Microbial responses to microgravity and other low-shear environments.

Microbial adaptation to environmental stimuli is essential for survival. While several of these stimuli have been studied in detail, recent studies have demonstrated an important role for a novel environmental parameter in which microgravity and the low fluid shear dynamics associated with microgravity globally regulate microbial gene expression, physiology, and pathogenesis. In addition to analyzing fundamental questions about microbial responses to spaceflight, these studies have demonstrated important applications for microbial responses to a ground-based, low-shear stress environment similar to that encountered during spaceflight. Moreover, the low-shear growth environment sensed by microbes during microgravity of spaceflight and during ground-based microgravity analogue culture is relevant to those encountered during their natural life cycles on Earth. While no mechanism has been clearly defined to explain how the mechanical force of fluid shear transmits intracellular signals to microbial cells at the molecular level, the fact that cross talk exists between microbial signal transduction systems holds intriguing possibilities that future studies might reveal common mechanotransduction themes between these systems and those used to sense and respond to low-shear stress and changes in gravitation forces. The study of microbial mechanotransduction may identify common conserved mechanisms used by cells to perceive changes in mechanical and/or physical forces, and it has the potential to provide valuable insight for understanding mechanosensing mechanisms in higher organisms. This review summarizes recent and future research trends aimed at understanding the dynamic effects of changes in the mechanical forces that occur in microgravity and other low-shear environments on a wide variety of important microbial parameters.     

Microbial Responses to Microgravity and Other Low-Shear Environments

Microbial adaptation to environmental stimuli is essential for survival. While several of these stimuli have been studied in detail, recent studies have demonstrated an important role for a novel environmental parameter in which microgravity and the low fluid shear dynamics associated with microgravity globally regulate microbial gene expression, physiology, and pathogenesis. In addition to analyzing fundamental questions about microbial responses to spaceflight, these studies have demonstrated important applications for microbial responses to a ground-based, low-shear stress environment similar to that encountered during spaceflight. Moreover, the low-shear growth environment sensed by microbes during microgravity of spaceflight and during ground-based microgravity analogue culture is relevant to those encountered during their natural life cycles on Earth. While no mechanism has been clearly defined to explain how the mechanical force of fluid shear transmits intracellular signals to microbial cells at the molecular level, the fact that cross talk exists between microbial signal transduction systems holds intriguing possibilities that future studies might reveal common mechanotransduction themes between these systems and those used to sense and respond to low-shear stress and changes in gravitation forces. The study of microbial mechanotransduction may identify common conserved mechanisms used by cells to perceive changes in mechanical and/or physical forces, and it has the potential to provide valuable insight for understanding mechanosensing mechanisms in higher organisms. This review summarizes recent and future research trends aimed at understanding the dynamic effects of changes in the mechanical forces that occur in microgravity and other low-shear environments on a wide variety of important microbial parameters.     

Forcing a connection: impacts of single-molecule force spectroscopy on in vivo tension sensing

Mechanical tension plays a large role in cell development ranging from morphology to gene expression. On the molecular level, the effects of tension can be seen in the dynamic arrangement of membrane proteins as well as the recruitment and activation of intracellular proteins. Forces applied to biopolymers during in vitro force measurements offer greater understanding of the effects of tension on molecules in live cells, and experimental techniques involving test tubes and live cells can often overlap. Indeed, when forces exerted on cellular components can be calibrated ex vivo with force spectroscopy, a powerful tool is available for researchers in probing cellular mechanotransduction on the molecular scale. This review will discuss the techniques used in measuring both cellular traction forces and single-molecule force spectroscopy. Emphasis will be placed on the use of fluorescence reporter systems for the development of in vivo tension sensors that can be used for calibration with single molecule force methods.     

T cell activation responses are differentially regulated during clinorotation and in spaceflight.

Studies of T lymphocyte activation with mitogenic lectins during spaceflight have shown a dramatic inhibition of activation as measured by DNA synthesis at 72 h, but the mechanism of this inhibition is unknown. We have investigated the progression of cellular events during the first 24 h of activation using both spaceflight microgravity culture and a ground-based model system that relies on the low shear culture environment of a rotating clinostat (clinorotation). Stimulation of human peripheral blood mononuclear cells (PBMCs) with soluble anti-CD3 (Leu4) in clinorotation and in microgravity culture shows a dramatic reduction in surface expression of the receptor for IL-2 (CD25) and CD69. An absence of bulk RNA synthesis in clinorotation indicates that stimulation with soluble Leu4 does not induce transition of T cells from G0 to the G1 stage of the cell cycle. However, internalization of the TCR by T cells and normal levels of IL-1 synthesis by monocytes indicate that intercellular interactions that are required for activation occur during clinorotation. Complementation of TCR-mediated signaling by phorbol ester restores the ability of PBMCs to express CD25 in clinorotation, indicating that a PKC-associated pathway may be compromised under these conditions. Bypassing the TCR by direct activation of intracellular pathways with a combination of phorbol ester and calcium ionophore in clinorotation resulted in full expression of CD25; however, only partial expression of CD25 occurred in microgravity culture. Though stimulation of purified T cells with Bead-Leu4 in microgravity culture resulted in the engagement and internalization of the TCR, the cells still failed to express CD25. When T cells were stimulated with Bead-Leu4 in microgravity culture, they were able to partially express CD69, a receptor that is constitutively stored in intracellular pools and can be expressed in the absence of new gene expression. Our results suggest that the inhibition of T cell proliferative response in microgravity culture is a result of alterations in signaling events within the first few hours of activation, which are required for the expression of important regulatory molecules.     

Low-shear-modeled microgravity-grown Penicillium chrysogenum -mediated biosynthesis of silver nanoparticles with enhanced antimicrobial activity and its anticancer effect in human liver cancer and fibroblast cells

Gravitational force and shear forces induce various changes in gene expression and metabolite production of microorganisms. Previous reports have shown that there are differences in the expression of different sets of proteins and enzymes under microgravity conditions compared to normal gravity. The aim of this study is to utilize culture filtrates of Penicillium chrysogenum grown under microgravity and normal conditions to synthesize silver nanoparticles and to examine whether there is any difference between their physiochemical and biological function. Synthesized nanoparticles were characterized using UV–Vis spectroscopy, FTIR, XRD, and TEM. Biological functional studies such as antimicrobial activity, cytotoxic studies, and anticancer activity were carried out. Antimicrobial activity was tested using antibiotic susceptibility testing by Kirby–Bauer method and cytotoxicity tests were carried out using 3T3-L1 normal fibroblasts cells and Hep-G2 cancer cell lines. Interestingly, our results indicated that microgravity-synthesized silver nanoparticles possess enhanced antibacterial activity and cytotoxic effect against cancer cells compared to normal gravity-synthesized silver nanoparticle. This work highlighted the importance of gravitational vector on the fungal enzyme profiles and their role in silver nanoparticle synthesis with enhanced biological activity.     

Nanostructure and force spectroscopy analysis of human peripheral blood CD4+ T cells using atomic force microscopy

To date, nanoscale imaging of the morphological changes and adhesion force of CD4⁺ T cells during in vitro activation remains largely unreported. In this study, we used atomic force microscopy (AFM) to study the morphological changes and specific binding forces in resting and activated human peripheral blood CD4⁺ T cells. The AFM images revealed that the volume of activated CD4⁺ T cells increased and the ultrastructure of these cells also became complex. Using a functionalized AFM tip, the strength of the specific binding force of the CD4 antigen-antibody interaction was found to be approximately three times that of the unspecific force. The adhesion forces were not randomly distributed over the surface of a single activated CD4⁺ T cell, indicated that the CD4 molecules concentrated into nanodomains. The magnitude of the adhesion force of the CD4 antigen-antibody interaction did not change markedly with the activation time. Multiple bonds involved in the CD4 antigen-antibody interaction were measured at different activation times. These results suggest that the adhesion force involved in the CD4 antigen-antibody interaction is highly selective and of high affinity.     

Changes in gene expression and signal transduction in microgravity

Studies from space flights over the past three decades have demonstrated that basic physiological changes occur in humans during space flight. These changes include cephalic fluid shifts, loss of fluid and electrolytes, loss of muscle mass, space motion sickness, anemia, reduced immune response, and loss of calcium and mineralized bone. The cause of most of these manifestations is not known and until recently, the general approach was to investigate general systemic changes, not basic cellular responses to microgravity. This laboratory has recently studied gene growth and activation of normal osteoblasts (MC3T3-El) during spaceflight. Osteoblast cells were grown on glass coverslips and loaded in the Biorack plunger boxes. The osteoblasts were launched in a serum deprived state, activated in microgravity and collected in microgravity. The osteoblasts were examined for changes in gene expression and signal transduction. Approximately one day after growth activation significant changes were observed in gene expression in 0-G flight samples. Immediate early growth genes/growth factors cox-2, c-myc, bcl2, TGF beta1, bFGF and PCNA showed a significant diminished mRNA induction in microgravity FCS activated cells when compared to ground and 1-G flight controls. Cox-1 was not detected in any of the samples. There were no significant differences in the expression of reference gene mRNA between the ground, 0-G and 1-G samples. The data suggest that quiescent osteoblasts are slower to enter the cell cycle in microgravity and that the lack of gravity itself may be a significant factor in bone loss in spaceflight. Preliminary data from our STS 76 flight experiment support our hypothesis that a basic biological response occurs at the tissue, cellular, and molecular level in 0-G. Here we examine ground-based and space flown data to help us understand the mechanism of bone loss in microgravity.     

Dome formation and tubule morphogenesis by Xenopus kidney A6 cell cultures exposed to microgravity simulated with a 3D-clinostat and to hypergravity

Confluent high-density cell cultures of A6 cells derived from adult male Xenopus kidney exhibit spontaneous dome-formation at 1 g. To determine whether this morphogenetic property is altered by gravity, we used a three-dimensional (3D) clinostat to subject the cells to simulated microgravity, and a centrifuge to subject them to hypergravity. We used the generation orbit control method as the new rotation control system of the 3D-clinostat, not the random method. The growth of A6 cells was significantly enhanced by hypergravity, but significantly reduced by simulated microgravity. Dome formation by A6 cells at high confluence was inhibited under simulated microgravity conditions, whereas hypergravity promoted dome formation and induced tubule morphogenesis, compared to the control at 1 g. These results indicated that changes in gravity influence the morphogenetic properties of A6 cells, such as dome formation and tubule morphogenesis. When dome formation by A6 cells at high confluence was induced spontaneously in the control 1 g culture, the gene expression of the HGF family of pleiotropic factors, such as HGF-like protein (HLP) and growth factor-Livertine (GF-l.ivertine), an epithelial serine protease of channel activating protease 1 (CAP1), and Na+, K+-adenosine triphosphatase (ATPase), increased. Simulated microgravity increased the gene expression of activin A and reduced the gene expression of HLP, GF-Livertine, CAP1, and Na+, K+-ATPase. Hypergravity, on the other hand, decreased the gene expression of activin A and increased the gene expression of HLP, GF-Livertine, CAP1, and Na+, K+-ATPase. These results suggest that the effects of gravitational changes on expression of the HGF family member gene, CAP1, and Na+, K+-ATPase gene may be important for the cell growth, tubule morphogenesis, and dome formation of A6 cells in altered     

模拟失重对白假丝酵母菌增殖和毒性的影响

【背景】近年来研究发现,失重条件可对一些致病微生物的增殖和毒性产生影响,白假丝酵母菌(Candida albicans)是典型的条件性致病真菌,在太空环境和人体中普遍存在,研究失重条件下白假丝酵母菌的增殖和毒性意义重大。【目的】利用旋转细胞培养系统(Rotary cell culture system,RCCS)模拟失重环境对白假丝酵母菌进行连续传代培养,检测模拟失重环境对白假丝酵母菌增殖情况、毒性以及基因表达的变化。【方法】将白假丝酵母菌接种在旋转生物反应器(High aspect rotating vessel,HARV)中,利用旋转细胞培养系统连续传代培养14 d,然后对菌株进行增殖速率测定、不同pH条件下增殖能力测定、生物膜相对形成能力测定和细胞毒性和动物毒力测定;利用转录组测序技术找出差异表达基因,结合性状分析模拟失重可能对白假丝酵母菌增殖和毒力的影响。【结果】与对照组相比,模拟失重组白假丝酵母菌对数期提前,增殖速率加快,在适宜pH条件下的增殖能力普遍提高,但其生物膜形成能力相对减弱,对LoVo细胞和小鼠的毒性减弱;转录组测序发现,模拟失重组共有280个基因表达差异达1.5倍以上(P0.05),其中248个上调、32个下调。差异基因经基因功能注释(Gene ontology,GO)和京都基因及基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)富集分析发现,相关胞膜形成及细胞分裂基因表达上调,生物膜形成、细胞黏附及共生粘连宿主基因表达下调。【结论】模拟失重环境可引起白假丝酵母菌增殖和毒性水平发生变化,相关改变可为研究失重环境对微生物的影响提供参考。     

Substrate rigidity and force define form through tyrosine phosphatase and kinase pathways

Cell forces define cell morphology, alterations in which are caused by tyrosine kinase and phosphatase mutations, which implies a causal linkage. Recent studies have shown that phosphotyrosine signaling is involved in force sensing for cells on flat surfaces. Early force-dependent activation of Src family kinases by phosphatases or cytoskeleton stretch leads to the activation of downstream signaling. In addition, force generation by cells depends on a feedback mechanism between matrix rigidity or force generation and myosin contractility. Components of the force-sensing pathway are linked to the integrin-cytoskeleton complex at sites of force application and serve as scaffolds for signaling processes. Thus, early events in force detection are mechanically induced cytoskeletal changes that result in biochemical signals to mechanoresponsive pathways that then regulate cell form.     

Nuclear responses to protein kinase C signal transduction are sensitive to gravity changes

A number of studies have suggested that gravity changes may influence mammalian cell growth and differentiation. To obtain insight in the molecular mechanisms underlying these effects, we have studied immediate early gene expression in response to activation of cytoplasmic signal transduction under microgravity conditions. In this paper we show that epidermal growth factor (EGF)- and 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced expression of the c-fos and c-jun protooncogenes is decreased in microgravity, while no effect of gravity changes was observed on A23187- and forskolin-induced expression of these genes. These decrease in c-fos expression was not due to delayed kinetics under microgravity. These results demonstrate that gravity differentially modulates distinctive signal transduction pathways.     

Microarray analysis of genes differentially expressed in hepG2 cells cultured in simulated microgravity: Preliminary report

Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.     

Mechanical force regulation of myofibroblast differentiation in cardiac fibroblasts

The myocardium responds to chronic pressure or volume overload by activation and proliferation of cardiac fibroblasts and their differentiation into myofibroblasts. Because alpha-smooth muscle actin (SMA) expression is the classical marker for myofibroblast differentiation, we examined force-induced SMA expression and regulation by specific MAPK pathways. Rat cardiac fibroblasts were separated from myocytes and smooth muscle cells, cultured, and phenotyped by using the presence of SMA, vimentin, and ED-A fibronectin and the absence of desmin as myofibroblast markers. Static tensile forces (0.65 pN/microm2) were applied to fibroblasts via collagen-coated magnetite beads. In neonatal cardiac fibroblasts cultured for 1 day, immunostaining and Western and Northern blotting showed very low basal levels of SMA. After the application of force, there were 1.5- to 2-fold increases of SMA protein and mRNA within 4 h. Force-induced SMA expression was dependent on ERK phosphorylation and on intact actin filaments. In contrast to cells cultured for 1 day, cells grown for 3 days on rigid substrates showed prominent stress fibers and high basal levels of SMA, which were reduced by 32% within 4 h after force application. ERK was not activated by force, but p38 phosphorylation was required for force-induced inhibition of SMA expression. These results indicate that mechanical force-induced regulation of SMA content is dependent on myofibroblast differentiation and by selective activation of MAPKs.     

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.     

Simulated microgravity activates MAPK pathways in fibroblasts cultured on microgrooved surface topography

This study evaluated in vitro the differences in morphological behaviour between fibroblast cultured on smooth and microgrooved substrata (groove depth: 0.5 mum, width: 1 mum), which were subjected to simulated microgravity. The aim of the study was to clarify which of these parameters was more dominant to determine cell behaviour. Morphological characteristics were investigated using scanning electron microscopy and fluorescence microscopy in order to obtain qualitative information on cell alignment. Expression of collagen type I, and alpha1-, beta1-, beta3-integrin were investigated by QPCR. Finally, immunoblotting was applied to visualise MAPK signalling pathways. Microscopy and image analysis showed that the fibroblasts aligned along the groove direction on all textured surfaces. On the smooth substrata, cells had spread out in a random fashion. The alignment of cells cultured on grooved surfaces under simulated microgravity, after 48 h of culturing appeared similar to those cultured at 1g, although cell shape was different. Analysis of variance proved that all main parameters: topography, gravity force, and time were significant. In addition, gene levels were reduced by simulated microgravity particularly those of beta3-integrin and collagen, however alpha-1 and beta-1 integrin levels were up-regulated. ERK1/2 was reduced in RPM, however, JNK/SAPK and p38 remained active. The members of the small GTPases family were stimulated under microgravity, particularly RhoA and Cdc42. The results are in agreement that application of microgravity to fibroblasts promotes a change in their morphological appearance and their expression of cell-substratum proteins through the MAPK intracellular signalling pathways.