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At least 397 records · Page 22

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DOE OSTI2023

Cellulose Nanocrystals are a Renewable and Biocompatible Nanocarrier of Agrochemicals Directly to Plant Cells

More sustainable agricultural practices are critical to meeting humanity's food needs while minimizing adverse environmental impacts. Engineered nanomaterials used as nanocarriers promise to reduce the volume of agrochemicals required for crop production but concerns about nanocontamination of agricultural products necessitate the discovery of naturally occurring alternatives. Here, in this study, plant‐derived cellulose nanocrystals (CNC) as a “green” alternative to man‐made nanomaterials for the economical delivery of agrochemicals to plants are suggested. Using confocal microscopy and fluorescently labeled CNC, it is demonstrated that CNC can successfully penetrate the plant cell wall and enter cells without causing any negative effects on the overall plant phenotype, genome, and metabolome. The efficiency of CNC to deliver chemicals is demonstrated through in vitro tests involving CNC covalently conjugated with 2,4‐dichlorophenoxyacetic acid (2,4‐D). Plant cell culture experiments confirmed that CNC can be used as a 2,4‐D nanocarrier and reduces the volume of plant growth regulators needed to cultivate plant cells. This work creates a new platform for cyclical agricultural practices where CNC extracted from agricultural waste can be used for the direct delivery of agrochemicals to crops, thereby reducing the environmental burden created by both agricultural waste and excess herbicides.

Cellulose nanocrystals↗

NASA NTRS1972

Further observations on the phenomenon of secondary vacuolation in living cells.

The dynamics of secondary vacuole movement is studied in living hair cells of Tradescantia virginiana. The pattern of movement of these vacuoles is found to be similar to that described by the author previously for organelles in cultured cells. Evidence is presented in support of the thesis that the occurrence and dynamics of secondary vacuoles is a common phenomenon for plant cells.

Mahlberg, P.↗

NASA NTRSDate not supplied

The NASA Space Radiation Laboratory

The Space Radiation Element (SRE) of the Human Research Program (HRP) aims to establish a robust biospecimen and tissue sharing collection (BTSC) to improve sample collection, tracking, access, distribution, and usability to maximize scientific return with intention. Future missions to the Moon, Mars, and beyond will require the appropriate characterization and mitigation of the many of the health risks associated with cosmic radiation exposure. An ongoing partnership between the National Aeronautics and Space Agency (NASA) and the Office of Science for the U.S. Department of Energy (DOE) has established the NASA Space Radiation Laboratory (NSRL) at the DOE Brookhaven National Laboratory (BNL) to create a space radiation ground-analog. NSRL provides the capability to use simulated space radiation with high energy ion beams for assessment of biological outcomes relevant space radiation risks as well as electronics testing for avionics safety. Current NSRL irradiation capabilities include a variety of exposure variables available for experimental needs: - Up to approximately 10 Gy/min dose-rates with a 20 × 20 cm 2 beam area. - Down to several hundred particles within a 60 × 60 cm 2 beam area. - Dose fractionation across multiple days is also possible. - For most radiobiology experiments the exposure structure, referred to as “spills”, has a 4 second repetition time where ions are extracted uniformly during a 0.3-0.4 second spill, followed by a ~3.6 second recovery time when the beam is off. - Both single or mixed-beam field irradiations are possible including a full 33-beam GCRSim and 6-beam SimGCRSim. Biological experiment capabilities: Two labs for Cell/tissue culture procedures can be conducted in two lab spaces with a separate lab available for sample processing that contains two chemical fume hoods. Sixteen fully equipped cell culture laboratories are available in the NSRL’s Long Term Support area in the Medical Building. The current vivarium configuration can support approximately 8,000 rodents/year for animal-based experiments. The NSRL area animal husbandry system includes five caging racks with 48 spaces in each coupled to a ventilation/filtration system in largest room, and two smaller rooms with conventional caging systems. Schedule: The NSRL is nominally open for experiment scheduling during three runs per year; Spring (March – May), Summer (May - June), and Fall (Sept – Nov). NASA funding is not required for biological and electronic testing experiments at the NSRL.

Jason M Weeks↗

NASA NTRS2013

Lab-On-Chip Clinorotation System for Live-Cell Microscopy Under Simulated Microgravity

Cells in microgravity are subject to mechanical unloading and changes to the surrounding chemical environment. How these factors jointly influence cellular function is not well understood. We can investigate their role using ground-based analogues to spaceflight, where mechanical unloading is simulated through the time-averaged nullification of gravity. The prevailing method for cellular microgravity simulation is to use fluid-filled containers called clinostats. However, conventional clinostats are not designed for temporally tracking cell response, nor are they able to establish dynamic fluid environments. To address these needs, we developed a Clinorotation Time-lapse Microscopy (CTM) system that accommodates lab-on- chip cell culture devices for visualizing time-dependent alterations to cellular behavior. For the purpose of demonstrating CTM, we present preliminary results showing time-dependent differences in cell area between human mesenchymal stem cells (hMSCs) under modeled microgravity and normal gravity.

Yew, Alvin G.↗

NASA NTRS1980

Lab-On-Chip Clinorotation System for Live-Cell Microscopy Under Simulated Microgravity

Yew, Alvin G.↗

NASA NTRS1999

Model system for plant cell biology: GFP imaging in living onion epidermal cells

The ability to visualize organelle localization and dynamics is very useful in studying cellular physiological events. Until recently, this has been accomplished using a variety of staining methods. However, staining can give inaccurate information due to nonspecific staining, diffusion of the stain or through toxic effects. The ability to target green fluorescent protein (GFP) to various organelles allows for specific labeling of organelles in vivo. The disadvantages of GFP thus far have been the time and money involved in developing stable transformants or maintaining cell cultures for transient expression. In this paper, we present a rapid transient expression system using onion epidermal peels. We have localized GFP to various cellular compartments (including the cell wall) to illustrate the utility of this method and to visualize dynamics of these compartments. The onion epidermis has large, living, transparent cells in a monolayer, making them ideal for visualizing GFP. This method is easy and inexpensive, and it allows for testing of new GFP fusion proteins in a living tissue to determine deleterious effects and the ability to express before stable transformants are attempted.

NASA Discipline Plant Biology↗

NASA NTRS2001

Suppression of antigen-specific lymphocyte activation in modeled microgravity

Various parameters of immune suppression are observed in lymphocytes from astronauts during and after a space flight. It is difficult to ascribe this suppression to microgravity effects on immune cells in crew specimens, due to the complex physiological response to space flight and the resultant effect on in vitro immune performance. Use of isolated immune cells in true and modeled microgravity in immune performance tests, suggests a direct effect of microgravity on in vitro cellular function. Specifically, polyclonal activation of T-cells is severely suppressed in true and modeled microgravity. These recent findings suggest a potential suppression of oligoclonal antigen-specific lymphocyte activation in microgravity. We utilized rotating wall vessel (RWV) bioreactors as an analog of microgravity for cell cultures to analyze three models of antigen-specific activation. A mixed-lymphocyte reaction, as a model for a primary immune response, a tetanus toxoid response and a Borrelia burgdorferi response, as models of a secondary immune response, were all suppressed in the RWV bioreactor. Our findings confirm that the suppression of activation observed with polyclonal models also encompasses oligoclonal antigen-specific activation.

NASA Discipline Cell Biology↗

NASA NTRS1988

Non-contact temperature measurements for biotechnology discipline working group

In the biotechnology research areas, there is interest in measuring temperature changes over very small dimensions, such as the surface of a 10-micrometer diameter biological cell immersed in cell culture fluid. Non-interference measurements of other properties, such as chemical constituents and their concentrations, are also needed. Contacting probes for pH have recently been developed to penetrate a cell but questions have been raised about their accuracy and net value.

Snyder, Robert S.↗

NASA NTRS1998

Distributed Pore Chemistry in Porous Organic Polymers

A method for making a biocompatible polymer article using a uniform atomic oxygen treatment is disclosed. The sub-strate may be subsequently optionally grated with a compatibilizing compound. Compatibilizing compounds may include proteins, phosphorylcholine groups, platelet adhesion preventing polymers, albumin adhesion promoters, and the like. The compatibilized substrate may also have a living cell layer adhered thereto. The atomic oxygen is preferably produced by a flowing afterglow microwave discharge, wherein the substrate resides in a sidearm out of the plasma. Also, methods for culturing cells for various purposes using the various membranes are disclosed as well. Also disclosed are porous organic polymers having a distributed pore chemistry (DPC) comprising hydrophilic and hydrophobic region, and a method for making the DPC by exposing the polymer to atomic oxygen wherein the rate of hydrophilization is greater than the rate of mass loss.

Koontz, Steven L.↗

NASA NTRS1999

Distributed Pore Chemistry in Porous Organic Polymers

A method for making a biocompatible polymer article using a uniform atomic oxygen treatment is disclosed. The substrate may be subsequently optionally grated with a compatibilizing compound. Compatibilizing compounds may include proteins, phosphorylcholine groups, platelet adhesion preventing polymers, albumin adhesion promoters, and the like. The compatibilized substrate may also have a living cell layer adhered thereto. The atomic oxygen is preferably produced by a flowing afterglow microwave discharge. wherein the substrate resides in a sidearm out of the plasma. Also, methods for culturing cells for various purposes using the various membranes are disclosed as well. Also disclosed are porous organic polymers having a distributed pore chemistry (DPC) comprising hydrophilic and hydrophobic regions. and a method for making the DPC by exposing the polymer to atomic oxygen wherein the rate of hydrophilization is greater than the rate of mass loss.

Koontz, Steven L.↗

NASA NTRS1990

Iodine susceptibility of pseudomonads grown attached to stainless steel surfaces

Pseudomonads were adapted to grow in phosphate-buffered water and on stainless steel surfaces to study the iodine sensitivity of attached and planktonic cells. Cultures adapted to low nutrient growth were incubated at room temperature in a circulating reactor system with stainless steel coupons to allow biofilm formation on the metal surfaces. In some experiments, the reactor was partially emptied and refilled with buffer at each sampling time to simulate a "fill-and-draw" water system. Biofilms of attached bacteria, resuspended biofilm bacteria, and reactor suspension, were exposed to 1 mg l-1 iodine for 2 min. Attached bacterial populations which established on coupons within 3 to 5 days displayed a significant increase in resistance to iodine. Increased resistance was also observed for resuspended cells from the biofilm and planktonic bacteria in the system suspension. Generally, intact biofilms and resuspended biofilm cells were most resistant, followed by planktonic bacteria and phosphate buffer cultures. Thus, biofilm formation on stainless steel surfaces within water systems can result in significantly increased disinfection resistance of commonly-occurring water-borne bacteria that may enhance their ability to colonise water treatment and distribution systems.

NASA Program Environmental Health↗

DOE OSTI2020

Scalable, two–stage, autoinduction of recombinant protein expression in E. coli utilizing phosphate depletion

We report the scalable production of recombinant proteins in Escherichia coli, reliant on tightly controlled autoinduction, triggered by phosphate depletion in the stationary phase. The method, reliant on engineered strains and plasmids, enables improved protein expression across scales. Expression levels using this approach have reached as high as 55% of the total cellular protein. The initial use of the method in instrumented fed–batch fermentations enables cell densities of ~30 gCDW/L and protein titers up to 8.1 ± 0.7 g/L (~270 mg/gCDW). The process has also been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 μl (384–well plates), 100 μl (96–well plates), 20 ml, and 100 ml. In batch cultures, cell densities routinely reach ~5–7 gCDW/L, offering protein titers above 2 g/L. Here, the methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed–batch fermentation.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1989

Bio-reactor chamber

A bioreactor for cell culture is disclosed which provides for the introduction of fresh medium without excessive turbulent action. The fresh medium enters the bioreactor through a filter with a backwash action which prevents the cells from settling on the filter. The bioreactor is sealed and depleted medium is forced out of the container as fresh medium is added.

Chandler, Joseph A.↗

NASA NTRS2006

Three-dimensional Myoblast Aggregates--Effects of Modeled Microgravity

The overall objective of these studies is to elucidate the molecular and cellular alterations that contribute to muscle atrophy in astronauts caused by exposure to microgravity conditions in space. To accomplish this, a three-dimensional model test system was developed using mouse myoblast cells (C2C12). Myoblast cells were grown as three-dimensional aggregates (without scaffolding or other solid support structures) in both modeled microgravity (Rotary Cell Culture System, Synthecon, Inc.) and at unit gravity in coated Petri dishes. Evaluation of H&E stained thin sections of the aggregates revealed the absence of any necrosis. Confocal microscopy evaluations of cells stained with the Live/Dead assay (Molecular Probes) confirmed that viable cells were present throughout the aggregates with an average of only three dead cells observed per aggregate. Preliminary results from gene array analysis (Affymetrix chip U74Av2) showed that approximately 14% of the genes were down regulated (decreased more than 3 fold) and 4% were upregulated in cells exposed to modeled microgravity for 12 hours compared to unit gravity controls. Additional studies using fluorescent phallacidin revealed a decrease in F-actin in the cells exposed to modeled microgravity compared to unit gravity. Myoblast cells grown as aggregates in modeled microgravity exhibited spontaneous differentiation into syncitia while no differentiation was seen in the unit gravity controls. These studies show that 1)the model test system developed is suitable for assessing cellular and molecular alterations in myoblasts; 2) gene expression alterations occur rapidly (within 12 hours) following exposure to modeled microgravity; and 3) modeled microgravity conditions stimulated myoblast cell differentiation. Achieving a greater understanding of the molecular alterations leading to muscle atrophy will eventually enable the development of cell-based countermeasures, which may be valuable for treatment of muscle diseases on Earth and future space explorations.

Byerly, Diane↗

NASA NTRS2011

Intake of Fish and Omega-3 (n-3) Fatty Acids: Effect on Humans During Actual and Simulated Weightlessness

Space flight has many negative effects on human physiology, including bone and muscle loss. Bone and muscle are two systems that are positively affected by dietary intake of fish and n-3 fatty acids. The mechanism is likely to be related to inhibition by n-3 fatty acids of inflammatory cytokines (such as TNF) and thus inhibition of downstream NF-kB activation. We have documented this effect in a 3-dimensional cell culture model, where NF-kB activation in osteoclasts was inhibited by eicosapentaenoic acid, an n-3 fatty acid. We have also indentified that NF-kB activation in peripheral blood mononuclear cells of Space Shuttle crews. We found that after Shuttle flights of ~2 wk, expression of the protein p65 (evidence of NF-kB activation) was increased at landing (P less than 0.001). When evaluating the effects of n-3 fatty acid intake on bone breakdown after 60 d of bed rest (a weightlessness analog). We found that after 60 d of bed rest, greater intake of n-3 fatty acids was associated with less N-telopeptide excretion (Pearson r = -0.62, P less than 0.05). We also evaluated the relationship of fish intake and bone loss in astronauts after 4 to 6 mo missions on the International Space Station. Higher consumption of fish during flight was associated with higher bone mineral density (Pearson r = 0.46, P less than 0.05). Together, these findings provide evidence of the cellular mechanism by which n-3 fatty acids can inhibit bone loss, and preliminary human evidence of the potential for n-3 fatty acids to counteract bone loss associated with space flight. This study was supported by the NASA Human Research Program.

Smith, S. M.↗

NASA NTRS2015

Determination of Roles of Microgravity and Ionizing Radiation on the Reactivation of Epstein-Barr Virus In Vitro

Astronauts experience symptomatic and asymptomatic herpes virus reactivation during spaceflight. We have shown increases in reactivation of Epstein-Barr virus (EBV), cytomegalovirus (CMV) and varicella zoster virus (VZV) and shedding in body fluids (saliva and urine) in astronauts during space travel. Alterations in immunity, increased stress hormone levels, microgravity, increased radiation, and other conditions unique to spaceflight may promote reactivation of latent herpes viruses. Unique mechanico-physico forces associated with spaceflight can have profound effects on cellular function, especially immune cells. In space flight analog studies such as Antarctica, bed rest studies, and NASA's undersea habitat (Aquarius), reactivation of these viruses occurred, but to a lesser extent than spaceflight. Spaceflight analogs model some spaceflight factors, but none of the analogs recreates all factors experienced in space. Most notably, microgravity and radiation are not included in many analogs. Stress, processed through the HPA axis and SAM systems, induces viral reactivation. However, the respective roles of microgravity and increased space radiation levels or if any synergy exists are not known. Therefore, we studied the effect of modeled space radiation and/or microgravity, independent of the immune system on the changes in cellular gene expression that results in viral (EBV) reactivation. The effects of modeled microgravity and low shear on EBV replication and cellular and EBV gene expression were studied in human B-lymphocyte cell cultures. Latently infected B-lymphocytes were propagated in the rotating wall bioreactor and irradiated with the various dosages of gamma irradiation. At specific time intervals following exposure to modeled microgravity, the cells and supernatant were harvested and reactivation of EBV were assessed by measuring EBV and gene expression, DNA methylation, and infectious virus production.

Mehta, Satish K↗

NASA NTRS2001

Agonist activation of cytosolic Ca2+ in subfornical organ cells projecting to the supraoptic nucleus

The subfornical organ (SFO) is sensitive to both ANG II and ACh, and local application of these agents produces dipsogenic responses and vasopressin release. The present study examined the effects of cholinergic drugs, ANG II, and increased extracellular osmolarity on dissociated, cultured cells of the SFO that were retrogradely labeled from the supraoptic nucleus. The effects were measured as changes in cytosolic calcium in fura 2-loaded cells by using a calcium imaging system. Both ACh and carbachol increased intracellular ionic calcium concentration ([Ca2+]i). However, in contrast to the effects of muscarinic receptor agonists on SFO neurons, manipulation of the extracellular osmolality produced no effects, and application of ANG II produced only moderate effects on [Ca2+]i in a few retrogradely labeled cells. The cholinergic effects on [Ca2+]i could be blocked with the muscarinic receptor antagonist atropine and with the more selective muscarinic receptor antagonists pirenzepine and 4-diphenylacetoxy-N-methylpiperdine methiodide (4-DAMP). In addition, the calcium in the extracellular fluid was required for the cholinergic-induced increase in [Ca2+]i. These findings indicate that ACh acts to induce a functional cellular response in SFO neurons through action on a muscarinic receptor, probably of the M1 subtype and that the increase of [Ca2+]i, at least initially, requires the entry of extracellular Ca2+. Also, consistent with a functional role of M1 receptors in the SFO are the results of immunohistochemical preparations demonstrating M1 muscarinic receptor-like protein present within this forebrain circumventricular organ.

NASA Discipline Regulatory Physiology↗

NASA NTRS1992

Production and characterization of monoclonal antibodies to budgerigar fledgling disease virus major capsid protein VP

Eleven hybridoma cell lines producing monoclonal antibodies (MAbs) against intact budgerigar fledgling disease (BFD) virions were produced and characterized. These antibodies were selected for their ability to react with BFD virions in an enzyme-linked immunosorbent assay. Each of these antibodies was reactive in the immunofluorescent detection of BFD virus-infected cells. These antibodies immunoprecipitated intact virions and specifically recognized the major capsid protein, VP1, of the dissociated virion. The MAbs were found to preferentially recognize native BFD virus capsid protein when compared with denatured virus protein. These MAbs were capable of detecting BFD virus protein in chicken embryonated cell-culture lysates by dot-blot analysis.

Non-NASA Center↗