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Long-term space travel is a harsh experience for the human body. Astronauts exposed to the effects of space travel often experience bone and muscle loss, as well as an increased risk of diseases, including heart disease[1]. While the human heart has shown an impressive ability to adapt to spaceflight, many of the long-term effects on cardiovascular health are unknown [2]. To plan for missions requiring longer flight durations, understanding these effects is neccessary to assess the risk to the astronauts undertaking these missions. However, evaluating these risks is difficult due to the lack of a sufficiently accurate model. While animal models and studies of returned astronauts can be informative, these methods also have significant limitations. The development of an accurate in vitro model of the myocardium would be a beneficial tool to further investigate the underlying cellular mechanisms affecting cardiac health during spaceflight. This project seeks to create and validate an accurate three-dimensional model of the myocardium using novel bio-mimetic hagfish proteins. Hagfish protein threads that mimic the properties of the myocardium and support cardiomyocyte cell culture were produced. Upon further validation, this model will be exposed to an artificial spaceflight environment incorporating simultaneous microgravity and radiation to investigate the effects of spaceflight on the myocardium.

This discussion will explore the impact of the COVID-19 pandemic on scholarly research and publishing. Travel restrictions, retracted funding, delayed or halted projects, and an increase in caretaker and other personal responsibilities at home compound to create unprecedented challenges for producing and publishing research. Early indicators show women, those with significant unpaid care responsibilities, and members of minoritized groups have been disproportionately impacted. For graduate students and early career faculty who depend on research and publication for promotion and tenure, the stakes are especially high. Join our panelists for a conversation about the how the COVID-19 pandemic is impacting the research landscape. Watch the video to see the discussion. Click on the download button for a list of readings and resources., https://digitalcommons.usu.edu/inter_inclusion/1001/thumbnail.jpg

A major challenge for astronauts in long-duration space travel is combatting the hazardous spaceflight environment caused by microgravity and increased levels of ionizing radiation. Microgravity damages cellular DNA by increasing the production of harmful reactive oxygen species, while ionizing radiation damages DNA by creating double-stranded DNA (dsDNA) breaks. Cellular damage due to microgravity and radiation has been investigated using ground-based models, but most models consider microgravity and ionizing radiation alone, or asynchronously. Synchronous modeling better mimics spaceflight conditions and can be used to understand the combined effects of microgravity and ionizing radiation. However, commercially available devices to model microgravity and radiation are rare and costly, requiring both a rotary cell culture system and beam time at a national lab or an independent radiation source. While independent radiation sources are becoming less difficult to use or purchase, commercially available devices to simulate microgravity are still cost prohibitive to many researchers. In this study, we developed a low-cost, open source cell culture system for studying the role that synchronous ionizing radiation and microgravity play in pathophysiology during spaceflight conditions. Our system was validated by exposing C2C12 mouse myoblast cells to ionizing radiation at levels approximating 9-month and 10-year Mars missions while simulating microgravity. DNA damage to cells was quantified using -H2AX (a fluorescent marker for double-stranded breaks), while reactive oxygen species production was visualized using CellROX green. Cells exposed to long-term mission doses had statistically significant increases in DNA damage and ROS production compared to both the short-term and control conditions. Our proof of concept shows that the low-cost, open source mini-RCCS can be used to mimic the radiation and microgravity hazards of the spaceflight environment at the same level as expensive commercial systems.

Muscular atrophy is a serious issue for extended spaceflight. Understanding and preventing the role of ionizing radiation in skeletal muscle loss would preserve the strength and endurance of astronauts and enable longer duration space travel and exploration. Irradiation was performed in the USU material physics group's Space Suvivability Test Chamber. C2C12 and CRL-1999 cells were exposed to dosages ranging from 0.5 - 36.8 Gy. Cell viability and growth rate were measured immediately following irradiation.

Background The cervix must undergo significant biochemical remodeling to allow for successful parturition. This process is not fully understood, especially in instances of spontaneous preterm birth. In vivo Raman spectroscopy is an optical technique that can be used to investigate the biochemical composition of tissue longitudinally and noninvasively in human beings, and has been utilized to measure physiology and disease states in a variety of medical applications. Objective The purpose of this study is to measure in vivo Raman spectra of the cervix throughout pregnancy in women, and to identify biochemical markers that change with the preparation for delivery and postpartum repair. Study Design In all, 68 healthy pregnant women were recruited. Raman spectra were measured from the cervix of each patient monthly in the first and second trimesters, weekly in the third trimester, and at the 6-week postpartum visit. Raman spectra were measured using an in vivo Raman system with an optical fiber probe to excite the tissue with 785 nm light. A spectral model was developed to highlight spectral regions that undergo the most changes throughout pregnancy, which were subsequently used for identifying Raman peaks for further analysis. These peaks were analyzed longitudinally to determine if they underwent significant changes over the course of pregnancy (P < .05). Finally, 6 individual components that comprise key biochemical constituents of the human cervix were measured to extract their contributions in spectral changes throughout pregnancy using a linear combination method. Patient factors including body mass index and parity were included as variables in these analyses. Results Raman peaks indicative of extracellular matrix proteins (1248 and 1254 cm−1) significantly decreased (P < .05), while peaks corresponding to blood (1233 and 1563 cm–1) significantly increased (P < .0005) in a linear manner throughout pregnancy. In the postpartum cervix, significant increases in peaks corresponding to actin (1003, 1339, and 1657 cm–1) and cholesterol (1447 cm–1) were observed when compared to late gestation, while signatures from blood significantly decreased. Postpartum actin signals were significantly higher than early pregnancy, whereas extracellular matrix proteins and water signals were significantly lower than early weeks of gestation. Parity had a significant effect on blood and extracellular matrix protein signals, with nulliparous patients having significant increases in blood signals throughout pregnancy, and higher extracellular matrix protein signals in early pregnancy compared to patients with prior pregnancies. Body mass index significantly affected actin signal contribution, with low body mass index patients showing decreasing actin contribution throughout pregnancy and high body mass index patients demonstrating increasing actin signals. Conclusion Raman spectroscopy was successfully used to biochemically monitor cervical remodeling in pregnant women during prenatal visits. This foundational study has demonstrated sensitivity to known biochemical dynamics that occur during cervical remodeling, and identified patient variables that have significant effects on Raman spectra throughout pregnancy. Raman spectroscopy has the potential to improve our understanding of cervical maturation, and be used as a noninvasive preterm birth risk assessment tool to reduce the incidence, morbidity, and mortality caused by preterm birth.

As longer space missions become more desirable to public and private institutions, the physiological impact on astronauts must be considered. One of the primary concerns for those spending time in low gravity and high radiation environments is muscle atrophy. A major cause of muscular atrophy is oxidative stress which is amplified by increased levels of ionizing radiation during spaceflight. Additionally, high levels of radiation can damage DNA, increasing the risk of cancer. Utah State University’s Space Environment Test Facility was used to irradiate C2C12 myoblasts and human vascular endothelial cells with a dosage mimicking that on the International Space Station and a 3-year deep space mission. Cell changes due to increased levels of radiation were characterized with fluorescent imaging for H2AX, a marker of double stranded DNA damage, and Trypan blue viability staining.

PURPOSE: To investigate the role of RPE cell-cell contact in vascular endothelial growth factor (VEGF) protein expression in cultures of primary human RPE (hRPE) cells and a human RPE cell line (ARPE-19). METHODS: Two in vitro methods, scratching and micropatterning, were used to control the physical dissociation of RPE cell-cell junctions. Scratching was performed by scoring monolayers of RPE cells with a cell scraper. Micropatterning was achieved by using a stencil patterning method. Extracellular VEGF expression was assessed by using an enzyme-linked immunosorbent assay (ELISA) kit. Immunocytochemistry (ICC) was performed to visualize the expression and localization of VEGF and intercellular proteins zonula occludens-1 (ZO-1), N-cadherin, β-catenin, and claudin-1 in RPE cultures. RESULTS: Higher expression of VEGF protein by cells on the edges of the scratched RPE layers was confirmed with ICC in short-term (1 day after confluency) and long-term (4 weeks after confluency) cultures. According to the ICC results, ZO-1, N-cadherin, β-catenin, and claudin-1 successfully localized to cell-cell junctions in long-term cultures of ARPE-19 and hRPE cells. However, unlike N-cadherin, β-catenin, and claudin-1, only ZO-1 localized junctionally in short-term cultures of both cell types. Moreover, removing cell-cell junctions by scratching resulted in the delocalization of ZO-1 from tight junctions to the cytoplasm. The loss of tight junction formation and the accumulation of ZO-1 in the cytoplasm correlated with increased VEGF expression. Micropatterning RPE cells on different sized circular patterns produced varying concentrations of cells with lost cell-cell junctions. When fewer cells formed intercellular junctions, increased extracellular VEGF secretion was observed from the ARPE-19 and hRPE cells. CONCLUSIONS: VEGF expression increases after physical disruption of RPE cell-cell connections. This increase in VEGF expression correlates with the loss of intercellular junctions and the localization of ZO-1 in the cytoplasm of RPE cells.

Muscular atrophy, defined as the loss of muscle tissue, is a serious issue for immobilized patients on Earth and in human spaceflight, where microgravity prevents normal muscle loading. A major factor in muscular atrophy is oxidative stress, which is amplified not only by muscle disuse, but also by the increased levels of ionizing radiation in spaceflight. Additionally, elevated radiation exposure can damage DNA, increasing cancer risk. To model oxidative stress and DNA damage generated by conditions on the International Space Station, murine C2C12 myoblasts were cultured in a rotary cell culture system irradiated by cesium-137. Changes due to the spaceflight model were characterized with fluorescent imaging, amino acid analysis, and enzyme linked immunosorbent assay for heme oxygenase 1. Fluorescent imaging was performed to assess viability, lipid peroxidation, and DNA damage. Minor DNA damage was observed in cells exposed to 20 μCi cesium-137 for 15 days. No significant differences in viability or lipid peroxidation were noted. Exposure to radiation decreased intracellular heme oxygenase 1 and extracellular alanine, but did not affect branch chain amino acids. Investigation of stronger radiation sources and extended culture time is ongoing. We anticipate that radiation will exacerbate the atrophic effects of microgravity on muscle cells. Simulation of microgravity and spaceflight radiation will provide a valuable platform for drug discovery and an understanding of the progression from normal to disease state.

SERS enhancement of different Raman reporters on spherical gold nanoparticles. Raman spectra were acquired for five different Raman reporter molecules; DTTC iodide, Crystal Violet, Malachite Green, 4-ATP, and 4-MBA. Sixty nanometer spherical gold nanoparticles were labeled with each reporter at a concentration of 400nM. After labeling, the Raman spectrum of each nanoparticle solution was acquired on our custom Raman microscope system. The intensity of the major peak in each spectrum was determined and compared. The Raman intensity of DTTC iodide is much greater than for other reporter molecules. (PDF 103 kb)

Background: Surface-enhanced Raman scattering (SERS) is a powerful light scattering technique that can be used for sensitive immunoassay development and cell labeling. A major obstacle to using SERS is the complexity of fabricating SERS probes since they require nanoscale characterization and optical uniformity. The light scattering response of SERS probes may also be modulated by the substrate used for SERS analysis. A typical SERS substrate such as quartz can be expensive. Polystyrene is a cheaper substrate option but can decrease the SERS response due to interfering Raman emission peaks and high background fluorescence. The goal of this research is to develop an optimized process for fabricating Raman-labeled nanoparticles for a SERS-based immunoassay on a polystyrene substrate. Results: We have developed a method for fabricating SERS nanoparticle probes for use in a light scattering immunoassay on a polystyrene substrate. The light scattering profile of both spherical gold nanoparticle and gold nanorod SERS probes were characterized using Raman spectroscopy and optical absorbance spectroscopy. The effects of substrate interference and autofluorescence were reduced by selecting a Raman reporter with a strong light scattering response in a spectral region where interfering substrate emission peaks are minimized. Both spherical gold nanoparticles and gold nanorods SERS probes used in the immunoassay were detected at labeling concentrations in the low pM range. This analytical sensitivity falls within the typical dynamic range for direct labeling of cell-surface biomarkers using SERS probes. Conclusion: SERS nanoparticle probes were fabricated to produce a strong light scattering signal despite substrate interference. The optical extinction and inelastic light scattering of these probes was detected by optical absorbance spectroscopy and Raman spectroscopy, respectively. This immunoassay demonstrates the feasibility of analyzing strongly enhanced Raman signals on polystyrene, which is an inexpensive yet non-ideal Raman substrate. The assay sensitivity, which is in the low pM range, suggests that these SERS probe particles could be used for Raman labeling of cell or tissue samples in a polystyrene tissue culture plate. With continued development, this approach could be used for direct labeling of multiple cell surface biomarkers on strongly interfering substrate platforms.

SERS probe stability and optical response. The stability of the SERS probes was determined by measuring the optical response of the probes before and after the addition of the SH-PEG. SERS probes fabricated using the synthesis methods described previously were stable for at least 1 month when stored at 4 °C. When SH-PEG was added to the gold nanoparticle solution, it did not significantly displace the Raman reporter. In addition, the aggregation state, optical response, and protein function were not adversely affected by centrifugation. (PDF 163 kb)

OPSS-PEG-SVA conjugation and hydrolysis. Activated OPSS-PEG-SVA can undergo two separate and competing reactions, hydrolysis and conjugation. To promote conjugation, the concentration of the protein solution was maintained at greater than 1mg/ml during conjugation and the NHS ester molecule was added to the protein solution immediately upon suspension. In addition, the NHS ester reagent was stored at â 20 °C in a desiccator and under nitrogen to avoid possible hydrolysis due to ambient moisture. The hydrolysis reaction will release a NHS ester-leaving group with a maximum absorbance at 260nm. The quality of the stored NHS ester reagent was estimated by measuring the absorbance of a freshly prepared solution at 260nm and comparing that value to the initial absorbance from a newly opened vial of the reagent. (PDF 114 kb)

Design of a custom Raman microscope system. The optical response of the SERS probes was detected using a custom Raman microscope system, which was constructed with an inverted microscope base, Figure S1A. With its inverted base and long working distance objectives lenses, the system was built to analyze SERS probes on a microplate substrate. The custom Raman system had a spectral resolution of 1.6 cmâ 1, a laser wavelength of 785nm, and a maximum power of 40mW. The Raman system captured spectral images using a 1340x400 pixel CCD detector. A Raman spectral image was obtained (Figure S1B, background image) and was averaged across each column to produce a Raman spectrum (Figure S1B, red overlay). The custom Raman system was used to acquire the Raman spectra of SERS probes in the development of light scattering immunoassays. (PDF 1673 kb)

Raman spectroscopy has enabled researchers to map the specific chemical makeup of surfaces, solutions, and even cells. However, the inherent insensitivity of the technique makes it difficult to use and statistically complicated. When Raman active molecules are near gold or silver nanoparticles, the Raman intensity is significantly amplified. This phenomenon is referred to as surface-enhanced Raman spectroscopy (SERS). The extent of SERS enhancement is due to a variety of factors such as nanoparticle size, shape, material, and configuration. The choice of Raman reporters and protective coatings will also influence SERS enhancement. This review provides an introduction to how these factors influence signal enhancement and how to optimize them during synthesis of SERS nanoparticles.

Raman spectroscopy has enabled researchers to map the specific chemical makeup of surfaces, solutions, and even cells. However, the inherent insensitivity of the technique makes it difficult to use and statistically complicated. When Raman active molecules are near gold or silver nanoparticles, the Raman intensity is significantly amplified. This phenomenon is referred to as surface-enhanced Raman spectroscopy (SERS). The extent of SERS enhancement is due to a variety of factors such as nanoparticle size, shape, material, and configuration. The choice of Raman reporters and protective coatings will also influence SERS enhancement. This review provides an introduction to how these factors influence signal enhancement and how to optimize them during synthesis of SERS nanoparticles.