Hydrogel Formulation for Biomimetic Fibroblast Cell Culture: Exploring Effects of External Stresses and Cellular Responses.

In the process of tissue engineering, several types of stresses can influence the outcome of tissue regeneration. This outcome can be understood by designing hydrogels that mimic this process and studying how such hydrogel scaffolds and cells behave under a set of stresses. Here, a hydrogel formulation is proposed to create biomimetic scaffolds suitable for fibroblast cell culture. Subsequently, we examine the impact of external stresses on fibroblast cells cultured on both solid and porous hydrogels. These stresses included mechanical tension and altered-gravity conditions experienced during the 83rd parabolic flight campaign conducted by the European Space Agency. This study shows distinct cellular responses characterized by cell aggregation and redistribution in regions of intensified stress concentration. This paper presents a new biomimetic hydrogel that fulfills tissue-engineering requirements in terms of biocompatibility and mechanical stability. Moreover, it contributes to our comprehension of cellular biomechanics under diverse gravitational conditions, shedding light on the dynamic cellular adaptations versus varying stress environments.

Under pressure-the influence of hypergravity on electrocortical activity and neurocognitive performance.

The effects of hypergravity and the associated increased pressure on the human body have not yet been studied in detail, but are of great importance for the safety of astronauts on space missions and could have a long-term impact on rehabilitation strategies for neurological patients. Considering the plans of international space agencies with the exploration of Mars and Moon, it is important to explore the effects of both extremes, weightlessness and hypergravity. During parabolic flights, a flight manoeuvre that artificially creates weightlessness and hypergravity, electrocortical activity as well as behavioural parameters (error rate and reaction time) and neuronal parameters (event-related potentials P300 and N200) were examined with an electroencephalogram. Thirteen participants solved a neurocognitive task (mental arithmetic task as a primary task and oddball paradigm as a secondary task) within normal as well as hypergravity condition in fifteen consecutive parabolas for 22 s each. No changes between the different gravity levels could be observed for the behavioural parameters and cortical current density. A significantly lower P300 amplitude was observed in 1 G, triggered by the primary task and the target sound of the oddball paradigm. The N200, provoked by the sounds of the oddball paradigm, revealed a higher amplitude in 1.8 G. A model established by Kohn et al. (2018) describing changes in neural communication with decreasing gravity can be used here as an explanatory approach. The fluid shift increases the intracranial pressure, decreases membrane viscosity and influences the open state probability of ion channels. This leads to an increase in the resting membrane potential, and the threshold for triggering an action potential can be reached more easily. The question now arises whether the observed changes are linear or whether they depend on a specific threshold.

Motor imagery helps updating internal models during microgravity exposure.

Skilled movements result from a mixture of feedforward and feedback mechanisms conceptualized by internal models. These mechanisms subserve both motor execution and motor imagery. Current research suggests that imagery allows updating feedforward mechanisms, leading to better performance in familiar contexts. Does this still hold in radically new contexts? Here, we test this ability by asking participants to imagine swinging arm movements around shoulder in normal gravity condition and in microgravity in which studies showed that movements slow down. We timed several cycles of actual and imagined arm pendular movements in three groups of subjects during parabolic flight campaign. The first, control, group remained on the ground. The second group was exposed to microgravity but did not imagine movements inflight. The third group was exposed to microgravity and imagined movements inflight. All groups performed and imagined the movements before and after the flight. We predicted that a mere exposure to microgravity would induce changes in imagined movement duration. We found this held true for the group who imagined the movements, suggesting an update of internal representations of gravity. However, we did not find a similar effect in the group exposed to microgravity despite the fact that the participants lived the same gravitational variations as the first group. Overall, these results suggest that motor imagery contributes to update internal representations of the considered movement in unfamiliar environments, while a mere exposure proved to be insufficient.NEW & NOTEWORTHY Gravity strongly affects the way movements are performed. How internal models process this information to adapt behavior to novel contexts is still unknown. The microgravity environment itself does not provide enough information to optimally adjust the period of natural arm swinging movements to microgravity. However, motor imagery of the task while immersed in microgravity was sufficient to update internal models. These results show that actually executing a task is not necessary to update graviception.

Rapid Downregulation of H3K4me3 Binding to Immunoregulatory Genes in Altered Gravity in Primary Human M1 Macrophages.

The sensitivity of human immune system cells to gravity changes has been investigated in numerous studies. Human macrophages mediate innate and thus rapid immune defense on the one hand and activate T- and B-cell-based adaptive immune response on the other hand. In this process they finally act as immunoeffector cells, and are essential for tissue regeneration and remodeling. Recently, we demonstrated in the human Jurkat T cell line that genes are differentially regulated in cluster structures under altered gravity. In order to study an in vivo near system of immunologically relevant human cells under physically real microgravity, we performed parabolic flight experiments with primary human M1 macrophages under highly standardized conditions and performed chromatin immunoprecipitation DNA sequencing (ChIP-Seq) for whole-genome epigenetic detection of the DNA-binding loci of the main transcription complex RNA polymerase II and the transcription-associated epigenetic chromatin modification H3K4me3. We identified an overall downregulation of H3K4me3 binding loci in altered gravity, which were unequally distributed inter- and intrachromosomally throughout the genome. Three-quarters of all affected loci were located on the p arm of the chromosomes chr5, chr6, chr9, and chr19. The genomic distribution of the downregulated H3K4me3 loci corresponds to a substantial extent to immunoregulatory genes. In microgravity, analysis of RNA polymerase II binding showed increased binding to multiple loci at coding sequences but decreased binding to central noncoding regions. Detection of altered DNA binding of RNA polymerase II provided direct evidence that gravity changes can lead to altered transcription. Based on this study, we hypothesize that the rapid transcriptional response to changing gravitational forces is specifically encoded in the epigenetic organization of chromatin.

In Prostate Cancer Cells Cytokines Are Early Responders to Gravitational Changes Occurring in Parabolic Flights.

The high mortality in men with metastatic prostate cancer (PC) establishes the need for diagnostic optimization by new biomarkers. Mindful of the effect of real microgravity on metabolic pathways of carcinogenesis, we attended a parabolic flight (PF) mission to perform an experiment with the PC cell line PC-3, and submitted the resulting RNA to next generation sequencing (NGS) and quantitative real-time PCR (qPCR). After the first parabola, alterations of the F-actin cytoskeleton-like stress fibers and pseudopodia are visible. Moreover, numerous significant transcriptional changes are evident. We were able to identify a network of relevant PC cytokines and chemokines showing differential expression due to gravitational changes, particularly during the early flight phases. Together with differentially expressed regulatory lncRNAs and micro RNAs, we present a portfolio of 298 potential biomarkers. Via qPCR we identified IL6 and PIK3CB to be sensitive to vibration effects and hypergravity, respectively. Per NGS we detected five upregulated cytokines (CCL2, CXCL1, IL6, CXCL2, CCL20), one zink finger protein (TNFAIP3) and one glycoprotein (ICAM1) related to c-REL signaling and thus relevant for carcinogenesis as well as inflammatory aspects. We found regulated miR-221 and the co-localized lncRNA MIR222HG induced by PF maneuvers. miR-221 is related to the PC-3 growth rate and MIR222HG is a known risk factor for glioma susceptibility. These findings in real microgravity may further improve our understanding of PC and contribute to the development of new diagnostic tools.

The effect of hypergravity on upright balance and voluntary sway.

We compared voluntary oscillatory sway for eight subjects tested in 1.8-g and 1-g gravito-inertial force (GIF) levels of parabolic flight. Subjects performed voluntary forward-backward (FB) and lateral left-right (LR) swaying as the forces and moments under the soles of each foot were measured. We calculated the experimental values of three parameters: two ankle stiffness parameters KSAP and KSML acting in orthogonal FB and LR directions and one parameter KED related to leg pivot shifting. Simulations of the engaged leg model (Bakshi A, DiZio P, Lackner JR. J Neurophysiol 121: 2042-2060, 2019; Bakshi A, DiZio P, Lackner JR. J Neurophysiol 121: 2028-2041, 2019) correctly predicted the experimentally determined stability bounds of upright balance and also the scaling of the postural parameters as a function of GIF magnitude. The effective stiffness, KSAP, at the ankles played the primary role to prevent falling in FB swaying and both model predictions, and experimental data showed KSAP to scale up in proportion to GIF magnitude. For LR swaying, the model predicted a 3:4 scaling of anterior-posterior stiffness to change in GIF magnitude, which was borne out by the experimental data. Simulations predict stability (nonfalling) not to depend on lateral stiffness, KSML, which was experimentally found not to depend on the GIF magnitude. Both model and experiment showed that the geometry-dependent pivot shift parameter KED was invariant to a change in GIF magnitude. Thus the ELM explains voluntary sway and balance in altered GIF magnitude conditions, rotating environments with Coriolis perturbations of sway, as well as normal terrestrial conditions.NEW & NOTEWORTHY A nonparallel leg model of balance, the engaged leg model (ELM), was previously developed to characterize adaptive balance control in a rotating environment. Here we show the ELM also explains sway in hypergravity. It predicts the changes in balance control parameters with changes in gravity. ELM is currently the only balance model applicable to artificial and hypergravity conditions. ELM can also be applied to terrestrial clinical situations for pathologies that generate postural asymmetries.

Dynamic Changes of Heart Failure Biomarkers in Response to Parabolic Flight.

BACKGROUND: we aimed at investigating the influence of weightlessness and hypergravity by means of parabolic flight on the levels of the heart failure biomarkers H-FABP, sST2, IL-33, GDF-15, suPAR and Fetuin-A. METHODS: 14 healthy volunteers (males: eight; mean age: 28.9) undergoing 31 short-term phases of weightlessness and hypergravity were included. At different time points (baseline, 1 h/24 h after parabolic flight), venous blood was drawn and analyzed by the use of ELISA. RESULTS: sST2 evidenced a significant decrease 24 h after parabolic flight (baseline vs. 24, p = 0.009; 1 h vs. 24 h, p = 0.004). A similar finding was observed for GDF-15 (baseline vs. 24 h, p = 0.002; 1 h vs. 24 h, p = 0.025). The suPAR showed a significant decrease 24 h after parabolic flight (baseline vs. 24 h, p = 0.1726; 1 h vs. 24 h, p = 0.009). Fetuin-A showed a significant increase at 1 h and 24 h after parabolic flight (baseline vs. 24 h, p = 0.007; 1 h vs. 24 h, p = 0.04). H-FABP and IL-33 showed no significant differences at all time points. CONCLUSION: Our results suggest a reduction in cardiac stress induced by exposure to gravitational changes. Moreover, our findings indicate an influence of gravitational changes on proliferative processes and calcium homeostasis.

The influence of microgravity on cerebral blood flow and electrocortical activity.

Changes in gravity conditions have previously been reported to influence brain hemodynamics as well as neuronal activity. This paper attempts to identify a possible link between changes in brain blood flow and neuronal activity during microgravity. Middle cerebral artery flow velocity (MCAv) was measured using Doppler ultrasound. Brain cortical activity (i.e., cortical current density) was measured using electroencephalography. Finger blood pressure was recorded and exported to generate beat-by-beat systolic (SBP), diastolic (DBP) and mean arterial pressure (MAP), heart rate (HR), cardiac output (CO), and cerebrovascular conductance index (CVCi). Seventeen participants were evaluated under normal gravity conditions and microgravity conditions, during 15 bouts of 22-s intervals of weightlessness during a parabolic flight. Although MAP decreased and CO increased, MCAv remained unchanged in the microgravity condition. CVCi as the quotient of MCAv and MAP increased in microgravity. Cortical current density showed a global decrease. Our data support earlier data reporting a decrease in the amplitude of event-related potentials recorded during microgravity. However, the general decrease in neural excitability in microgravity seems not to be dependent on hemodynamic changes.

Responses of Bioconvection of Tetrahymena thermophila to Partial Gravity Created by Aircraft Flight.

Bioconvection is a form of collective pattern formation driven by negative gravitaxis of swimming microorganisms. In bioconvection, the interaction between individual swimmers results in self-organization leading to the development of a macroscopic structure typically 100-1000 times greater than an individual microorganism. To gain insight into the role of gravity in this self-organization phenomenon, we investigated the bioconvective behavior of the ciliate Tetrahymena thermophila under short-term partial gravity, i.e., gravitational acceleration < 1 g, achieved by quasiparabolic flight maneuvers of an aircraft. The bioconvective responses of T. thermophila were assessed by observing the collective motion simultaneously in two separate scales, which we call macroscale and microscale, using a newly designed "dual-objective" device with two different magnifications. Microscale analysis revealed that the magnitude of gravikinesis, i.e., active regulation of the propulsive thrust, decreased almost linearly with changes in gravitational acceleration, while gravitactic characteristics, assessed by the distribution of the swimming direction, did not change significantly during partial gravity. Macroscale analysis demonstrated that downward plumes of convection pattern gradually shortened from the lower end, and disappeared under partial gravity. The sustained time of the plumes decreased almost linearly with changes in gravitational acceleration. The response of downward plumes to partial gravity may be attributable to the accumulation of cells into blobs in downward migration, which increases the rate of downward migration enough to exceed the rate of upward movement, which is enhanced due to gravikinesis. This suggests that gravity may act on cells involved in collective pattern formation differently than on free-swimming cells.

Modeling human orthostatic responses on the Moon and on Mars.

PURPOSE: Since manned missions to the Moon and Mars are planned, we conducted active standing tests with lunar, Martian, terrestrial, and 1.8 loads of inertial resistance (+Gz) modeled through defined parabolic flight maneuvers. We hypothesized that the cardiovascular response to active standing is proportional to the +Gz load. METHODS: During partial-+Gz parabolic flights, 14 healthy test subjects performed active stand-up maneuvers under 1 +Gz, lunar (0.16 +Gz), Martian (0.38 +Gz), and hyper inertial resistance (1.8 +Gz) while heart rate and finger blood pressure were continuously monitored. We quantified amplitudes and timing of orthostatic response immediately following standing up. RESULTS: The maximum early heart rate increase was 21 (SD ± 10) bpm with lunar, 23 (± 11) bpm with Martian, 34 (± 17) bpm with terrestrial +Gz, and 40 (± 11) bpm hyper +Gz. The time to maximum heart rate increased gradually with increasing loads of inertial resistance. The transient blood pressure reduction was most pronounced with hyper +Gz but did not differ significantly between lunar and Martian +Gz. The mean arterial pressure nadir was reached significantly later with Martian and lunar compared to 1 +Gz. Paradoxically, the time for blood pressure to recover was shortest with terrestrial +Gz. CONCLUSION: While load of inertial resistance directly affects the magnitude of the transient blood pressure reduction and heart rate response to active standing, blood pressure stabilization is most rapidly attained during terrestrial +Gz. The observation might suggest that the human cardiovascular system is tuned to cope with orthostatic stress on earth.

Motion sickness and sopite syndrome associated with parabolic flights: a case report.

OBJECTIVE: The purpose of this paper was to provide an extensive description of a case of sopite syndrome, a manifestation of motion sickness scarcely described in the literature. DESIGN: The following questionnaires were used to assess sopite syndrome symptoms pre, during and post parabolic flight; Misery Scale rate, Positive and Negative Affect Scale, the Motion Sickness Assessment Questionnaire and the Epworth Sleepiness Scale. STUDY SAMPLE: Case report of a 35-year old female compared to a control group (n = 26). RESULTS: We describe of sopite syndrome during parabolic flight. The subject reported drowsiness and she noted mood changes such as irritation and annoyance. Furthermore, she was not able to perform her assigned task. Her symptoms escalated into extreme fatigue and the inability to stay awake. Nauseogenic symptoms improved, but soporific symptoms persisted for several hours after the motion stimulus (i.e. parabolic flight). CONCLUSIONS: This case points towards the need for controlled studies to assess the prevalence of this syndrome among the general population and select groups. Future research should focus on developing tests for personnel screening and explore treatment options.

Jugular venous flow dynamics during acute weightlessness.

During spaceflight, fluids shift headward, causing internal jugular vein (IJV) distension and altered hemodynamics, including stasis and retrograde flow, that may increase the risk of thrombosis. This study's purpose was to determine the effects of acute exposure to weightlessness (0-G) on IJV dimensions and flow dynamics. We used two-dimensional (2-D) ultrasound to measure IJV cross-sectional area (CSA) and Doppler ultrasound to characterize venous blood flow patterns in the right and left IJV in 13 healthy participants (6 females) while 1) seated and supine on the ground, 2) supine during 0-G parabolic flight, and 3) supine during level flight (at 1-G). On Earth, in 1-G, moving from seated to supine posture increased CSA in both left (+62 [95% CI: +42 to 81] mm2, P < 0.0001) and right (+86 [95% CI: +58 to 113] mm2, P < 0.00012) IJV. Entry into 0-G further increased IJV CSA in both left (+27 [95% CI: +5 to 48] mm2, P = 0.02) and right (+30 [95% CI: +0.3 to 61] mm2, P = 0.02) relative to supine in 1-G. We observed stagnant flow in the left IJV of one participant during 0-G parabolic flight that remained during level flight but was not present during any imaging during preflight measures in the seated or supine postures; normal venous flow patterns were observed in the right IJV during all conditions in all participants. Alterations to cerebral outflow dynamics in the left IJV can occur during acute exposure to weightlessness and thus, may increase the risk of venous thrombosis during any duration of spaceflight.NEW & NOTEWORTHY The absence of hydrostatic pressure gradients in the vascular system and loss of tissue weight during weightlessness results in altered flow dynamics in the left internal jugular vein in some astronauts that may contribute to an increased risk of thromboembolism during spaceflight. Here, we report that the internal jugular veins distend bilaterally in healthy participants and that flow stasis can occur in the left internal jugular vein during acute weightlessness produced by parabolic flight.

Shades of gravity - effects of planetary gravity levels on electrocortical activity and neurocognitive performance.

The plans of international space agencies to return to the Moon and explore deep space, including Mars, highlight the challenges of human adaptation and stress the need for a thorough analysis of the factors that facilitate, limit and modify human performance under extreme environments. This study investigates the influence of partial gravity on behavioural (error rate and reaction time) and neuronal parameters (event-related potentials) through parabolic flights. Brain cortical activity was assessed using EEG from 18 participants who solved a neurocognitive task, consisting of a mental arithmetic task and an auditory oddball paradigm, during Earth (1G), Lunar (0.16G + 0.25G) and Martian gravity (0.38G + 0.5G) for 15 consecutive parabolas. Data shows higher electrocortical activity in Earth gravity compared to Lunar and Martian gravity in the parietal lobe. No differences in participants' performance were found among the gravity levels. Event-related potentials displayed gravity-dependent variations, though limited stimuli recording suggests caution in interpretation. Data suggests a threshold between Earth and Martian gravity within the different gravities responsible for physiological changes, but it seems to vary greatly between individuals. The altered neuronal communication could be explained with a model developed by Kohn and Ritzmann in 2018. The increasing intracranial pressure in weightlessness changes the properties of the cell membrane of neurons and leads to a depolarisation of the resting membrane potential. The findings underscore the individuality of physiological changes in response to gravity alterations, signalling the need for further investigations in future studies.

Lunar and mars gravity induce similar changes in spinal motor control as microgravity.

Introduction: Once more, plans are underway to send humans to the Moon or possibly even to Mars. It is therefore, important to know potential physiological effects of a prolonged stay in space and to minimize possible health risks to astronauts. It has been shown that spinal motor control strategies change during microgravity induced by parabolic flight. The way in which spinal motor control strategies change during partial microgravity, such as that encountered on the Moon and on Mars, is not known. Methods: Spinal motor control measurements were performed during Earth, lunar, Mars, and micro-gravity conditions and two hypergravity conditions of a parabola. Three proxy measures of spinal motor control were recorded: spinal stiffness of lumbar L3 vertebra using the impulse response, muscle activity of lumbar flexors and extensors using surface electromyography, and lumbar curvature using two curvature distance sensors placed at the upper and lower lumbar spine. The participants were six females and six males, with a mean age of 33 years (standard deviation: 7 years). Results: Gravity condition had a statistically significant (Friedmann tests) effect spinal stiffness (p < 0.001); on EMG measures (multifidus (p = 0.047), transversus abdominis (p < 0.001), and psoas (p < 0.001) muscles) and on upper lumbar curvature sensor (p < 0.001). No effect was found on the erector spinae muscle (p = 0.063) or lower curvature sensor (p = 0.170). Post hoc tests revealed a significant increase in stiffness under micro-, lunar-, and Martian gravity conditions (all p's < 0.034). Spinal stiffness decreased under both hypergravity conditions (all p's ≤ 0.012) and decreased during the second hypergravity compared to the first hypergravity condition (p = 0.012). Discussion: Micro-, lunar-, and Martian gravity conditions resulted in similar increases in spinal stiffness, a decrease in transversus abdominis muscle activity, with no change in psoas muscle activity and thus modulation of spinal motor stabilization strategy compared to those observed under Earth's gravity. These findings suggest that the spine is highly sensitive to gravity transitions but that Lunar and Martian gravity are below that required for normal modulation of spinal motor stabilization strategy and thus may be associated with LBP and/or IVD risk without the definition of countermeasures.

Back to the future-revisiting Skylab data on ocular counter-rolling and motion sickness.

In the early 1970s, nine astronauts participated in missions to the Skylab space station. During two preflight testing sessions at the Naval Aerospace Medical Research Laboratory in Pensacola, the amplitudes of their ocular counter-rolling (OCR) during body tilts were assessed to determine if their vestibular functions were within normal ranges. We recently re-evaluated this data to determine asymmetry of each astronaut's OCR response and their OCR slope from sigmoid fits during static leftward and rightward body tilts, which we then compared with their Coriolis sickness susceptibility index (CSSI) on the ground, their motion sickness symptom scores during 0 g maneuvers in parabolic flight, and the severity of the symptoms of space motion sickness (SMS) they reported during their spaceflights. We arranged the astronauts in rank order for SMS severity based on the SMS symptoms they reported during spaceflight and the amount of anti-motion sickness medication they used. As previously reported, the OCR amplitudes of these astronauts were within the normal range. We determined that the OCR amplitudes were not correlated with SMS severity ranking, CSSI, or motion sickness symptoms experienced during parabolic flight. Indices of asymmetry in the OCR reflex were generally small and poorly correlated with SMS scores; however, the only subject with a high index of asymmetry also ranked highly for SMS. Although OCR slope, CSSI, and motion sickness symptoms induced during parabolic flight were each only moderately correlated with SMS severity ranking (rho = 0.41-0.44), a combined index that included all three parameters with equal weighting was significantly correlated with SMS severity ranking (rho = 0.71, p = 0.015). These results demonstrate the challenge of predicting an individual's susceptibility to SMS by measuring a single test parameter in a terrestrial environment and from a limited sample size.

Rational and design of the REMOTE trial: An exploratory, pilot study to analyze REtinal MicrOcirculaTion in wEightlessness.

BACKGROUND: "Spaceflight associated neuro-ocular syndrome" (SANS) represents a challenging health condition in modern space medicine. Forty-eight percent of astronauts are diagnosed with SANS after long-term space missions. The pathophysiological mechanism seems to be multifactorial, and yet remains unknown. In this proof-of-concept study we plan to investigate retinal microcirculatory changes in weightlessness and aim to identify their role in the development of SANS. METHODS AND DESIGN: Healthy individuals will take part in a parabolic flight campaign, which recreates fractioned total weightlessness periods. The airplane is specifically equipped, and designed for the execution of parabolic flight maneuvers and scientific research in microgravity. Retinal microcirculation will be assessed with a modified fundus camera, which allows dynamic vessel analysis. We will additionally measure intra-ocular pressure and hemodynamic changes during each phase of the flight. Blood samples will be analyzed at baseline, one hour and 24 hours after exposure to weightlessness. CONCLUSIONS: This pilot study aims to investigate the feasibility of retinal microcirculation assessment during varying gravity. Results of this study may generate insights whether venous stasis in the eye, surrogated by the dilatation of retinal vessels and increase in intraocular pressure as signs of venous insufficiency, may potentially contribute to the development of SANS.

Corrigendum: Impact of different ground-based microgravity models on human sensorimotor system.

[This corrects the article DOI: 10.3389/fphys.2023.1085545.].

Impact of different ground-based microgravity models on human sensorimotor system.

This review includes current and updated information about various ground-based microgravity models and their impact on the human sensorimotor system. All known models of microgravity are imperfect in a simulation of the physiological effects of microgravity but have their advantages and disadvantages. This review points out that understanding the role of gravity in motion control requires consideration of data from different environments and in various contexts. The compiled information can be helpful to researchers to effectively plan experiments using ground-based models of the effects of space flight, depending on the problem posed.

Retrograde Analysis of Calcium Signaling by CaMPARI2 Shows Cytosolic Calcium in Chondrocytes Is Unaffected by Parabolic Flights.

Calcium (Ca2+) elevation is an essential secondary messenger in many cellular processes, including disease progression and adaptation to external stimuli, e.g., gravitational load. Therefore, mapping and quantifying Ca2+ signaling with a high spatiotemporal resolution is a key challenge. However, particularly on microgravity platforms, experiment time is limited, allowing only a small number of replicates. Furthermore, experiment hardware is exposed to changes in gravity levels, causing experimental artifacts unless appropriately controlled. We introduce a new experimental setup based on the fluorescent Ca2+ reporter CaMPARI2, onboard LED arrays, and subsequent microscopic analysis on the ground. This setup allows for higher throughput and accuracy due to its retrograde nature. The excellent performance of CaMPARI2 was demonstrated with human chondrocytes during the 75th ESA parabolic flight campaign. CaMPARI2 revealed a strong Ca2+ response triggered by histamine but was not affected by the alternating gravitational load of a parabolic flight.

Proteomic Response of Deinococcus radiodurans to Short-Term Real Microgravity during Parabolic Flight Reveals Altered Abundance of Proteins Involved in Stress Response and Cell Envelope Functions.

Rapidly evolving space exploration makes understanding the short- and long- term effects of microgravity on humans, plants, and microorganisms an important task. The ubiquitous presence of the gravitational force has had an influence on the development of all living entities on Earth, and short- and long-term changes in perceived gravitational force can induce notable changes within cells. Deinococcus radiodurans is the Gram-positive bacterium that is best known for its extreme resistance to UV-C and gamma radiation, oxidation stress, and desiccation. Thus increased interest has been placed on this species in the context of space research. The present study aims to elucidate the short-term proteomic response of this species to real microgravity during parabolic flight. Overnight cultures of D. radiodurans were subjected to microgravity during a single parabola, and metabolic activity was quenched using methanol. Proteins were extracted and subsequently measured using HPLC nESI MS/MS. The results, such as the enrichment of the peptidoglycan biosynthesis pathway with differentially abundant proteins and altered S-layer protein abundance, suggested molecular rearrangements in the cell envelope of D. radiodurans. Altered abundance of proteins involved in energy metabolism and DNA repair could be linked with increased endogenous ROS production that contributes to the stress response. Moreover, changes in protein abundance in response to microgravity show similarities with previously reported stress responses. Thus, the present results could be used to further investigate the complex regulation of the remarkable stress management of this bacterium.