Factors limiting the duration of artificially induced torpor in mice.

The possibility of artificial induction of a torpid state in animals that do not naturally do so, as well as in humans, offers a great potential in biomedicine and in human spaceflight. However, the mechanisms of action that provide a coordinated and concomitant downregulation with a safe recovery from this state are poorly understood. In our previous study, we demonstrated that the metabolic rate of mice can be reduced by nearly 94% and can remain stable under hypothermic conditions for a prolonged period of up to 11 h. The present study was carried out in order to test the limitations and identify potential factors that can enable the safe and reversible arousal of non-hibernating mice from deep artificially-induced torpor to an active state. Results demonstrate that the energy budget may be a limiting factor for the prolongation and safe recovery from the hypometabolic state. While the continuation of torpor may be possible for additional hours, we found that a reduction of 40% or more in the plasma glucose level increases the risk of heart fibrillation, which results in death during arousal. Therefore, the plasma glucose level could be a component of the criteria indicating the reversibility of torpor. Another important factor complementing the energetic necessity that may limit the duration of torpor in mice is a gradual reduction in body mass during torpor. Under the conditions of our experiment, body mass declines by nearly 15% after 16 h from the initiation of torpor and may continue to decline if the mice are allowed to remain in torpor longer. Extrapolation of this data suggests that there may be a critical mass relating to animal mortality and thus limiting the duration of torpor. Control and maintenance of the body mass and glucose level in a torpid animal may extend the longevity of torpor and mitigate the risk of cardiac failure during rewarming to the metabolically active state. The cardiac complications that occur during arousal from torpor in many cases could be mitigated and even avoided by applying appropriate temperature-arising kinetics and providing a sufficient dynamic range to maintain cardiac output.

Multi-analyte biochip (MAB) based on all-solid-state ion-selective electrodes (ASSISE) for physiological research.

Lab-on-a-chip (LOC) applications in environmental, biomedical, agricultural, biological, and spaceflight research require an ion-selective electrode (ISE) that can withstand prolonged storage in complex biological media (1-4). An all-solid-state ion-selective-electrode (ASSISE) is especially attractive for the aforementioned applications. The electrode should have the following favorable characteristics: easy construction, low maintenance, and (potential for) miniaturization, allowing for batch processing. A microfabricated ASSISE intended for quantifying H(+), Ca(2+), and CO3(2-) ions was constructed. It consists of a noble-metal electrode layer (i.e. Pt), a transduction layer, and an ion-selective membrane (ISM) layer. The transduction layer functions to transduce the concentration-dependent chemical potential of the ion-selective membrane into a measurable electrical signal. The lifetime of an ASSISE is found to depend on maintaining the potential at the conductive layer/membrane interface (5-7). To extend the ASSISE working lifetime and thereby maintain stable potentials at the interfacial layers, we utilized the conductive polymer (CP) poly(3,4-ethylenedioxythiophene) (PEDOT) (7-9) in place of silver/silver chloride (Ag/AgCl) as the transducer layer. We constructed the ASSISE in a lab-on-a-chip format, which we called the multi-analyte biochip (MAB) (Figure 1). Calibrations in test solutions demonstrated that the MAB can monitor pH (operational range pH 4-9), CO3(2-) (measured range 0.01 mM - 1 mM), and Ca(2+) (log-linear range 0.01 mM to 1 mM). The MAB for pH provides a near-Nernstian slope response after almost one month storage in algal medium. The carbonate biochips show a potentiometric profile similar to that of a conventional ion-selective electrode. Physiological measurements were employed to monitor biological activity of the model system, the microalga Chlorella vulgaris. The MAB conveys an advantage in size, versatility, and multiplexed analyte sensing capability, making it applicable to many confined monitoring situations, on Earth or in space. Biochip Design and Experimental Methods The biochip is 10 x 11 mm in dimension and has 9 ASSISEs designated as working electrodes (WEs) and 5 Ag/AgCl reference electrodes (REs). Each working electrode (WE) is 240 µm in diameter and is equally spaced at 1.4 mm from the REs, which are 480 µm in diameter. These electrodes are connected to electrical contact pads with a dimension of 0.5 mm x 0.5 mm. The schematic is shown in Figure 2. Cyclic voltammetry (CV) and galvanostatic deposition methods are used to electropolymerize the PEDOT films using a Bioanalytical Systems Inc. (BASI) C3 cell stand (Figure 3). The counter-ion for the PEDOT film is tailored to suit the analyte ion of interest. A PEDOT with poly(styrenesulfonate) counter ion (PEDOT/PSS) is utilized for H(+) and CO3(2-), while one with sulphate (added to the solution as CaSO4) is utilized for Ca(2+). The electrochemical properties of the PEDOT-coated WE is analyzed using CVs in redox-active solution (i.e. 2 mM potassium ferricyanide (K3Fe(CN)6)). Based on the CV profile, Randles-Sevcik analysis was used to determine the effective surface area (10). Spin-coating at 1,500 rpm is used to cast ~2 µm thick ion-selective membranes (ISMs) on the MAB working electrodes (WEs). The MAB is contained in a microfluidic flow-cell chamber filled with a 150 µl volume of algal medium; the contact pads are electrically connected to the BASI system (Figure 4). The photosynthetic activity of Chlorella vulgaris is monitored in ambient light and dark conditions.

Genomic response of the nematode Caenorhabditis elegans to spaceflight.

On Earth, it is common to employ laboratory animals such as the nematode Caenorhabditis elegans to help understand human health concerns. Similar studies in Earth orbit should help understand and address the concerns associated with spaceflight. The "International Caenorhabditis elegans Experiment FIRST" (ICE FIRST), was carried out onboard the Dutch Taxiflight in April of 2004 by an international collaboration of laboratories in France, Canada, Japan and the United States. With the exception of a slight movement defect upon return to Earth, the result of altered muscle development, no significant abnormalities were detected in spaceflown C. elegans. Work from Japan revealed apoptosis proceeds normally and work from Canada revealed no significant increase in the rate of mutation. These results suggest that C. elegans can be used to study non-lethal responses to spaceflight and can possibly be developed as a biological sensor. To further our understanding of C. elegans response to spaceflight, we examined the gene transcription response to the 10 days in space using a near full genome microarray analysis. The transcriptional response is consistent with the observed normal developmental timing, apoptosis, DNA repair, and altered muscle development. The genes identified as altered in response to spaceflight are enriched for genes known to be regulated, in C. elegans, in response to altered environmental conditions (Insulin and TGF-beta regulated). These results demonstrate C. elegans can be used to study the effects of altered gravity and suggest that C. elegans responds to spaceflight by altering the expression of at least some of the same metabolic genes that are altered in response to differing terrestrial environments.