Monitoring functional immune responses with a cytokine release assay: ISS flight hardware design and experimental protocol for whole blood cultures executed under microgravity conditions.

Introduction: Long-term space missions trigger a prolonged neuroendocrine stress response leading to immune system dysregulation evidenced by susceptibility to infections, viral reactivation, and skin irritations. However, due to existing technical constraints, real-time functional immune assessments are not currently available to crew inflight. The in vitro cytokine release assay (CRA) has been effectively employed to study the stimulated cytokine response of immune cells in whole blood albeit limited to pre- and post-flight sessions. A novel two-valve reaction tube (RT) has been developed to enable the execution of the CRA on the International Space Station (ISS). Methods: In a comprehensive test campaign, we assessed the suitability of three materials (silicone, C-Flex, and PVC) for the RT design in terms of biochemical compatibility, chemical stability, and final data quality analysis. Furthermore, we thoroughly examined additional quality criteria such as safety, handling, and the frozen storage of antigens within the RTs. The validation of the proposed crew procedure was conducted during a parabolic flight campaign. Results: The selected material and procedure proved to be both feasible and secure yielding consistent and dependable data outcomes. This new hardware allows for the stimulation of blood samples on board the ISS, with subsequent analysis still conducted on the ground. Discussion: The resultant data promises to offer a more accurate understanding of the stress-induced neuroendocrine modulation of immunity during space travel providing valuable insights for the scientific community. Furthermore, the versatile nature of the RT suggests its potential utility as a testing platform for various other assays or sample types.

European space agency's hibernation (torpor) strategy for deep space missions: Linking biology to engineering.

Long-duration space missions to Mars will impose extreme stresses of physical and psychological nature on the crew, as well as significant logistical and technical challenges for life support and transportation. Main challenges include optimising overall mass and maintaining crew physical and mental health. These key scopes have been taken up as the baseline for a study by the European Space Agency (ESA) using its Concurrent Design Facility (CDF). It focussed on the biology of hibernation in reducing metabolism and hence stress, and its links to the infrastructure and life support. We concluded that torpor of crew members can reduce the payload with respect to oxygen, food and water but will require monitoring and artificial intelligence (AI) assisted monitoring of the crew. These studies additionally offer new potential applications for patient care on Earth. Keywords: Space flight, concurrent design facility, metabolic reduction.

'White Mars' - nearly two decades of biomedical research at the Antarctic Concordia station.

NEW FINDINGS: What is the topic of this review? Biomedical research at the Antarctic Concordia Station. What advances does it highlight? Overview of findings in psychology, neuroscience, sleep, cardiovascular physiology and immune system, relevant in isolated, confined and extreme environments and spaceflight. ABSTRACT: Extended stays in isolated, confined and extreme (ICE) environments like Antarctica are associated with a whole set of psychological and physiological challenges for the crew. As such, winter-over stays at Antarctica provide an important opportunity to acquire knowledge into the physiological and psychological changes that ICE environments inevitably bring. The European Space Agency (ESA) is particularly interested in conducting research in such an environment, as it is a unique opportunity to translate these results to space crews experiencing very similar issues. In the past two decades, the ESA has supported a total of 36 biomedical research projects at the Concordia station in collaboration with the French and Italian polar institutes. More specifically, studies in the areas of psychology, neuroscience, sleep physiology, cardiovascular physiology and immunology were performed. The outcomes of these studies are directly relevant for people working in ICE environments, but also help to better understand the biomedical challenges of those environments. Consequently, they can help to better prepare for human space exploration and to identify countermeasures to minimize the adverse effects of space environments on astronaut health. The aim of this review is to provide an overview of the biomedical studies that have taken place in the past two decades at the Antarctic Concordia station and to summarize the results and their implication for human spaceflight.

Barium titanate nanoparticles and hypergravity stimulation improve differentiation of mesenchymal stem cells into osteoblasts.

BACKGROUND: Enhancement of the osteogenic potential of mesenchymal stem cells (MSCs) is highly desirable in the field of bone regeneration. This paper proposes a new approach for the improvement of osteogenesis combining hypergravity with osteoinductive nanoparticles (NPs). MATERIALS AND METHODS: In this study, we aimed to investigate the combined effects of hypergravity and barium titanate NPs (BTNPs) on the osteogenic differentiation of rat MSCs, and the hypergravity effects on NP internalization. To obtain the hypergravity condition, we used a large-diameter centrifuge in the presence of a BTNP-doped culture medium. We analyzed cell morphology and NP internalization with immunofluorescent staining and coherent anti-Stokes Raman scattering, respectively. Moreover, cell differentiation was evaluated both at the gene level with quantitative real-time reverse-transcription polymerase chain reaction and at the protein level with Western blotting. RESULTS: Following a 20 g treatment, we found alterations in cytoskeleton conformation, cellular shape and morphology, as well as a significant increment of expression of osteoblastic markers both at the gene and protein levels, jointly pointing to a substantial increment of NP uptake. Taken together, our findings suggest a synergistic effect of hypergravity and BTNPs in the enhancement of the osteogenic differentiation of MSCs. CONCLUSION: The obtained results could become useful in the design of new approaches in bone-tissue engineering, as well as for in vitro drug-delivery strategies where an increment of nanocarrier internalization could result in a higher drug uptake by cell and/or tissue constructs.

Small-conductance Ca2+-activated K+ channel type 2 (SK2) modulates hippocampal learning, memory, and synaptic plasticity.

Apamin-sensitive, small-conductance, Ca2+-activated K+ channels (SK channels) modulate neuronal excitability in CA1 neurons. Blocking all SK channel subtypes with apamin facilitates the induction of hippocampal synaptic plasticity and enhances hippocampal learning. In CA1 dendrites, SK channels are activated by Ca2+ through NMDA receptors and restrict glutamate-mediated EPSPs. Studies of SK channel knock-out mice reveal that of the three apamin-sensitive SK channel subunits (SK1-SK3), only SK2 subunits are necessary for the apamin-sensitive currents in CA1 hippocampal neurons. To determine the specific influence of SK2 channels on hippocampal synaptic plasticity, learning, and memory, we used gene targeting through homologous recombination in embryonic stem cells to generate transgenic mice that overexpress SK2 subunits by 10-fold (SK2+/T). In these mice, the apamin-sensitive current in CA1 neurons was increased by approximately fourfold, relative to wild-type (WT) littermates. In addition, the amplitude of synaptically evoked EPSPs recorded from SK2+/T CA1 neurons increased twice as much in response to SK channel blockade relative to EPSPs recorded from WT CA1 neurons. Consistent with this, SK2 overexpression reduced long-term potentiation after high-frequency stimulation compared with WT littermates and severely impaired learning in both hippocampus- and amygdala-dependent tasks. We conclude that SK2 channels regulate hippocampal synaptic plasticity and play a critical role in modulating mechanisms of learning and memory.

SK channels and NMDA receptors form a Ca2+-mediated feedback loop in dendritic spines.

Small-conductance Ca(2+)-activated K(+) channels (SK channels) influence the induction of synaptic plasticity at hippocampal CA3-CA1 synapses. We find that in mice, SK channels are localized to dendritic spines, and their activity reduces the amplitude of evoked synaptic potentials in an NMDA receptor (NMDAR)-dependent manner. Using combined two-photon laser scanning microscopy and two-photon laser uncaging of glutamate, we show that SK channels regulate NMDAR-dependent Ca(2+) influx within individual spines. SK channels are tightly coupled to synaptically activated Ca(2+) sources, and their activity reduces the amplitude of NMDAR-dependent Ca(2+) transients. These effects are mediated by a feedback loop within the spine head; during an excitatory postsynaptic potential (EPSP), Ca(2+) influx opens SK channels that provide a local shunting current to reduce the EPSP and promote rapid Mg(2+) block of the NMDAR. Thus, blocking SK channels facilitates the induction of long-term potentiation by enhancing NMDAR-dependent Ca(2+) signals within dendritic spines.

Small conductance Ca2+-activated K+ channels and calmodulin: cell surface expression and gating.

Small conductance Ca2+-activated K+ channels (SK channels) are heteromeric complexes of pore-forming alpha subunits and constitutively bound calmodulin (CaM). The binding of CaM is mediated in part by the electrostatic interaction between residues Arg-464 and Lys-467 of SK2 and Glu-84 and Glu-87 of CaM. Heterologous expression of the double charge reversal in SK2, SK2 R464E/K467E (SK2:64/67), did not yield detectable surface expression or channel activity in whole cell or inside-out patch recordings. Coexpression of SK2:64/67 with wild type CaM or CaM1,2,3,4, a mutant lacking the ability to bind Ca2+, rescued surface expression. In patches from cells coexpressing SK2:64/67 and wild type CaM, currents were recorded immediately following excision into Ca2+-containing solution but disappeared within minutes after excision or immediately upon exposure to Ca2+-free solution and were not reactivated upon reapplication of Ca2+-containing solution. Channel activity was restored by application of purified recombinant Ca2+-CaM or exposure to Ca2+-free CaM followed by application of Ca2+-containing solution. Coexpression of the double charge reversal E84R/E87K in CaM (CaM:84/87) with SK2:64/67 reconstituted stable Ca2+-dependent channel activity that was not lost with exposure to Ca2+-free solution. Therefore, Ca2+-independent interactions with CaM are required for surface expression of SK channels, whereas the constitutive association between the two channel subunits is not an essential requirement for gating.