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At least 145 records · Page 8

Covisualization by computational optical-sectioning microscopy of integrin and associated proteins at the cell membrane of living onion protoplasts

Using higher-resolution wide-field computational optical-sectioning fluorescence microscopy, the distribution of antigens recognized by antibodies against animal beta 1 integrin, fibronectin, and vitronectin has been visualized at the outer surface of enzymatically protoplasted onion epidermis cells and in depectinated cell wall fragments. On the protoplast all three antigens are colocalized in an array of small spots, as seen in raw images, in Gaussian filtered images, and in images restored by two different algorithms. Fibronectin and vitronectin but not beta 1 integrin antigenicities colocalize as puncta in comparably prepared and processed images of the wall fragments. Several control visualizations suggest considerable specifity of antibody recognition. Affinity purification of onion cell extract with the same anti-integrin used for visualization has yielded protein that separates in SDS-PAGE into two bands of about 105-110 and 115-125 kDa. These bands are again recognized by the visualization antibody, which was raised against the extracellular domain of chicken beta 1 integrin, and are also recognized by an antibody against the intracellular domain of chicken beta 1 integrin. Because beta 1 integrin is a key protein in numerous animal adhesion sites, it appears that the punctate distribution of this protein in the cell membranes of onion epidermis represents the adhesion sites long known to occur in cells of this tissue. Because vitronectin and fibronection are matrix proteins that bind to integrin in animals, the punctate occurrence of antigenically similar proteins both in the wall (matrix) and on enzymatically prepared protoplasts reinforces the concept that onion cells have adhesion sites with some similarity to certain kinds of adhesion sites in animals.

NASA Discipline Plant Biology↗

Food Fortification Stability Study

NASA has established the goal of traveling beyond low-Earth orbit and extending manned exploration to Mars. The extended length of a Mars mission, along with the lack of resupply missions increases the importance of nutritional content in the food system. The purpose of this research is to assess the stability of vitamin supplementation in traditionally processed spaceflight foods. It is expected that commercially available fortificants will remain stable through long-duration missions if proper formulation, processing, and storage temperatures are all achieved. Five vitamins (vitamin E, vitamin K, pantothenic acid, folic acid, and thiamin) were blended into a vitamin premix (DSM, Freeport, TX); premixes were formulated to be compatible with current processing techniques (retort or freeze-dried), varied water activities (high or low), and packaging material. The overall goal of this process is to provide 25% of the recommended daily intake of each vitamin (per serving), following processing and two years of ambient storage. Four freeze-dried foods (Scrambled Eggs, Italian Vegetables, Potatoes Au Gratin, Noodles and Chicken) and four thermostabilized foods (Curry Sauce with Vegetables, Chicken Noodle Soup, Grilled Pork Chop, Rice with Butter) were produced (with and without the vitamin premix), to assess the impact of the added fortificant on color and taste, and to determine the stability of supplemental vitamins in spaceflight foods. The use of fortification in spaceflight foods appears to be a plausible mitigation step to inadequate nutrition. This is due to the ease of vitamin addition as well as the sustainability of the premixes through initial processing steps. Postprocessing analysis indicated that vitamin fortification with this premix did not immediately impact organoleptic properties of the food. At this stage, the largest hurdle to fortification is the preciseness to which vitamins can be added; the total amount of vitamins required for production is 10 - 20 grams, a minor percentage of the formulation. As demonstrated by the over-fortification measured in Italian Vegetables and Grilled Pork Chop, homogeneity may be difficult to achieve with such small amounts. Thus, pouch-to-pouch variability, over-fortification, and underfortification may ensue if a method for precise addition is not identified. Stability will continue to be evaluated over two years of storage at three temperatures, and future analysis should reveal the extent to which this issue should be a concern

Abdulmalik, T. O.↗

Grouse and Land-Based Wind Energy Development in the United States

Several species of grouse occupy prairie and sagebrush habitat across the western United States. Sharp-tailed grouse (Tympanuchus phasianellus), greater prairie chicken (Tympanuchus cupido), and lesser prairie-chicken (Tympanuchus pallidicinctus) reside in the grassland prairies of the Great Plains. In the Intermountain West, greater sage-grouse (Centrocercus urophasianus) and Gunnison sage-grouse (Centrocercus minimus) inhabit the expansive sagebrush plains. The continued deployment of wind energy across the United States, particularly in prairie and sagebrush habitats, may result in a myriad of impacts to grouse species, although there is a high degree of uncertainty regarding the extent of the impact.

avoidance↗

Dietary microalgae on poultry meat and eggs: explained versus unexplained effects

Different types and sources of microalgae are used to feed broiler chickens and laying hens. This report provides a concise update on various impacts of feeding these novel ingredients on physical, chemical, and nutritional attributes of the resultant meat and eggs. Some of the observed effects may be associated with biochemical and molecular mechanisms derived from unique chemical compositions and nutritional values of microalgae. However, the full potential and the accurate mechanism of microalgae in producing health-promoting poultry foods remain to be explored.

59 BASIC BIOLOGICAL SCIENCES↗

Caspase inhibitors promote vestibular hair cell survival and function after aminoglycoside treatment in vivo

The sensory hair cells of the inner ear undergo apoptosis after acoustic trauma or aminoglycoside antibiotic treatment, causing permanent auditory and vestibular deficits in humans. Previous studies have demonstrated a role for caspase activation in hair cell death and ototoxic injury that can be reduced by concurrent treatment with caspase inhibitors in vitro. In this study, we examined the protective effects of caspase inhibition on hair cell death in vivo after systemic injections of aminoglycosides. In one series of experiments, chickens were implanted with osmotic pumps that administrated the pan-caspase inhibitor z-Val-Ala-Asp(Ome)-fluoromethylketone (zVAD) into inner ear fluids. One day after the surgery, the animals received a 5 d course of treatment with streptomycin, a vestibulotoxic aminoglycoside. Direct infusion of zVAD into the vestibule significantly increased hair cell survival after streptomycin treatment. A second series of experiments determined whether rescued hair cells could function as sensory receptors. Animals treated with streptomycin displayed vestibular system impairment as measured by a greatly reduced vestibulo-ocular response (VOR). In contrast, animals that received concurrent systemic administration of zVAD with streptomycin had both significantly greater hair cell survival and significantly increased VOR responses, as compared with animals treated with streptomycin alone. These findings suggest that inhibiting the activation of caspases promotes the survival of hair cells and protects against vestibular function deficits after aminoglycoside treatment.

NASA Discipline Neuroscience↗

Avian dark cells

Dark cells (DCs) of mammalian and non-mammalian species help to maintain the homeostasis of the inner ear fluids in vivo. Although the avian cochlea is straight and the mammalian cochlea is coiled, no significant difference in the morphology and/or function of mammalian and avian DCs has been reported. The mammalian equivalent of avian DCs are marginal cells and are located in the stria vascularis along a bony sheet. Avian DCs hang free from the tegmentum vasculosum (TV) of the avian lagena between the perilymph and endolymph. Frame averaging was used to image the fluorescence emitted by several fluorochromes applied to freshly isolated dark cells (iDCs) from chickens (Gallus domesticus) inner ears. The viability of iDCs was monitored via trypan blue exclusion at each isolation step. Sodium Green, BCECF-AM, Rhodamine 123 and 9-anthroyl ouabain molecules were used to test iDC function. These fluorochromes label iDCs ionic transmembrane trafficking function, membrane electrogenic potentials and Na+/K+ ATPase pump's activity. Na+/K+ ATPase pump sites, were also evaluated by the p-nitrophenyl phosphatase reaction. These results suggest that iDCs remain viable for several hours after isolation without special culturing requirements and that the number and functional activity of Na+/K+ ATPase pumps in the iDCs were indistinguishable from in vivo DCs. Primary cultures of freshly iDCs were successfully maintained for 28 days in plastic dishes with RPMI 1640 culture medium. The preparation of iDCs overcomes the difficulty of DCs accessability in vivo and the unavoidable contamination that rupturing the inner ear microenvironments induces.

NASA Discipline Neuroscience↗

Compound gravity receptor polarization vectors evidenced by linear vestibular evoked potentials

The utricle and saccule are gravity receptor organs of the vestibular system. These receptors rely on a high-density otoconial membrane to detect linear acceleration and the position of the cranium relative to Earth's gravitational vector. The linear vestibular evoked potential (VsEP) has been shown to be an effective non-invasive functional test specifically for otoconial gravity receptors (Jones et al., 1999). Moreover, there is some evidence that the VsEP can be used to independently test utricular and saccular function (Taylor et al., 1997; Jones et al., 1998). Here we characterize compound macular polarization vectors for the utricle and saccule in hatchling chickens. Pulsed linear acceleration stimuli were presented in two axes, the dorsoventral (DV, +/- Z axis) to isolate the saccule, and the interaural (IA, +/- Y axis) to isolate the utricle. Traditional signal averaging was used to resolve responses recorded from the surface of the skull. Latency and amplitude of eighth nerve components of the linear VsEP were measured. Gravity receptor responses exhibited clear preferences for one stimulus direction in each axis. With respect to each utricular macula, lateral translation in the IA axis produced maximum ipsilateral response amplitudes with substantially greater amplitude intensity (AI) slopes than medially directed movement. Downward caudal motions in the DV axis produced substantially larger response amplitudes and AI slopes. The results show that the macula lagena does not contribute to the VsEP compound polarization vectors of the sacculus and utricle. The findings suggest further that preferred compound vectors for the utricle depend on the pars externa (i.e. lateral hair cell field) whereas for the saccule they depend on pars interna (i.e. superior hair cell fields). These data provide evidence that maculae saccule and utricle can be selectively evaluated using the linear VsEP.

NASA Discipline Developmental Biology↗

Sequence, molecular properties, and chromosomal mapping of mouse lumican

PURPOSE. Lumican is a major proteoglycan of vertebrate cornea. This study characterizes mouse lumican, its molecular form, cDNA sequence, and chromosomal localization. METHODS. Lumican sequence was determined from cDNA clones selected from a mouse corneal cDNA expression library using a bovine lumican cDNA probe. Tissue expression and size of lumican mRNA were determined using Northern hybridization. Glycosidase digestion followed by Western blot analysis provided characterization of molecular properties of purified mouse corneal lumican. Chromosomal mapping of the lumican gene (Lcn) used Southern hybridization of a panel of genomic DNAs from an interspecific murine backcross. RESULTS. Mouse lumican is a 338-amino acid protein with high-sequence identity to bovine and chicken lumican proteins. The N-terminus of the lumican protein contains consensus sequences for tyrosine sulfation. A 1.9-kb lumican mRNA is present in cornea and several other tissues. Antibody against bovine lumican reacted with recombinant mouse lumican expressed in Escherichia coli and also detected high molecular weight proteoglycans in extracts of mouse cornea. Keratanase digestion of corneal proteoglycans released lumican protein, demonstrating the presence of sulfated keratan sulfate chains on mouse corneal lumican in vivo. The lumican gene (Lcn) was mapped to the distal region of mouse chromosome 10. The Lcn map site is in the region of a previously identified developmental mutant, eye blebs, affecting corneal morphology. CONCLUSIONS. This study demonstrates sulfated keratan sulfate proteoglycan in mouse cornea and describes the tools (antibodies and cDNA) necessary to investigate the functional role of this important corneal molecule using naturally occurring and induced mutants of the murine lumican gene.

NASA Discipline Cell Biology↗

Osteoclast cytosolic calcium, regulated by voltage-gated calcium channels and extracellular calcium, controls podosome assembly and bone resorption

The mechanisms of Ca2+ entry and their effects on cell function were investigated in cultured chicken osteoclasts and putative osteoclasts produced by fusion of mononuclear cell precursors. Voltage-gated Ca2+ channels (VGCC) were detected by the effects of membrane depolarization with K+, BAY K 8644, and dihydropyridine antagonists. K+ produced dose-dependent increases of cytosolic calcium ([Ca2+]i) in osteoclasts on glass coverslips. Half-maximal effects were achieved at 70 mM K+. The effects of K+ were completely inhibited by dihydropyridine derivative Ca2+ channel blocking agents. BAY K 8644 (5 X 10(-6) M), a VGCC agonist, stimulated Ca2+ entry which was inhibited by nicardipine. VGCCs were inactivated by the attachment of osteoclasts to bone, indicating a rapid phenotypic change in Ca2+ entry mechanisms associated with adhesion of osteoclasts to their resorption substrate. Increasing extracellular Ca2+ ([Ca2+]e) induced Ca2+ release from intracellular stores and Ca2+ influx. The Ca2+ release was blocked by dantrolene (10(-5) M), and the influx by La3+. The effects of [Ca2+]e on [Ca2+]i suggests the presence of a Ca2+ receptor on the osteoclast cell membrane that could be coupled to mechanisms regulating cell function. Expression of the [Ca2+]e effect on [Ca2+]i was similar in the presence or absence of bone matrix substrate. Each of the mechanisms producing increases in [Ca2+]i, (membrane depolarization, BAY K 8644, and [Ca2+]e) reduced expression of the osteoclast-specific adhesion structure, the podosome. The decrease in podosome expression was mirrored by a 50% decrease in bone resorptive activity. Thus, stimulated increases of osteoclast [Ca2+]i lead to cytoskeletal changes affecting cell adhesion and decreasing bone resorptive activity.

NASA Discipline Musculoskeletal↗

Comparison and validation of the QuEChERSER mega-method for determination of per- and polyfluoroalkyl substances in foods by liquid chromatography with high-resolution and triple quadrupole mass spectrometry

Instances of food contamination with per- and polyfluoroalkyl substances (PFAS) continue to occur globally, but sample preparation and analytical methods are quite limited and often monitor for a small percentage of known PFAS. This study aimed to evaluate, validate, and compare performance of two instruments with the recently developed “quick, easy, cheap, effective, rugged, safe, efficient, and robust” (QuEChERSER) sample preparation mega-method – a method developed to monitor chemicals over a broad range of physicochemical properties. Initial evaluation of the QuEChERSER mega-method for determination of PFAS in food demonstrated recoveries, matrix interferences, and co-extractive removal comparable to (or better than) US Food and Drug Administration (FDA) and USDA Food Safety and Inspection Service (FSIS) methods. Subsequent validation of QuEChERSER in beef, catfish, chicken, pork, liquid eggs, and powdered eggs on a high-resolution mass spectrometer achieved acceptable recoveries (70–120%) and precision (RSDs ≤20%) for all 33 target analytes at the 1 and 5 ng g –1 levels and 67–88% of analytes at the 0.1 ng g –1 level, depending on the matrix. Additional validation was performed by tandem mass spectrometry on a triple quadrupole instrument. This approach provided no non-detects and better recoveries at the 0.1 ng g –1 level than the HRMS method but exhibited more variability at 1 and 5 ng g –1 spiking levels. Analysis of NIST SRMs 1946 and 1947 gave accuracies of 70–117%. Furthermore, these results demonstrate the capability of combining PFAS analysis with a mega-method previously validated for 350 analytes, while collecting non-target data for future retrospective analysis of emerging alternatives with a high-resolution mass spectrometry method.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Cracking vs. surface reactivity in high-nickel cathodes for lithium-ion batteries

High-nickel layered oxide cathodes LiNi x Mn y Co z O 2 (NMC) experience microcracks during cycling. This can expose fresh cathode surfaces for parasitic reactions and isolate active cathode material from the conductive electrode matrix, resulting in impedance increase and capacity fade. The commonly held belief attributes microcracks to anisotropic lattice volume changes of primary particles during cycling. Nevertheless, recent reports suggest that certain electrolytes might reduce microcracks in NMC cathodes during deep cycling. This raises a crucial question on the origin of microcracks: do microcracks exacerbate surface stability, or does poor surface stability contribute to microcrack formation? This perspective aims to provide context and expound this “chicken or egg” dilemma. We contend that the consequence of surface reactivity on the cycle life of high-Ni cathodes is more pronounced than that of particle cracking. Here, we hypothesize that particle cracking is more of a symptom of severe surface reactivity rather than a cause of capacity fade.

25 ENERGY STORAGE↗

Metabolic responses to increased temperatures in three semi-aquatic turtle species from the southeastern United States

Physiological maintenance of ectotherms is largely dependent on temperature. Abrupt changes in environmental conditions can cause shifts in energy budgets, with higher temperatures requiring more energy be devoted to maintenance functions, making less energy available for other activities. As a group, freshwater turtles are of conservation concern; thus, it is important to understand their physiological responses to temperature shifts, particularly given current climate change predictions. Here, we quantified metabolic rates (MR) for three species of semi-aquatic turtles that vary in their degree of terrestriality and propensity to aestivate: chicken turtles (Deirochelys reticularia), eastern mud turtles (Kinosternon subrubrum), and eastern musk turtles (Sternotherus odoratus). Metabolic trials were performed using a flow-through respirometer at three environmentally-relevant temperatures: 25°C, 30°C, and 35°C. As expected, MRs of turtles were significantly and positively associated with increases in mass and temperature. Pairwise comparisons revealed that D. reticularia exhibited significantly higher oxygen consumption rates (VO 2 ) relative to K. subrubrum, which had significantly higher VO 2 than S. odoratus. The combination of higher metabolic rates, the tendency to terrestrially aestivate, and declining quantity of high quality thermal habitats may place some semi-aquatic turtles at a disadvantage when considering future climate scenarios. Our findings, when combined with knowledge of other ecological traits, may be a useful tool for predicting relative sensitivities of turtle species to predicted climate change, particularly those species inhabiting seasonal wetlands.

Sternotherus odoratus↗