Influence of shear waves on transcranial ultrasound propagation in cortical brain regions

Transcranial ultrasound applications require accurate simulations to predict intracranial acoustic pressure fields. The current gold standard typically consists of calculating a longitudinal ultrasound wave propagation using a fluid skull model, which is based on full head CT images for retrieving the skull's geometry and elastic constants. Although this approach has extensively been validated for deep brain targets and routinely used in transcranial ultrasound ablation procedures, its accuracy in shallow cortical regions remains unexplored. In this study, we explore the shear wave effects associated with transcranial focused ultrasound propagation, both numerically and experimentally. The intracranial acoustic pressure was measured at different incidence angles at the parietal and frontal regions in an ex vivo human skull. The fluid-like skull model was then compared to the solid model comprising both longitudinal and shear waves. The results consistently show a larger error and variability for both models when considering an oblique incidence, reaching a maximum of 125% mean deviation of the focal area when employing the fluid skull model. Statistical assessments further revealed that ignoring shear waves results in an average ~40% overestimation of the intracranial acoustic pressure and inability to obtain an accurate intracranial acoustic pressure distribution. Moreover, the solid model has a more stable performance, even when small variations in the skull-transducer relative position are introduced. Our results could contribute to the refinement of the transcranial ultrasound propagation modeling methods thus help improving the safety and outcome of transcranial ultrasound therapy in the cortical brain areas

Post-Transcriptional Dynamics is Involved in Rapid Adaptation to Hypergravity in Jurkat T Cells

The transcriptome of human immune cells rapidly reacts to altered gravity in a highly dynamic way. We could show in previous experiments that transcriptional patterns show profound adaption after seconds to minutes of altered gravity. To gain further insight into these transcriptional alteration and adaption dynamics, we conducted a highly standardized RNA-Seq experiment with human Jurkat T cells exposed to 9xg hypergravity for 3 and 15 min, respectively. We investigated the frequency with which individual exons were used during transcription and discovered that differential exon usage broadly appeared after 3 min and became less pronounced after 15 min. Additionally, we observed a shift in the transcript pool from coding towards non-coding transcripts. Thus, adaption of gravity-sensitive differentially expressed genes followed a dynamic transcriptional rebound effect. The general dynamics were compatible with previous studies on the transcriptional effects of short hypergravity on human immune cells and suggest that initial up-regulatory changes mostly result from increased elongation rates. The shift correlated with a general downregulation of the affected genes. All chromosome bands carried homogenous numbers of gravity-sensitive genes but showed a specific tendency towards up- or downregulation. Altered gravity affected transcriptional regulation throughout the entire genome, whereby the direction of differential expression was strongly dependent on the structural location in the genome. A correlation analysis with potential mediators of the early transcriptional response identified a link between initially upregulated genes with certain transcription factors. Based on these findings, we have been able to further develop our model of the transcriptional response to altered gravity

Cytoskeletal stability and metabolic alterations in primary human macrophages in long-term microgravity

The immune system is one of the most affected systems of the human body during space flight. The cells of the immune system are exceptionally sensitive to microgravity. Thus, serious concerns arise, whether space flight associated weakening of the immune system ultimately precludes the expansion of human presence beyond the Earth's orbit. For human space flight, it is an urgent need to understand the cellular and molecular mechanisms by which altered gravity influences and changes the functions of immune cells. The CELLBOX-PRIME (= CellBox-Primary Human Macrophages in Microgravity Environment) experiment investigated for the first time microgravity-associated long-term alterations in primary human macrophages, one of the most important effector cells of the immune system. The experiment was conducted in the U.S. National Laboratory on board of the International Space Station ISS using the NanoRacks laboratory and Biorack type I standard CELLBOX EUE type IV containers. Upload and download were performed with the SpaceX CRS-3 and the Dragon spaceship on April 18th, 2014 / May 18th, 2014. Surprisingly, primary human macrophages exhibited neither quantitative nor structural changes of the actin and vimentin cytoskeleton after 11 days in microgravity when compared to 1g controls. Neither CD18 or CD14 surface expression were altered in microgravity, however ICAM-1 expression was reduced. The analysis of 74 metabolites in the cell culture supernatant by GC-TOF-MS, revealed eight metabolites with significantly different quantities when compared to 1g controls. In particular, the significant increase of free fucose in the cell culture supernatant was associated with a significant decrease of cell surface-bound fucose. The reduced ICAM-1 expression and the loss of cell surface-bound fucose may contribute to functional impairments, e.g. the activation of T cells, migration and activation of the innate immune response. We assume that the surprisingly small and non-significant cytoskeletal alterations represent a stable "steady state" after adaptive processes are initiated in the new microgravity environment. Due to the utmost importance of the human macrophage system for the elimination of pathogens and the clearance of apoptotic cells, its apparent robustness to a low gravity environment is crucial for human health and performance during long-term space missions

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

Cytoskeletal stability and metabolic alterations in primary human macrophages in long-term microgravity

The immune system is one of the most affected systems of the human body during space flight. The cells of the immune system are exceptionally sensitive to microgravity. Thus, serious concerns arise, whether space flight associated weakening of the immune system ultimately precludes the expansion of human presence beyond the Earth's orbit. For human space flight, it is an urgent need to understand the cellular and molecular mechanisms by which altered gravity influences and changes the functions of immune cells. The CELLBOXPRIME (= CellBox-Primary Human Macrophages in Microgravity Environment) experiment investigated for the first time microgravity-associated long-term alterations in primary human macrophages, one of the most important effector cells of the immune system. The experiment was conducted in the U. S. National Laboratory on board of the International Space Station ISS using the NanoRacks laboratory and Biorack type I standard CELLBOX EUE type IV containers. Upload and download were performed with the SpaceX CRS- 3 and the Dragon spaceship on April 18th, 2014 / May 18th, 2014. Surprisingly, primary human macrophages exhibited neither quantitative nor structural changes of the actin and vimentin cytoskeleton after 11 days in microgravity when compared to 1g controls. Neither CD18 or CD14 surface expression were altered in microgravity, however ICAM-1 expression was reduced. The analysis of 74 metabolites in the cell culture supernatant by GC-TOF-MS, revealed eight metabolites with significantly different quantities when compared to 1g controls. In particular, the significant increase of free fucose in the cell culture supernatant was associated with a significant decrease of cell surface-bound fucose. The reduced ICAM-1 expression and the loss of cell surface-bound fucose may contribute to functional impairments, e.g. the activation of T cells, migration and activation of the innate immune response. We assume that the surprisingly small and non-significant cytoskeletal alterations represent a '' stable-steady state '' after adaptive processes are initiated in the new microgravity environment. Due to the utmost importance of the human macrophage system for the elimination of pathogens and the clearance of apoptotic cells, its apparent robustness to a low gravity environment is crucial for human health and performance during long-term space missions

Evolutionary Relationships of Wild Hominids Recapitulated by Gut Microbial Communities

Although bacteria are continually acquired over the lifetime of an individual, the phylogenetic relationships of great ape species is mirrored in the compositions of their gut microbial communities

Time course of cellular and molecular regulation in the immune system in altered gravity: progressive damage or adaptation?

We summarized the current knowledge about adaptation processes of isolated immune cells, animal models and the human body to altered gravity conditions. Many studies indicate an adaptation reaction of the immune system to the new microgravity environment, at least for the T cell system. Animal and human studies indicated adaptation processes starting after two weeks and continuing until 6 month or longer, which was reflected by cytokine concentrations in blood plasma or in stimulation assays. Adaptive reactions regarding IFN-c, TNF-a and IL-2 concentrations were detected after 12 days spaceflight in animal studies and after 2–4 months in human studies, whereas adaptive reactions regarding IL-4, IL-6, IL-8 and IL-10 were found after 6 months spaceflight. Cellular studies were performed mainly as short-term studies, and only a few studies addressed alterations longer than 3 days. However, cross validation between studies is often not possible or indicated conflicting results. Many in vitro studies, mostly done with T lymphocytes, demonstrated extensive cellular and molecular alterations. In contrast, long-term studies with animals and humans are completely lacking this dramatic picture of short-term cellular effects, which indicates a very efficient adaptation process, partially evidenced by new steady state of adaptive response in the human immune system after weeks until months. Therefore, we assume that the human body and its cells are equipped with a robust and efficient adaptation potential when challenged with low gravitational environments

Rapid Transient Transcriptional Adaptation to Hypergravity in Jurkat T Cells Revealed by Comparative Analysis of Microarray and RNA-Seq Data

Cellular responses to micro- and hypergravity are rapid and complex and appear within the first few seconds of exposure. Transcriptomic analyses are a valuable tool to analyze these genome-wide cellular alterations. For a better understanding of the cellular dynamics upon altered gravity exposure, it is important to compare different time points. However, since most of the experiments are designed as endpoint measurements, the combination of cross-experiment meta-studies is inevitable. Microarray and RNA-Seq analyses are two of the main methods to study transcriptomics. In the field of altered gravity research, both methods are frequently used. However, the generation of these data sets is difficult and time-consuming and therefore the number of available data sets in this research field is limited. In this study, we investigated the comparability of microarray and RNA-Seq data and applied the results to a comparison of the transcriptomics dynamics between the hypergravity conditions during two real flight platforms and a centrifuge experiment to identify temporal adaptation processes. We performed a comparative study on an Affymetrix HTA2.0 microarray and a paired-end RNA-Seq data set originating from the same Jurkat T cell RNA samples from a short-term hypergravity experiment. The overall agreeability was high, with better sensitivity of the RNA-Seq analysis. The microarray data set showed weaknesses on the level of single upregulated genes, likely due to its normalization approach. On an aggregated level of biotypes, chromosomal distribution, and gene sets, both technologies performed equally well. The microarray showed better performance on the detection of altered gravity-related splicing events. We found that all initially altered transcripts fully adapted after 15 min to hypergravity and concluded that the altered gene expression response to hypergravity is transient and fully reversible. Based on the combined multiple-platform meta-analysis, we could demonstrate rapid transcriptional adaptation to hypergravity, the differential expression of the ATPase subunits ATP6V1A and ATP6V1D, and the cluster of differentiation (CD) molecules CD1E, CD2AP, CD46, CD47, CD53, CD69, CD96, CD164, and CD226 in hypergravity. We could experimentally demonstrate that it is possible to develop methodological evidence for the meta-analysis of individual data

Effects of Spaceflight on the Immune System

The immune system belongs to the most affected systems during spaceflight, and sensitivity of cells of the human immune system to reduced gravity has been confirmed in numerous studies in real and simulated microgravity. Immune system dysfunction during spaceflight represents a substantial risk for exploration class mission knowledge about the clinical, cellular, and genetic basis of immune system response, and adaptation to altered gravity will provide key information for appropriate risk management, efficient monitoring, and countermeasures against existing limiting factors for human health and performance during manned exploration of the solar system. In spite of the immune system dysregulation, studies indicate an adaptation reaction of the immune system to the new microgravity environment, at least for the T-cell system, starting after 2 weeks and continuing until 6 months or longer, reflected by cytokine concentrations in blood plasma or in stimulation assays. At the cellular level, rapid adaptation responses could be detected as early as after seconds until minutes in T cells and macrophages. Therefore, adaptive responses of cells and the whole organism could be expected under microgravity and altered gravity in general. Preventive countermeasures should therefore consider support and stabilization of the endogenous adaptation programs. Potential countermeasures for risk mitigation are summarized in this chapter. We assume that the immune systems not only have a significant adaptation potential when challenged with low gravitational environments but also provide interesting preventive and therapeutic options for long-term space missions

Rapid cellular perception of gravitational forces in human jurkat T cells and transduction into gene expression regulation

Cellular processes are influenced in many ways by changes in gravitational force. In previous studies, we were able to demonstrate, in various cellular systems and research platforms that reactions and adaptation processes occur very rapidly after the onset of altered gravity. In this study we systematically compared differentially expressed gene transcript clusters (TCs) in human Jurkat T cells in microgravity provided by a suborbital ballistic rocket with vector-averaged gravity (vag) provided by a 2D clinostat. Additionally, we included 9× g centrifuge experiments and rigorous controls for excluding other factors of influence than gravity. We found that 11 TCs were significantly altered in 5 min of flight-induced and vector-averaged gravity. Among the annotated clusters were G3BP1, KPNB1, NUDT3, SFT2D2, and POMK. Our results revealed that less than 1% of all examined TCs show the same response in vag and flight-induced microgravity, while 38% of differentially regulated TCs identified during the hypergravity phase of the suborbital ballistic rocket flight could be verified with a 9× g ground centrifuge. In the 2D clinostat system, doing one full rotation per second, vector effects of the gravitational force are only nullified if the sensing mechanism requires 1 s or longer. Due to the fact that vag with an integration period of 1 s was not able to reproduce the results obtained in flight-induced microgravity, we conclude that the initial trigger of gene expression response to microgravity requires less than 1 s reaction time. Additionally, we discovered extensive gene expression differences caused by simple handling of the cell suspension in control experiments, which underlines the need for rigorous standardization regarding mechanical forces during cell culture experiments in general