Increased filamentous growth of Candida albicans in simulated microgravity

Knowledge of simulated microgravity （SMG）-induced changes in the pathogenicity of microorganisms is important for success of long-term spaceflight. In a previous study using the high aspect ratio vessel bioreactor, we showed that the yeast species Saccharomyces cerevisiae underwent a significant phenotypic response when grown in modeled microgravity, which was reflected in the analysis of gene expression profiles. In this study, we establish that Candida albicans responds to SMG in a similar fashion, demonstrating that there is a conserved response among yeast to this environmental stress. We also report that the growth of C. albicans in SMG results in a morphogenic switch that is consistent with enhanced pathogenicity. Specifically, we observed an increase in filamentous forms of the organism and accompanying changes in the expression of two genes associated with the yeasthyphal transition. The morphological response may have significant implications for astronauts＇ safety, as the fungal pathogen may become more virulent during spaceflight.     

模拟微重力对骨髓间充质干细胞增殖及其向脂肪方向分化能力的影响

目的：探讨模拟微重力（SMG）对骨髓间充质干细胞（MSCs）的增殖及向脂肪方向分化能力的影响。方法：第一部分将第三代的MSCs分为两组,分别在正常重力下（NG组）及微重力下（SMG组,采用回转模拟装置以30r/min回转模拟微重力）,培养72h后,采用BrdU标记法检测两组细胞的增殖情况,细胞计数法绘制细胞生长曲线。Western Blot检测干细胞标志物Oct4、SSEA4的表达情况,第二部分将第三代MSCs分为三组：第一组在NG条件下培养后,加入脂肪方向诱导剂在NG条件下诱导、第二组在SMG条件下培养,在NG条件下诱导,第三组在SMG条件下培养,在SMG条件下诱导。7天后,油红O染色观察脂肪方向的诱导率,Western Blot检测过氧化物酶增殖物激活受体γ2（PPARγ2）以及Oct4的表达。结果：第一部分：流式细胞仪检测SMG组BrdU标记阳性率明显高于NG组,表明细胞增殖较快,Western Blot结果显示SMG组细胞中Oct4、SSEA4的表达量明显高于NG组,有统计学意义。第二部分：脂肪方向诱导后第一组细胞油红O染色阳性,Western Blot显示PPARγ2呈阳性表达,Oct4仅有微量表达,第二组油红O染色阳性表达率明显高于第一组,且PPARγ2表达较第一组增多,几乎未见Oct4的表达,第三组细胞油红O染色阴性,且几乎不表达PPARγ2,而Oct4表达较前两组升高。结论：模拟微重力可促进骨髓间充质干细胞增殖,提高其向脂肪方向分化的能力可能与微重力保持其未分化状态相关。     

Genome-wide gene expression profiling of microgravity effect on human liver cells

Human exposure to microgravity is considered the major environmental factor of space flight that affects cells and tissues causing adverse effects to human health. Ground-based gravity-simulation experiments at the cellular and molecular levels have gained some insight into the underlying molecular and cellular alterations induced by microgravity. However, systematic study and detailed molecular mechanisms of the adverse effect of microgravity on living cells are still lacking. The main objective of this study was to apply DNA microarray technology in time-course experiments for genome-wide search of genes whose expression are altered by microgravity, as part of the effort in the identification of major space genes. In this study, we analyzed global gene expression profiles for a human liver cell line exposed to a ground-based modeled microgravity system for 1, 3, and 4 days using the rotary cell culture system (RCCS) and the Agilent 22k human oligo DNA microarrays. We have found that 139 genes' mRNA levels were significantly (P < or = 0.01) altered by the microgravity exposures. Some of these identified genes were further verified by Northern analysis.     

Coordinate expression of amplified metallothionein I and II genes in cadmium-resistant Chinese hamster cells.

Recombinant DNA probes complementary to Chinese hamster metallothionein (MT)-1 and MT-2 mRNAs were used to compare MT gene copy numbers, zinc-induced MT mRNA levels, and uninduced MT mRNA levels in cadmium-resistant (Cdr) Chinese hamster ovary cell lines. Quantitative hybridization analyses determined that the MT-1 and MT-2 genes are each present at approximately single-copy levels in the genome of cell line Cdr2C10 and are coordinately amplified approximately 7, 3, and 12 times over the Cdr2C10 value in the genomes of cell lines Cdr20F4, Cdr30F9, and Cdr200T1, respectively. The maximum zinc-induced MT-1 mRNA concentrations in cell lines Cdr20F4, Cdr30F9, and Cdr200T1 were equal to 1, 3, and 15 times that measured in Cdr2C10, respectively. Similarly, the maximum zinc-induced MT-2 mRNA concentrations were equal to 1, 3, and 14 times that measured in Cdr2C10, respectively, and in each instance they were 90 to 150 times greater than their respective concentrations in uninduced cells. Thus, relative MT gene numbers are closely correlated with both zinc-induced and uninduced MT mRNA levels in Cdr2C10, Cdr30F9, and Cdr200T1, but not in Cdr20F4. Each of the latter two lines possesses structurally altered chromosomes whose breakpoints are near the MT locus. Nonetheless, the ratio of the levels of MT-1 to MT-2 mRNAs was constant in each of the four cell lines, including Cdr20F4. These results demonstrate that MT-1 and MT-2 mRNAs are induced coordinately in each Cdr cell line. Therefore, the coordination of the induction of MT-1 and MT-2 mRNA is independent of MT gene amplification, MT gene rearrangement, and the relative inducibilities of amplified MT genes. However, MT mRNA and protein levels each indicate that MT-1 and MT-2 expression is non-coordinate in uninduced cells. Thus, regulation of MT expression may involve two different mechanisms which are differentially operative in induced and uninduced cells.     

Androgen dependence of specific kallikrein gene family members expressed in rat prostate

We have used oligonucleotide probes specific for members of the rat kallikrein/tonin gene family (PS, S1, S2, S3, K1, and P1) to establish which arginyl esteropeptidase (kallikrein-like) genes are expressed in the prostate. We have also compared the expression and androgen dependence of these genes in prostate, submaxillary gland (SMG) and kidney. Only S3 (tonin-like) and P1 (kallikrein-like) are expressed in the prostate, with S3 very much more abundant. Prostatic S3 mRNA disappears after 8 days castration and is restored to intact levels by dihydrotestosterone (DHT) but not estradiol benzoate (EB) for 8 days. Prostate P1 mRNA levels were similarly but not identically affected. All six genes are expressed in the SMG, with PS (true kallikrein) the most abundant. Levels of PS mRNA in SMG are unaffected by castration, DHT, or EB treatment, although mRNA levels of other kallikrein-like (S1, K1, and P1), tonin (S2), and tonin-like (S3) genes fall 40-60% after castration, and are unaffected or partially restored by DHT and/or EB administration. Only PS and K1 are expressed in the kidney, at much lower levels than in the SMG and unaffected by castration or steroids. These studies thus confirm and extend the concept of tissue specificity of arginyl esteropeptidase gene expression, and further demonstrate that the same gene(s) is differentially regulated by androgens in the rat prostate, SMG, and kidney.     

Cytoskeleton structure and adhesion properties of human stromal precursors under conditions of simulated microgravity

During spaceflight and in simulated microgravity (SMG), cytoskeleton rearrangements were observed in lymphocytes, glial cells and osteoblasts. One potential mechanism for the cytoskeletal gravisensitivity of cells is the disruption of the extracellular matrix and integrin interactions. We investigated the effect of SMG on the structure of the actin cytoskeleton, distribution of cellular vinculin, the expression of some integrin subtypes and cellular adhesion molecules in cultured mesenchymal stem cells (hMSCs) derived from human bone marrow in vitro. Simulated microgravity was produced by desktop RPM equipment (Dutch Space, Netherlands). Cells were exposed to simulated microgravity for 30 min to 120 h. The results showed that the actin cytoskeleton was reorganized very quickly (30 min). Later (6, 24, and 48 h), the number of cells with disrupted actin cytoskeletons was increased; however, after 120 h of exposure, cells partly regained their F-actin structures. RPM exposure augmented the number of cells that express integrin-α2. We also observed a decrease in the number of VCAM-1-positive cells and changes in the expression of ICAM-1. Our findings indicate that SMG induces reversible microfilament reorganization in hMSCs and alters their adhesion properties.     

Gravity, a regulation factor in the differentiation of rat bone marrow mesenchymal stem cells

Background

Stem cell therapy has emerged as a potential therapeutic option for tissue engineering and regenerative medicine, but many issues remain to be resolved, such as the amount of seed cells, committed differentiation and the efficiency. Several previous studies have focused on the study of chemical inducement microenvironments. In the present study, we investigated the effects of gravity on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into force-sensitive or force-insensitive cells.

Methods and results

Rat BMSCs (rBMSCs) were cultured under hypergravity or simulated microgravity (SMG) conditions with or without inducement medium. The expression levels of the characteristic proteins were measured and analyzed using immunocytochemical, RT-PCR and Western-blot analyses. After treatment with 5-azacytidine and hypergravity, rBMSCs expressed more characteristic proteins of cardiomyocytes such as cTnT, GATA4 and β-MHC; however, fewer such proteins were seen with SMG. After treating rBMSCs with osteogenic inducer and hypergravity, there were marked increases in the expression levels of ColIA1, Cbfa1 and ALP. Reverse results were obtained with SMG. rBMSCs treated with adipogenic inducer and SMG expressed greater levels of PPARgamma. Greater levels of Cbfa1- or cTnT-positive cells were observed under hypergravity without inducer, as shown by FACS analysis. These results indicate that hypergravity induces differentiation of rBMSCs into force-sensitive cells (cardiomyocytes and osteoblasts), whereas SMG induces force-insensitive cells (adipocytes).

Conclusion

Taken together, we conclude that gravity is an important factor affecting the differentiation of rBMSCs; this provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated or undifferentiated cells.     

Light Control of Arabidopsis Development Entails Coordinated Regulation of Genome Expression and Cellular Pathways

An expressed sequence tag-based microarray was used to profile genome expression underlying light control of Arabidopsis development. Qualitatively similar gene expression profiles were observed among seedlings grown in different light qualities, including far-red, red, and blue light, which are mediated primarily by phytochrome A, phytochrome B, and the cryptochromes, respectively. Furthermore, light/dark transitions also triggered similar differential genome expression profiles. Most light treatments also resulted in distinct expression profiles in small fractions of the expressed sequence tags examined. The similarly regulated genes in all light conditions were estimated to account for approximately one-third of the genome, with three-fifths upregulated and two-fifths downregulated by light. Analysis of those light-regulated genes revealed more than 26 cellular pathways that are regulated coordinately by light. Thus, light controls Arabidopsis development through coordinately regulating metabolic and regulatory pathways.     

Global expression of simulated microgravity-responsive genes in Xenopus liver cells

The random positioning machine (RPM) is a method used to generate a simulated-microgravity environment at approximately 0 g. Using an RPM, we analyzed the global gene expression of A8 cells derived from the liver of adult Xenopus laevis. A range of genes on a Xenopus 44K-scale microarray were up- or downregulated two-fold or more: 43 genes (up, 36 genes; down, 7 genes) on culture day 5 in RPM, 74 genes (up, 48 genes; down, 26 genes) on day 8, 105 genes (up, 71 genes; down, 34 genes) on day 10, and 132 genes (up, 98 genes; down, 34 genes) on day 15. Five genes were upregulated two-fold or more throughout culturing in RPM, while only one gene was downregulated over the entire time. We then compared the expression patterns of the RPM-dependent genes in the A8 cells with those in A6 cells established from the kidney of adult Xenopus laevis. Six upregulated genes and three downregulated genes showed the same expression patterns throughout the culturing of A6 and A8 cells in RPM. Such globally responsive genes may play a common role in the cell response to simulated microgravity. We were particularly interested in the downregulation of SPARC in both cell types in RPM, which supported previous observations from simulated-microgravity experiments on earth or microgravity in space. We conclude that SPARC is plays a key role in the response of a cell to microgravity.     

Role and regulation of sigma S in general resistance conferred by low-shear simulated microgravity in Escherichia coli

Life on Earth evolved in the presence of gravity, and thus it is of interest from the perspective of space exploration to determine if diminished gravity affects biological processes. Cultivation of Escherichia coli under low-shear simulated microgravity (SMG) conditions resulted in enhanced stress resistance in both exponential- and stationary-phase cells, making the latter superresistant. Given that microgravity of space and SMG also compromise human immune response, this phenomenon constitutes a potential threat to astronauts. As low-shear environments are encountered by pathogens on Earth as well, SMG-conferred resistance is also relevant to controlling infectious disease on this planet. The SMG effect resembles the general stress response on Earth, which makes bacteria resistant to multiple stresses; this response is sigma s dependent, irrespective of the growth phase. However, SMG-induced increased resistance was dependent on sigma s only in stationary phase, being independent of this sigma factor in exponential phase. sigma s concentration was some 30% lower in exponential-phase SMG cells than in normal gravity cells but was twofold higher in stationary-phase SMG cells. While SMG affected sigma s synthesis at all levels of control, the main reasons for the differential effect of this gravity condition on sigma s levels were that it rendered the sigma protein less stable in exponential phase and increased rpoS mRNA translational efficiency. Since sigma s regulatory processes are influenced by mRNA and protein-folding patterns, the data suggest that SMG may affect these configurations.     

Structural analysis of 5'-flanking regions of rat, mouse and human renin genes reveals the presence of a transposable-like element in the two mouse genes

The two renin genes of the mouse (Ren1 and Ren2) are expressed at different levels in the submaxillary gland (SMG). In contrast to mice, there is no detectable renin gene expression in the rat SMG. To determine the molecular basis for these different levels of renin expression, we have compared the 5'-flanking regions of the rat and mouse genes. The sequence of mouse, but not rat, genes reveals the presence in Ren1 and Ren2 of a large insertion, probably a new class of transposable elements. A second, apparently unrelated shorter insertion is present only in Ren2. Otherwise, the mouse and rat 5'-flanking sequences are well conserved and resemble the corresponding region of the human Ren gene, indicating that the insertions occurred after the separation of the rat and mouse species but before the duplication of the mouse Ren gene. We suggest that these structural differences may have a role in the differential expression of the Ren genes in mice and other animals.     

The simulated microgravity enhances multipotential differentiation capacity of bone marrow mesenchymal stem cells

Multi-differentiation capability is an essential characteristic of bone marrow mesenchymal stem cells (BMSCs). Method on obtaining higher-quality stem cells with an improved differentiation potential has gained significant attention for the treatment of clinical diseases and developmental biology. In our study, we investigated the multipotential differentiation capacity of BMSCs under simulated microgravity (SMG) condition. F-actin staining found that cytoskeleton took on a time-dependent change under SMG condition, which caused spindle to round morphological change of the cultured cells. Quantitative PCR and Western Blotting showed the pluripotency marker OCT4 was up-regulated in the SMG condition especially after SMG of 72 h, which we observed would be the most appropriate SMG duration for enhancing pluripotency of BMSCs. After dividing BMSCs into normal gravity (NG) group and SMG group, we induced them respectively in endothelium oriented, adipogenic and neuronal induction media. Immunostaining and Western Blotting found that endothelium oriented differentiated BMSCs expressed higher VWF and CD31 in the SMG group than in the NG group. The neuron-like cells derived from BMSCs in the SMG group also expressed higher level of MAP2 and NF-H. Furthermore, the quantity of induced adipocytes increased in the SMG group compared to the NG group shown by Oil Red O staining, The expression of PPARγ2 increased significantly under SMG condition. Therefore, we demonstrated that SMG could promote BMSCs to differentiate into many kinds of cells and predicted that enhanced multi-potential differentiation capacity response in BMSCs following SMG might be relevant to the changes of cytoskeleton and the stem cell marker OCT4.     

Transcriptional analysis of normal human fibroblast responses to microgravity stress

To understand the molecular mechanism（s） of how spaceflight affects cellular signaling pathways, quiescent normal human WI-38 fibroblasts were flown on the STS-93 space shuttle mission. Subsequently, RNA samples from the spaceflown and ground-control cells were used to construct two cDNA libraries, which were then processed for suppression subtractive hybridization （SSH） to identify spaceflight-specific gene expression. The SSH data show that key genes related to oxidative stress, DNA repair, and fatty acid oxidation are activated by spaceflight, suggesting the induction of cellular oxidative stress. This is further substantiated by the up-regulation of neuregulin 1 and the calcium-binding protein calmodulin 2. Another obvious stress sign is that spaceflight evokes the Ras/mitogen-activated protein kinase and phosphatidylinositol-3 kinase signaling pathways, along with up-regulating several Gl-phase cell cycle traverse genes. Other genes showing upregulation of expression are involved in protein synthesis and pro-apoptosis, as well as pro-survival. Interactome analysis of functionally related genes shows that c-Myc is the ＂hub＂ for those genes showing significant changes. Hence, our results suggest that microgravity travel may impact changes in gene expression mostly associated with cellular stress signaling, directing cells to either apoptotic death or premature senescence.     

Gene expression changes induced by space flight in single-cells of the fern <Emphasis Type="Italic">Ceratopteris richardii</Emphasis>

This work describes a rare high-throughput evaluation of gene expression changes induced by space flight in a single plant cell. The cell evaluated is the spore of the fern Ceratopteris richardii, which exhibits both perception and response to gravity. cDNA microarray and Q RT-PCR analysis of spores germinating in microgravity onboard NASA space shuttle flight STS-93 revealed changes in the mRNA expression of roughly 5% of genes analyzed. These gene expression changes were compared with gene expression changes that occur during gravity perception and response in animal cells and multicellular plants. Our data contribute to a better understanding of the impact of space flight conditions, including microgravity, on cellular growth and development, and provide insights into the adaptive strategies of individual cells in response to these conditions.