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NASA NTRS1998

Evaluation of radiation effects against C6 glioma in combination with vaccinia virus-p53 gene therapy

The primary objective of this study was to evaluate the antitumor effects of recombinant vaccinia virus-p53 (rVV-p53) in combination with radiation therapy against the C6 rat glioma, a p53 deficient tumor that is relatively radioresistant. VV-LIVP, the parental virus (Lister strain), was used as a control. Localized treatment of subcutaneous C6 tumors in athymic mice with either rVV-p53 or VV-LIVP together with tumor irradiation resulted in low tumor incidence and significantly slower tumor progression compared to the agents given as single modalities. Assays of blood and spleen indicated that immune system activation may account, at least partly, for the enhance tumor inhibition seen with combined treatment. No overt signs of treatment-related toxicity were noted.

NASA Discipline Radiation Health↗

DOE OSTI2024

Electrothermal vaporization as a new method for pre-inspection check samples

The use of electrothermal vaporization (ETV) has not been readily applied to nuclear safeguards or pre inspection check (PIC) sample analysis, even though the technique is ideally suited for low level (pg) isotopic quantification due to its high analyte transport efficiency. Here demonstrate the ETV method for PIC safeguard analysis. The ETV system is an automated, commercial of the shelf (COTS) technology, with the capability to introduce solid samples to a mass spectrometer platform. The system has been shown to simultaneously determine uranium assay, and the major isotopic ratios for uranium and plutonium. The ETV unit has demonstrated analysis up to 50 samples a day autonomously. The utilization of this ETV methodology provide a secondary option to the current PIC analysis and offers high throughput, enhanced sensitivity, and broader isotopic detection platform.

Hexel, Cole↗

DOE OSTI2024

Utilizing Advanced Statistics to Determine Anomalistic Conditions in Pebble-Bed Reactors

Pebble-bed reactors (PBRs) utilize hundreds of thousands of fuel pebbles, which continuously circulate through the core, in lieu of traditional fuel assemblies to generate fissions and produce power. The use of unmarked fuel pebbles presents a challenge for international safeguards verification that nuclear material is not being diverted. To ensure pebble diversion is not taking place, new methods for accounting for and monitoring the pebbles should be examined to determine an appropriate methodology for performing bulk accountancy with pebbles. Here, this work examines the use of statistical methods for determining if the reactor is within a declared range of operation by examining the statistical distribution of pebble burnup as they are discharged from the core. Using this methodology, we created a model that detects diversion over 95% of the time, over multiple diversion pathways, if the reactor core maintains a constant power density during the diversion process and only falsely labels a diversion case nominal 2% of the time. For a diversion scenario where the reactor is maintained at a constant power, the statistical analysis can correctly identify if diversion is occurring over 80% of the time; however, nearly 20% of specific diversion pathways are mislabeled nominal. These results provide a basis and framework for exploring the further use of statistical methods to determine where these methods could be most useful and where additional methods, such as machine learning, could be used to capture if diversion is occurring in pebble-bed reactors.

11 NUCLEAR FUEL CYCLE AND FUEL MATERIALS↗

NASA NTRS1988

Biochemical assays on plasminogen activators and hormones from kidney sources

Investigations were established for the purpose of analyzing the conditioned media from human embryonic kidney cell subpopulations separated in space by electrophoresis. This data is based on the experiments performed on STS-8 on the continuous flow electrophoresis system. The primary biological activity that was analyzed was plasminogen activator activity, but some assays for erythropoeitin and human granulocyte colony stimulating activity were also performed. It is concluded that a battery of assays are required to completely define the plasminogen activator profile of a conditioned media from cell culture. Each type of assay measures different parts of the mixture and are influenced by different parameters. The functional role of each assay is given along with an indication of which combination of assays are required to answer specific questions. With this type of information it is possible by combinations of assays with mathematical analysis to pinpoint a specific component of the system.

Barlow, Grant H.↗

DOE OSTI2021

Biocompatible Electrostatic Layered Systems for Viral Elimination in the Nose/Throat

An anti-viral coating for the nose & throat is needed to quickly address the spread of COVID-19 infections and to aid future pandemics. Current nasal delivery systems are typically a 1-spray homogenous solution, which is convenient but may not be as efficient or durable as a multi-spray solution that contains a binding layer to anchor the anti-viral components. Many of the current nasal solutions being investigated to aid in pandemic response have solution-based mechanisms of action and are quickly cleared from the nose/mouth limiting their efficacy lifetime. A multi-spray approach utilizing strong intermolecular forces between polymeric materials and anti-viral agents to provide a robust biocompatible coating is expected to have increased physical and chemical properties to combat viral infection. This work evaluated which tailored biocompatible materials are needed to generate a layered system to combat viral infection. This layered system takes advantage of electrostatic interactions to bind anti-viral components, shown in Figure 1. Proof of concept success was demonstrated through cell toxicity studies and anti-viral assays on both the individual layer components and the complete layered system. Project work began with chemical modifications of Xantham Gum to hydrolyzing xanthan gum to give a negatively charged polymer with varying degrees of ionic character and investigate bonding efficiencies. However, this was abandoned in favor of comparing COTS available materials listed in Table 1 (Results section). Concentration and adhesion studies were performed on layers created using the listed polymeric materials and anti-viral agents. Finally, biocompatibility of materials, layers, and coating system were confirmed through cytotoxicity studies and the efficacy of the anti-viral properties tested with phi6 bacteriophage as a SARS-CoV2 surrogate.

36 MATERIALS SCIENCE↗

NASA NTRS2017

Coastal California's Fog as a Unique Habitable Niche: Design for Autonomous Sampling and Preliminary Aerobiological Characterization

Just as on the land or in the ocean, atmospheric regions may be more or less hospitable to life. The aerobiosphere, or collection of living things in Earth's atmosphere, is poorly understood due to the small number and ad hoc nature of samples studied. However, we know viable airborne microbes play important roles, such as providing cloud condensation nuclei. Knowing the distribution of such microorganisms and how their activity can alter water, carbon, and other geochemical cycles is key to developing criteria for planetary habitability, particularly for potential habitats with wet atmospheres but little stable surface water. Coastal California has regular, dense fog known to play a major transport role in the local ecosystem. In addition to the significant local (1 km) geographical variation in typical fog, previous studies have found that changes in height above surface of as little as a few meters can yield significant differences in typical concentrations, populations and residence times. No single current sampling platform (ground-based impactors, towers, balloons, aircraft) is capable of accessing all of these regions of interest.A novel passive fog and cloud water sampler, consisting of a lightweight passive impactor suspended from autonomous aerial vehicles (UAVs), is being developed to allow 4D point sampling within a single fog bank, allowing closer study of small-scale (100 m) system dynamics. Fog and cloud droplet water samples from low-altitude aircraft flights in nearby coastal waters were collected and assayed to estimate the required sample volumes, flight times, and sensitivity thresholds of the system under design.125 cloud water samples were collected from 16 flights of the Center for Interdisciplinary Remotely Piloted Aircraft Studies (CIRPAS) instrumented Twin Otter, equipped with a sampling tube collector, occurring between 18 July and 12 August 2016 below 1 km altitude off the central coast. The collector was flushed first with 70 ethanol, then with sterile DI water, between sampling regions. Collected volumes ranged from 100 L to 12 mL. All samples were diluted serially and plated on two different types of agar, nutrient-dense (PCA) and sparse (R-2A). Plates were incubated at room temperature and counted when colonies first appeared and again at 2 weeks.Preliminary results from seven flights are consistent with generally reported colony-forming unit (CFU) values for terrestrial fog water (e.g., [4]). The PCA assay ranged from 400 to 125,000 CFU/mL, R-2A from 700 to 130,000 CFU/mL. PCA and R-2A counts were not significantly different from each other at I^ plus or minus +/- 0.05, although observationally, the R2A plates had more pigmented colonies. CFU counts from the majority of flights were not different from each other in mean at the same level of significance, but about half differed in median, indicating differences in underlying distribution. These results validate the presence of viable microorganisms in coastal California fog at levels that should be easily detectable by our sampling system. The indicated distribution differences underscore the need for small-scale, long-term sampling surveys. Future planned work includes ion chromatography for limiting nutrients, ATP quantification, and qPCR for several microbial classes of interest.

Coastal↗

NASA NTRS2016

On-orbit RNA Purification and qRT-PCR Capabilities of the WetLab-2 System

NASA Ames Research Center’s WetLab-2 system brings new capabilities to the ISS for researchers. The system can lyse cells and extract RNA on-orbit from different sample types ranging from microbial cultures to animal tissues. Our purification method has the advantage of using non-toxic chemicals and does not require alcohols or other organics. The purified RNA can then either be stabilized for return to Earth or can be used to conduct on-orbit quantitative Reverse Transcriptase PCR (qRT-PCR) analysis without the need for sample return. qRT-PCR reactions are performed by dispensing the RNA into reaction tubes that contain all lyophilized reagents needed to perform the analysis. The system uses a Cepheid® SmartCycler that allows for multiplexing of assays, this can be used to normalize for RNA concentration and integrity and to study multiple genes of interest in each tube. There are a total of 16 independent PCR modules each capable of detecting up to four fluorescent channels. The WetLab- 2 system can downlink data from the ISS to the ground after a completed run and uplink new thermal cycling programs. The ability to purify and stabilize RNA on-orbit can eliminate the confounding effects of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of some samples. It also has the benefit of minimizing the needed downmass. Conducting qRT-PCR and generating results on-orbit is also an important step towards utilizing the ISS as a National Laboratory facility. Specifically, the ability to get on-orbit data will provide investigators with the opportunity to adjust experimental parameters in real time without the need for sample return and re-flight. On orbit gene expression analysis can also provide benchmarking prior to sample return. The system can also be used for analysis of air, surface, water, and clinical samples to monitor environmental pathogens and crew health. The validation flight of the WetLab-2 system using E. coli bacteria and mouse liver is scheduled to launch on SpaceX-8 this spring. Pending operations, the preliminary results from the validation flight will be presented. To support the needs of future researchers, we are adapting our system to purify RNA from two additional sample types: fibrous tissue such as muscle and mammalian adherent cells grown on alginate beads. Progress of this work will also be presented. The WetLab-2 Project is supported by the Research Integration Office in the ISS Program.

Parra, Macarena↗

DOE OSTI2023

Devices and systems for material transportation

Various embodiments relate to devices for transporting high-assay low-enriched uranium (HALEU). A device may include at least one section, wherein each section of the at least one section includes a number of storage tubes. Each storage tube, which is configured to receive and hold a container, extends from adjacent a first end of the section toward a second, opposite end of the section. Each section further includes a number of flux traps, wherein each storage tube of the number of storage tubes is at least partially surrounded by a flux trap of the number of flux traps Associated systems are also disclosed.

Eidelpes, Elmar F.↗

DOE OSTI2022

Using Cosmic Ray Muons to Assess Geological Characteristics in the Subsurface

Cosmic rays are energetic nuclei and elementary particles that originate from stars and intergalactic events. The interaction of these particles with the upper atmosphere produces a wide range of secondary particles that reach the surface of the earth, of which muons are the most prominent. With enough energy, muons can travel up to a few kilometers beneath the surface of the earth before being stopped completely. The terrestrial muon flux profile and associated zenith angle can be utilized to determine geological characteristics of a location (e.g., rock overburden and density) without having to use conventional methods such as boreholes. This work uses a low-power plastic scintillator-based muon detection system as a prototype for this non-destructive geological assay methodology. Four custom designed 102 cm x 51 cm x 5 cm plastic scintillation panels are used to realize two orthogonal detection planes. Optical photons from each scintillation panel are read using OnSemi J-Series 4x4 silicon photomultiplier (SiPM) arrays in conjunction with preamplifiers. Simultaneous triggers between detectors from two planes indicate a coincidence event which is recorded using the QuarkNet data acquisition system (DAQ) from Fermi National Accelerator Laboratory. A custom detector holder was designed to securely mount the detection system and rotate the panels along the zenith to collect data at variable angles. In order to quantify the systematic uncertainties associated with the detector, such as energy depositions and angular resolution of the detector design, a Monte Carlo (MC) simulation using Geant4 is being developed. Cosmic ray flux prediction will be included in the project by adding the CORSIKA MC code to the simulation toolchain. Simulated and experimental data will drive the development and validation of a reconstruction algorithm that, upon completion, is expected to predict average overburden and rock density. Extended detector exposure to muons can be used as a means to understand changes in the surrounding environment like rock porosity. On the experimental front, muons will initially be measured at the surface, establishing the baseline flux. This is followed by recording the muon flux at variable depths and zenith angles, where the data will be used by the reconstruction algorithm to predict the overburden. The result will be benchmarked against geological surveys. The measured flux data will also be used to benchmark independent and established models. Successful proof-of-concept demonstration of this technology can open doors for long term non-invasive geological monitoring. The detector design, experimental methodology, and the benchmarking efforts are detailed in this work.

Gadey, Harish Reddy↗

NASA NTRSDate not supplied

Low Cost Micro-Spectrometer for Resource Mapping on Mars

Scientific measurements within craters and along steep embankments on Mars pose extreme challenges since astronauts and rovers cannot access them. However, these areas offer the potential for thegreatest scientific return. NASA Langley Research Center (LaRC) has invented a deployable wireless micro-spectrometer [1-13] capable of accessing thosechallenging areas and, using telemetry, send measurements to a receiver safely outside those extreme environments. A prototype of the micro-spectrometer concept was fabricated and successfullytested for measurement of dopamine as a neural probe [14]. The bullet-sized micro-spectrometer concept can be designed for deployment by an astronaut, a rover, an aerial drone, or a lander, and the measurements broadcasted back to a receiver [13,15,16]. This device can also be installed on rovertires, under the astronaut’s boots, or a cane stick. The bullet-like consumable micro-spectrometer can penetrate soil to spectrally identify the components ofthe soil, such as water or minerals. The signals of the soil assay data are transmitted to a mother station through a telemetry system. The LaRC developed micro-spectrometer bullet consists of micro-spectrometer optics with an all imbedded burst-mode LED UV light source, a super-capacitor with control electronics, and telemetry electronics. Prototypes have been fabricated to demonstrate spectral assay of soil components. Further maturation of this technology would be necessary for demonstration on Mars. Future work includes a potential R&D program that includes other NASA Centers and industry partners interested in developing a business case that requires knowledge of the locations of extraterrestrial ice, for instance.

micro-spectrometer, mineral assay, resource mappin↗

NASA NTRS1997

Effects of Spaceflight on Drosophila Neural Development

The major goal from the animal side, however, has been achieved, namely to develop Drosophila lines where we can assay individual neuromuscular endings directly without dissection. This was achieved by means of using the GAL4-UAS system, where we have succeeded in establishing stocks of flies where the key neuromuscular connections can be assayed directly in undissected larvae by means of the expression of endogenously fluorescent reporters in the specific motor endings. The green fluorescent protein (GFP) as a reporter allows scoring of neural anatomy en-masse in whole mount using fluorescent microscopy without the need for either dissection or specific labeling. Two stocks have been developed. The first, which we developed first, uses the S65T mutant form, which has a dramatically brighter expression than the native protein. This animal will use GAL4 drivers with expression under the control of the elav gene, and which will ensure expression in all neurons of the embryo and larva. The second transgenic animal we have developed is of a novel kind, and makes use of dicistronic design, so that two copies of the protein will be expressed per insert. We have also developed a tricistronic form, but this has not yet been transformed into flies, and we do not imagine that this third line will be ready in time for the flight.

Keshishian, Haig S.↗

DOE OSTI2026

Improvements to In-Canal Assay of High Specific Activity 60Co at the ATR

Improvements to the quality of measurements in the assaying of High Specific Activity (HSA) cobalt 60 produced at the Advanced Test Reactor at the Idaho National Laboratory are presented. Such assays are conducted regularly between cycles at the ATR, and due to the radiological safety concerns, they are performed within the ATR canal. A standard ion chamber detector was placed in a collimated assay tube within the canal. Another tube, parallel to the detector, was used to position various cobalt 60 standards and targets. The collimated port between these tubes allows for the targets to be pulled across via a winch system. Previous measurements were performed at discrete 1.5-inch steps using a hand-cranked winch system. Charge was collected and integrated for 10 seconds at each discrete step during a scan. Two substantial improvements to the assay include an electronically controlled, motor-driven winch system and a software controlled continuous charge collection. Combined, these changes improved the quality of measurements as well as streamline the assay process. Further examination of these charge profiles indicated a reasonable amount of photon scattering, or shine, before a line-of-sight is established. This created inaccuracies in the reported activity of the cobalt targets. A series of MCNP-6.3 calculations were performed in an effort to quantify and correct for this shine effect. The results of the calculations and how they were employed to a create shine correction factor for the in-canal cobalt assay at the ATR are presented. The overall bias showed an overestimation of the HSA cobalt 60 activity by around 7-8%.

07 - ISOTOPES AND RADIATION SOURCES↗

NASA NTRS2015

Risk of Crew Adverse Health Event Due to Altered Immune Response

Determining the effect of space travel on the human immune system has proven to be extremely challenging. Limited opportunities for in-flight studies, varying mission durations, technical and logistical obstacles, small subject numbers, and a broad range of potential assays have contributed to this problem. Additionally, the inherent complexity of the immune system, with its vast array of cell populations, sub-populations, diverse regulatory molecules, and broad interactions with other physiological systems, makes determining precise variables to measure very difficult. There is also the challenge of determining the clinical significance of any observed immune alterations. Will such a change lead to disease, or is it a transient subclinical observation related to short-term stress? The effect of this problem may be observed by scanning publications associated with immunity and spaceflight, which began to appear during the 1970s. Although individually they are each valid studies, the comprehensive literature to date suffers from widely varying sampling methods and assay techniques, low subject counts, and sometimes a disparate focus on narrow aspects of immunity. The most clinically relevant data are derived from in-flight human studies, which have demonstrated altered cell-mediated immunity and reactivation of latent herpes viruses. Much more data are available from post-flight testing of humans, with clear evidence of altered cytokine production patterns, altered leukocyte distribution, continued latent viral reactivation, and evidence of dramatically altered virus-specific immunity. It is unknown if post-flight assessments relate to the in-flight condition or are a response to landing stress and readaptation. In-flight culture of cells has clearly demonstrated that immune cells are gravity-sensitive and display altered functional characteristics. It is unknown if these data are related to in vivo immune cell function or are an artifact of microgravity culture. Ground analog testing of humans and animals, as well as microgravity-analog cell culture, has demonstrated utility. However, in all cases, it is not known with certainty if these data would reflect similar testing during space travel. Given their ready availability, ground analogs may be extremely useful for assay development and the evaluation of potential countermeasures. In general, the evidence base suffers from widely disparate studies on small numbers of subjects that do not directly correlate well with each other or spaceflight itself. Also lacking are investigations of the effect of gender on adaption to spaceflight. This results in significant knowledge 'gaps' that must be filled by future studies to completely determine any clinical risk related to immunity for human exploration-class space missions. These gaps include a significant lack of in-flight data, particularly during long-duration space missions. The International Space Station represents an excellent science platform with which to address this knowledge gap. Other knowledge gaps include lack of a single validated ground analog for the phenomenon and a lack of flight-compatible laboratory equipment capable of monitoring astronauts (for either clinical or research purposes). However, enough significant data exist, as described in this manuscript, to warrant addressing this phenomenon during the utilization phase of the ISS. A recent Space Shuttle investigation has confirmed the 31 in-flight nature of immune dysregulation, demonstrating that it is not merely a post-flight phenomenon. Several current studies are ongoing onboard the ISS that should thoroughly characterize the phenomenon. NASA recognizes that if spaceflight-associated immune dysregulation persists during exploration flights in conjunction with other dangers, such as high-energy radiation, the result may be a significant clinical risk. This emphasizes the need for a continued integrated comprehensive approach to determining the effect of prolonged spaceflight, separated from transient launch and landing stresses, on human immunity. After such studies, the phenomenon will be understood, and, hopefully, a monitoring strategy will have been developed that could be used to monitor the effectiveness of countermeasure

Crucian, Brian↗

DOE OSTI2020

Efficiency of Room Air Cleaners for Removal of Bioaerosols From Ambient Air

The COVID-19 pandemic necessitated the evaluation of commercial air purification units to protect worker safety in indoor spaces. This project evaluated the efficacy of commercial off-the-shelf air purification systems that utilize electrostatic precipitation for the collection and removal of particulates (including bioaerosols) from ambient air. An aerosol mist containing Escherichia coli (E. coli) and bacteriophage surrogate virus, MS2, was produced using a nebulizer and delivered to the front intake of the commercial system with the ionizer ON and OFF. Removal efficiency was evaluated by colony/plaque formation assays using a small agar plate (60 X 15 mm) to passively collect E. coli and MS2 exhausting from the electrostatic precipitation cell. The system was determined to remove 98-99% of the microorganism introduced to the system. Testing was performed with all pre- and post-electrostatic cell filters removed so further removal is anticipated.

32 ENERGY CONSERVATION, CONSUMPTION, AND UTILIZATI↗

NASA NTRS1978

Rapid, quantitative determination of bacteria in water

A bioluminescent assay for ATP in water borne bacteria is made by adding nitric acid to a water sample with concentrated bacteria to rupture the bacterial cells. The sample is diluted with sterile, deionized water, then mixed with a luciferase-luciferin mixture and the resulting light output of the bioluminescent reaction is measured and correlated with bacteria present. A standard and a blank also are presented so that the light output can be correlated to bacteria in the sample and system noise can be substracted from the readings. A chemiluminescent assay for iron porphyrins in water borne bacteria is made by adding luminol reagent to a water sample with concentrated bacteria and measuring the resulting light output of the chemiluminescent reaction.

Chappelle, E. W.↗

NASA NTRSDate not supplied

Micro-Spectrometer for Resource Mapping in Extreme Environments

Measurements and analyses of resources within permanently shadowed craters and along steep embankments on extraterrestrial surfaces pose extreme challenges since astronauts and rovers cannot access them. However, these areas offer the potential for the greatest scientific returns. Researchers at the NASA Langley Research Center (LaRC) invented a deployable wireless micro-spectrometer that can access those challenging areas and deliver telemetry data to a receiver safely outside those extreme environments. The micro-spectrometers are designed for deployment by an astronaut, a rover, or a lander flying overhead. This device can also be installed on rover tires, under the astronaut’s shoes, or on a cane stick. The bullet-like, expendable micro-spectrometer can penetrate into soil to spectrally identify the components of soil, such as water, He-3, or other minerals. The signals from the soil assay data are transmitted to a mother station through a telemetry system. This LaRC developed micro-spectrometer bullet consists of micro-spectrometer optics with an all-imbedded, burst-mode, light-emitting-diode ultraviolet (LED UV) light source, a super-capacitor with control electronics, and telemetry electronics. Prototypes have been fabricated to demonstrate a spectral assay of soil components. Further maturation of this technology would be necessary for demonstrations on the Moon.

micro-spectrometer↗

DOE OSTI2022

Using high-throughput amplicon sequencing to determine diet of generalist lady beetles in agricultural landscapes

Determining feeding relationships is central to understanding biological control potential in the field. However, methods to differentiate actual (or realized) feeding relationships from potential feeding relationships is lacking especially for small, generalist predators such as lady beetles. In this study, we used DNA metabarcoding approaches to characterize actual feeding relationships of lady beetles (Coccinellidae) in the field and validated our methods with a lab study. We first asked whether high-throughput amplicon sequencing (HTS) can characterize diets of lady beetles ranging from monotypic diets to diverse diet mixtures in the lab. We then examined whether diet composition and breadth of lady beetles collected from different habitat types in southern WI varied between monocultures of soybean and corn, diverse tallgrass prairie, and urban habitats. Lastly, we asked whether different body or tissue types (partial-body versus whole-body specimens) would change the likelihood of prey detection for both studies. In our controlled lab study, we found that HTS can accurately assess diet composition and diet breadth for lady beetle populations, but at the individual level, HTS has limitation for individuals feeding on more than three species of prey at any given time. In our field study, we documented lower prey richness and diet breadth in corn compared to soybean and grassland, and greater prey DNA in regurgitants than in whole-body specimens. Finally, we found that reduced diet diversity was associated with an increased prevalence of intraguild predation, but that habitat and prey diversity are not necessarily correlated. The prey detection rates (proportion of specimens with prey DNA) in our study were comparable to other studies (55% in the lab study, 33% in field study) and varied with diet composition and habitat type. Furthermore, the relatively low detection rates suggest that many consumer individuals would need to be assayed to fully assess diet diversity, especially in diverse systems.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Functional characterization of a cellulose synthase, CtCESA1, from the marine red alga Calliarthron tuberculosum (Corallinales)

In land plants and algae, cellulose is important for strengthening cell walls and preventing breakage due to physical forces. Though our understanding of cellulose production by cellulose synthases (CESAs) has seen significant advances for several land plant and bacterial species, functional characterization of this fundamental protein is absent in red algae. Here we identify CESA gene candidates in the calcifying red alga Calliarthron tuberculosum using sequence similarity-based approaches, and elucidate their phylogenetic relationship with other CESAs from diverse taxa. One gene candidate, CtCESA1, was closely related to other putative red algal CESA genes. To test if CtCESA1 encoded a true cellulose synthase, CtCESA1 protein was expressed and purified from insect and yeast expression systems. CtCESA1 showed glucan synthase activity in glucose tracer assays. CtCESA1 activity was relatively low when compared with plant and bacterial CESA activity. In an in vitro assay, a predicted N-terminal starch-binding domain from CtCESA1 bound red algal floridean starch extracts, representing a unique domain in red algal CESAs not present in CESAs from other lineages. When the CtCESA1 gene was introduced into Arabidopsis thaliana cesa mutants, the red algal CtCESA1 partially rescued the growth defects of the primary cell wall cesa6 mutant, but not cesa3 or secondary cell wall cesa7 mutants. A fluorescently tagged CtCESA1 localized to the plasma membrane in the Arabidopsis cesa6 mutant background. This study presents functional evidence validating the sequence annotation of red algal CESAs. Furthermore, the relatively low activity of CtCESA1, partial complementation in Arabidopsis, and presence of unique protein domains suggest that there are probably functional differences between the algal and land plant CESAs.

59 BASIC BIOLOGICAL SCIENCES↗