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At least 325 records · Page 18

Microarray analysis of genes differentially expressed in HepG2 cells cultured in simulated microgravity: preliminary report

Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.

Non-NASA Center↗

Temperature dependence of nitrate-reducing Fe(II) oxidation by Acidovorax strain BoFeN1 – evaluating the role of enzymatic vs. abiotic Fe(II) oxidation by nitrite

ABSTRACT Fe(II) oxidation coupled to nitrate reduction is a widely observed metabolism. However, to what extent the observed Fe(II) oxidation is driven enzymatically or abiotically by metabolically produced nitrite remains puzzling. To distinguish between biotic and abiotic reactions, we cultivated the mixotrophic nitrate-reducing Fe(II)-oxidizing Acidovorax strain BoFeN1 over a wide range of temperatures and compared it to abiotic Fe(II) oxidation by nitrite at temperatures up to 60°C. The collected experimental data were subsequently analyzed through biogeochemical modeling. At 5°C, BoFeN1 cultures consumed acetate and reduced nitrate but did not significantly oxidize Fe(II). Abiotic Fe(II) oxidation by nitrite at different temperatures showed an Arrhenius-type behavior with an activation energy of 80±7 kJ/mol. Above 40°C, the kinetics of Fe(II) oxidation were abiotically driven, whereas at 30°C, where BoFeN1 can actively metabolize, the model-based interpretation strongly suggested that an enzymatic pathway was responsible for a large fraction (ca. 62%) of the oxidation. This result was reproduced even when no additional carbon source was present. Our results show that at below 30°C, i.e. at temperatures representing most natural environments, biological Fe(II) oxidation was largely responsible for overall Fe(II) oxidation, while abiotic Fe(II) oxidation by nitrite played a less important role.

Microbiology↗

Zone-forming fungi experiment MA-147

Streptomyces levoris was used as an experimental microorganism during the Apollo Soyuz Test Project to study specific biological considerations that may be influenced by space flight factors. Preflight, inflight, and postflight growth rates of the cultures were compared by photographing the specimens at regular intervals. Preliminary results based on visual comparison of the photographic data indicate that an increased growth rate occurred during space flight in two of eight flight specimens. The increased growth rate continued in the two specimens during the postflight period until termination of the experiment. Radiation effects may be responsible for the absence of spores in two areas of the last spore ring that was formed during the inflight period in one of the flight cultures; however, the radiation studies related to this experiment have not been completed. Distinct morphological differences in spore rings were observed when postflight spore rings were compared with inflight spore rings. Factors that are related to space flight recovery and reentry into earth gravity may have effected these alterations.

Rogers, T. D.↗

Effect of ultrasonic irradiation on mammalian cells and chromosomes in vitro

Human peripheral blood and HeLa cells were irradiated in vitro at the ultrasonic frequency of 65 kHz. The whole blood and HeLa cell suspensions were exposed to continuous and pulsed ultrasonic power levels of 0.12, 0.16, 0.72, 1.12 and 2.24 W for a period of one minute. The method of ultrasonic irradiation was carried out with the whole blood or HeLa cell suspensions coupled directly to a cylindrical transducer while heating of the cell suspensions in excess of 41 C was avoided. Irradiated and unirradiated peripheral blood lymphocyte chromosome cultures were prepared and scored for selected numerical and morphological aberrations. There was no significant difference in the frequency of chromosomal aberrations between irradiated and unirradiated cells.

Roseboro, J. A.↗

NASA/DOD Aerospace Knowledge Diffusion Research Project. Paper 61: The Technical Communications Practices of ESL Aerospace Engineering Students in the United States: Results of a National Survey

When engineering students graduate and enter the world of work, they make the transition from an academic to a professional community of knowledge. The importance of oral and written communication to the professional success and advancement of engineers is well documented. For example, studies such as those conducted by Mailloux (1989) indicate that communicating data, information, and knowledge takes up as much as 80% of an engineer's time. However, these same studies also indicate that many engineering graduates cannot (a) write technical reports that effectively inform and influence decisionmaking, (b) present their ideas persuasively, and (c) communicate with their peers. If these statements are true, how is learning to communicate effectively in their professional knowledge community different for engineering students educated in the United States but who come from other cultures-cultures in which English is not the primary language of communication? Answering this question requires adequate and generalizable data about these students' communications abilities, skills, and competencies. To contribute to the answer, we undertook a national (mail) survey of 1,727 student members of the American Institute of Aeronautics and Astronautics (AIAA). The focus of our analysis and this paper is a comparison of the responses of 297 student members for whom English is a second language with the responses of 1,430 native English speaking students to queries regarding career choice, bilingualism and language fluency, communication skills, collaborative writing, computer use, and the use of electronic (computer) networks.

Webb, John R.↗

Ecology and life history of an amoebomastigote, Paratetramitus jugosus, from a microbial mat: new evidence for multiple fission

Five microbial habitats (gypsum crust, gypsum photosynthetic community, Microcoleus mat, Thiocapsa scum, and black mud) were sampled for the presence of the euryhaline, rapidly growing amoebomastigote, Paratetramitus jugosus. Field investigations of microbial mats from Baja California Norte, Mexico, and Salina Bido near Matanzas, Cuba, reveal that P. jugosus is most frequently found in the Thiocapsa layer of microbial mats. Various stages of the life history were studied using phase-contrast, differential-interference, and transmission electron microscopy. Mastigote stages were induced and studied by electron microscopy; mastigotes that actively feed on bacteria bear two or more undulipodia. A three-dimensional drawing of the kinetid ("basal apparatus") based on electron micrographs is presented. Although promitoses were occasionally observed, it is unlikely that they can account for the rapid growth of P. jugosus populations on culture media. Dense, refractile, spherical, and irregular-shaped bodies were seen at all times in all cultures along with small mononucleate (approximately 2-7 micrometers diameter) amoebae. Cytochemical studies employing two different fluorescent stains for DNA (DAPI, mithramycin) verified the presence of DNA in these small bodies. Chromatin-like material seen in electron micrographs within the cytoplasm and blebbing off nuclei were interpreted to the chromatin bodies. Our interpretation, consistent with the data but not proven, is that propagation by multiple fission of released chromatin bodies that become small amoebae may occur in Paratetramitus jugosus. These observations are consistent with descriptions of amoeba propagules in the early literature (Hogue, 1914).

Non-NASA Center↗

Comparison of the Microbial Diversity and Abundance Between the Freshwater Land-Locked Lakes of Schirmacher Oasis and the Perennially Ice-Covered Lake Untersee in East Antarctica

Extreme conditions such as low temperature, dryness, and constant UV-radiation in terrestrial Antarctica are limiting factors of the survival of microbial populations. The objective of this study was to investigate the microbial diversity and enumeration between the open water lakes of Schirmacher Oasis and the permanently ice-covered Lake Untersee. The lakes in Schirmacher Oasis possessed abundant and diverse group of microorganisms compared to the Lake Untersee. Furthermore, the microbial diversity between two lakes in Schirmacher Oasis (Lake L27C and L47) was compared by culture-based molecular approach. It was determined that L27Chad a richer microbial diversity representing 5 different phyla and 7 different genera. In contrast L47 consisted of 4 different phyla and 6 different genera. The difference in microbial community could be due to the wide range of pH between L27C (pH 9.1) and L47 (pH 5.7). Most of the microbes isolated from these lakes consisted of adaptive biological pigmentation. Characterization of the microbial community found in the freshwater lakes of East Antarctica is important because it gives a further glimpse into the adaptation and survival strategies found in extreme conditions.

Huang, Jonathan↗

Transduction-like gene transfer in the methanogen Methanococcus voltae

Strain PS of Methanococcus voltae (a methanogenic, anaerobic archaebacterium) was shown to generate spontaneously 4.4-kbp chromosomal DNA fragments that are fully protected from DNase and that, upon contact with a cell, transform it genetically. This activity, here called VTA (voltae transfer agent), affects all markers tested: three different auxotrophies (histidine, purine, and cobalamin) and resistance to BES (2-bromoethanesulfonate, an inhibitor of methanogenesis). VTA was most effectively prepared by culture filtration. This process disrupted a fraction of the M. voltae cells (which have only an S-layer covering their cytoplasmic membrane). VTA was rapidly inactivated upon storage. VTA particles were present in cultures at concentrations of approximately two per cell. Gene transfer activity varied from a minimum of 2 x 10(-5) (BES resistance) to a maximum of 10(-3) (histidine independence) per donor cell. Very little VTA was found free in culture supernatants. The phenomenon is functionally similar to generalized transduction, but there is no evidence, for the time being, of intrinsically viral (i.e., containing a complete viral genome) particles. Consideration of VTA DNA size makes the existence of such viral particles unlikely. If they exist, they must be relatively few in number;perhaps they differ from VTA particles in size and other properties and thus escaped detection. Digestion of VTA DNA with the AluI restriction enzyme suggests that it is a random sample of the bacterial DNA, except for a 0.9-kbp sequence which is amplified relative to the rest of the bacterial chromosome. A VTA-sized DNA fraction was demonstrated in a few other isolates of M. voltae.

Methanococcus/genetics/growth & development/metabo↗

Potent Antiviral Activity against HSV-1 and SARS-CoV-2 by Antimicrobial Peptoids

Viral infections, such as those caused by Herpes Simplex Virus-1 (HSV-1) and SARS-CoV-2, affect millions of people each year. However, there are few antiviral drugs that can effectively treat these infections. The standard approach in the development of antiviral drugs involves the identification of a unique viral target, followed by the design of an agent that addresses that target. Antimicrobial peptides (AMPs) represent a novel source of potential antiviral drugs. AMPs have been shown to inactivate numerous different enveloped viruses through the disruption of their viral envelopes. However, the clinical development of AMPs as antimicrobial therapeutics has been hampered by a number of factors, especially their enzymatically labile structure as peptides. We have examined the antiviral potential of peptoid mimics of AMPs (sequence-specific N-substituted glycine oligomers). These peptoids have the distinct advantage of being insensitive to proteases, and also exhibit increased bioavailability and stability. Our results demonstrate that several peptoids exhibit potent in vitro antiviral activity against both HSV-1 and SARS-CoV-2 when incubated prior to infection. In other words, they have a direct effect on the viral structure, which appears to render the viral particles non-infective. Visualization by cryo-EM shows viral envelope disruption similar to what has been observed with AMP activity against other viruses. Furthermore, we observed no cytotoxicity against primary cultures of oral epithelial cells. These results suggest a common or biomimetic mechanism, possibly due to the differences between the phospholipid head group makeup of viral envelopes and host cell membranes, thus underscoring the potential of this class of molecules as safe and effective broad-spectrum antiviral agents. We discuss how and why differing molecular features between 10 peptoid candidates may affect both antiviral activity and selectivity.

60 APPLIED LIFE SCIENCES↗

Functional analysis of Salix purpurea genes support roles for ARR17 and GATA15 as master regulators of sex determination

Abstract The Salicaceae family is of growing interest in the study of dioecy in plants because the sex determination region (SDR) has been shown to be highly dynamic, with differing locations and heterogametic systems between species. Without the ability to transform and regenerate Salix in tissue culture, previous studies investigating the mechanisms regulating sex in the genus Salix have been limited to genome resequencing and differential gene expression, which are mostly descriptive in nature, and functional validation of candidate sex determination genes has not yet been conducted. Here, we used Arabidopsis to functionally characterize a suite of previously identified candidate genes involved in sex determination and sex dimorphism in the bioenergy shrub willow Salix purpurea . Six candidate master regulator genes for sex determination were heterologously expressed in Arabidopsis, followed by floral proteome analysis. In addition, 11 transcription factors with predicted roles in mediating sex dimorphism downstream of the SDR were tested using DAP‐Seq in both male and female S. purpurea DNA. The results of this study provide further evidence to support models for the roles of ARR17 and GATA15 as master regulator genes of sex determination in S. purpurea , contributing to a regulatory system that is notably different from that of its sister genus Populus . Evidence was also obtained for the roles of two transcription factors, an AP2 / ERF family gene and a homeodomain‐like transcription factor, in downstream regulation of sex dimorphism.

60 APPLIED LIFE SCIENCES↗

Laboratory tank studies of a single species of phytoplankton using a remote sensing fluorosensor

Phytoplankton were grown in the laboratory for the purpose of testing a remote fluorosensor. The fluorosensor uses a unique four-wavelength dye laser system to excite phytoplankton bearing chlorophyll and to measure the chlorophyll fluorescence generated by this excitation. Six different species were tested, one at a time, and each was grown two to four times. Fluorescence measured by the fluorosensor provides good quantitative measurement of chlorophyll concentrations for all species tested while the cultures were in log phase growth. Fluorescene cross section ratios obtained in the single species tank tests support the hypothesis that the shape of the fluorescence cross section curve remains constant with the species (differences in fluorescence cross section ratios are a basis for determining composition of phytoplankton according to color group when a multiwavelength source of excitation is used. Linear relationships exist between extracted chlorophyll concentration and fluorescence measured by the remote fluorosensor during the log phase growth of phytoplankton cultures tested.

Brown, C. A., Jr.↗

Photointerpretation of Skylab photography

The author has identified the following significant results. In terms of film grain texture and object definition, the S190B color positive film is distinctly superior to the S190A films, when both are compared in the 9 x 9 inch format. Within the six S190A films, the panchromatic black and white films are superior to the infrared black and white, and the color positive film is superior to the color infrared. Minimum relief differences on the order of 500 to 100 feet could be detected by stereoscopic study, however, it is not possible to determine to what extent vegetation and cultural practices assist in such delineations. Water and wind gaps through major ridges were easily seen. Streams of third order and larger were clearly visible and easy to trace; second order streams were not identified with consistency. Differences in color, tone, and textural patterns rarely supplied clues for differentiating soils or bedrock. The separation of naturally forested areas from areas of cultivation and pasture was effective and a valuable clue to the underlying geology. Suburban and industrial developments were clearly differentiated from urban areas and surrounding agricultural fields. Soil associations could be identified on a regional basis, but no sharp boundary could be drawn separating soil associations.

Mcmurtry, G. J.↗

Identifying a novel pathway for extracellular electron uptake in Methanosarcina barkeri using shotgun

The overarching goal of this project was to identify the genetic and/or enzymatic basis for extracellular electron uptake in Methanosarcina barkeri—an organism we previously characterized to be capable of this process using poised potential electrodes and a range of electrochemical techniques. In our first aim, we will utilized proteomic techniques that labels extracellular proteins with free amine groups with biotin and perform a selective purification of these proteins from the rest of the proteome. These were identified using mass spectrometry, providing us insight into the identity of the cell surface proteins, including redox active proteins present during extracellular electron uptake. We will also used an isobaric tagging technique (iTRAQ labeling) of proteomes from different experiment conditions to look at differential proteins expression and better understand the total pathway involved in extracellular electron uptake coupled to methanogenesis. In our final aim, we will investigated the electrochemical nature of electron uptake in co-cultures, compared to our pure culture data sets. We predict the process of electron uptake from electrodes replicates what goes on in nature when these organisms for co-cultures with electrogenic microbes such as Geobacter, and to test this hypothesis we have begun to probe co-cultures electrochemically during direct interspecies electron transfer. Our final aim took much longer than expected due to challenges replicating co-cultures between Geobacter metallireducens and Methanosarcina barkeri. We found the major problem with this work was the inability of the M. barkeri strain we used (and we tried strains from three different labs) to successfully grow on acetate in the absence of hydrogen. As such, we began to use the strain Methanosarcina acetivorans—another close relative that specializes on acetate growth. We are currently in the process of comparing electrochemical observations of this microorganism between pure and co-culture experiments.

59 BASIC BIOLOGICAL SCIENCES↗

Interaction of amphiphilic lipoarabinomannan with host carrier lipoproteins in tuberculosis patients: Implications for blood-based diagnostics

Lipoarabinomannan (LAM), an amphiphilic lipoglycan of the Mycobacterium tuberculosis cell wall, is a diagnostic target for tuberculosis. Previous work from our laboratory and others suggests that LAM is associated with host serum lipoproteins, which may in turn have implications for diagnostic assays. Our team has developed two serum assays for amphiphile detection: lipoprotein capture and membrane insertion. The lipoprotein capture assay relies on capture of the host lipoproteins, exploiting the biological association of host lipoprotein with microbial amphiphilic biomarkers to “concentrate” LAM. In contrast, the membrane insertion assay is independent of the association between pathogen amphiphiles and host lipoprotein association, and directly captures LAM based on its thermodynamic propensity for association with a supported lipid membrane, which forms the functional surface of an optical biosensor. In this manuscript, we explored the use of these assays for the detection of LAM in sera from adults whose tuberculosis status had been well-characterized using conventional microbiological tests, and endemic controls. Using the lipoprotein capture assay, LAM signal/noise ratios were >1.0 in 29/35 (83%) individuals with culture-confirmed active tuberculosis, 8/13 (62%) individuals with tuberculosis symptoms, but no positive culture for M . tuberculosis , and 0/6 (0%) symptom-free endemic controls. To evaluate serum LAM levels without bias associated with potential differences in circulating host lipoprotein concentrations between individuals, we subsequently processed available samples to liberate LAM from associated host lipoprotein assemblies followed by direct detection of the pathogen biomarker using the membrane insertion approach. Using the membrane insertion assay, signal/noise for detection of serum LAM was greater than that observed using the lipoprotein capture method for culture-confirmed TB patients (6/6), yet remained negative for controls (2/2). Taken together, these results suggest that detection of serum LAM is a promising TB diagnostic approach, but that further work is required to optimize assay performance and to decipher the implications of LAM/host lipoprotein associations for diagnostic assay performance and TB pathogenesis.

59 BASIC BIOLOGICAL SCIENCES↗

Changes in pituitary growth hormone cells prepared from rats flown on Spacelab 3

The effect of exposure to microgravity on pituitary gland was investigated by examining cells isolated from anterior pituitaries of rats flown on the 7-day Spacelab 3 mission and, subsequently, cultured for 6 days. Compared with ground controls, flight cells contained more intracellular growth hormone (GH); however, the flight cells released less GH over the 6-day culture period and after implantation into hypophysectomized rats than did the control cells. Compared with control rats, glands from large rats (400 g) contained more somatotrophs (44 percent compared with 37 percent in control rats); small rats (200 g) showed no difference. No major differences were found in the somatotroph ultrastructure (by TEM) or in the pattern of the immunoactive GH variants. However, high-performance liquid chromatography fractionation of culture media indicated that flight cells released much less of a biologically active high-molecular weight GH variant, suggesting that space flight may lead to secretory dysfunction.

Grindeland, R.↗

The Antaeus Project - An orbital quarantine facility for analysis of planetary return samples

A design is presented for an earth-orbiting facility for the analysis of planetary return samples under conditions of maximum protection against contamination but minimal damage to the sample. The design is keyed to a Mars sample return mission profile, returning 1 kg of documented subsamples, to be analyzed in low earth orbit by a small crew aided by automated procedures, tissue culture and microassay. The facility itself would consist of Spacelab shells, formed into five modules of different sizes with purposes of power supply, habitation, supplies and waste storage, the linking of the facility, and both quarantine and investigation of the samples. Three barriers are envisioned to protect the biosphere from any putative extraterrestrial organisms: sealed biological containment cabinets within the Laboratory Module, the Laboratory Module itself, and the conditions of space surrounding the facility.

Sweet, H. C.↗

Outer space law: A problem of astronautics

The theory of space law is discussed from the point of view of similarities and differences between hypothetical space law and current (1932) aviation law. International legal aspects and economic and cultural effects are also addressed.

Mandl, V.↗

Kidney cell electrophoresis in space flight: Rationale, methods, results and flow cytometry applications

Cultures of human embryonic kidney cells consistently contain an electrophoretically separable subpopulation of cells that produce high levels of urokinase and have an electrophoretic mobility about 85 percent as high as that of the most mobile human embryonic kidney cells. This subpopulation is rich in large epithelioid cells that have relatively little internal structure. When resolution and throughput are adequate, free fluid electrophoresis can be used to isolate a broad band of low mobility cells which also produces high levels of plasminogen activators (PAs). In the course of performing this, it was discovered that all electrophoretic subpopulations of cultured human embryonic kidney cells produce some PAs and that separate subpopulations produce high quantities of different types of PA's. This information and the development of sensitive assays for this project have provided new insights into cell secretion mechanisms related to fibrinolysis. These advances would probably not have been made without the NASA program to explore fundamental questions of free fluid electrophoresis in space.

Todd, P.↗