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At least 883 records · Page 49

A Biochemical Magic Frequency

Life is composed principally of four classes of biomolecules - protein, nucleic acid, polysaccharide and lipid. Using 1) estimates of the reducing equivalents (electron pairs) needed to synthesize these biomolecules from carbon dioxide, and 2) measurements of the molecular composition of different organisms, we calculated the average number of electron pairs required for the reduction of carbon dioxide to biological carbon (electron pairs/carbon atom). These calculations showed that the carbon of the Earths biosphere is at the reduction level of formaldehyde that requires 2 electron pairs/carbon atom to be synthesized from carbon dioxide. This was also the reduction level of carbon of individual organisms, except for those that stored large amounts of fuel as lipid. Since this chemical property of life is easily discovered and probably universal, it's most likely known by other intelligent life in the universe. It could be the one thing we know about other carbon-based life in the universe, and the one thing that other intelligent life knows about us. We believe this common knowledge that formaldehyde represents the reduction level of life's carbon could lead to the selection of the 72.83814 GHz line of the 0,0,0,1,0,1 ground-state rotational transition of formaldehyde as a frequency for interstellar communication.

Weber, Arthur L.↗

Evolution of the Earth and Origin of Life: The Role of Gas/Fluid Interactions with Rocks

The work under the Cooperative Agreement will be centered on questions of the evolution of Life on the early Earth and possibly on Mars. It is still hotly debated whether the essential organic molecules were delivered to the early Earth from space (by comets, meteorites or interplanetary dust particles) or were generated in situ on Earth. Prior work that has shown that the matrix of igneous minerals is a medium in which progenitors of organic molecules assemble from H2O, C02 and N2 incorporated as minority "impurities" in minerals of igneous rocks during crystallization from H2O/CO2/N2-laden magmas. The underlying processes involve a redox. conversion whereby C, H, and N become chemically reduced, while 0 becomes oxidized to the peroxy state. During Year 02 the work will be divided into three tasks. Task 1: After carboxylic (fatty) acids and N-bearing compounds have been identified, other extractable organic molecules including lipids, oily substances and amino acids will be studied. Dedicated lipid analysis will be combined with gas chromatographic-mass spectroscopic (GCMS) analysis of organic compounds extracted from minerals and rocks. Task 2: Using infrared (IR) spectroscopy, C-H entities that are indicators for the organic progenitors in mineral matrices will be studied. A preliminary heating experiment with MgO single crystals has shown that the C-H entities can be pyrolyzed, causing the IR bands to disappear, but at room temperature the IR bands reappear in a matter of days to weeks. This work will be expanded, both by studying synthetic MgO crystals and olivine crystals from the Earth's upper mantle. The C-H bands will be compared to the published "organic" IR feature of dust in the interstellar medium (ISM) and interplanetary dust particles (IDP). Task 3: A paradox marks the evolution of early Life: Oxygen is highly toxic to primitive life, yet early organisms "learned" to detoxify reactive oxygen species, to utilize oxygen, and even produce it. Why would organisms on the early anaerobic Earth be under evolutionary pressure to evolve defenses against reactive oxygen species? Minerals in igneous rocks are now known to contain peroxy. When such minerals weather, the peroxy hydrolyzes to H2O2. The hypothesis will be tested whether organisms living in intimate contact with rock surfaces are subjected to a constant trickle of H202 and thus under stress to develop strategies to either detoxify the reactive oxygen species or repair the molecular damage that they cause. Understanding these processes is central to the Astrobiology mission. It opens new avenues toward understanding the evolution of early life on Earth, and the potential for aerobic life elsewhere. This Cooperative Agreement also has a strong educational and public outreach component involving high school, undergraduate students, and high school teachers.

Freund, Friedemann↗

Discrimination of Single Base Pair Differences Among Individual DNA Molecules Using a Nanopore

The protein toxin alpha-hemolysin form nanometer scale channels across lipid membranes. Our lab uses a single channel in an artificial lipid bilayer in a patch clamp device to capture and examine individual DNA molecules. This nanopore detector used with a support vector machine (SVM) can analyze DNA hairpin molecules on the millisecond time scale. We distinguish duplex stem length, base pair mismatches, loop length, and single base pair differences. The residual current fluxes also reveal structural molecular dynamics elements. DNA end-fraying (terminal base pair dissociation) can be observed as near full blockades, or spikes, in current. This technique can be used to investigate other biological processes dependent on DNA end-fraying, such as the processing of HIV DNA by HIV integrase.

Vercoutere, Wenonah↗

Up-regulation of phosphoinositide metabolism in tobacco cells constitutively expressing the human type I inositol polyphosphate 5-phosphatase

To evaluate the impact of suppressing inositol 1,4,5-trisphosphate (InsP(3)) in plants, tobacco (Nicotiana tabacum) cells were transformed with the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme which specifically hydrolyzes InsP(3). The transgenic cell lines showed a 12- to 25-fold increase in InsP 5-ptase activity in vitro and a 60% to 80% reduction in basal InsP(3) compared with wild-type cells. Stimulation with Mas-7, a synthetic analog of the wasp venom peptide mastoparan, resulted in an approximately 2-fold increase in InsP(3) in both wild-type and transgenic cells. However, even with stimulation, InsP(3) levels in the transgenic cells did not reach wild-type basal values, suggesting that InsP(3) signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)), the lipid precursor of InsP(3), was greatly reduced in the transgenic cells. In vitro assays of enzymes involved in PtdInsP(2) metabolism showed that the activity of the PtdInsP(2)-hydrolyzing enzyme phospholipase C was not significantly altered in the transgenic cells. In contrast, the activity of the plasma membrane PtdInsP 5 kinase was increased by approximately 3-fold in the transgenic cells. In vivo labeling studies revealed a greater incorporation of (32)P into PtdInsP(2) in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP(2) synthesis was increased. These studies show that the constitutive expression of the human type I InsP 5-ptase in tobacco cells leads to an up-regulation of the phosphoinositide pathway and highlight the importance of PtdInsP(2) synthesis as a regulatory step in this system.

NASA Discipline Cell Biology↗

Evidence for gammacerane as an indicator of water column stratification

A new route for the formation of gammacerane from tetrahymanol is proposed; in addition to dehydration and hydrogenation, sulphurisation and early C-S cleavage are shown to be important in the pathway of formation, especially in marine sediments. Evidence is twofold. First, relatively large amounts of the gammacerane skeleton are sequestered in S-rich macromolecular aggregates formed by natural sulphurisation of functionalised lipids. Selective cleavage of polysulphide linkages with MeLi/MeI led to formation of 3-methylthiogammacerane, indicating that the gammacerane skeleton is primarily bound via sulphur at position 3, consistent with the idea that tetrahymanol (or the corresponding ketone) is the precursor for gammacerane. Second, upon mild artificial maturation of two sediments using hydrous pyrolysis, gammacerane is released from S-rich macromolecular aggregates by cleavage of the relatively weak C-S bonds. The stable carbon isotopic compositions of gammacerane and lipids derived from primary producers and green sulphur bacteria in both the Miocene Gessoso-solfifera and Upper Jurassic Allgau Formations indicate that gammacerane is derived from bacterivorous ciliates which were partially feeding on green sulphur bacteria. This demonstrates that anaerobic ciliates living at or below the chemocline are important sources for gammacerane, consistent with the fact that ciliates only biosynthesize tetrahymanol if their diet is deprived of sterols. This leads to the conclusion that gammacerane is an indicator for water column stratification, which solves two current enigmas in gammacerane geochemistry. Firstly, it explains why gammacerane is often found in sediments deposited under hypersaline conditions but is not necessarily restricted to this type of deposits. Secondly, it explains why lacustrine deposits may contain abundant gammacerane since most lakes in the temperate climatic zones are stratified during summer.

NASA Discipline Exobiology↗

Molecular and bulk isotopic analyses of organic matter in marls of the Mulhouse Basin (Tertiary, Alsace, France)

Contents of 13C in kerogens and carbonates in 21 samples from a core of the MAX borehole, Mulhouse Evaporite Basin, range from -27.3 to -23.5 and -3.7 to -1.8% vs PDB, respectively. Organic nitrogen in the same samples is enriched in 15N relative to atmospheric N2 by 12.2-15.7%. Hydrogen indices and delta values for kerogens vary systematically with facies, averaging 493 mg HC/g Corg and -25.7% in the most saline facies (dominated by inputs from aquatic sources) and 267 mg HC/g Corg and -23.7% in the least saline facies (50/50 aquatic/terrigenous). Values of delta were measured for individual aliphatic hydrocarbons from three samples representing three different organic facies. For all samples, terrigenous inputs were unusually rich in 13C, the estimated delta value for bulk terrigenous debris, apparently derived partly from CAM plants, being -22.5%. In the most saline facies, isotopic evidence indicates the mixing of 13C-depleted products of photosynthetic bacteria with 13C-enriched products of halotolerant eukaryotic algae. At lower salinities, a change in the producer community is marked by a decrease in the 13C content of algal lipids. The content of 13C in algal lipids increases in the least saline facies, due either to succession of different organisms or to decreased concentrations of dissolved CO2.

NASA Discipline Exobiology↗

Simulated conditions of microgravity suppress progesterone production by luteal cells of the pregnant rat

The purpose of this study was to assess whether simulated conditions of microgravity induce changes in the production of progesterone by luteal cells of the pregnant rat ovary using an in vitro model system. The microgravity environment was simulated using either a high aspect ratio vessel (HARV) bioreactor with free fall or a clinostat without free fall of cells. A mixed population of luteal cells isolated from the corpora lutea of day 8 pregnant rats was attached to cytodex microcarrier beads (cytodex 3). These anchorage dependent cells were placed in equal numbers in the HARV or a spinner flask control vessel in culture conditions. It was found that HARV significantly reduced the daily production of progesterone from day 1 through day 8 compared to controls. Scanning electron microscopy showed that cells attached to the microcarrier beads throughout the duration of the experiment in both types of culture vessels. Cells cultured in chamber slide flasks and placed in a clinostat yielded similar results when compared to those in the HARV. Also, when they were stained by Oil Red-O for lipid droplets, the clinostat flasks showed a larger number of stained cells compared to control flasks at 48 h. Further, the relative amount of Oil Red-O staining per milligram of protein was found to be higher in the clinostat than in the control cells at 48 h. It is speculated that the increase in the level of lipid content in cells subjected to simulated conditions of microgravity may be due to a disruption in cholesterol transport and/or lesions in the steroidogenic pathway leading to a fall in the synthesis of progesterone. Additionally, the fall in progesterone in simulated conditions of microgravity could be due to apoptosis of luteal cells.

Progesterone/metabolism↗

Biomass of the cryptoendolithic microbiota from the Antarctic desert

Extractable lipid phosphate was used to determine the biomass of the cryptoendolithic microbiota that colonizes sandstone rocks in the Ross Desert region of Antarctica. The mean amount of lipid phosphate was 0.053 micromole/cm2 (n = 9), which equals 2.54 g of C per m2 (range, 1.92 to 3.26 g of C per m2) of biomass in the biotic zone of these rocks. The turnover of phospholipids was comparable to that of temperate sediments and soils (t1/2, 6 to 9 days) at 0 degrees C and a light intensity of 305 micromoles of photons per m2 per s, indicating that this was a good method to measure viable biomass. The biomass was 0.3 to 9.6% of the total carbon content of the biotic zone and was about 2 orders of magnitude smaller than the epilithic lichen dry weight at a location some 7 degrees north in latitude. The cryptoendolithic microbiota had a uniform density throughout the biotic zone under the rock surface. The results indicate that the cryptoendolithic microbial biomass is small but viable in this unique, extreme ecosystem.

NASA Discipline Exobiology↗

Novel effects of nitric oxide

Nitric oxide (NO), a simple free radical gas, elicits a surprisingly wide range of physiological and pathophysiological effects. NO interacts with soluble guanylate cyclase to evoke many of these effects. However, NO can also interact with molecular oxygen and superoxide radicals to produce reactive nitrogen species that can modify a number of macromolecules including proteins, lipids, and nucleic acids. NO can also interact directly with transition metals. Here, we have reviewed the non--3',5'-cyclic-guanosine-monophosphate-mediated effects of NO including modifications of proteins, lipids, and nucleic acids.

Non-NASA Center↗

Metabolic myopathies

Metabolic myopathies are disorders of muscle energy production that result in skeletal muscle dysfunction. Cardiac and systemic metabolic dysfunction may coexist. Symptoms are often intermittent and provoked by exercise or changes in supply of lipid and carbohydrate fuels. Specific disorders of lipid and carbohydrate metabolism in muscle are reviewed. Evaluation often requires provocative exercise testing. These tests may include ischemic forearm exercise, aerobic cycle exercise, and 31P magnetic resonance spectroscopy with exercise.

Review↗

Computer Modeling of Protocellular Functions: Peptide Insertion in Membranes

Lipid vesicles became the precursors to protocells by acquiring the capabilities needed to survive and reproduce. These include transport of ions, nutrients and waste products across cell walls and capture of energy and its conversion into a chemically usable form. In modem organisms these functions are carried out by membrane-bound proteins (about 30% of the genome codes for this kind of proteins). A number of properties of alpha-helical peptides suggest that their associations are excellent candidates for protobiological precursors of proteins. In particular, some simple a-helical peptides can aggregate spontaneously and form functional channels. This process can be described conceptually by a three-step thermodynamic cycle: 1 - folding of helices at the water-membrane interface, 2 - helix insertion into the lipid bilayer and 3 - specific interactions of these helices that result in functional tertiary structures. Although a crucial step, helix insertion has not been adequately studied because of the insolubility and aggregation of hydrophobic peptides. In this work, we use computer simulation methods (Molecular Dynamics) to characterize the energetics of helix insertion and we discuss its importance in an evolutionary context. Specifically, helices could self-assemble only if their interactions were sufficiently strong to compensate the unfavorable Free Energy of insertion of individual helices into membranes, providing a selection mechanism for protobiological evolution.

Rodriquez-Gomez, D.↗

Progress in Development of Improved Ion-Channel Biosensors

Further improvements have recently been made in the development of the devices described in Improved Ion-Channel Biosensors (NPO-30710), NASA Tech Briefs, Vol. 28, No. 10 (October 2004), page 30. As discussed in more detail in that article, these sensors offer advantages of greater stability, greater lifetime, and individual electrical addressability, relative to prior ion-channel biosensors. In order to give meaning to a brief description of the recent improvements, it is necessary to recapitulate a substantial portion of the text of the cited previous article. The figure depicts one sensor that incorporates the recent improvements, and can be helpful in understanding the recapitulated text, which follows: These sensors are microfabricated from silicon and other materials compatible with silicon. Typically, the sensors are fabricated in arrays in silicon wafers on glass plates. Each sensor in the array can be individually electrically addressed, without interference with its neighbors. Each sensor includes a well covered by a thin layer of silicon nitride, in which is made a pinhole for the formation of a lipid bilayer membrane. In one stage of fabrication, the lower half of the well is filled with agarose, which is allowed to harden. Then the upper half of the well is filled with a liquid electrolyte (which thereafter remains liquid) and a lipid bilayer is painted over the pinhole. The liquid contains a protein that forms an ion channel on top of the hardened agarose. The combination of enclosure in the well and support by the hardened agarose provides the stability needed to keep the membrane functional for times as long as days or even weeks. An electrode above the well, another electrode below the well, and all the materials between the electrodes together constitute a capacitor. What is measured is the capacitive transient current in response to an applied voltage pulse. One notable feature of this sensor, in comparison with prior such sensors, is a relatively thick dielectric layer between the top of the well and the top electrode. This layer greatly reduces the capacitance of an aperture across which the ion channels are formed, thereby increasing the signal-to-noise ratio. The use of a relatively large aperture with agarose support makes it possible to form many ion channels instead of only one, thereby further increasing the signal-to-noise ratio and effectively increasing the size of the available ionic reservoir. The relatively large reservoir makes it possible to measure AC rather than DC. This concludes the recapitulation from the cited previous article.

Nadeau, Jay L.↗

Diagnosis and Treatment of Neurological Disorders by Millimeter-Wave Stimulation

Increasingly, millimeter waves are being employed for telecomm, radar, and imaging applications. To date in the U.S, however, very few investigations on the impact of this radiation on biological systems at the cellular level have been undertaken. In the beginning, to examine the impact of millimeter waves on cellular processes, researchers discovered that cell membrane depolarization may be triggered by low levels of integrated power at these high frequencies. Such a situation could be used to advantage in the direct stimulation of neuronal cells for applications in neuroprosthetics and diagnosing or treating neurological disorders. An experimental system was set up to directly monitor cell response on exposure to continuous-wave, fixed-frequency, millimeter-wave radiation at low and modest power levels (0.1 to 100 safe exposure standards) between 50 and 100 GHz. Two immortalized cell lines derived from lung and neuronal tissue were transfected with green fluorescent protein (GFP) that locates on the inside of the cell membrane lipid bi-layer. Oxonol dye was added to the cell medium. When membrane depolarization occurs, the oxonal bound to the outer wall of the lipid bi-layer can penetrate close to the inner wall where the GFP resides. Under fluorescent excitation (488 nm), the normally green GFP (520 nm) optical signal quenches and gives rise to a red output when the oxonol comes close enough to the GFP to excite a fluorescence resonance energy transfer (FRET) with an output at 620 nm. The presence of a strong FRET signature upon exposures of 30 seconds to 2 minutes at 5-10 milliwatts per square centimeter RF power at 50 GHz, followed by a return to the normal 520-nm GFP signal after a few minutes indicating repolarization of the membrane, indicates that low levels of RF energy may be able to trigger non-destructive membrane depolarization without direct cell contact. Such a mechanism could be used to stimulate neuronal cells in the cortex without the need for invasive electrodes as millimeter waves penetrate skin and bone on the order of 15 mm in depth. Although 50 GHz could not readily penetrate from the outer skull to the center of the cortex, implants on the outer skull or even on the scalp could reach the outer layer of the cerebral cortex where substantial benefit could be realized from such non-contact type excitation.

Siegel, Peter H.↗

Deployment of an Advanced Electrocardiographic Analysis (A-ECG) to Detect Cardiovascular Risk in Career Firefighters

INTRODUCTION: Sudden cardiac death is the leading cause of line of duty death among firefighters, accounting for approximately 45% of fatalities annually. Firefighters perform strenuous muscular work while wearing heavy, encapsulating personal protective equipment in high ambient temperatures, under chaotic and emotionally stressful conditions. These factors can precipitate sudden cardiac events like myocardial infarction, serious dysrhythmias, or cerebrovascular accidents in firefighters with underlying cardiovascular disease. PURPOSE: The purpose of this study was to deploy and then evaluate the contribution of resting advanced ECG (A-ECG) in addition to other screening tools (family history, lipid profiles, and cardiopulmonary exercise tests, XT) in assessment of an individual fs cardiac risk profile. METHODS: Forty-four career firefighters were recruited to perform comprehensive baseline assessments including tests of aerobic performance, fasting lipids and glucose. Five-min resting 12-lead A-ECGs were obtained in a subset of firefighters (n=21) and transmitted over a secure networked system to a NASA physician collaborator. Using myocardial perfusion and other imaging as the gold standard, A-ECG scoring has been proven useful in accurately identifying a number of cardiac pathologies including coronary artery disease (CAD), left ventricular hypertrophy, hypertrophic cardiomyopathy, and non-ischemic and ischemic cardiomyopathy. RESULTS: Subjects f mean (SD) age was 43 (8) years, weight 91 (13) kg, and BMI 28 (3) kg/m2. Fifty-one percent of subjects had .3 cardiovascular risk factors. One subject had ST depression on XT ECG, at least one positive A-ECG score for CAD, and documented CAD based on cardiology referral. While all other subjects, including those with fewer risk factors, higher aerobic fitness, and normal exercise ECGs, were classified as healthy by A-ECG, there was no trend for association between risk factors and any of 20 A-ECG parameters in the grouped data.

Dolezal, B. A.↗

Microorganism Utilization for Synthetic Milk Production

A desired architecture for long duration spaceflight, such as aboard the International Space Station (ISS) or for future missions to Mars, is to provide a supply of fresh food crops for the astronauts. However, some crops can create a high proportion of inedible plant waste. The main goal of this project was to produce the components of milk (sugar, lipid, protein) from inedible plant waste by utilizing microorganisms (fungi, yeast, bacteria). Of particular interest was utilizing the valuable polysaccharide, cellulose, found in plant waste, to naturally fuel- through microorganism cellular metabolism- the creation of sugar (glucose), lipid (milk fat), and protein (casein) to produce a synthetic edible food product. Environmental conditions such as pH, temperature, carbon source, aeration, and choice microorganisms.

Microorganism↗

The Effect of Fatty Acids to Protect Forward Osmosis Membranes from Damage

NASA has conducted research and development on forward osmosis (FO) membranes for wastewater reclamation in space since 1993. The lessons learned during operation of the International Space Station and FO based technologies on the ground taught us that reliability is a key limitation. Membranes are susceptible to organic fouling, oxidation and calcium scaling, and these factors tend to damage the membrane reducing their operating life and performance. The development of a Synthetic Biological Membrane (SBM), a membrane that mimics naturally occurring biological processes, will mitigate membrane damage and improve reliability. The SBM is a lipid-based membrane with a protective fatty acid layer configured for use in a FO water purification system. In this configuration, the protective layer on the surface of the lipid membrane is composed of fatty acids (FA). The FA interact with the chemicals found in the wastewater feed, and protect the membrane from damage. In this study, we conducted preliminary experiments to determine the feasibility of using fatty acids to alleviate damage from calcium scaling, oxidation and organic fouling.

Romero Mangado, Jaione↗

Searching for Evidence of Life in Deep Time and Space

Cyanobacterial mats provide insights into ancient benthic microbial communities and their biosignatures. Thick mats occupy hypersaline saltern ponds at Guerrero Negro, Baja California, Mexico. Mat biota maintains rapid rates of biogeochemical processes under steep and rapidly changing environmental gradients. Cycling of C, O, and S all increased identically with temperature, indicating the tight coupling of these cycles. An enormous microbial diversity exhibits a highly structured spatial distribution of populations. Combined universal clone libraries from all mat layers indicated Bacteria/Archaea/Eukarya ratios of 57:7:1. More than 10,000 unique bacterial sequences were present. The relative abundance of Archaea increased with depth - below 10 cm, solvent-extractable archaeal lipids were twice as abundant as bacterial lipids. Only 15 species of Eukarya were found among 890 clones analyzed. Degradation of the mats’ insoluble macromolecular organic fraction (IMOM) by hydropyrolysis released a complex variety of linear, branched and polycyclic alkane structures, e.g., hopanes, methylhopanes and steranes. Covalent binding of these biosignatures into IMOM aids their long-term geological preservation. Mars rover missions revealed evidence of long-lived fluvial lacustrine systems and organics in associated mudstones. NASA’s Mars 2020 rover mission will examine sediments in Jezero crater, including a delta and shoreline carbonate deposits, environments that on Earth have sustained microbial mats.

Des Marais, David J.↗

Nutrient Stability in NASA Spaceflight Experiment Rodent Food Bars

Nutrient-upgraded Rodent Food Bars are the standard diet for mice involved in spaceflight experiments aboard the International Space Station as part of NASA’s Rodent Research Project. These Food Bars are a twin-screw extruded AIN-93G based product with nutrient additives to preserve a healthy and complete diet for the animals, and the bar form factor is necessary for use in the microgravity environment. Due to the nature of NASA flight projects and the lengthy production process of these Food Bars, a shelf-life assessment was undertaken to determine nutrient stability over an extended storage time. To assess palatability and stability over an extended duration, a panel of lipid oxidation markers and both fat- and water-soluble vitamins were assessed under various time and temperature conditions via AOAC International standardized methodology. Thiamine was the least stable vitamin at room temperature. Bars that had been continually kept at 4C (t=6, 12, 27 months) showed significantly higher levels of thiamine (via Student’s t-test) compared to all other bars that underwent some degree of ambient temperature storage. This suggests that refrigeration is a key component in maintaining adequate nutrient levels in these relatively high moisture (average 29.3%) food bars. Also of note was the level of Vitamin A in the form of retinol. There was no correlation to storage temperature, but rather the only factor having an effect on retinol levels was time. As low, yet consistent decrease in retinol was observed over the course of the experiment, though at no time did the retinol levels drop below the rodent Nutrient Requirements for Laboratory Animals (NRC). In conclusion, over the 27-month time course, all analyzed vitamins and lipid oxidation factors remained relatively stable and at levels which provide fully sufficient nutrition to rodents during spaceflight. Some groups however, displayed higher-than-expected aerobic and anaerobic bacteria colony forming units, which is believed to stem from a since-revised handling technique.

Food↗