Metagenomic technologies have revolutionized our understanding of microbes in different spheres of life, revealing the massive diversity and complex functionalities of microbial communities across various environments. S...Metagenomic technologies have revolutionized our understanding of microbes in different spheres of life, revealing the massive diversity and complex functionalities of microbial communities across various environments. Shotgun metagenomics, which involves sequencing the DNA of all the organisms in a sample, is emerging as a powerful tool in assessing the microbial content. Unlike the traditional culturing approach, the shotgun metagenomic technology provides a comprehensive view of the entire microbial community, including potential functions that the organisms could be performing. In this chapter, we describe a typical bioinformatics workflow to generate the taxonomic profiles from metagenomic sequencing data and demonstrate a few basic statistical analyses that can be performed from this data to generate insights. In addition, we discuss the experimental and analytical considerations that must be taken into account while generating and making inferences from metagenomic data. Lastly, we provide insights on automating the workflow for consistent and reproducible large-scale analyses.
The field of space medicine has served the population of professional astronauts well over the many decades of human space travel, which is reflected in the considerable success of astronauts executing missions and retur...The field of space medicine has served the population of professional astronauts well over the many decades of human space travel, which is reflected in the considerable success of astronauts executing missions and returning to Earth in good health (rare comorbidities notwithstanding). At the same time, there are considerable gaps in our biomedical knowledge that will only scale as less fit individuals embark upon spaceflight, and we enter more extreme environments such as the Moon and Mars for longer duration missions. One means to partially fill this knowledge gap is to expand the practice of precision medicine, which we define as comprehensive genotyping and molecular phenotyping-coupled with derived countermeasures tailored to individuals and crews-to improve health and mitigate risk of disease. Improving the methods and tools of spaceflight precision medicine (PM) will improve our ability to optimize astronaut health and performance before entering space, while providing additional insight into the needs of specific astronauts while in space and upon return to Earth. A critical feature of PM in human spaceflight rests upon the foundation of complex molecular profiling, as the basis for developing these tailored countermeasures. This chapter provides an introductory overview of one means to use systems thinking to explore principles and molecular targets that are fundamental to PM in space.
Space biology research increasingly requires integrated platforms capable of managing complex datasets. This chapter introduces the NASA Open Science Data Repository (OSDR), designed to unify multiomics, physiological, e...Space biology research increasingly requires integrated platforms capable of managing complex datasets. This chapter introduces the NASA Open Science Data Repository (OSDR), designed to unify multiomics, physiological, environmental telemetry, and radiation exposure data from spaceflight and analog experiments. By strictly adhering to FAIR (Findable, Accessible, Interoperable, Reusable) principles, OSDR ensures standardized data curation and facilitates integrated analyses critical for understanding space-induced biological responses. The integration of AI technologies and federated learning frameworks will transform OSDR into a dynamic predictive modeling platform, enhancing discoveries and astronaut health research.
Regenerative medicine is vital for counteracting astronaut bone loss in microgravity, where reduced mechanical loading accelerates osteopenia and impairs fracture healing. Current countermeasures like exercise and drugs...Regenerative medicine is vital for counteracting astronaut bone loss in microgravity, where reduced mechanical loading accelerates osteopenia and impairs fracture healing. Current countermeasures like exercise and drugs help, but advanced regenerative solutions are needed. Bone-on-chip (BOC) technology, a microfluidic system mimicking bone physiology, enables precise studies of bone formation and degeneration in space. Adipose-derived stem cells (ASCs) committed to osteogenic differentiation (osASCs) offer a promising alternative to primary osteoblasts due to their ease of isolation, expansion potential, and strong regenerative properties. Integrating osASCs into BOC models facilitates research on bone loss mechanisms and novel osteoporosis treatments. This chapter details ASC isolation, osteogenic differentiation, and application in microgravity-simulating platforms, with the aim of contributing to progress in both space medicine and bone regeneration on Earth.
Thanks to advancements in innovative technologies, it is now possible to conduct testing for new drugs and medical treatments without using animals. This significant development not only saves time and costs but also eli...Thanks to advancements in innovative technologies, it is now possible to conduct testing for new drugs and medical treatments without using animals. This significant development not only saves time and costs but also eliminates the need for animal sacrifice. In this context, we present a 3D-printed construct made from glioblastoma U87-MG cells combined with biomimetic keratin-coated gold nanoparticles (Ker-AuNPs). By utilizing the thermoplasmonic properties of these Ker-AuNPs, we can test photothermal therapy (PTT)-a minimally invasive treatment designed to destroy cancer cells-using a more realistic representation of the tumor environment. Additionally, our research represents a significant turning point for potential applications in microgravity conditions, enabling us to speculate on the efficacy of PTT in a controlled gravity environment.
Microbiome-integrated Caenorhabditis elegans cultivation methods enable investigation of host-microbiome interactions in the context of space-relevant stresses using three key innovations: introduction of live bacterial...Microbiome-integrated Caenorhabditis elegans cultivation methods enable investigation of host-microbiome interactions in the context of space-relevant stresses using three key innovations: introduction of live bacterial communities replacing chemically defined media, implementation of auxin-inducible degradation systems to prevent progeny production, and development of complementary hardware platforms. Polyethylene bags provide gas-permeable cultivation environments for large populations with complex microbiomes supporting downstream molecular analyses, while NemaCapsules with micropillar arrays and passive culturing chambers allow real-time phenotypic assessment through on-orbit imaging, transforming our ability to correlate molecular signatures with physiological outcomes in microgravity.
Long-term space missions expose astronauts to an altered gravitational field (microgravity) that significantly affects human physiology. Although it is critical to assess the effect of microgravity on female reproductive...Long-term space missions expose astronauts to an altered gravitational field (microgravity) that significantly affects human physiology. Although it is critical to assess the effect of microgravity on female reproductive health, this aspect remains a poorly investigated one. To date, interest in the effects on the main ovary hormones (estrogens) has been in depth for its role in bone loss, while the impact of altered estrogen availability on fertility and ovarian function is unclear. The female reproductive apparatus depends on a finely tuned hormonal network involving the hypothalamic-pituitary-ovarian axis. At the heart of this system are the ovarian follicles, composed of granulosa and theca cells, which cooperate in the synthesis of steroid hormones in supporting oocyte maturation. Folliculogenesis and estrogen production are tightly regulated by gonadotropins-FSH and LH-which act on these two distinct cell types. Any disruption in this delicate endocrine cross talk may impair oocyte quality, ovulatory function, and overall reproductive health. New evidence suggests that microgravity may interfere with this delicate hormonal balance, potentially reducing estrogen synthesis and impairing follicular development mostly by downregulating aromatase activity. As the regulation of ovarian function is distributed across different tiers, investigation is particularly delicate. Nevertheless, molecular assessment of relevant parameters can be performed upon cells/tissues cultures growing in real and simulated weightlessness in order to collect data and drawing explicative models.
Approaches based on analytical technologies of molecular histology are of basic importance, among the analytical algorithms for studying the functional morphology of tissue microenvironment in space biology. This is due...Approaches based on analytical technologies of molecular histology are of basic importance, among the analytical algorithms for studying the functional morphology of tissue microenvironment in space biology. This is due to both the unique ability to visualize the phenomena under study in situ in the spatial aspect, and the great potential to develop promising innovative protocols for the detection of cells and extracellular structures. This chapter is dedicated to the possible applications of histochemical and immunohistochemical analysis of the tissue microenvironment in space biology.
We present an experimental strategy to dissect the effects of simulated microgravity on the interaction between cells and extracellular matrix. The protocol encompasses morphological and transcriptomic analyses on human...We present an experimental strategy to dissect the effects of simulated microgravity on the interaction between cells and extracellular matrix. The protocol encompasses morphological and transcriptomic analyses on human neural progenitor cells (hNSPCs) cultured in flasks coated with poly-L-ornithine and laminin matrix at low density and subjected to microgravity simulation. High-density cultures subjected to microgravity or grown under normal gravity without a matrix are also analyzed as controls. The experimental approach could be adapted to different cell types by changing the extracellular matrix composition and the culture medium accordingly.
Space exploration is moving toward the next milestone: space colonization. The risks for human health, associated to the permanence in the space, are already object of studies by several experiments both in orbit and, un...Space exploration is moving toward the next milestone: space colonization. The risks for human health, associated to the permanence in the space, are already object of studies by several experiments both in orbit and, under simulated space conditions, on hearth. Now, these assessments will need to be extended over the increased permanence, in view of the stable life on other planets. It is well known that the environmental conditions present in the space, primarily related to the altered gravity, introduce a physical stress and the lack of a fundamental bond that contributes to the spatial organization of the cells. This status further induces possible alteration in cell metabolism and in gene expression. Unexpectedly, despite their fundamental involvement in the regulation of gene expression and their responsiveness to stresses, epigenetics modifications have been poorly studied in space and in altered gravity environment. Epigenetic modifications, such as DNA methylation, histones modifications, chromatin remodeling, and non-coding RNAs, can exert the role of mediator of the specific environmental stimuli encountered in space, resulting in modulations of the epigenome, possibly leading to altered cell response. Increasing evidence point out that this three-way interaction-environment-epigenome-cell-is associated to cell and tissue dyshomeostasis and possibly to disease onset. For this reason, increasing attention to the epigenetic modifications induced by the spatial environment and more comprehensive epigenetic studies are warranted. Unfortunately, the study of the epigenetic modifications in space presents some technical difficulty mainly due to the lability of some of these marks, particularly the most studied, i.e., the DNA methylation. Profiling the epigenetic modification often requires long and complex protocols that implies the use of specific instruments. These studies can be, on the other hand, performed on biological specimens collected during the space missions and returned to hearth. For the most labile epigenetic marks, using simulated microgravity on ground may represent a valid alternative approach. This chapter is therefore aimed at introducing the basics of the epigenetic modifications, the already known epigenetic changes associated to the space environment and the techniques used to study these marks.
This chapter aims to synthesize the various methods and technologies employed in space-based research missions to study the cellular cytoskeleton alteration in the spaceflight environmental conditions. We describe interd...This chapter aims to synthesize the various methods and technologies employed in space-based research missions to study the cellular cytoskeleton alteration in the spaceflight environmental conditions. We describe interdisciplinary approaches combining cell imaging and live-cell imaging, mechanical assays, and molecular analyses.
When living organisms are exposed to the space environment, numerous changes occur that affect virtually all cellular functions and molecular pathways. One class of understudied molecules in space biomedicine is noncodin...When living organisms are exposed to the space environment, numerous changes occur that affect virtually all cellular functions and molecular pathways. One class of understudied molecules in space biomedicine is noncoding RNAs (ncRNAs), particularly microRNAs (miRNAs). Given that spaceflight induces systemic health risks, miRNAs represent natural candidates for key regulators in space, as they can control hundreds to thousands of genes and influence central biological pathways. Transcriptomic data and pathway analyses are employed to identify the key genes driving specific physiological states, which can in turn be used to predict the miRNAs regulating those states. MiRNAs that act as upstream regulators of these driver genes are selected as candidates for a spaceflight-associated miRNA signature. Once these candidate miRNAs are identified, they must be experimentally validated. Depending on the health risk or biological process under investigation, miRNAs are validated either in circulating fluids (e.g., serum, plasma, blood) or in tissue samples using droplet digital polymerase chain reaction (ddPCR). Compared to conventional PCR methods, ddPCR enables absolute quantification of miRNA copy numbers and overcomes several limitations associated with traditional miRNA expression assays. This approach provides an unbiased prediction of candidate miRNAs, followed by precise and specific verification of their expression profiles, and can be broadly applied to identify miRNA signatures associated with various diseases and physiological stressors. Once validated, miRNAs that are overexpressed in response to spaceflight can be targeted for inhibition as potential countermeasures to mitigate health risks. Harnessing the power of miRNAs not only deepens our understanding of space biology but also accelerates the development of targeted interventions, ultimately enabling safe and sustained human exploration of deep space.
Space life sciences have been revolutionized by the paradigm shift of biology into a "big data" era. Spaceflight experiments are now routinely generating large-scale molecular ("omics") datasets across different tissues...Space life sciences have been revolutionized by the paradigm shift of biology into a "big data" era. Spaceflight experiments are now routinely generating large-scale molecular ("omics") datasets across different tissues and organisms to help better understand the mechanisms of health decline in space. Large-scale study of gene expression ("transcriptomics") has proven to be a particularly useful for producing new insights into the signaling pathways and key molecules that underpin maladaptation to spaceflight. Technologically, bulk RNA-sequencing provides a simple yet powerful platform for studying transcriptome-wide dysregulation in space. This chapter outlines a basic pipeline for analyzing bulk RNA-sequencing data using R, a well-established programming tool for transcriptomics. The pipeline is demonstrated throughout via application to RNA-sequencing data from a real-life spaceflight experiment, and is provided as a single-resource code script (available at: https://github.com/williscrg/MiMB-Space-RNAseq-Pipeline ) that the reader can use to run the pipeline from start to finish "as is" or with modification if/as desired or required.
Spaceflight imposes a unique combination of environmental stressors including ionizing radiation, microgravity, and prolonged isolation that disrupt biological systems from the genome to the proteome. These stressors con...Spaceflight imposes a unique combination of environmental stressors including ionizing radiation, microgravity, and prolonged isolation that disrupt biological systems from the genome to the proteome. These stressors contribute to a range of health effects such as musculoskeletal degradation, neuroimmune dysfunction, and psychological challenges. Advances in countermeasure development, including biomedical devices and therapeutics, have already yielded terrestrial benefits for conditions like osteoporosis and cardiovascular disease. To systematically characterize spaceflight-induced biological alterations, researchers employ a wide array of omics techniques transcriptomics, proteomics, metabolomics alongside silico modeling approaches such as molecular dynamics simulations. Microgravity research is conducted both in true spaceflight environments (e.g., aboard the ISS and suborbital vehicles) and via terrestrial analogs (e.g., clinostats and random positioning machines), using in vitro and in vivo models to capture cellular and organismal responses. Open-access platforms like NASA GeneLab facilitate data sharing and integrative analysis of multiomics datasets generated under spaceflight conditions. In this chapter, we describe computational pipelines for processing space-derived transcriptomic data, with a focus on quality control, alignment, quantification, and downstream functional analysis. These methods support the construction of multiscale models that bridge molecular insights with physiological outcomes, enabling a deeper understanding of space biology and guiding countermeasure development.
This chapter has the aim to propose a theoretical framework for describing the qualitative changes in the deformation behavior of isolated human breast cancer cells under varying gravitational conditions. We describe int...This chapter has the aim to propose a theoretical framework for describing the qualitative changes in the deformation behavior of isolated human breast cancer cells under varying gravitational conditions. We describe interdisciplinary principles of statistical thermodynamics and nonlinear dynamics of eukaryotic cells. The framework aims to capture how microgravity-induced alterations in cytoskeletal organization lead to distinct mechanobiological phenotypes.
Gravity plays a significant role in both life and physical science processes. In life sciences, at the level of a whole organism, gravity as unit gravity on Earth (9.81 ms), the lack of gravity during free fall, or the i...Gravity plays a significant role in both life and physical science processes. In life sciences, at the level of a whole organism, gravity as unit gravity on Earth (9.81 ms), the lack of gravity during free fall, or the increase of gravity while, e.g., during centrifugation is reflected in an organism's metabolism, anatomy, and behavior. In this chapter, the impact of near weightlessness and hypo- and hypergravity on animals related to buoyancy, sedimentation, and hydrostatic pressure will be addressed. It will provide a concise historical overview of these processes for in-flight and ground-based studies on animals and the experimental modalities that have been or could be used in future investigations.
As humanity continues to push toward interplanetary travel and beyond, short- and long-term adaptation of the human body to extreme environmental conditions in space remains a fundamental concern. An astronaut experience...As humanity continues to push toward interplanetary travel and beyond, short- and long-term adaptation of the human body to extreme environmental conditions in space remains a fundamental concern. An astronaut experiences physically and mentally enduring situations like microgravity, extreme heat or cold, radiation, isolation, and other unforeseen technical challenges. The assessment of clinical conditions-the specific task of Clinical Pathology-during spaceflights is becoming a relevant priority. Defining a strategy to establish a sound analytical approach is among the topics of the present volume. Namely, we are trying to assess those parameters to identify the "fingerprint" of specific biological (complex) processes, including metabolic, immunologic, protein/genetic and signaling pathways, in order to depict a comprehensive, systemic model able to provide clear explanations and preventative health scenarios. To address such a challenge, an impressive technological endeavor is going on to develop innovative diagnostic tools, medical devices and infrastructures for developing remote sensing and medical support, given that medical capabilities are inherently limited during spaceflights.
Shaped by evolution, human skin cells have acquired a remarkable capacity to detect and react to sunlight photons through a wide array of photochemical processes. These reactions trigger intricate intracellular signaling...Shaped by evolution, human skin cells have acquired a remarkable capacity to detect and react to sunlight photons through a wide array of photochemical processes. These reactions trigger intricate intracellular signaling cascades, ultimately resulting in photobiological effects that help regulate skin cell homeostasis. Some of these photobiological responses (also so-called photobiomodulation or low-level light therapy) are capable of initiating profound and beneficial therapeutic effects. To identify the regimes for these light-based therapeutic solutions, one needs to carefully decipher the physical, optical, biological, and chemical conditions that all need to be fulfilled to facilitate such positive photobiological effects. Here, we provide the protocols specifically developed to investigate multidimensional parameter space driving photobiological interactions triggered by light in the cells of human integumentary system. The approach also includes the so-called design of experiment (DoE), a statistical method, which allows for the investigation of multidimensional parameters landscapes. This goes hand in hand with sharing practical tips for the design of light-based devices for experimental work. We illustrate practical applications of our methods and light-based devices by sharing comprehensive experimental datasets, highlighting both the robustness and reproducibility of the results.
Irradiation of skin cells with ultraviolet radiation is a central procedure in photobiology and related fields. A detailed description of UV irradiation protocols can be found in this chapter, with the consideration of i...Irradiation of skin cells with ultraviolet radiation is a central procedure in photobiology and related fields. A detailed description of UV irradiation protocols can be found in this chapter, with the consideration of important variations, including choice of cells, growth state of cells, UV sources, and dosing. A few examples of assays to study UVR-induced cellular effects are mentioned and briefly discussed, but not described in detail.
Two-stage chemical carcinogenesis method is widely used to elucidate genetic and molecular changes that lead to skin cancer development, as well as to test chemotherapeutic properties of novel drugs. This protocol allows...Two-stage chemical carcinogenesis method is widely used to elucidate genetic and molecular changes that lead to skin cancer development, as well as to test chemotherapeutic properties of novel drugs. This protocol allows researchers to reliably induce benign papilloma development and their conversion to squamous cell carcinoma in the skin of susceptible mouse strains in response to a single dose of carcinogen 2,4-dimethoxybenzaldehyde (DMBA) and repetitive applications of tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA).