Changes in Gravitaxis and Gene-Expression in an Euglena gracilis Culture over Time.

Age-dependent changes in the transcription levels of 5-day-old Euglena gracilis cells, which showed positive gravitaxis, 6-day-old cells without gravitactic orientation, and older cells (9- and 11-day-old, which displayed a precise negative gravitaxis) were determined through microarray analysis. Hierarchical clustering of four independent cell cultures revealed pronounced similarities in transcription levels at the same culture age, which proves the reproducibility of the cultivation method. Employing the non-oriented cells from the 6-day-old culture as a reference, about 2779 transcripts were found to be differentially expressed. While positively gravitactic cells (5-day-old culture) showed only minor differences in gene expression compared to the 6-day reference, pronounced changes of mRNAs (mainly an increase) were found in older cells compared to the reference culture. Among others, genes coding for adenylyl cyclases, photosynthesis, and metabolic enzymes were identified to be differentially expressed. The investigated cells were grown in batch cultures, so variations in transcription levels most likely account for factors such as nutrient depletion in the medium and self-shading. Based on these findings, a particular transcript (e.g., transcript 19556) was downregulated using the RNA interference technique. Gravitaxis and phototaxis were impaired in the transformants, indicating the role of this transcript in signal transduction. Results of the experiment are discussed regarding the increasing importance of E. gracilis in biotechnology as a source of valuable products and the possible application of E. gracilis in life-support systems.

Current Knowledge about the Impact of Microgravity on Gene Regulation.

Microgravity (µg) has a massive impact on the health of space explorers. Microgravity changes the proliferation, differentiation, and growth of cells. As crewed spaceflights into deep space are being planned along with the commercialization of space travelling, researchers have focused on gene regulation in cells and organisms exposed to real (r-) and simulated (s-) µg. In particular, cancer and metastasis research benefits from the findings obtained under µg conditions. Gene regulation is a key factor in a cell or an organism's ability to sustain life and respond to environmental changes. It is a universal process to control the amount, location, and timing in which genes are expressed. In this review, we provide an overview of µg-induced changes in the numerous mechanisms involved in gene regulation, including regulatory proteins, microRNAs, and the chemical modification of DNA. In particular, we discuss the current knowledge about the impact of microgravity on gene regulation in different types of bacteria, protists, fungi, animals, humans, and cells with a focus on the brain, eye, endothelium, immune system, cartilage, muscle, bone, and various cancers as well as recent findings in plants. Importantly, the obtained data clearly imply that µg experiments can support translational medicine on Earth.

Polyethylenimine Increases Antibacterial Efficiency of Chlorophyllin.

Polyethylenimines (PEIs), a group of polycationic molecules, are known to impair the outer membrane of Gram-negative bacteria and exhibit antimicrobial activity. The outer membrane of Gram-negative strains hinders the uptake of photosensitizer chlorophyllin. In this study, we report chlorophyllin and branched PEI combinations' activity against Escherichia coli strains DH5α and RB791, Salmonella enterica sv. Typhimurium LT2, and Bacillus subtilis 168. The minimal bactericidal concentration (MBC) was determined by plating cells treated with different concentrations of PEI and chlorophyllin on agar and monitoring their growth after 24 h. All tested combinations of PEI and chlorophyllin were lethal for S. enterica after 240 min of incubation in light, whereas PEI alone (<100 µg mL−1) was ineffective. In the darkness, complete inhibition was noted with a combination of ≥2.5 µg mL−1 chlorophyllin and 50 µg mL−1 PEI. If applied alone, PEI alone of ≥800 µg mL−1 of PEI was required to completely inactivate E. coli DH5α cells in light, whereas with ≥5 µg mL−1 chlorophyllin, only ≥100 µg mL−1 PEI was needed. No effect was detected in darkness with PEI alone. However, 1600 µg mL−1 PEI in combination with 2.5 µg mL−1 resulted in complete inactivation after 4 h dark incubation. PEI alone did not inhibit E. coli strain RB791, while cells were inactivated when treated with 10 µg mL−1 chlorophyllin in combination with ≥100 µg mL−1 (in light) or ≥800 µg mL−1 PEI (in darkness). Under illumination, B. subtilis was inactivated at all tested concentrations. In the darkness, 1 µg mL−1 chlorophyllin and 12.5 µg mL−1 PEI were lethal for B. subtilis. Overall, PEI can be used as an antimicrobial agent or potentiating agent for ameliorating the antimicrobial activity of chlorophyllin.

Latest knowledge about changes in the proteome in microgravity.

INTRODUCTION: A long-term stay of humans in space causes health problems and changes in protists and plants. Deep space exploration will increase the time humans or rodents will spend in microgravity (µg). Moreover, they are exposed to cosmic radiation, hypodynamia, and isolation. OMICS investigations will increase our knowledge of the underlying mechanisms of µg-induced alterations in vivo and in vitro. AREAS COVERED: We summarize the findings over the recent 3 years on µg-induced changes in the proteome of protists, plants, rodent, and human cells. Considering the thematic orientation of microgravity-related publications in that time frame, we focus on medicine-associated findings, such as the µg-induced antibiotic resistance of bacteria, the myocardial consequences of µg-induced calpain activation, and the role of MMP13 in osteoarthritis. All these point to the fact that µg is an extreme stressor that could not be evolutionarily addressed on Earth. EXPERT OPINION: In conclusion, when interpreting µg-experiments, the direct, mostly unspecific stress response, must be distinguished from specific µg-effects. For this reason, recent studies often do not consider single protein findings but place them in the context of protein-protein interactions. This enables an estimation of functional relationships, especially if these are supported by epigenetic and transcriptional data (multi-omics).

Natural Products in Modern Biology: Ancient Wisdom for Today's Challenges.

Nature provides a unique diversity of primary and secondary metabolites [...].

Influence of Microgravity on Apoptosis in Cells, Tissues, and Other Systems In Vivo and In Vitro.

All life forms have evolved under the constant force of gravity on Earth and developed ways to counterbalance acceleration load. In space, shear forces, buoyance-driven convection, and hydrostatic pressure are nullified or strongly reduced. When subjected to microgravity in space, the equilibrium between cell architecture and the external force is disturbed, resulting in changes at the cellular and sub-cellular levels (e.g., cytoskeleton, signal transduction, membrane permeability, etc.). Cosmic radiation also poses great health risks to astronauts because it has high linear energy transfer values that evoke complex DNA and other cellular damage. Space environmental conditions have been shown to influence apoptosis in various cell types. Apoptosis has important functions in morphogenesis, organ development, and wound healing. This review provides an overview of microgravity research platforms and apoptosis. The sections summarize the current knowledge of the impact of microgravity and cosmic radiation on cells with respect to apoptosis. Apoptosis-related microgravity experiments conducted with different mammalian model systems are presented. Recent findings in cells of the immune system, cardiovascular system, brain, eyes, cartilage, bone, gastrointestinal tract, liver, and pancreas, as well as cancer cells investigated under real and simulated microgravity conditions, are discussed. This comprehensive review indicates the potential of the space environment in biomedical research.

Exploration of space to achieve scientific breakthroughs.

Living organisms adapt to changing environments using their amazing flexibility to remodel themselves by a process called evolution. Environmental stress causes selective pressure and is associated with genetic and phenotypic shifts for better modifications, maintenance, and functioning of organismal systems. The natural evolution process can be used in complement to rational strain engineering for the development of desired traits or phenotypes as well as for the production of novel biomaterials through the imposition of one or more selective pressures. Space provides a unique environment of stressors (e.g., weightlessness and high radiation) that organisms have never experienced on Earth. Cells in the outer space reorganize and develop or activate a range of molecular responses that lead to changes in cellular properties. Exposure of cells to the outer space will lead to the development of novel variants more efficiently than on Earth. For instance, natural crop varieties can be generated with higher nutrition value, yield, and improved features, such as resistance against high and low temperatures, salt stress, and microbial and pest attacks. The review summarizes the literature on the parameters of outer space that affect the growth and behavior of cells and organisms as well as complex colloidal systems. We illustrate an understanding of gravity-related basic biological mechanisms and enlighten the possibility to explore the outer space environment for application-oriented aspects. This will stimulate biological research in the pursuit of innovative approaches for the future of agriculture and health on Earth.

Changes of Gene Expression in Euglena gracilis Obtained During the 29th DLR Parabolic Flight Campaign.

Parabolic flight maneuvers of Novespace's Airbus A310 ZERO-G produce subsequent phases of hypergravity (about 20 s), microgravity (about 22 s) and another 20 s hypergravity on experiments located in the experiment area of the aircraft. The 29th DLR parabolic flight campaign consisted of four consecutive flight days with thirty-one parabolas each day. Euglena gracilis cells were fixed with TRIzol during different acceleration conditions at the first and the last parabola of each flight. Samples were collected and analyzed with microarrays for one-color gene expression analysis. The data indicate significant changes in gene expression in E. gracilis within short time. Hierarchical clustering shows that changes induced by the different accelerations yield reproducible effects at independent flight days. Transcription differed between the first and last parabolas indicating adaptation effects in the course of the flight. Different gene groups were found to be affected in different phases of the parabolic flight, among others, genes involved in signal transduction, calcium signaling, transport mechanisms, metabolic pathways, and stress-response as well as membrane and cytoskeletal proteins. In addition, transcripts of other areas, e.g., DNA and protein modification, were altered. The study contributes to the understanding of short-term effects of microgravity and different accelerations on cells at a molecular level.

Using Colistin as a Trojan Horse: Inactivation of Gram-Negative Bacteria with Chlorophyllin.

Colistin (polymyxin E) is a membrane-destabilizing antibiotic used against Gram-negative bacteria. We have recently reported that the outer membrane prevents the uptake of antibacterial chlorophyllin into Gram-negative cells. In this study, we used sub-toxic concentrations of colistin to weaken this barrier for a combination treatment of Escherichia coli and Salmonella enterica serovar Typhimurium with chlorophyllin. In the presence of 0.25 µg/mL colistin, chlorophyllin was able to inactivate both bacteria strains at concentrations of 5-10 mg/L for E. coli and 0.5-1 mg/L for S. Typhimurium, which showed a higher overall susceptibility to chlorophyllin treatment. In accordance with a previous study, chlorophyllin has proven antibacterial activity both as a photosensitizer, illuminated with 12 mW/cm2, and in darkness. Our data clearly confirmed the relevance of the outer membrane in protection against xenobiotics. Combination treatment with colistin broadens chlorophyllin's application spectrum against Gram-negatives and gives rise to the assumption that chlorophyllin together with cell membrane-destabilizing substances may become a promising approach in bacteria control. Furthermore, we demonstrated that colistin acts as a door opener even for the photodynamic inactivation of colistin-resistant (mcr-1-positive) E. coli cells by chlorophyllin, which could help us to overcome this antimicrobial resistance.

Disproval of the Starch-Amyloplast Hypothesis?

In a recent publication, Edelmann (Protoplasma 2018; 255,1877-1881) refuted the well-established starch-amyloplast hypothesis of gravitropism in plants. Gravitropic curvatures of shoots and roots were still present after amyloplast-containing tissues (in sheath of vascular bundles and root caps) were dissected. Here, we discuss Edelmann's data in the light of Popper's falsification principle.

What an Escherichia coli Mutant Can Teach Us About the Antibacterial Effect of Chlorophyllin.

Due to the increasing development of antibiotic resistances in recent years, scientists search intensely for new methods to control bacteria. Photodynamic treatment with porphyrins such as chlorophyll derivatives is one of the most promising methods to handle bacterial infestation, but their use is dependent on illumination and they seem to be more effective against Gram-positive bacteria than against Gram-negatives. In this study, we tested chlorophyllin against three bacterial model strains, the Gram-positive Bacillus subtilis 168, the Gram-negative Escherichia coli DH5α and E. coli strain NR698 which has a deficient outer membrane, simulating a Gram-negative "without" its outer membrane. Illuminated with a standardized light intensity of 12 mW/cm², B. subtilis showed high sensitivity already at low chlorophyllin concentrations (≤105 cfu/mL: ≤0.1 mg/L, 106⁻108 cfu/mL: 0.5 mg/L), whereas E. coli DH5α was less sensitive (≤105 cfu/mL: 2.5 mg/L, 106 cfu/mL: 5 mg/L, 107⁻108 cfu/mL: ineffective at ≤25 mg/L chlorophyllin). E. coli NR698 was almost as sensitive as B. subtilis against chlorophyllin, pointing out that the outer membrane plays a significant role in protection against photodynamic chlorophyllin impacts. Interestingly, E. coli NR698 and B. subtilis can also be inactivated by chlorophyllin in darkness, indicating a second, light-independent mode of action. Thus, chlorophyllin seems to be more than a photosensitizer, and a promising substance for the control of bacteria, which deserves further investigation.

Current knowledge about the impact of microgravity on the proteome.

Introduction: Microgravity (µg) is an extreme stressor for plants, animals, and humans and influences biological systems. Humans in space experience various health problems during and after a long-term stay in orbit. Various studies have demonstrated structural alterations and molecular biological changes within the cellular milieu of plants, bacteria, microorganisms, animals, and cells. These data were obtained by proteomics investigations applied in gravitational biology to elucidate changes in the proteome occurring when cells or organisms were exposed to real µg (r-µg) and simulated µg (s-µg). Areas covered: In this review, we summarize the current knowledge about the impact of µg on the proteome in plants, animals, and human cells. The literature suggests that µg impacts the proteome and thus various biological processes such as angiogenesis, apoptosis, cell adhesion, cytoskeleton, extracellular matrix proteins, migration, proliferation, stress response, and signal transduction. The changes in cellular function depend on the respective cell type. Expert commentary: This data is important for the topics of gravitational biology, tissue engineering, cancer research, and translational regenerative medicine. Moreover, it may provide new ideas for countermeasures to protect the health of future space travelers.

Long term stability of Oligo (dT) 25 magnetic beads for the expression analysis of Euglena gracilis for long term space projects.

The unicellular freshwater flagellate Euglena gracilis has a highly developed sensory system. The cells use different stimuli such as light and gravity to orient themselves in the surrounding medium to find areas for optimal growth. Due to the ability to produce oxygen and consume carbon dioxide, Euglena is a suitable candidate for life support systems. Participation in a long-term space experiment would allow for the analysis of changes and adaptations to the new environment, and this could bring new insights into the mechanism of perception of gravity and the associated signal transduction chain. For a molecular analysis of transcription patterns, an automated system is necessary, capable of performing all steps from taking a sample, processing it and generating data. One of the developmental steps is to find long-term stable reagents and materials and test them for stability at higher-than-recommended temperature conditions during extended storage time. We investigated the usability of magnetic beads in an Euglena specific lysis buffer after addition of the RNA stabilizer Dithiothreitol over 360 days and the lysis buffer with the stabilizer alone over 455 days at the expected storage temperature of 19 °C. We can claim that the stability is not impaired at all after an incubation period of over one year. This might be an interesting result for researchers who have to work under non-standard lab conditions, as in biological or medicinal fieldwork.

Influence of different light-dark cycles on motility and photosynthesis of Euglena gracilis in closed bioreactors.

Abstract The unicellular photosynthetic freshwater flagellate Euglena gracilis is a promising candidate as an oxygen producer in biological life-support systems. In this study, the capacity of Euglena gracilis to cope with different light regimes was determined. Cultures of Euglena gracilis in closed bioreactors were exposed to different dark-light cycles (40 W/m(2) light intensity on the surface of the 20 L reactor; cool white fluorescent lamps in combination with a 100 W filament bulb): 1 h-1 h, 2 h-2 h, 4 h-4 h, 6 h-6 h, and 8 h-16 h, respectively. Motility and oxygen development in the reactors were measured constantly. It was found that, during exposure to light-dark cycles of 1 h-1 h, 2 h-2 h, 4 h-4 h, and 6 h-6 h, precision of gravitaxis as well as the number of motile cells increased during the dark phase, while velocity increased in the light phase. Oxygen concentration did not yet reach a plateau phase. During dark-light cycles of 8 h-16 h, fast changes of movement behavior in the cells were detected. The cells showed an initial decrease of graviorientation after onset of light and an increase after the start of the dark period. In the course of the light phase, graviorientation increased, while motility and velocity decreased after some hours of illumination. In all light profiles, Euglena gracilis was able to produce sufficient oxygen in the light phase to maintain the oxygen concentration above zero in the subsequent dark phase.

The impact of microgravity-based proteomics research.

Proteomics is performed in microgravity research in order to determine protein alterations occurring qualitatively and quantitatively, when single cells or whole organisms are exposed to real or simulated microgravity. To this purpose, antibody-dependent (Western blotting, flow cytometry, Luminex(®) technology) and antibody-independent (mass spectrometry, gene array) techniques are applied. The anticipated findings will help to understand microgravity-specific behavior, which has been observed in bacteria, as well as in plant, animal and human cells. To date, the analyses revealed that cell cultures are more sensitive to microgravity than cells embedded in organisms and that proteins changing under microgravity are highly interactive. Furthermore, one has to distinguish between primary gravity-induced and subsequent interaction-dependent changes of proteins, as well as between direct microgravity-related effects and indirect stress responses. Progress in this field will impact on tissue engineering and medicine and will uncover possibilities of counteracting alterations of protein expression at lowered gravity.

Influence of temperature and UVR on photosynthesis and morphology of four species of cyanobacteria.

During the late austral spring of 2009 we carried out experiments (4days of duration) with four cyanobacteria species, Anabaena sp., Nostoc sp., Arthrospira platensis and Microcystis sp., to assess the combined effects of temperature and solar radiation on photosynthesis performance and morphology. Two experimental temperatures (18°C and 23°C, simulating a 5°C increase under a scenario of climate change) and three radiation treatments (by using different filters/materials) were implemented: (i) P (PAR, 400-700nm), (ii) PA (PAR+UV-A, 320-700nm) and, (iii) PAB (PAR+UV-A+UV-B, 280-700nm). In general, samples under the P treatment had less decrease/higher recovery rates of effective photochemical quantum yield (Y) than those receiving UV-A or UV-A+UV-B. The effects of increased temperature were species-specific: At the end of the experiments, it was seen that increased temperature benefited photosynthetic performance of Anabaena sp. and Nostoc sp. but not of Microcystis sp. and A. platensis. Higher temperature was also associated to an increase in the chain area of Anabaena sp., and to bigger trichomes in A. platensis; however, no morphological effects were observed in Microcystis sp. In addition, in Nostoc sp. the increase in temperature counteracted the UVR impact on the reduction of the chain area. How these effects and mechanisms will affect the trophodynamics and production of aquatic ecosystems is still uncertain, but the specificity of the responses suggests that not all cyanobacteria would be equally benefited by temperature increases therefore affecting the balance and interaction among species in the water column.