer space biological science questions.

space biology↗

Augmented Computer Mouse Would Measure Applied Force

Proposed computer mouse measures force of contact applied by user. Adds another dimension to two-dimensional-position-measuring capability of conventional computer mouse; force measurement designated to represent any desired continuously variable function of time and position, such as control force, acceleration, velocity, or position along axis perpendicular to computer video display. Proposed mouse enhances sense of realism and intuition in interaction between operator and computer. Useful in such applications as three-dimensional computer graphics, computer games, and mathematical modeling of dynamics.

Li, Larry C. H.↗

Radiogenic cell transformation and carcinogenesis

Radiation carcinogenesis is one of the major biological effects considered important in the risk assessment for space travel. Various biological model systems, including both cultured cells and animals, have been found useful for studying the carcinogenic effects of space radiations, which consist of energetic electrons, protons and heavy ions. The development of techniques for studying neoplastic cell transformation in culture has made it possible to examine the cellular and molecular mechanisms of radiation carcinogenesis. Cultured cell systems are thus complementary to animal models. Many investigators have determined the oncogenic effects of ionizing and nonionizing radiation in cultured mammalian cells. One of the cell systems used most often for radiation transformation studies is mouse embryonic cells (C3H10T1/2), which are easy to culture and give good quantitative dose-response curves. Relative biological effectiveness (RBE) for heavy ions with various energies and linear energy transfer (LET) have been obtained with this cell system. Similar RBE and LET relationship was observed by investigators for other cell systems. In addition to RBE measurements, fundamental questions on repair of sub- and potential oncogenic lesions, direct and indirect effect, primary target and lesion, the importance of cell-cell interaction and the role of oncogenes and tumor suppressor genes in radiogenic carcinogenesis have been studied, and interesting results have been found. Recently several human epithelial cell systems have been developed, and ionizing radiation have been shown to transform these cells. Oncogenic transformation of these cells, however, requires a long expression time and/or multiple radiation exposures. Limited experimental data indicate high-LET heavy ions can be more effective than low-LET radiation in inducing cell transformation. Cytogenetic and molecular analyses can be performed with cloned transformants to provide insights into basic genetic mechanism(s) of radiogenic transformation of human epithelial cells.

NASA Discipline Radiation Health↗

Quirky Circling Behavior in Mice Informs Research on Humans in Space

As interest in long duration effects of space habitation increases, understanding the behavior of model organisms living within the habitats engineered to fly them is vital for designing, validating, and interpreting future spaceflight studies. We previously conducted a detailed phenotypic analysis of mouse behavior during long duration (33-day) spaceflight in the NASA Rodent Habitat (Rodent Research-1; RR1 mission). Notably, we documented the emergence within the 8-10 days of launch of spontaneous ambulatory behavior in the form of circling or ‘race-tracking’ behavior in spaceflight but not in an identical ground control condition. Circling is unique to the NASA RH not having been reported to occur in other mouse habitats that provide limited opportunity for grasping, and/or are characterized by smaller habitable volumes viz., Italian Mice Drawer System (MDS) flown on ISS; Russian Block Obespecheniya Soderzhaniya (BOS) flown on Bion M-1, or JAXA Habitat Cage Unit (HCU) flown in the ISS Kibo module. Over time, circling became the primary dark cycle activity of FLT mice, occurring in individuals then as a coordinated group activity. Here we discuss possible interpretations of circling behavior including: (1) Stereotypic or abnormal repetitive behaviors (ARBs) that are unvarying, and apparently functionless behavior patterns documented numerous species in laboratory and zoo settings, possibility related to insufficient environmental enrichment and stress, (2) Rewarding effects of physical activity that are well-documented in terrestrial studies of rodents given the opportunity to run in wheels, and (3) Vestibular self-stimulation, i.e., the generation biologically-relevant amounts of vestibular sensory input to reduce the effects of microgravity. Affording mice the opportunity to grab and run in the RH resembles physical activities that the crew participate in routinely. Our approach is yielding an interesting analogue for better understanding human responses to spaceflight, and providing the opportunity to begin to address how physical movement influences responses to microgravity.

spaceflight↗

Estrous Cyclicity of Mice During Simulated Weightlessness

Hindlimb unloading (HU) is a rodent model system used to simulate weightlessness experienced in space. However, some effects of this approach on rodent physiology are under-studied, specifically the effects on ovarian estrogen production which drives the estrous cycle. To resolve this deficiency, we conducted a ground-based validation study using the HU model, while monitoring estrous cycles in 16-weeks-old female C57BL6 mice. Animals were exposed to HU for 12 days following a 3 day HU cage acclimation period, and estrous cycling was analyzed in HU animals (n22), normally loaded HU Cage Pair-Fed controls (CPF; n22), and Vivarium controls fed ad libitum (VIV; n10). Pair feeding was used to control for potential nutritional deficits on ovarian function. Vaginal cells were sampled daily in all mice via saline lavage. Cells were dried and stained with crystal violet, and the smears evaluated using established vaginal cytology techniques by two individuals blinded to the animal treatment group. Estrous cyclicity was disrupted in nearly all HU and CPF mice, while those maintained in VIV had an average normal cycle length of 4.8 0.5 days, with all stages in the cycle visibly observed. CPF and HU animals arrested in the diestrous phase, which precedes the pre-ovulatory estrogen surge. Additionally, infection-like symptoms characterized by vaginal discharge and swelling arose in several HU animals, which we suspect was due to an inability of these mice to properly groom themselves, andor due to the change in the gravity vector relative to the vaginal opening, which prevented drainage of the lavage solution. Pair-feeding resulted in similar weight gains of HU and CPF (1.5 vs 3.0, respectively). The current results indicate that pair-feeding controlled weight gain and that the HU cage alone influenced estrous cyclicity. Thus, longer acclimation needs to be tested to determine if and when normal estrous cycling resumes in non-loaded mice in HU cages prior to HU testing. Future studies might also examine whether modifications to the vaginal lavage procedure might prevent the onset of the infection-like symptoms, and allow estrous cyclicity to be measured in this model system.

fertilit↗

The Integrated Mode Management Interface

Mode management is the processes of understanding the character and consequences of autoflight modes, planning and selecting the engagement, disengagement and transitions between modes, and anticipating automatic mode transitions made by the autoflight system itself. The state of the art is represented by the latest designs produced by each of the major airframe manufacturers, the Boeing 747-400, the Boeing 777, the McDonnell Douglas MD-11, and the Airbus A320/A340 family of airplanes. In these airplanes autoflight modes are selected by manipulating switches on the control panel. The state of the autoflight system is displayed on the flight mode annunciators. The integrated mode management interface (IMMI) is a graphical interface to autoflight mode management systems for aircraft equipped with flight management computer systems (FMCS). The interface consists of a vertical mode manager and a lateral mode manager. Autoflight modes are depicted by icons on a graphical display. Mode selection is accomplished by touching (or mousing) the appropriate icon. The IMMI provides flight crews with an integrated interface to autoflight systems for aircraft equipped with flight management computer systems (FMCS). The current version is modeled on the Boeing glass-cockpit airplanes (747-400, 757/767). It runs on the SGI Indigo workstation. A working prototype of this graphics-based crew interface to the autoflight mode management tasks of glass cockpit airplanes has been installed in the Advanced Concepts Flight Simulator of the CSSRF of NASA Ames Research Center. This IMMI replaces the devices in FMCS equipped airplanes currently known as mode control panel (Boeing), flight guidance control panel (McDonnell Douglas), and flight control unit (Airbus). It also augments the functions of the flight mode annunciators. All glass cockpit airplanes are sufficiently similar that the IMMI could be tailored to the mode management system of any modern cockpit. The IMMI does not replace the functions of the FMCS control and display unit. The purpose of the INMI is to provide flight crews with a shared medium in which they can assess the state of the autoflight system, take control actions on it, reason about its behavior, and communicate with each other about its behavior. The design is intended to increase mode awareness and provide a better interface to autoflight mode management. This report describes the IMMI, the methods that were used in designing and developing it, and the theory underlying the design and development processes.

Hutchins, Edwin↗

Virtual Keyboard for Hands-Free Operations

The measurement of direction of gaze (d.o.g.) has been used for clinical purposes to detect illness, such as nystagmus, unusual fixation movements and many others. It also is used to determine the points of interest in objects. In this study we employ a measurement of d.o.g. as a computer interface. The interface provides a full keyboard as well as a mouse function. Such an interface is important to computer users with paralysis or in environments where hand-free machine interface is required. The study utilizes the commercially available (ISCAN Model RK426TC) headset which consists of an InfraRed (IR) source and an IR camera to sense deflection of the illuminating beam. It also incorporates image processing package that provides the position of the pupil as well as the pupil size. The study shows the ability of implementing a full keyboard, together with some control functions, imaged on a head mounted monitor screen. This document is composed of four sections: (1) The Nature of the Equipment; (2) The Calibration Process; (3) Running Process; and (4) Conclusions.

Abou-Ali, Abdel-Latief↗

Systemic immunological responses are dependent on sex and ovarian hormone presence following acute inhaled woodsmoke exposure

Background: Rural regions of the western United States have experienced a noticeable surge in both the frequency and severity of acute wildfire events, which brings significant challenges to both public safety and environmental conservation efforts, with impacts felt globally. Identifying factors contributing to immune dysfunction, including endocrinological phenotypes, is essential to understanding how hormones may influence toxicological susceptibility. Methods: This exploratory study utilized male and female C57BL/6 mice as in vivo models to investigate distinct responses to acute woodsmoke (WS) exposure with a focus on sex-based differences. In a second set of investigations, two groups were established within the female mouse cohort. In one group, mice experienced ovariectomy (OVX) to simulate an ovarian hormone-deficient state similar to surgical menopause, while the other group received Sham surgery as controls, to investigate the mechanistic role of ovarian hormone presence in driving immune dysregulation following acute WS exposure. Each experimental cohort followed a consecutive 2-day protocol with daily 4-h exposure intervals under two conditions: control HEPA-filtered air (FA) and acute WS to simulate an acute wildfire episode. Results: Metals analysis of WS particulate matter (PM) revealed significantly increased levels of 63 Cu, 182 W, 208 Pb, and 238 U, compared to filtered air (FA) controls, providing insights into the specific metal components most impacted by the changing dynamics of wildfire occurrences in the region. Male and female mice exhibited diverse patterns in lung mRNA cytokine expression following WS exposure, with males showing downregulation and females displaying upregulation, notably for IL-1β, TNF-α, CXCL-1, CCL-5, TGF-β, and IL-6. After acute WS exposure, there were notable differences in the responses of macrophages, neutrophils, and bronchoalveolar lavage (BAL) cytokines IL-10, IL-6, IL-1β, and TNF-α. Significant diverse alterations were observed in BAL cytokines, specifically IL-1β, IL-10, IL-6, and TNF-α, as well as in the populations of immune cells, such as macrophages and polymorphonuclear leukocytes, in both Sham and OVX mice, following acute WS exposure. These findings elucidated the profound influence of hormonal changes on inflammatory outcomes, delineating substantial sex-related differences in immune activation and revealing altered immune responses in OVX mice due to ovarian hormone deficiency. In addition, the flow cytometry analysis highlighted the complex interaction between OVX surgery, acute WS exposure, and their collective impact on immune cell populations within the hematopoietic bone marrow niche. Conclusions: In summary, both male and female mice, alongside females subjected to OVX and those who had sham surgery, exhibit significant variations in the expression of proinflammatory cytokines, chemokines, lung mRNA gene expression, and related functional networks linked to signaling pathways. These differences potentially act as mediators of sex-specific and hormonal influences in the systemic inflammatory response to acute WS exposure during a wildfire event. Understanding the regulatory roles of genes expressed differentially under environmental stressors holds considerable implications, aiding in identifying sex-specific therapeutic targets for addressing acute lung inflammation and injury.

59 BASIC BIOLOGICAL SCIENCES↗

Activity Scratchpad Prototype: Simplifying the Rover Activity Planning Cycle

The Mars Exploration Rover mission depends on the Science Activity Planner as its primary interface to the Spirit and Opportunity Rovers. Scientists alternate between a series of mouse clicks and keyboard inputs to create a set of instructions for the rovers. To accelerate planning by minimizing mouse usage, a rover planning editor should receive the majority of inputted commands from the keyboard. Thorough investigation of the Eclipse platform's Java editor has provided the understanding of the base model for the Activity Scratchpad. Desirable Eclipse features can be mapped to specific rover planning commands, such as auto-completion for activity titles and content assist for target names. A custom editor imitating the Java editor's features was created with an XML parser for experimenting purposes. The prototype editor minimized effort for redundant tasks and significantly improved the visual representation of XML syntax by highlighting keywords, coloring rules, folding projections, and providing hover assist, templates and an outline view of the code.

Abramyan, Lucy↗

The current status of REH theory

A response is made to the evaluation of Fitch (1980) of REH (random evolutionary hits) theory for the evolutionary divergence of proteins and nucleic acids. Correct calculations for the beta hemoglobin mRNAs of the human, mouse and rabbit in the absence and presence of selective constraints are summarized, and it is shown that the alternative evolutionary analysis of Fitch underestimates the total fixed mutations. It is further shown that the model used by Fitch to test for the completeness of the count of total base substitutions is in fact a variant of REH theory. Considerations of the variance inherent in evolutionary estimations are also presented which show the REH model to produce no more variance than other evolutionary models. In the reply, it is argued that, despite the objections raised, REH theory applied to proteins gives inaccurate estimates of total gene substitutions. It is further contended that REH theory developed for nucleic sequences suffers from problems relating to the frequency of nucleotide substitutions, the identity of the codons accepting silent and amino acid-changing substitutions, and estimate uncertainties.

Holmquist, R.↗