Effects of Microparticle Size and Fc Density on Macrophage Phagocytosis

Controlled induction of phagocytosis in macrophages offers the ability to therapeutically regulate the immune system as well as improve delivery of chemicals or biologicals for immune processing. Maximizing particle uptake by macrophages through Fc receptor-mediated phagocytosis could lead to new delivery mechanisms in drug or vaccine development. Fc ligand density and particle size were examined independently and in combination in order to optimize and tune the phagocytosis of opsonized microparticles. We show the internalization efficiency of small polystyrene particles (0.5 µm to 2 µm) is significantly affected by changes in Fc ligand density, while particles greater than 2 µm show little correlation between internalization and Fc density. We found that while macrophages can efficiently phagocytose a large number of smaller particles, the total volume of phagocytosed particles is maximized through the non-specific uptake of larger microparticles. Therefore, larger microparticles may be more efficient at delivering a greater therapeutic payload to macrophages, but smaller opsonized microparticles can deliver bio-active substances to a greater percentage of the macrophage population. This study is the first to treat as independent variables the physical and biological properties of Fc density and microparticle size that initiate macrophage phagocytosis. Defining the physical and biological parameters that affect phagocytosis efficiency will lead to improved methods of microparticle delivery to macrophages.     

Immuno-Regulatory Function of Indoleamine 2,3 Dioxygenase through Modulation of Innate Immune Responses

Successful long-term treatment of type-1 diabetes mainly relies on replacement of β-cells via islet transplantation. Donor shortage is one of the main obstacles preventing transplantation from becoming the treatment of choice. Although animal organs could be an alternative source for transplantation, common immunosuppressive treatments demonstrate low efficacy in preventing xenorejection. Immunoprotective effects of indoleamine 2,3-dioxygenase (IDO) on T-cell mediated allorejection has been extensively studied. Our studies revealed that IDO expression by fibroblasts, induced apoptosis in T-cells while not affecting non-immune cell survival/function. Since macrophages play a pivotal role in xenograft rejection, herein we investigated the effect of IDO-induced tryptophan deficiency/kynurenine accumulation on macrophage function/survival. Moreover, we evaluated the local immunosuppressive effect of IDO on islet-xenograft protection. Our results indicated that IDO expression by bystander fibroblasts significantly reduced the viability of primary macrophages via apoptosis induction. Treatment of peritoneal macrophages by IDO-expressing fibroblast conditioned medium significantly reduced their proinflammatory activity through inhibition of iNOS expression. To determine whether IDO-induced tryptophan starvation or kynurenine accumulation is responsible for macrophage apoptosis and inhibition of their proinflammatory activity, Raw264.7 cell viability and proinflammatory responses were evaluated in tryptophan deficient medium or in the presence of kynurenine. Tryptophan deficiency, but not kynurenine accumulation, reduced Raw264.7 cell viability and suppressed their proinflammatory activity. Next a three-dimensional islet-xenograft was engineered by embedding rat islets within either control or IDO–expressing fibroblast-populated collagen matrix. Islets morphology and immune cell infiltration were then studied in the xenografts transplanted into the C57BL/6 mouse renal sub-capsular space. Local IDO significantly decreased the number of infiltrating macrophages (11±1.47 vs. 70.5±7.57 cells/HPF), T-cells (8.75±1.03 vs. 75.75±5.72 cells/HPF) and iNOS expression in IDO-expressing xenografts versus controls. Islet morphology remained intact in IDO-expressing grafts and islets were strongly stained for insulin/glucagon compared to control. These findings support the immunosuppressive role of IDO on macrophage-mediated xeno-rejection.     

Effect of culture in a rotating wall bioreactor on the physiology of differentiated neuron-like PC12 and SH-SY5Y cells.

A variety of evidence suggests that nervous system function is altered during microgravity, however, assessing changes in neuronal physiology during space flight is a non-trivial task. We have used a rotating wall bioreactor with a high aspect ratio vessel (HARV), which simulates the microgravity environment, to investigate the how the viability, neurite extension, and signaling of differentiated neuron-like cells changes in different culture environments. We show that culture of differentiated PC12 and SH-SY5Y cells in the simulated microgravity HARV bioreactor resulted in high cell viability, moderate neurite extension, and cell aggregation accompanied by NO production. Neurite extension was less than that seen in static cultures, suggesting that less than optimal differentiation occurs in simulated microgravity relative to normal gravity. Cells grown in a mixed vessel under normal gravity (a spinner flask) had low viability, low neurite extension, and high glutamate release. This work demonstrates the feasibility of using a rotating wall bioreactor to explore the effects of simulated microgravity on differentiation and physiology of neuron-like cells.     

Stage Specific Assessment of Candida albicans Phagocytosis by Macrophages Identifies Cell Wall Composition and Morphogenesis as Key Determinants

Candida albicans is a major life-threatening human fungal pathogen. Host defence against systemic Candida infection relies mainly on phagocytosis of fungal cells by cells of the innate immune system. In this study, we have employed video microscopy, coupled with sophisticated image analysis tools, to assess the contribution of distinct C. albicans cell wall components and yeast-hypha morphogenesis to specific stages of phagocytosis by macrophages. We show that macrophage migration towards C. albicans was dependent on the glycosylation status of the fungal cell wall, but not cell viability or morphogenic switching from yeast to hyphal forms. This was not a consequence of differences in maximal macrophage track velocity, but stems from a greater percentage of macrophages pursuing glycosylation deficient C. albicans during the first hour of the phagocytosis assay. The rate of engulfment of C. albicans attached to the macrophage surface was significantly delayed for glycosylation and yeast-locked morphogenetic mutant strains, but enhanced for non-viable cells. Hyphal cells were engulfed at a slower rate than yeast cells, especially those with hyphae in excess of 20 µm, but there was no correlation between hyphal length and the rate of engulfment below this threshold. We show that spatial orientation of the hypha and whether hyphal C. albicans attached to the macrophage via the yeast or hyphal end were also important determinants of the rate of engulfment. Breaking down the overall phagocytic process into its individual components revealed novel insights into what determines the speed and effectiveness of C. albicans phagocytosis by macrophages.     

Circadian clock genes promote glioma progression by affecting tumour immune infiltration and tumour cell proliferation

ObjectivesCircadian rhythm controls complicated physiological activities in organisms. Circadian clock genes have been related to tumour progression, but its role in glioma is unknown. Therefore, we explored the relationship between dysregulated circadian clock genes and glioma progression.Materials and MethodsSamples were divided into different groups based on circadian clock gene expression in training dataset (n = 672) and we verified the results in other four validating datasets (n = 1570). The GO and GSEA enrichment analysis were conducted to explore potential mechanism of how circadian clock genes affected glioma progression. The single‐cell RNA‐Seq analysis was conducted to verified previous results. The immune landscape was evaluated by the ssGSEA and CIBERSORT algorithm. Cell proliferation and viability were confirmed by the CCK8 assay, colony‐forming assay and flow cytometry.ResultsThe cluster and risk model based on circadian clock gene expression can predict survival outcome. Samples were scoring by the least absolute shrinkage and selection operator regression analysis, and high scoring tumour was associated with worse survival outcome. Samples in high‐risk group manifested higher activation of immune pathway and cell cycle. Tumour immune landscape suggested high‐risk tumour infiltrated more immunocytes and more sensitivity to immunotherapy. Interfering TIMELESS expression affected circadian clock gene expression, inhibited tumour cell proliferation and arrested cell cycle at the G0/G1 phase.ConclusionsDysregulated circadian clock gene expression can affect glioma progression by affecting tumour immune landscape and cell cycle. The risk model can predict glioma survival outcome, and this model can also be applied to pan‐cancer.     

Corrected Super-Resolution Microscopy Enables Nanoscale Imaging of Autofluorescent Lung Macrophages

Observing the cell surface and underlying cytoskeleton at nanoscale resolution using super-resolution microscopy has enabled many insights into cell signaling and function. However, the nanoscale dynamics of tissue-specific immune cells have been relatively little studied. Tissue macrophages, for example, are highly autofluorescent, severely limiting the utility of light microscopy. Here, we report a correction technique to remove autofluorescent noise from stochastic optical reconstruction microscopy (STORM) data sets. Simulations and analysis of experimental data identified a moving median filter as an accurate and robust correction technique, which is widely applicable across challenging biological samples. Here, we used this method to visualize lung macrophages activated through Fc receptors by antibody-coated glass slides. Accurate, nanoscale quantification of macrophage morphology revealed that activation induced the formation of cellular protrusions tipped with MHC class I protein. These data are consistent with a role for lung macrophage protrusions in antigen presentation. Moreover, the tetraspanin protein CD81, known to mark extracellular vesicles, appeared in ring-shaped structures (mean diameter 93 ± 50 nm) at the surface of activated lung macrophages. Thus, a moving median filter correction technique allowed us to quantitatively analyze extracellular secretions and membrane structure in tissue-derived immune cells.     

Impairment of antigen-specific cellular immune responses under simulated microgravity conditions

Summary Microgravity has been implicated to play a role in the observed immune dysfunction of astronauts and cosmonauts after either short-term or long-term space travel. These reports, together with studies describing increased levels of microorganisms in the space cabin environment suggest potential risk for in-flight incidences of infectious diseases. In order to understand the mechanism underlying these immune defects, it is important to have a ground-based model that would reliably mimic the effects of microgravity on antigen-specific immune function. We tested the utility of the rotating wall vessel (RWV) technology developed at NASA as a model system because in the RWV the culture medium and the cells rotate synchronously with the vessel, thereby creating simulated microgravity conditions. We compared the RWV to the conventional tissue culture flask (T-flask), for culturing immune precursor cells with cytotoxic T lymphocyte (CTL) activity against synthetic viral peptides. We observed a significant loss of antigen-specific CTL activity in RWV cultures, but not in those from the T-flask, irrespective of the peptide immunogen used for inducing the primary immune response in different mouse strains. Loss of CTL activity in RWV cultures coincided with a significant reduction in CD8⁺ cells as well as CD4⁺ cells and DEC205⁺ dendritic cells, suggesting adverse effects of RWV culturing on both the effector and accessory cells for the loss of antigen-specific CTL function. These results provide a strong parallel to the reported defects in cell-mediated immunity during space travel and strongly support the utility of the RWV technology as an effective ground-based model for identifying key steps in immune cell dysfunction related to microgravity.     

Flow Cytometry Approach to Quantify the Viability of Milk Somatic Cell Counts after Various Physico-Chemical Treatments

Flow cytometry has been used as a routine method to count somatic cells in milk, and to ascertain udder health and milk quality. However, few studies investigate the viability of somatic cells and even fewer at a subpopulation level to follow up how the cells can resist to various stresses that can be encountered during technological processes. To address this issue, a flow cytometry approach was used to simultaneously identify cell types of bovine milk using cell-specific antibodies and to measure the cell viability among the identified subpopulations by using a live/dead cell viability kit. Confirmation of the cell viability was performed by using conventional microscopy. Different physico-chemical treatments were carried out on standardized cell samples, such as heat treatment, various centrifugation rates and storage in milk or in PBS pH 7.4 for three days. Cytometry gating strategy was developed by using blood cell samples stored at 4°C in PBS and milk cell samples heat-treated at 80°C for 30 min as a control for the maximum (95.9%) and minimum (0.7%) values of cell viability respectively. Cell viability in the initial samples was 39.5% for all cells and varied for each cell population from 26.7% for PMNs, to 32.6% for macrophages, and 58.3% for lymphocytes. Regarding the physico-chemical treatments applied, somatic cells did not sustain heat treatment at 60°C and 80°C in contrast to changes in centrifugation rates, for which only the higher level, i.e. 5000×g led to a cell viability decrease, down to 9.4%, but no significant changes within the cell subpopulation distribution were observed. Finally, the somatic cells were better preserved in milk after 72h storage, in particular PMNs, that maintained a viability of 34.0 ± 2.9% compared to 4.9±1.9% in PBS, while there was almost no changes for macrophages (41.7 ± 5.7% in milk vs 31.2 ± 2.4% in PBS) and lymphocytes (25.3 ± 3.0% in milk vs 11.4 ± 3.1% in PBS). This study provides a new array to better understand milk cell biology and to establish the relationship between the cell viability and the release of their endogenous enzymes in dairy matrix.     

A Hypomagnetic Field Aggravates Bone Loss Induced by Hindlimb Unloading in Rat Femurs

A hypomagnetic field is an extremely weak magnetic field—it is considerably weaker than the geomagnetic field. In deep-space exploration missions, such as those involving extended stays on the moon and interplanetary travel, astronauts will experience abnormal space environments involving hypomagnetic fields and microgravity. It is known that microgravity in space causes bone loss, which results in decreased bone mineral density. However, it is unclear whether hypomagnetic fields affect the skeletal system. In the present study, we aimed to investigate the complex effects of a hypomagnetic field and microgravity on bone loss. To study the effects of hypomagnetic fields on the femoral characteristics of rats in simulated weightlessness, we established a rat model of hindlimb unloading that was exposed to a hypomagnetic field. We used a geomagnetic field-shielding chamber to generate a hypomagnetic field of <300 nT. The results show that hypomagnetic fields can exacerbate bone mineral density loss and alter femoral biomechanical characteristics in hindlimb-unloaded rats. The underlying mechanism might involve changes in biological rhythms and the concentrations of trace elements due to the hypomagnetic field, which would result in the generation of oxidative stress responses in the rat. Excessive levels of reactive oxygen species would stimulate osteoblasts to secrete receptor activator of nuclear factor-κB ligand and promote the maturation and activation of osteoclasts and thus eventually cause bone resorption.     

Alginate Microencapsulated Hepatocytes Optimised for Transplantation in Acute Liver Failure

MethodsHuman hepatocyte microbeads (HMBs) were prepared using sterile GMP grade materials. We determined physical stability, cell viability, and hepatocyte metabolic function of HMBs using different polymerisation times and cell densities. The immune activation of peripheral blood mononuclear cells (PBMCs) after co-culture with HMBs was studied. Rats with ALF induced by galactosamine were transplanted intraperitoneally with rat hepatocyte microbeads (RMBs) produced using a similar optimised protocol. Survival rate and biochemical profiles were determined. Retrieved microbeads were evaluated for morphology and functionality.ResultsThe optimised HMBs were of uniform size (583.5±3.3 µm) and mechanically stable using 15 min polymerisation time compared to 10 min and 20 min (p<0.001). 3D confocal microscopy images demonstrated that hepatocytes with similar cell viability were evenly distributed within HMBs. Cell density of 3.5×10⁶ cells/ml provided the highest viability. HMBs incubated in human ascitic fluid showed better cell viability and function than controls. There was no significant activation of PBMCs co-cultured with empty or hepatocyte microbeads, compared to PBMCs alone. Intraperitoneal transplantation of RMBs was safe and significantly improved the severity of liver damage compared to control groups (empty microbeads and medium alone; p<0.01). Retrieved RMBs were intact and free of immune cell adherence and contained viable hepatocytes with preserved function.ConclusionAn optimised protocol to produce GMP grade alginate-encapsulated human hepatocytes has been established. Transplantation of microbeads provided effective metabolic function in ALF. These high quality HMBs should be suitable for use in clinical transplantation.     

Leukocyte Activity Is Altered in a Ground Based Murine Model of Microgravity and Proton Radiation Exposure

Immune system adaptation during spaceflight is a concern in space medicine. Decreased circulating leukocytes observed during and after space flight infer suppressed immune responses and susceptibility to infection. The microgravity aspect of the space environment has been simulated on Earth to study adverse biological effects in astronauts. In this report, the hindlimb unloading (HU) model was employed to investigate the combined effects of solar particle event-like proton radiation and simulated microgravity on immune cell parameters including lymphocyte subtype populations and activity. Lymphocytes are a type of white blood cell critical for adaptive immune responses and T lymphocytes are regulators of cell-mediated immunity, controlling the entire immune response. Mice were suspended prior to and after proton radiation exposure (2 Gy dose) and total leukocyte numbers and splenic lymphocyte functionality were evaluated on days 4 or 21 after combined HU and radiation exposure. Total white blood cell (WBC), lymphocyte, neutrophil, and monocyte counts are reduced by approximately 65%, 70%, 55%, and 70%, respectively, compared to the non-treated control group at 4 days after combined exposure. Splenic lymphocyte subpopulations are altered at both time points investigated. At 21 days post-exposure to combined HU and proton radiation, T cell activation and proliferation were assessed in isolated lymphocytes. Cell surface expression of the Early Activation Marker, CD69, is decreased by 30% in the combined treatment group, compared to the non-treated control group and cell proliferation was suppressed by approximately 50%, compared to the non-treated control group. These findings reveal that the combined stressors (HU and proton radiation exposure) result in decreased leukocyte numbers and function, which could contribute to immune system dysfunction in crew members. This investigation is one of the first to report on combined proton radiation and simulated microgravity effects on hematopoietic, specifically immune cells.     

Tumour microenvironment‐based molecular profiling reveals ideal candidates for high‐grade serous ovarian cancer immunotherapy

ObjectiveDue to limited immunological profiles of high‐grade serous ovarian cancer (HGSOC), we aimed to characterize its molecular features to determine whether a specific subset that can respond to immunotherapy exists.Materials and MethodsA training cohort of 418 HGSOC samples from TCGA was analysed by consensus non‐negative matrix factorization. We correlated the expression patterns with the presence of immune cell infiltrates, immune regulatory molecules and other genomic or epigenetic features. Two independent cohorts containing 482 HGSOCs and in vitro experiments were used for validation.ResultsWe identified immune and non‐immune groups where the former was enriched in signatures that reflect immune cells, infiltration and PD‐1 signalling (all, P < 0.001), and presented with a lower chromosomal aberrations but increased neoantigens, tumour mutation burden, and microsatellite instability (all, P < 0.05); this group was further refined into two microenvironment‐based subtypes characterized by either immunoactivation or carcinoma‐associated fibroblasts (CAFs) and distinct prognosis. CAFs‐immune subtype was enriched for factors that mediate immunosuppression and promote tumour progression, including highly expressed stromal signature, TGF‐β signalling, epithelial‐mesenchymal transition and tumour‐associated M2‐polarized macrophages (all, P < 0.001). Robustness of these immune‐specific subtypes was verified in validation cohorts, and in vitro experiments indicated that activated‐immune subtype may benefit from anti‐PD1 antibody therapy (P < 0.05).ConclusionOur findings revealed two immune subtypes with different responses to immunotherapy and indicated that some HGSOCs may be susceptible to immunotherapies or combination therapies.     

Cholesterol and sphingomyelin are critical for Fcγ receptor–mediated phagocytosis of Cryptococcus neoformans by macrophages

Cryptococcus neoformans is a fungal pathogen that causes life-threatening meningoencephalitis in lymphopenic patients. Pulmonary macrophages comprise the first line of host defense upon inhalation of fungal spores by aiding in clearance but can also potentially serve as a niche for their dissemination. Given that macrophages play a key role in the outcome of a cryptococcal infection, it is crucial to understand factors that mediate phagocytosis of C. neoformans. Since lipid rafts (high-order plasma membrane domains enriched in cholesterol and sphingomyelin [SM]) have been implicated in facilitating phagocytosis, we evaluated whether these ordered domains govern macrophages'' ability to phagocytose C. neoformans. We found that cholesterol or SM depletion resulted in significantly deficient immunoglobulin G (IgG)-mediated phagocytosis of fungus. Moreover, repletion of macrophage cells with a raft-promoting sterol (7-dehydrocholesterol) rescued this phagocytic deficiency, whereas a raft-inhibiting sterol (coprostanol) significantly decreased IgG-mediated phagocytosis of C. neoformans. Using a photoswitchable SM (AzoSM), we observed that the raft-promoting conformation (trans-AzoSM) resulted in efficient phagocytosis, whereas the raft-inhibiting conformation (cis-AzoSM) significantly but reversibly blunted phagocytosis. We observed that the effect on phagocytosis may be facilitated by Fcγ receptor (FcγR) function, whereby IgG immune complexes crosslink to FcγRIII, resulting in tyrosine phosphorylation of FcR γ-subunit (FcRγ), an important accessory protein in the FcγR signaling cascade. Correspondingly, cholesterol or SM depletion resulted in decreased FcRγ phosphorylation. Repletion with 7-dehydrocholesterol restored phosphorylation, whereas repletion with coprostanol showed FcRγ phosphorylation comparable to unstimulated cells. Together, these data suggest that lipid rafts are critical for facilitating FcγRIII-mediated phagocytosis of C. neoformans.     

Studies on the Growth and Indole-3-Acetic Acid and Abscisic Acid Content of Zea mays Seedlings Grown in Microgravity

Measurements were made of the fresh weight, dry weight, dry weight-fresh weight ratio, free and conjugated indole-3-acetic acid, and free and conjugated abscisic acid in seedlings of Zea mays grown in darkness in microgravity and on earth. Imbibition of the dry kernels was for 17 h prior to launch. Growth was for 5 d at ambient orbiter temperature and at a chronic accelerational force of the order of 3 × 10⁻⁵ times earth gravity. Weights and hormone content of the microgravity seedlings were, with minor exceptions, not statistically different from seedlings grown in normal gravity. The tissues of the shuttle-grown plants appeared normal and the seedlings differed only in the lack of orientation of roots and shoots. These findings, based upon 5 d of growth in microgravity, cannot be extrapolated to growth in microgravity for weeks, months, and years, as might occur on a space station. Nonetheless, it is encouraging, for prospects of bioregeneration of the atmosphere and food production in a space station, that no pronounced differences in the parameters measured were apparent during the 5 d of plant seedling growth in microgravity.     

Suppression of antigen-specific lymphocyte activation in modeled microgravity

Various parameters of immune suppression are observed in lymphocytes from astronauts during and after a space flight. It is difficult to ascribe this suppression to microgravity effects on immune cells in crew specimens, due to the complex physiological response to space flight and the resultant effect on in vitro immune performance. Use of isolated immune cells in true and modeled microgravity in immune performance tests, suggests a direct effect of microgravity on in vitro cellular function. Specifically, polyclonal activation of T-cells is severely suppressed in true and modeled microgravity. These recent findings suggest a potential suppression of oligoclonal antigen-specific lymphocyte activation in microgravity. We utilized rotating wall vessel (RWV) bioreactors as an analog of microgravity for cell cultures to analyze three models of antigen-specific activation. A mixed-lymphocyte reaction, as a model for a primary immune response, a tetanus toxoid response and a Borrelia burgdorferi response, as models of a secondary immune response, were all suppressed in the RWV bioreactor. Our findings confirm that the suppression of activation observed with polyclonal models also encompasses oligoclonal antigen-specific activation.     

SUBPLASMALEMMAL MICROFILAMENTS AND MICROTUBULES IN RESTING AND PHAGOCYTIZING CULTIVATED MACROPHAGES

The subplasmalemmal organization of the free and glass-attached surfaces of resting and phagocytizing cultivated macrophages were examined in an attempt to define specific membrane-associated structures related to phagocytosis. From analysis of serial thin sections of oriented cells it was found that the subplasmalemmal region of the attached cell surface has a complex microfilament and microtubule organization relative to the subplasmalemmal area of the free surface. A filamentous network composed of 40–50-Å microfilaments extended for a depth of 400–600 Å from the attached plasma membrane. Immediately subjacent to the filamentous network was a zone of oriented bundles of 40–50-Å microfilaments and a zone of microtubules. Additional microtubules were found to extend from the plasma membrane to the interior of the cell in close association with electron-dense, channellike structures. In contrast, the free aspect of the cultivated macrophage contained only the subplasmalemmal filamentous network. However, after a phagocytic pulse with polystyrene particles (14 µm diam) microtubules and oriented filaments similar to those found on the attached surface were observed surrounding the ingested particles. The observations reported in this paper provide support for the hypothesis that microfilaments and/or microtubules play a role in the translocation of plasma membrane required for the functionally similar processes of phagocytosis and cell attachment to glass.     

Full-Length Recombinant Human SCF1-165 Is More Thermostable than the Truncated SCF1-141 Form

Human stem cell factor initiates a diverse array of cellular responses, including hematopoiesis, cell proliferation, differentiation, migration and survival. To explore the relationship between its structure and function, we produced recombinant soluble human stem cell factor^1–165 (wild type) and human stem cell factor^1–141 (C-terminal truncated) in a yeast expression system and compared their biological activities and thermal stabilities. The biological activity of the two proteins was measured as a function of TF-1 cell viability and effects on downstream signaling targets after incubation. We found that these proteins enhanced cell viability and downstream signaling to a similar extent, in a dose-dependent manner. The biological activity of recombinant human stem cell factor^1–165 was significantly greater than that of recombinant human stem cell factor^1–141 after heating the proteins (100 ng/mL) at 25–110°C for 10 minutes (P<0.05 for all temperatures). In addition, circular dichroism spectral analysis indicated that β-sheet structures were altered in recombinant human stem cell factor^1–141 but not recombinant human stem cell factor^1–165 after heating at 90°C for 15 or 30 min. Molecular modeling and limited proteolytic digestion were also used to compare the thermo stability between human stem cell factor^1–165 and human stem cell factor^1–141. Together, these data indicate that stem cell factor^1–165 is more thermostable than stem cell factor^1–141.     

Altered Baseline Brain Activity with 72 h of Simulated Microgravity – Initial Evidence from Resting-State fMRI

To provide the basis and reference to further insights into the neural activity of the human brain in a microgravity environment, we discuss the amplitude changes of low-frequency brain activity fluctuations using a simulated microgravity model. Twelve male participants between 24 and 31 years old received resting-state fMRI scans in both a normal condition and after 72 hours in a −6° head down tilt (HDT). A paired sample t-test was used to test the amplitude differences of low-frequency brain activity fluctuations between these two conditions. With 72 hours in a −6° HDT, the participants showed a decreased amplitude of low-frequency fluctuations in the left thalamus compared with the normal condition (a combined threshold of P<0.005 and a minimum cluster size of 351 mm³ (13 voxels), which corresponded with the corrected threshold of P<0.05 determined by AlphaSim). Our findings indicate that a gravity change-induced redistribution of body fluid may disrupt the function of the left thalamus in the resting state, which may contribute to reduced motor control abilities and multiple executive functions in astronauts in a microgravity environment.