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A novel method is reported for determining bacterial levels in urine samples, which method depends on the quantitative determination of bacterial adenosine triphosphate (ATP) in the presence of non-bacterial ATP. After the removal of non-bacterial ATP, the bacterial ATP is released by cell rupture and is measured by an enzymatic bioluminescent assay using an enzyme obtained from the firefly.
An automated apparatus is reported for sequentially assaying urine samples for the presence of bacterial adenosine triphosphate (ATP) that comprises a rotary table which carries a plurality of sample containing vials and automatically dispenses fluid reagents into the vials preparatory to injecting a light producing luciferase-luciferin mixture into the samples. The device automatically measures the light produced in each urine sample by a bioluminescence reaction of the free bacterial adenosine triphosphate with the luciferase-luciferin mixture. The light measured is proportional to the concentration of bacterial adenosine triphosphate which, in turn, is proportional to the number of bacteria present in the respective urine sample.
The ubiquity of adenosine triphosphate (ATP) in terrestrial organisms provides the basis for proposing the assay of this vital metabolic intermediate for detecting extraterrestrial biological activity. If an organic carbon chemistry is present on the planets, the occurrence of ATP is possible either from biosynthetic or purely chemical reactions. However, ATP's relative complexity minimizes the probability of abiogenic synthesis. A sensitive technique for the quantitative detection of ATP was developed using the firefly bioluminescent reaction. The procedure was used successfully for the determination of the ATP content of soil and bacteria. This technique is also being investigated from the standpoint of its application in clinical medicine.
An improved method is provided for determining bacterial levels, especially in samples of aqueous physiological fluids. The method depends on the quantitative determination of bacterial adenosine triphosphate (ATP) in the presence of nonbacterial ATP. The bacterial ATP is released by cell rupture and is measured by an enzymatic bioluminescent assay. A concentration technique is included to make the method more sensitive. It is particularly useful where the fluid to be measured contains an unknown or low bacteria count.
The DUMAND (Deep Underwater Muon and Neutrino Detector) array, a hexagon 800 m on a side, 673 m high, and consisting of 22,698 sensor modules, is designed to detect neutrinos in the TeV range, hadronic cascades, muons and Cerenkov radiation. Its engineering, signal processing, and logistic aspects are considered, as are its optical detection (photomultiplier tubes) system and electronics. Geological and bottom current surveys were made at two proposed sites for the array (the Maui and Keahole Point basins of Hawaii), and a study of the steady-state response of a sensor string to current drag forces is reported. Biological interference with the DUMAND array, including mechanical entanglement by large animals, bioluminescence, and especially biofouling are considered, as well as the deployment, implantment and maintenance of the array.
The primary sequence of the 5S ribosomal RNA isolated from the free-living bioluminescent marine bacterium Beneckea harveyi is reported and discussed in regard to indications of phylogenetic relationships with the bacteria Escherichia coli and Photobacterium phosphoreum. Sequences were determined for oligonucleotide products generated by digestion with ribonuclease T1, pancreatic ribonuclease and ribonuclease T2. The presence of heterogeneity is indicated for two sites. The B. harveyi sequence can be arranged into the same four helix secondary structures as E. coli and other prokaryotic 5S rRNAs. Examination of the 5S-RNS sequences of the three bacteria indicates that B. harveyi and P. phosphoreum are specifically related and share a common ancestor which diverged from an ancestor of E. coli at a somewhat earlier time, consistent with previous studies.
The mean intensity of Cerenkov emission from the products of K(40) decay and bioluminescence was measured at depths to 5 km. The intensity of ocean light background is found to depend upon depth and at the 5 km level is equal on averaged to 300 + or - 60 quanta/sq cms into spatial angle of 2 pi sterradian in transparency window. The amplitudes, duration and number of BL flashes were measured at various depths. The intensive flashes due to BL are shown to be observed rather seldom at depths over 4 km.
The imaging instrumentation for the Dynamics Explorer Mission was designed primarily to obtain global auroral images. The instrument, however, was also used successfully to study marine bioluminescence, the geocorona, and the global distribution of atmospheric ozone. The imager has considerable potential for the study of astronomical sources of ultraviolet radiation as well. The data produced by the imager is used to study the brightness and isotrophy of the diffuse ultraviolet background.
From February 1986 to the present, the AOL participated in six interagency flight missions. (1) Shelf Edge Exchange Processes (SEEP II) (Department of Energy). The SEEP experiments are designed to assess the assimilative capacity of the Continental Shelf to absorb the energy by-products introduced into the near-shore ocean environment from coastal communities and marine activities such as energy production plants and offshore oil operations. (2) BIOWATT II (Office of Naval Research). The major objective of this study was to provide a better understanding of the relationships between ocean physics, biology, bioluminescence, and optics in oligotrophic portions of the Atlantic Ocean. (3) Fall Experiment (FLEX) (Department of Energy). The FLEX studies were designed to determine the fate of low salinity water in the coastal boundary zone that is advected south towards the Florida coast during autumn. (4) Greenland Sea and Icelandic Marine Biological Experiments (NASA). The investigations were designed to evaluate the distribution of surface layer chlorophyll in the Greeland Sea and in the coastal waters in the vicinity of Iceland. (5) Submerged Oceanic Scattering Layer Experiment (Naval Ocean Systems Center). This flight experiment demonstrated for the first time the feasibility of detecting and metrically measuring the depth to submerged layers of particulate matter in the shelf break region and in the inner coastal zone. (6) Microbial Exchanges and Coupling in Coastal Atlantic Systems (National Science Foundation). This investigation was designed to study the transportation and fate of particulates in coastal waters and in particular the Chesapeake Bay/coastal Atlantic Ocean. Shortly after the conduct of the flight experiments, airborne laser-induced chlorophyll a and phycoerythrin fluorescence data, as well as sea surface temperature and airborne expendable bathythermograph water column temperature profiles are supplied to cooperating institutions.
Light emitted from high temperature black smokers (350 C) at mid-ocean ridge spreading centers has been documented, but the source of this light and its photochemical and biological consequences have yet to be investigated. Preliminary studies indicate that thermal radiation alone might account for the 'glow' and that a novel photoreceptor in shrimp colonizing black smoker chimneys may detect this 'glow.' A more controversial question is whether there may be sufficient photon flux of appropriate wavelengths to support geothermally-driven photosynthesis (GDP) by microorganisms. Although only a very low level of visible and near infrared light may be emitted from any single hydrothermal vent, several aspects of the light make it of more than enigmatic interest. First, the light is clearly linked to geophysical (and perhaps geochemical) processes; its attributes may serve as powerful index parameters for monitoring change in these processes. Second, while the glow at a vent orifice is a very local phenomenon, more expansive subsurface environments may be illuminated, thereby increasing the spatial scale at which biological consequences of this light might be considered. Third, in contrast to intermittent bioluminescent light sources in the deep sea, the light emitted at vents almost certainly glows or flickers continuously over the life of the individual black smokers (years to decades); collectively, light emitted from black smokers along the ocean's spreading centers superimposed on background Cerenkov radiation negates the concept of the deep sea as an environment devoid of abiotic light. Finally, the history of hydrothermal activity predates the origin of life; light in the deep sea has been a continuous phenomenon on a geological time scale and may have served either as a seed or refugium for the evolution of biological photochemical reactions or adaptations.
The two Dynamics Explorer spacecraft, DE-1 and DE-2, were launched on August 3, 1981, into polar coplanar orbits at different altitudes for the purpose of studying interactive processes within the atmosphere-ionosphere-magnetosphere system. The DE-1 spacecraft (high-altitude mission) used an elliptical orbit that was selected to allow: (1) measurements extending from the hot magnetospheric plasma through the plasmasphere to the cool ionosphere; (2) global auroral imaging, wave measurements in the heart of the magnetosphere, and crossing of auroral field lines at several earth radii; and (3) measurements for significant periods of time along a magnetic field flux tube. The orbit of Dynamics Explorer 1 offered an opportunity to obtain global images of Earth's dayglow and auroral luminosities and to acquire consecutive images of the entire auroral oval during the growth, onset, expansion, and recovery phases of substorms. The University of Iowa's Spin-scan Auroral Imaging (SAI) instrument, was on-board DE-1. SAI was activated in orbit and placed in routine operation on September 23, 1981, and has provided outstanding new contributions in the fields of auroral, magnetospheric and geocoronal physics, introduced a powerful tool for the study of global atmospheric ozone, and initiated the first search from space for marine bioluminescence on the surface of the global ocean. The SAI instrumentation consists of three imaging photometers, two for visible wavelengths and the third for vacuum-ultraviolet wavelengths equipped with primary catoptric optics with superpolished mirror surfaces. The primary focusing element is an off-axis section of a parabolic mirror that is used to provide an optical path completely free of support structures for the mirrors.
A new firefly luciferase bioluminescence assay method that differentiates free extracellular ATP (dead cells, etc.) from intracellular ATP (viable microbes) was used to determine the viable microbial cleanliness of various clean-room facilities. For comparison, samples were taken from both clean-rooms, where the air was filtered to remove particles >0.5 microm, and ordinary rooms with unfiltered air. The intracellular ATP was determined after enzymatically degrading the sample's free ATP. Also for comparison, cultivable microbial populations were counted on nutrient-rich trypticase soy agar (TSA) plates. Both the cultivable and ATP-based determinations indicate that the microbial burden was lower in clean-room facilities than in ordinary rooms. However, there was no direct correlation between the two sets of measurements because the two assays measured very different populations. A large fraction of the samples yielded no colony formers on TSA, but were positive for intracellular ATP. Subsequently, genomic DNA was isolated directly from selected samples and 16S rDNA fragments were cloned and sequenced, identifying nearest neighbors, many of which are known to be noncultivable in the media employed. It was concluded that viable microbial contamination can be reliably monitored by measurement of intracellular ATP, and that this method may be considered superior to cultivable colony counts due to its speed and its ability to report the presence of viable but noncultivable organisms. When the detection of nonviable microbes is of interest, the ATP assay can be supplemented with DNA analysis.
Acute cardiovascular events exhibit a circadian rhythm in the frequency of occurrence. The mechanisms underlying these phenomena are not yet fully understood, but they may be due to rhythmicity inherent in the cardiovascular system. We have begun to characterize rhythmicity of the clock gene mPer1 in the rat cardiovascular system. Luciferase activity driven by the mPer1 gene promoter is rhythmic in vitro in heart tissue explants and a wide variety of veins and arteries cultured from the transgenic Per1-luc rat. The tissues showed between 3 and 12 circadian cycles of gene expression in vitro before damping. Whereas peak per1-driven bioluminescence consistently occurred during the late night in the heart and all arteries sampled, the phases of the rhythms in veins varied significantly by anatomical location. Varying the time of the culture procedure relative to the donor animal's light:dark cycle revealed that, unlike some other rat tissues such as liver, the phases of in vitro rhythms of arteries, veins, and heart explants were affected by culture time. However, phase relationships among tissues were consistent across culture times; this suggests diversity in circadian regulation among components of the cardiovascular system.
A method of sensitive detection of bacterial spores within delays of no more than a few hours has been developed to provide an alternative to a prior three-day NASA standard culture-based assay. A capability for relatively rapid detection of bacterial spores would be beneficial for many endeavors, a few examples being agriculture, medicine, public health, defense against biowarfare, water supply, sanitation, hygiene, and the food-packaging and medical-equipment industries. The method involves the use of a commercial rapid microbial detection system (RMDS) that utilizes a combination of membrane filtration, adenosine triphosphate (ATP) bioluminescence chemistry, and analysis of luminescence images detected by a charge-coupled-device camera. This RMDS has been demonstrated to be highly sensitive in enumerating microbes (it can detect as little as one colony-forming unit per sample) and has been found to yield data in excellent correlation with those of culture-based methods. What makes the present method necessary is that the specific RMDS and the original protocols for its use are not designed for discriminating between bacterial spores and other microbes. In this method, a heat-shock procedure is added prior to an incubation procedure that is specified in the original RMDS protocols. In this heat-shock procedure (which was also described in a prior NASA Tech Briefs article on enumerating sporeforming bacteria), a sample is exposed to a temperature of 80 C for 15 minutes. Spores can survive the heat shock, but nonspore- forming bacteria and spore-forming bacteria that are not in spore form cannot survive. Therefore, any colonies that grow during incubation after the heat shock are deemed to have originated as spores.
This candidate solution proposes to use the night-imaging capabilities of the HSTC from SAC-C and of the HSC from SAC-D/Aquarius to detect bioluminescent events associated with HABs (harmful algal blooms). Once detected, this information could be fed to the NOAA CSCOR (Center for Sponsored Coastal Ocean Research) Harmful Algal Bloom Event Response Program, which acts quickly to fund the mobilization of research teams and to engage local agencies in a response. The HSC/HSTC data can serve as input to the HABSOS decision support system to provide information on location, extent, and duration of HAB events. Society will benefit from improved protection of the health of humans beings, aquatic ecosystems, and coastal economies. This work supports coastal management, public health, and homeland security applications.
Quantum ghost imaging is a method that utilizes the correlated detection of two photons to generate an image. One photon is detected by an imaging sensor and the other by a single-element bucket detector. The selection of the imaging sensor and its capabilities relative to the bucket detector impact the quality of the ghost images. This work examines a SPAD array and a photocathode detector as imaging sensors for quantum ghost imaging. We discuss how to achieve optimal images using these two technologies. We also demonstrate that these devices are able to generate ghost images at 1Hz frame rates, expanding the technique to biologically relevant time scales.
Bioinspired photocatalysis has resulted in efficient solutions for many areas of science and technology spanning from solar cells to medicine. Here we show a new bioinspired semiconductor nanocomposite (nanoTiO 2 DOPA-luciferase, TiDoL) capable of converting light energy within cancerous tissues into chemical species that are highly disruptive to cell metabolism and lead to cell death. This localized activity of semiconductor nanocomposites is triggered by cancer-generated activators. Adenosine triphosphate (ATP) is produced in excess in cancer tissues only and activates nearby immobilized TiDoL composites, thereby eliminating its off-target toxicity. The interaction of TiDoL with cancerous cells was probed in situand in real-time to establish a detailed mechanism of nanoparticle activation, triggering of the apoptotic signaling cascade, and finally, cancer cell death. Activation of TiDoL with non-cancerous cells did not result in cell toxicity. Exploring the activation of antibody-targeted semiconductor conjugates using ATP is a step toward a universal approach to single-cell-targeted medical therapies with more precision, efficacy, and potentially fewer side effects.
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