STEM Today

STEM TODAY December 2016, No.15

Microbial Research

STEM TODAY December 2016 , No.15

CONTENTS MICRO­02: We need to determine if spaceflight induces changes in diversity, concentration, and/or characteristics of medically significant microorganisms associated with the crew and environment aboard the International Space Station that could affect crew health.

Editorial Editor: Mr. Abhishek Kumar Sinha Editor / Technical Advisor: Mr. Martin Cabaniss

STEM Today, December 2016, No.15

Cover Page Earth Behold one of the more detailed images of the Earth yet created. This Blue Marble Earth montage shown above – created from photographs taken by the Visible/Infrared Imager Radiometer Suite (VIIRS) instrument on board the new Suomi NPP satellite – shows many stunning details of our home planet. The Suomi NPP satellite was launched last October and renamed last week after Verner Suomi, commonly deemed the father of satellite meteorology. The composite was created from the data collected during four orbits of the robotic satellite taken earlier this month and digitally projected onto the globe. Many features of North America and the Western Hemisphere are particularly visible on a high resolution version of the image.

Image Credit: NASA

Back Cover View of ISS as it Rotates 90 Degrees S135-E-011814 (19 July 2011) — This picture of the International Space Station was photographed from the space shuttle Atlantis as the orbiting complex and the shuttle performed their relative separation in the early hours of July 19, 2011. Onboard the station were Russian cosmonauts Andrey Borisenko, Expedition 28 commander; Sergei Volkov and Alexander Samokutyaev, both flight engineers; Japan Aerospace Exploration astronaut Satoshi Furukawa, and NASA astronauts Mike Fossum and Ron Garan, all flight engineers. Onboard the shuttle were NASA astronauts Chris Ferguson, STS-135 commander; Doug Hurley, pilot; and Sandy Magnus and Rex Walheim, both mission specialists.

Image Credit: NASA

STEM Today , December 2016

Editorial Dear Reader

STEM Today, December 2016, No.15

All young people should be prepared to think deeply and to think well so that they have the chance to become the innovators, educators, researchers, and leaders who can solve the most pressing challenges facing our world, both today and tomorrow. But, right now, not enough of our youth have access to quality STEM learning opportunities and too few students see these disciplines as springboards for their careers.

According to Marillyn Hewson, "Our children - the elementary, middle and high school students of today - make up a generation that will change our universe forever. This is the generation that will walk on Mars, explore deep space and unlock mysteries that we can’t yet imagine". "They won’t get there alone. It is our job to prepare, inspire and equip them to build the future - and that’s exactly what Generation Beyond is designed to do."

STEM Today will inspire and educate people about Spaceflight and effects of Spaceflight on Astronauts.

Editor Mr. Abhishek Kumar Sinha

Editorial Dear Reader The Science, Technology, Engineering and Math (STEM) program is designed to inspire the next generation of innovators, explorers, inventors and pioneers to pursue STEM careers.

According to President Barack Obama, "[Science] is more than a school subject, or the periodic table, or the properties of waves. It is an approach to the world, a critical way to understand and explore and engage with the world, and then have the capacity to change that world..."

STEM Today addresses the inadequate number of teachers skilled to educate in Human Spaceflight. It will prepare , inspire and educate teachers about Spaceflight. STEM Today will focus on NASA’S Human Research Roadmap. It will research on long duration spaceflight and put together latest research in Human Spaceflight in its monthly newsletter.

Editor / Technical Advisor Mr. Martin Cabaniss

STEM Today, December 2016, No.15

MICROBIAL RESEARCH MICRO-02

We need to determine if space ight induces changes in diversity, concentration, and/or characteristics of medically signi cant microorganisms associated with the crew and environment aboard the International Space Station that could a ect crew health.

These tasks are aimed at identifying and characterizing the crew health related microbial hazards in ight aboard the International Space Station. Research has shown that there are changes in the microbial characteristics during space ight and future e orts will be aimed at de ning the changes in medically signi cant organisms associated with crewmembers.

Special Edition on Microbial Research

STEM Today, December 2016, No.15

Dysbiosis and Immune Dysregulation in Outer Space

The compositions of intestinal,oral and nasal flora have been shown to change even during short spaceflights.The perturbation to the structure and composition of complex bacterial communities is called dybiosis . Spaceflights can induce dysbiosis in the human microflora, which can result in the reduction of the defense group of mi-

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Special Edition on Microbial Research croorganisms (bifidobacteria and lactobacilli) and appearance of opportunistic pathogens such as Escherichia coli, Enterobacteria and Clostridia . Subsequently, this procedure can lead to accumulation of the potentially pathogenic species and their long-term persistence.

Studies revealed a reduction in the number of nonpathogenic bacteria and an increase in the number of opportunistic pathogens in the nasal flora of cosmonauts. A significant reduction in the number of bacterial species in the intestine after spaceflight and terrestrial analogs has also been reported . In addition, a decrease in lactobacilli, with concurrent replacement with pathogens, has been recently observed, utilizing three different models emulating space environment.

In the gut, two major phyla, Bacteroides and Firmicutes are dominating in composition of the microbiota at this site . Furthermore, butyrate-producing bacteria, including Faecalibacterium prausnitzii, Roseburia intestinalis, and Bacteroides uniformis, were implicated to play a key role in adult gut, yet their relative abundance varied. Nevertheless, these variations can be useful and adapted to design personalized medicine for astronaut’s unique health condition.

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Microgravity Increases Bacterial Virulence Reduction in the diversity of the gastrointestinal microbiota may give rise to an increase in the presence of the drug-resistant bacteria. This process constitutes the pathogenesis of conditions such as antibiotic-associated diarrhea caused by Clostridium difficile . Such a phenomenon could occur during spaceflight and the emergence of resistant clones could be facilitated by the administration of antibiotics either before or during the spaceflight.

Certain bacteria acquire increased pathogenic features, such as changes in growth modulation and alterations in response to antibiotics after exposure to simulated microgravity or spaceflight . Salmonella typhimurium grown in spaceflight analogs exhibits increased virulence, increased resistance to environmental stress and increased survival in macrophages.The Hfq pathway, which is required for virulence in several bacterial pathogens, appears to be a key for enhanced virulence in S. typhimurium and extracellular matrix accumulation consistent with biofilm formation. E. coli becomes more adherent to mammalian gastrointestinal epithelial-like cell line, Caco-2 , and increases production of the heat-labile enterotoxin in microgravity. Candida albicans exhibited increased filamentation and formation of biofilmcommunities, and amphotericin B resistance in microgravity conditions.

Biofilm-based infections have been identified in virtually all tissue and organ systems of the human body, including oropharyngeal soft tissues, teeth and dental implants, the middle ear, eye, endobronchial and pulmonary parenchymal tissues, cardiac valves, the gastrointestinal tract and the urogenital tract. Biofilm formation linked to chronic diseases are difficult to treat, as microorganisms within a biofilm tend to be far more difficult to eradicate with antimicrobial agents, and are particularly difficult for the host immune system to recognize and respond to in an appropriate manner. Bacterial biofilm provides superior resistance to oxidative, osmolarity, pH and antibiotic stresses.These changes are assumed to enhance bacterial survivalby resistance to the immune system and antimicrobial agents. Diminished gravity has been shown to stimulate bacterial biofilm formation in both E. coli and Pseudomonas aeruginosa . Spaceflight increases not only the biofilm biomass and thickness but in the case of P. aeruginosa promotes the display of a novel architecture. Hfq is involved in the global transcriptional and proteomic responses of P. aeruginosa to simulated microgravity and to actual spaceflight.

The issue of a confined compartment that could facilitate transmission of a resistant strain among all crewmembers on a mission is of great concern. This can have serious implications as the efficacy of antibiotics may be diminished during space missions. Although bacterial mutations occur more frequently in long-term spaceflights , there is concern that antibiotic resistance may also increase during short-term spaceflights . The humanmicroorganism ecosystem in an enclosed environment, such as spacecrafts, has the potential for pathogenicity, which along with a possible weakened immune system during spaceflight carries the risk of increased severity of infection in long-term space missions.

Immune Dysregulation in Microgravity The human immune system, similar to all human cell biological systems, has developed in the continuous presence of the earth’s gravitational field. An acute absence of this external force, as occurs during spaceflight, would affect the functionality of this system. Microgravity has been associated with impaired human immune response , and with reactivation of latent infections, such as herpes virus. In spite of a poorly controlled lymphocyte immune response, no increase in virus infection nor latent virus infection reactivation were observed in a recent study of simulated long-term isolation in an enclosed habitat on the earth , suggesting that microgravity may be the crucial factor for the occurrence of these conditions. Conjunctivitis, dental infections, upper respiratory infections, influenza, viral gastroenteritis, rhinitis, pharyngitis or mild dermatologic problems during spaceflights

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Special Edition on Microbial Research have been documented.

Even though physiological stress has been associated with changes in specific immune parameters associated with spaceflight , ground analogs for immune dysregulation in long-duration spaceflights trying to replicate remote deployment, mission stress, environmental hazards, prolonged isolation and disrupted circadian rhythms still lack the microgravity variable into the equation. Furthermore, data about human immunity during longduration spaceflight might differ from post-flight data that has the potential to be influenced by landing and readaptation stress.

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Immune Cell Alteration in Spaceflight Several important immune parameters are decreased during spaceflight. Reductions in the number of lymphocytes, monocytes, their cytokine production, depression of dendritic cells (DCs) function and T-cell activation have been reported. Immune cells subjected to conditions of altered gravity manifest a number of changes instructure and function (Figure 1b). Studies have revealed a decrease in total white blood cell (WBC) count, lymphocytes, monocytes, and eosinophils, and a slight increase in neutrophils in rats flown on a nine-day mission . Similar results showing a decrease in total leukocytes, lymphocytes, monocytes and elevated neutrophils were later reported in rats flown on a 14-day mission. Both studies demonstrated a decrease in the absolute number of CD4 and CD8 as well as B-lymphocytes.

A later study comparing nine-day mission versus 16-day mission found that the number of WBCs, polymorphonuclear leukocytes and CD4+ T cells were significantly increased after astronauts’ postflight . This study also showed that after the nine-day missions, monocytes were increased, while natural killer cells were decreased, with a subsequent decrease inmonocytes after the 16-day missions, while no change occurred in natural killer cells.These findings suggest that mission length may be an important factor in the variability of immune alterations observed after spaceflight. Even though spaceflight strongly alters human immunity, it is in general well tolerated. The observed decrease in the number of leukocytes and lymphocytes at the immediate postflight period appears to be transient and all values return to the control levels by seven to nine days postflight.

Neutrophils Elevated neutrophils along with reduction in phagocytosis and oxidative bursts are associated with spaceflight, and this relationship may depend on duration of mission . No significant differences have been observed in neutrophil bacterial phagocytosis and oxidative burst capacity after a five-day mission. Longer missions (9-11 days), however, rendered significant lower phagocytosis and oxidative burst capacities, and decreased chemotaxis.

Monocytes/macrophages Although monocyte numbers may increase after 4-16-day flights, they do not seem to change in number in longer flights ; however, they exhibited reduced phagocytic activity, and lower levels of Fc receptors CD32 and CD64 . The effect of microgravity on macrophages appears to be immediate . Spaceflight enhances cellular proliferation but inhibited differentiation and secretion of IL-6 in murine bone marrow-derived macrophages .

A very recent study, utilizing an in − vitro monocyte system exposed to microgravity, has shown that the effect of microgravity on immunological signal transduction is highly specific. This study demonstrates that in human monocytes, lipopolysaccharide (LPS)-dependent Jun-N-terminal kinase activation is sensitive to spaceflightassociated microgravity, while the corresponding LPS-dependent activation of p38 MAP kinase remains unaffected. Macrophages also show changes in cytokine production in space, with increased secretion of IL-1 and Tumor Necrosis Factor (TNF)-α . Elevated TNF-α has been reported in peripheral blood mononuclear cells (PBMCs) under simulatedmicrogravity conditions.

Dendritic cells Generation of an effective immune response requires that antigens be processed and presented to T lymphocytes. Dendritic cells are efficient antigen-presenting cells(APC). Because of their influence on both the innate and the acquired arms of immunity, a defect in DC would be expected to result in a broad impairment of immune function. This is in fact what is observed using microgravity analogs on a DC generation culture system, a lower yield of DCs, which were also less phagocytic and expressed a lower density of surface HLA-DR, and CD80 was obtained. These DCs produced lower levels of IL-12 and failed to upregulate some costimulatory/adhesion molecules involved in antigen presentation. IL-12 is naturally produced by APCs (DCs, macrophages, and human B-lymphoblastoid cells) in response to antigenic stimulation, and it is involved in the differentiation of naive T cells into Th1 cells.

Lymphocytes Lymphocytes are critical for adaptive immune responses, and T lymphocytes are regulators of cell-mediated immunity, controlling the entire immune response. Short-term flights (8-15 days) may present a slight decrease

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in lymphocytes . Long-term isolation of 520 days, aiming to simulate chronic stress involved in future missions such as those to Mars, have shown increased lymphocyte amounts, specifically CD19 B cells and CD8 T cells, along with heightened immune responses. Decline in lymphocyte growth with increased apoptosis , chromosomal aberrations , inhibited locomotion , altered cytokine production , and changes in energy metabolism have been described under altered gravity conditions . Chromosomal damage observed in lymphocytes correlates with estimated absorbed cosmic radiation doses . The improper activation of lymphocytes, an apoptotic effect of elevated levels of TNF-α , and damage to their DNA could be responsible for the observed apoptosis in these cells in microgravity . A recent study, combining proton radiation and simulated microgravity, confirmed a decrease in lymphocyte numbers along with other leukocytes, and a decrease in T cell proliferation .This same study also showed that expression of early activation marker CD69 was decreased in T cells 21 days post-exposure.

Interestingly, microgravity-associated CD69 and IL-2 decrease can be corrected with short-oxidized multi-walled carbon nanotubes. This novel nanotechnology approach, developed initially as drug carries, was found to provide a boosting effect on the activation of human monocyte when screened for potential immune cytotoxicity.

Microgravity exposure to resting T cells inhibits activation of T cells .The inhibition of T cell activation by a decrease in IL-2 and IL-2R expressions caused bymicrogravity has been observed in vitro , and has been confirmed recently in mice after spaceflight .A decrease in IL-2R has also been observed innatural killer (NK) cells when exposed to conditions mimicking microgravity . NK cell numbers, as well as their cell cytotoxicity, have been reported to decrease after spaceflight . Microgravity affects lymphocyte redistribution among organs, which in turn influences the activation potential of the cells . Rat CD4+ CD8+ splenocytes, and CD2+ lymphocytes increased while CD5+ lymphocytes decreased after 10-day flight. Lymphocyte function-associated antigen 1, LFA-1, the integrin receptor for intercellular adhesion molecule ICAM-1, is a receptor found on leukocytes. When activated, leukocytes bind to endothelial cells via ICAM-1/LFA-1 and then transmigrate into tissues. LFA-1 is increased in splenocytes but decreased in lymphocytes , while no change in ICAM-1 expression was observed in rats after spaceflight. In humans, both CD4+ helper T cells and CD8+ cytotoxic T cells increase after spaceflight.

Lack of immune response in microgravity occurs at the cellular level. It is hypothesized that reduced cell-cell interactions between T cells and monocytes could account for the observed depression of human lymphocytes activation by microgravity in vitro . Since locomotion is essential for cell-cell contacts, an impaired locomotion of monocytes and cytoskeletal changes, both linked to cell contacts, could be responsible for their reduced interactionwith T lymphocytes . Although lymphocytes in suspension are highly motile in microgravity, they exhibit alterations in their cytoskeletal organization by gravity, which appears to underlie mechanisms of microgravityassociated apoptosis. Multiple investigators have observed actin and microtubule cytoskeletal modifications in microgravity. Microarray studies of gene expression in lymphocytes have shown changes in cytoskeleton-related genes . Studies in osteoblasts have pointed out a major alteration in anabolic signal transduction under microgravity conditions, most probably through associated kinase pathways that are connected to the cytoskeleton .

Osteoblast activity can also be compromised during spaceflight. Various types of stress influence proliferation of osteoblasts , including a lack of mechanical stimulation, affecting the bone remodeling constant process of mass retention and loss.

CD19+ B cells have been reported to increase after spaceflight . Recently, an increase in resting memory B cells (CD21+) but a decrease in activated and memory like tissue B cells has been shown , which may translate in a decrease in B cell response to antigens during spaceflight. In humans, increase in total immunoglobulin A (IgA) and immunoglobulin M (IgM) has been found after long-term but not short-term missions . Total IgE was significantly increased after 16-day missions and correlated with urinary cortisol . The clinical relevance of these findings as a risk for atopy or a hypothalamus-pituitary-adrenal axis imbalance still remains to be studied.

Cytokines The combination of space travel-related environmental conditions results in an aberrant innate immune activation, with increased Nuclear Factor κB (NF-κB) cytokines like TNF-α and IL-6, and Type I Interferon (IFN), IFNα. A very recent study aiming to characterize the immune system dysregulation associated with long-duration spaceflight has shown that TNF-α, IL-8, IL-1ra, CCL2, CCL4 and CXCL5 along with vascular endothelial growth factor (VEGF) and thrombopoietin were increased due to spaceflight. These findings suggest that the inflammation, leukocyte recruitment, angiogenesis and thrombocyte generation are persisting processes among the multiple physiological adaptations during space flight. Although the levels of other NF-κB-dependent cytokines were not elevated, the increased IL-8 and TNF-α are indicative of mild inflammation that could be derived from flight-associated alterations in the gut microbiome or the consistent exposure to increased environmental radiation.

Elevation of IL-1ra, an inhibitor of IL-1, may represent an adaptive response to inflammatory stress . Another study provided information on the source of these cytokines, investigating alterations in human immune cell

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Special Edition on Microbial Research distribution and changes in cytokines during head-down bed rest, one of the several ground-based spaceflight analogs . This study showed a decrease in IFN-α and IL-17 secretion by activated T cells, an increase in IL-1α and IL-18 by activated B and myeloid cells, and an increase in memory T and B cells and regulatory T cells (T-regs). T-regs presumably may be suppressing conventional T cell activation, leading to an impaired memory T cell function.This would translate into a decreased protective T cell immunity and enhanced pro-inflammatory cytokines. A DNA array study of activated T cells demonstrated that microgravity-exposed T cells were deficient in transcription of early genes associated with the TNF-α pathway. However, T cells stimulated in an on-board centrifuge, artificially generating a 1 g condition, did not result in the same effect. This again points out that the effects of spaceflight on the immune system are related to microgravity rather than a result of stress. Lack of immune response in microgravity occurs at a molecular level as well. Altered gravity environment-impaired inductions of early genes were regulated primarily by transcription factors NF-κB,CREB , ELK, AP-1 and STAT. These transcription factors were regulated by the Protein kinase A (PKA) pathway, a key regulator in T cell activation, and were downregulated in vectorless gravity.

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Type I interferons Although typically considered to be most important in the control of viral infections, Type I IFNs are also important in the response to bacterial pathogens. Murine macrophages produced increased IFN-β, along with IL-1 and TNF-α, after spaceflight . Whereas simulation studies in animals showed inhibition of Type I IFN, in humans, Type I IFNs are increased when leukocyte cultures were subjected to spaceflight. However, the same subjects also showed a decrease in their ability to produce Type I IFNs immediately after spaceflight. The latter could possibly explain the occurrence of bacterial and viral infections during missions or after return.

PROBIOTICS AND DIETARY SUPPLEMENTS Diet, lifestyle, antibiotic therapy and various environmental stresses can exert alterations in an astronaut’s gut microbiome in space and impair their immune system. The administration and/or consumption of probiotics is supposed to have immuneenhancing effects , hinder alterations in the human microbiome to a large extent, and prevent colonization of potential pathogens (Figure 1c).

It has been suggested that consumption of soy-based fermented products (containing lactic acid bacteria) can prevent the health problems of astronauts associated with long-term space travel . Oral administration of lactobacilli and bacteria from the genus Bifidobacterium increases the systemic and mucosal IgA response to antigens.

Lactobacilli also enhance the response of live oral vaccines against viruses such as rotavirus . Ingestion of lactobacilli potentiates IFN-γ production by PBMCs and activates macrophages. In the gut, lactobacilli stimulates Peyer’s patches macrophages and/or DCs to release inflammatory cytokines such as TNF-α, IFN-γ and IL-12, and regulatory cytokines such as IL-4 and IL-10 .

Probiotics can modulate the gut immune system . Lactobacillus rhamnosus is able to reduce elevated fecal concentration of TNF-α in patients with atopic dermatitis and cow milk allergy . Lactic acid bacteria can downregulate production of the proinflammatory cytokine IL-8 , which has been reported to increase during longterm spaceflight. A gut-stabilizing effect seems to occur through a balance between proinflammatory and antiinflammatory cytokines. Microgravity, magnetic field and cosmic radiation exposure, and stress have immunedysregulatory effects, affecting an astronaut’s ability to prevent acquisition of infectious agents or reactivation of latent infection. In microgravity, there are alterations in potentialmicrobial pathogens regarding virulence, growth kinetics and biofilm formation, which may increase the risk posed to astronauts.

REACTIVATION OF LATENT HERPES VIRUSES Latent viruses are used as an early predictor of changes in immune system of astronauts due to space flight.

Humans host 8 different herpes viruses. Following primary infection, these viruses establish a permanent presence with the host called latency. The virus may remain latent and unnoticed for years or decades.

Cell mediated immunity (CMI) is the immune element most responsible for controlling latent viruses.

During times of stress, latent viruses may reactivate and cause disease. Stress is processed through the Hypothermus pituitary adrenal (HPA) axis and sypamathetic adrenalmedullary axis, resulting in increased secretion of stress hormones including cortisol and catecholamines. Increased levels of stress hormones reduce the immune response, specifically the CMI resulting in proliferation of latent viruses and disease at a later stage.

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Special Edition on Microbial Research In this research, authors present the results from studies of reactivation of three latent herpes viruses (Epstein Barr Virus, Cytomegalovirus, Varicella Zoster Virus) in astronauts .

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Epstein-Barr Virus Epstein-Barr virus (EBV), a DNA virus is highly infectious and can be transmitted by microdroplets and by direct contact with saliva. When the acute infectious phase subsides, EBV can become latent in B lymphocytes.

EBV is the causative agent of infectious mononucleosis and is associated with several malignancies, including Burkitt’s lymphoma, nasopharyngeal carcinoma, and diffuse oligoclonal B-cell lymphoma . Latent EBV may be reactivated by a range of physical and psychosocial stress factors and shed in saliva. EBV was used as a model for latent virus reactivation in astronauts. Approximately 95% of the adult population is infected by EBV . This makes EBV ideal for studying virus reactivation in the relatively small astronaut population. Pierson et al., collected and used a PCR assay to detect EBV DNA and demonstrate EBV reactivation in shuttle astronauts. Pierson et al. demonstrated EBV DNA shed in saliva from astronauts before, during, and after space flight. EBV copies were about 10-fold higher during the flight phase than shed either before or after space flight ( Figure 1). In addition, the number of EBV copies shed during space flight seemed to increase as a function of time in space.

A significant increase in EBV (VCA) antibody titers before launch, at landing, and after landing was observed (p < 0.001) from the baseline values taken 5 to 24 months before flight.

Cytomegalovirus To determine if the effects of space flight observed in astronauts were limited to EBV, authors studied another human herpes virus, cytomegalovirus (CMV). When CMV reactivates, it is shed in urine. CMV DNA was found in astronaut urine collected before and after flight.

Figure 2 shows that 27% (15/55) of astronauts shed CMV in their urine while less than 2 per cent (1 of 61) of control subjects shed CMV . Plasma IgG antibodies to CMV increased significantly as compared to the controls in astronauts who shed CMV, confirming reactivation of the virus (Figure 3).

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Varicella Zoster Virus Subsequently, varicella zoater virus (VZV) was found in saliva of shuttle astronauts during and after space flight (Figure 3). This was the first report of shedding of VZV with no clinical symptoms. As expected, no shedding was found in control subjects. VZV antibodies also increased over control values substantiating occurrence of VZV reactivation. Consistent with findings from EBV and CMV studies, urinary cortisol levels increased in astronauts at landing. All available data suggest that stress, and perhaps other factors, are processed through the HPA axis.

Levels of stress hormones, including cortisol and catecholamines, in astronauts were consistently elevated at landing. Elevations in these hormones result in diminished CMI response, and decreased CMI is followed by increased reactivation and shedding of latent herpes viruses.

Having shown that either stress or environmental factors associated with space flight can trigger VZV reactivation detected as viral DNA in saliva, authors refocused research to determine if VZV reactivation constitutes a health risk to astronauts. Their approach was to demonstrate the propagation of infectious VZV from astronautsâ&#x20AC;&#x2122; saliva after space flight. First, however, authors asked if VZV can remain infectious after being exposed to human saliva. Saliva taken 26 days after landing from 3 subjects was cultured on human fetal lung cells (Figure 4).

Infectious VZV was recovered from saliva of subjects 1 and 2 on the second day after landing.

Virus specificity was confirmed by antibody staining and DNA analysis which showed it to be VZV of European descent, common in the US. Further, both antibody staining and DNA PCR demonstrated that no HSV-1 was detected in any infected culture.

IgG antibody against VZV was determined by enzyme immunoassay (EIA). Serum titers of anti-VZV IgG were determined in 5 control subjects and in 6 astronauts 10 days before flight and again 2 to 3 hours after landing (Figure 5). Authors found two- to threefold greater levels of circulating anti-VZV IgG in astronauts than in control subjects.

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Special Edition on Microbial Research However, the combination of VZV DNA in saliva and a larger specific antibody response in serum from astronauts than in serum from control subjects further indicates sub-clinical reactivation of VZV. This study adds VZV to the list of human herpes viruses capable of reactivation in response to acute non-surgical stress. HSV types 1 and 2 were detected in 2 to 3% of pre- and postflight and in 8% of in-flight saliva samples collected from space shuttle crew members. HHV-6 was detected in 29% of preflight, 2% of in-flight, and 15% of postflight samples.

Selected neuropeptides, including SP, CGRP, Neuropeptide Y, and VIP, were also measured in plasma from 5 astronauts before and after space flight. They were elevated immediately after landing (Figure 6). This is consistent with the decrease in cell-mediated immunity (CMI) reported earlier in astronauts and Antarctic expeditioners , and therefore is consistent with an increase in viral reactivation .

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However, until neuropeptide data can be obtained from a larger number of astronauts, the significance of changes in astronaut neuropeptide levels cannot be adequately assessed.

Medical Significance of Latent Virus Reactivation in Space Flight The medical significance of asymptomatic viral shedding in astronauts remains unknown. During the flight phase, the mean number of EBV DNA copies shed by astronauts was 417/mL, with a maximum of 738/mL. In the saliva of acquired immunodeficiency syndrome (AIDS) patients, authors found 3700 copies/mL (mean value) of EBV DNA. However, some AIDS patients had levels as low as 600 copies. Kimura , who used a similar PCR assay, reported that a group of patients with infectious mononucleosis had a mean number of 158 copies of EBV DNA per mL of saliva. In renal transplant recipients with active CMV infection, Stagno et al. found that the number of CMV genomes per mL of urine was 100-fold greater in symptomatic patients than in asymptomatic patients.

The number of EBV copies found in astronaut saliva indicated that the diminishment of immune response is very mild on short shuttle flights and cannot be compared to that of patients with severely impaired immunity (such as AIDS patients).

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Special Edition on Microbial Research However, lengthy stays in space may result in substantial reductions in immunity, and the number of EBV copies in saliva of cosmonauts aboard the Mir space station is consistent with this scenario. The increased amount of EBV DNA in saliva, coupled with the propensity of large and small saliva droplets to float in the microgravity environment of the crew compartment, may lead to increased risk of crossinfection among crew members. One would expect minimal medical effects of such events in healthy individuals, but viral reactivation is more likely to have clinical significance for astronauts if their immune responses are impaired.

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In Shuttle astronauts, a pattern of immediate-early and early viral gene transcription (the first step in gene production) was observed indicative of EBV reactivation. Altogether, there was a significant increase in the number of immediate early and early gene replications in Shuttle astronaut samples as compared to healthy control samples. Although EBV reactivation did occur in Shuttle astronauts, productive EBV replication in peripheral blood B-lymphocytes did not.

In contrast, samples from three ISS astronauts after flight show strong evidence that complete productive EBV replication is occurring in the peripheral blood B-cells of these astronauts, and data clearly show activation of the full cascade of replicative EBV gene expression in the B-cells of ISS astronauts. For these crewmembers who flew longer (6 month) in space, latent gene expression and late lytic (involves the destruction of the host cell) gene transcripts were both more frequent and diverse. Overall, there was a significant increase in the number of immediate early and early gene transcripts in ISS astronaut samples as compared to healthy control samples and Shuttle astronauts at landing.

The authors acknowledged there were limitations to this study. First, the sample size was limited (the results, however, were quite striking). Second, data from other potential stressors (e.g., sleep deprivation, changes in circadian rhythms, etc.), known to affect immune function, were not available. Additionally, increased radiation exposure on long-duration missions may also affect cellular immunity. Another consideration is that only postflight samples were analyzed and do not necessarily reflect EBV gene expression during flight (Stowe et.al.).

Researchers also found increases in the stress hormone cortisol in Shuttle crewmembers, presumably due to the rigors of preflight training, which were accompanied by significant changes in white blood cells even days prior to launch. It is important to note, however, that for short-duration crewmembers, these immune system changes do not appear to linger beyond a few days after mission completion. Further understanding that unique mission variables and stress levels can elicit specific immune responses in space flight crewmembers is crucial for mission planners as they strive to maintain a balance between optimum crew health, mission requirements, and stress inducing factors especially on prolonged missions. While the clinical significance of these findings remain to be determined, they are potentially significant for the health of astronauts who will spend long periods in space and could pose a health risk for crewmembers following prolonged space travel, in particular due to the lack of specialized medical facilities in case of injury or illness. (Stowe et.al., Crucian et.al.).

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SIMBOX(Science in Microgravity Box) mission

During the SIMBOX mission on Shenzhou-8, authors investigated microgravity-associated long-term alterations in macrophages, the most important effector cells of the immune system, which are responsible for attacking and killing bacteria and other foreign and pathogenic intruders in the human body. Authors analyzed surface molecules, which are required for recognition of bacteria and cell-cell-communication, and investigated the cytoskeletal architecture after several days in microgravity.

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The material of the sample slides (polycarbonate) required biocompatibility tests and optimization of the cell culture and differentiation conditions in order to achieve an optimal density of the adherent macrophageal human U937 cells on the samples slide surfaces. Different culture volumes and stimulation times with PMA were tested, and density of adherent macrophageal human U937 on polycarbonate slides was analyzed (Fig. 3A).

Macrophageal differentiation was induced in a culture of 0.5 X 106 cells/ml in human serum and RPMI-1640 medium without HEPES, 5% CO2 and 37◦ C by 25 nM PMA in a volume between 3.0 and 5.5 ml and for a stimulation time of either 72 h or 5 days. As shown in fig. 3A, macrophageal differentiation on polycarbonate slides was possible and the number of adherent macrophageal cells increased significantly with the culture volume (0.24677 ± 0.08819 X 106 cells with 3 ml volume vs. 0.5733 ± 0.09387 X 106 cells with 4.5 ml volume vs. 1.150 ± 0.1443 X 106 cells with 5.5 ml volume ). An almost confluent monolayer of adherent macrophageal cells was achieved by using 5.5 ml of cell suspension (Fig. 3A top). Additionally, no significant loss of cells after 5 days of cultivation could be observed observed(1.150 ± 0.1443 X 106 cells after 5 days vs. 1.063 ± 0.1674 X 106 cells after 72 h).

Macrophageal differentiated human U937 cells were cultured for 5 or 8 days in the test culture chamber (TCC) under flight conditions (23 ◦ C, no gas exchange) and under standard cell culture conditions (con, 37 ◦ C, 5% CO2 ). The medium was supplemented with 10% human or fetal calf serum (Fig. 3B), with or without 20 mM HEPES (Fig. 3C and D). The number of adherent cells on polycarbonate slides (Fig. 3B and C) and cell viability (Fig. 3D) were analyzed. As shown in Fig. 3B and C, the number of adherent macrophageal cells differed significantly between normal conditions (con) and in-flight culture conditions in the test culture chambers (TCC).

After 5 days of culture under flight conditions (23 ◦ C, no gas exchange) with no additional medium supplementation, the number of adherent macrophageal cells was reduced from 0.2900 ± 0.03512 to 0.1167 ± 0.02404 X

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106 cells compared to the control cell culture conditions (with CO2 supplementation, 37 ◦ C). Even under optimum culture conditions, cell density on polycarbonate slides was only 35% after 8 days of culture compared to 5 days of culture (0.9250 ± 0.01500 X 106 cells after 5 days vs. 0.2650 ± 0.03374 X 106 cells after 8 days).

To improve the culture conditions, human serum was tested against fetal calf serum (Fig. 3B). Supplementation of the culture medium with human serum significantly increased the number of adherent macrophageal cells on the sample slides after 5 days of cultivation under standard culture conditions as well as under flight conditions in the TCCs (0.9250 ± 0.01500 X 106 cells vs. 0.2900 ± 0.03512 X 106 cells under standard conditions and 0.2650 ± 0.03374 X 106 cells vs. 0.1167 ± 0.02404 X 106 cells in the TCCs. After 8 days in culture, human serum improved long-term survival of adherent macrophageal cells under standard culture conditions, but not under flight conditions in the TCCs (0.02533 ± 0.008253 X 106 cells vs. 0.02137 ± 0.01144 X 106 cells).

Due to the lack of gas exchange during flight conditions, a buffer system capable of keeping the pH constant under flight conditions is necessary. HEPES buffers are known to maintain the pH very well and to dissociate less in decreased temperatures, rendering HEPES a more effective buffer for maintaining enzyme and protein structures at a lower temperature. Authors thus cultured adherent macrophageal cells (1) under standard conditions and (2) under flight conditions in the TCC with supplementation of 20 mM HEPES (Fig. 3C). Surprisingly, the number of adherent cells decreased significantly under standard culture conditions and did not change in the TCCs did not change in the TCCs (0.9000 ± 0.00042 X 106 cells vs. 0.1068 ± 0.01106 X 106 cells during standard culture conditions and 0.2597 ± 0.03454 X 106 cells vs. 0.2200 ± 0.04531 X 106 cells in the TCCs , Fig.3C).

In further experiments it was investigated whether a medium exchange after 5 days of cultivation could enhance cell number and viability after 8 days of total cultivation time, but revealed no significant differences with or without medium exchange after 5 days (data not show). Because addition of HEPES buffer kept a higher risk of cell loss and viability (Fig. 3CD), no HEPES buffer was used in flight conditions in the SIMBOX experiment. Additionally, medium exchange after 5 days was not advantageous, though the two possible in-flight fluid injections were used for addition of the fixative and for the subsequent removal of the fixative

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in order to avoid prolonged fixation that could damage epitopes for immu-nocytochemical detection. Human macrophageal U937 cells were cultured in human serum instead of calf serum, beginning from the time point of macrophageal differen-tiation due to the increased viability and cell number. According to viability and cell number, the time point of fixation was set to 5 days. To exclude dead cells from analysis, authors applied a staining method, which was utilized to differentiate between living and apoptotic/necrotic cells at the time point of fixation (TUNEL-CellMask staining) and combined this with digital image analysis. The final protocol was: U937 cells were differentiated into the macrophageal phenotype on polycarbonate slides by 256nM PMA in 0.1% DMSO at a cell density of 0.5 X 106 cells/m in RPMI-1640 medium supplemented with 10% human serum and 200 mM glutamine for 72 h at 37 ◦ C with 5% CO2 . Inside the EUEs the slides were incubated in 0.5 ml fully CO2 saturated RPMI-1640 medium with 10% human serum and 200 mM glutamine at 23 ◦ C without CO2 . Fixation was performed after 5 days incubation with 1% PFA and 0.6% sucrose at 23 ◦ C for 2 h. Fixed cells were stored on board in PBS for 12 days at 23◦ C until landing.

Severe disturbance of the cytoskeleton in microgravity U937 cells are a human monocytic cell line and preserves the main monoblastic characteristics of mono-cytes including the ability to differentiate into a macro-phageal phenotype, which expresses specific surface molecules (MHCI, MHCII, CD11, CD14) and has the ability of phagocytosis. The maximum of adherence is reached 72 h after induction with PMA, whereas after removal of PMA, retro-differentiation starts around day 9-10 . During PMA-induced differentiation, monocytic U937 cells changed their morphology from a round shape with short microvilli and kidney-shaped nuclei to adherent, flat cells with long pseudopodia. Because cytoskeletal functions are not only indispensable for the differentiation process but also for crucial macrophageal functions such as migration and phagocytosis, authors investigated the main cytoskeletal structures actin, tubulin and vimentin, which are all required for migration and adhesion, intracellular trans-port and structural stability. In Fig. 4A the confocal micro-scopy analysis of actin staining is shown. Exclusively living CellMask-positive and TUNELnegative cells were analyzed regarding the mean fluorescence intensity of the whole cell: A slightly decreased actin fluorescence could be detected in Imaris analysis of the 1g ground hardware control cells integrated in the EUEs (1g) compared to standard cell culture control (con) (Fig. 4A). Importantly, an excessive loss of actin fluorescence staining could be observed in the microgravity sample (mg) compared to both controls samples (con and 1g). A morphological view of the actin cytoskeleton in all three groups is depicted in Fig. 4B, I-VI (I-III: single actin stain [green]; IV-VI: overlay of Aktin [green], cell mask-deep red [red] and TUNEL [blue]).

In standard culture condition (I, IV), long pseudo-podia, a flat shape and intense actin signals were present.

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High fluorescent signals and long pseudopodia could be seen also in the 1g ground hardware control sample (II,V), however some cells rounded up. After 5 days microgravity (mg) actin fluorescence was almost lost, whereas some pseudopodia could be still detected (III,VI). In CellMask staining, indicating regular cellular structures, no changes were evident between the groups. The results of the tubulin staining are demonstrated in Fig. 4C and D (I-III: single stain alpha tubulin [green]; IV-VI overlay of tubulin [green], CellMask [red] and TUNEL [blue]). In standard culture conditions (con), a normal distribution of fine fibrillary tubulin filaments with missing signals in the nucleus region and an organized structure beginning in the centrosome could be detected (I, IV).

In 1g ground hardware control samples (II,V), tubulin staining was reduced and some cells lost the fine fibrillary structure of tubulin, whereas the nuclear region was still free of tubulin and the structural integrity and organization appeared intact. In contrast to the control groups (con, 1g), increased fluorescent signal and disorganized tubulin skeleton could be observed in the microgravity sample (mg) (III,VI). Tubulin protein appeared to be accumulated and clumped, and the whole cell including the nuclear region was filled with tubulin fibers, with the exception only of vacuole-like regions. Vimentin staining demonstrated no change in structure or concentration (data not shown).

Down-regulation of CD18 in microgravity Cell migration, adhesion and communication are indi-spensable functions of macrophages and required cytoskeletal elements and integrins (such as LFA-1, Mac-1 or ICAM-1), which mediate the cell-cell-contact or the cellmatrix-contact. The integrins LFA1 (CD11a/CD18) and CR3/ Mac1 (CD11b/CD18) share a common beta-chain (CD18) and bind to ICAM-1 on endothelial cells and T cells. CR3/ Mac-1 is mainly expressed on macrophages, dendritic cells and neutrophils, where it fulfills a function as a pattern recognition receptor and therefore plays a key role in the innate immune response. In Fig. 5, the analysis of CD11a, CD11b and CD18 during standard cell culture conditions at 1g (con) and during microgravity conditions (mg) is shown. All chains of the LFA-1 and CR3/Mac-1 receptor decreased in microgravity (mg), but while the alpha chain of the receptors (CD11a and CD11b) were only slightly decreased ( 916.4 ± 26.5 vs. 723.2 ± 12.20 for CD11a , 500.7 ± 4.441 vs. 360.7 ± 5.129 for CD11b ) ,down-regulation of the beta chain CD18 ( 652.6 ± 13.51 vs. 332.5 ± 7.639) was very distinct. Authors thus suppose that key macrophageal functions such as adhesion and pattern recognition could be disturbed in microgravity through the down-regulation of CD18.

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Down-regulation of CD36 and MHC-II in microgravity Finally, authors analyzed macrophageal surface molecules, which are responsible for antigen-presentation (MHCI/II) and cell-cell-communication with T lymphocytes, as well as CD36, an important macrophageal scavenger receptor, binding many ligands such as oxidized low density lipo-protein (oxLDL), oxidized phospholipids, long chain fatty acids and collagen. In Fig. 6 the expression of CD36a and MHC-I in standard cell culture conditions at 1g (con), in 1g ground hardware control (1g) and in microgravity conditions (mg) is displayed. MHC-II expression as present in standard cell culture conditions at 1g (con) and in microgravity condi-tions (mg) is compared. CD36 expression was slightly reduced in 1g ground hardware control samples (1g) compared to standard cell culture conditions (con), but decreased distinctly under microgravity (mg) (Fig. 6A). Images of CD36 expression and distribution in all three groups are shown in Fig. 6B (I-III: single actin stain [green]; IV-VI: overlay of CD36 [green], CellMask [red] and TUNEL [blue]).

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During standard culture conditions CD36 is homogenously distributed between different cells (I, IV), whereas in 1g ground hardware control samples (1g), CD36 expression is more heterogeneously (II, V) expressed. CD36 expression during microgravity was reduced highly significant (III, VI). Image analysis quantified the loss of CD36 expression in microgravity (539.7 ± 18.27 in mg sample vs. 1445 ± 9.331 in standard culture control vs.

1012 ± 21.28 in 1g hardware controls). MHC-II expression was decreased in microgravity compared to standard culture controls (751.5 ± 6.474 in standard culture controls vs. 262.8 ± 2.245 in microgravity sample), whereas expression of MHC-I was increased in microgravity compared to standard culture controls and 1g ground hardware controls.

Impact of the space flight environment on bacterial physiology Although bacteria have evolved to survive in sometimes hostile terrestrial niches and will not have previously encountered the environment within the confines of spacecraft traveling beyond Earth’s gravitational field, they are able to sense, respond, and adapt to changes in their surroundings. In addition to low or zero gravity, they will be exposed to vibration, acceleration, and radiation in the form of galactic cosmic rays and solar energetic particle events at levels not encountered elsewhere. There is general agreement that microgravity represents the major influence on bacterial growth kinetics and bacterial cell behavior during short orbital flights, although radiation may increase microbial mutation rates during flight: after 40 days aboard Mir, mutation rates for a cloned bacterial gene carried by a yeast were two to three times higher than the ground control.

Investigations conducted during short orbital flights suggest that a range of bacteria display increased metabolic activity in space, manifest as a shorter lag phase, increased biomass, and increased production of secondary metabolites, although some comparable studies reported no differences between flight cultures and terrestrial controls. Some of these observations have been confirmed aboard multiple Space Shuttle flights: the consistency of the data obtained makes it unlikely that disparities of outcome are due to a lack of reproducibility resulting from the technical difficulties inherent in conducting scientific experiments in low gravity, implying that differences in growth media, culture conditions, strain-to-strain variations, and the nature of the bioreactors inside the spacecraft habitat account for differing responses to the space flight environment. Differential impact of bacterial cell behavior in microgravity could be exploited for the production of pharmaceutical compounds, secondary metabolites, and vaccines.

Klaus et al, and Benoit and Klaus have suggested that bacteria are affected only indirectly by microgravity due to the quiescent fluid environment surrounding the cells in liquid suspension culture. The settling of cells through liquid media and the potential for buoyant convection of less dense fluid in the vicinity of suspended bacteria are massively reduced in microgravity, and diffusion becomes the predominant means of nutrient transport toward and of metabolic waste away from the cell. The view that fluid dynamics and extracellular transport phenomena rather than cellular dynamics contribute to microgravity-induced differences in liquid-culture growth kinetics is supported by observations that bacteria such as Escherichia coli and Bacillus subtilis cultured on solid medium during flight grow at the same rate and to the same extent as terrestrial controls. A strong correlation has been noted between the impact of space flight on growth kinetics and bacterial motility, which goes a long way toward explaining differences between flown experiments in this area.

Thus, differences between microgravity-induced growth effects and ground controls seem to be, in the main, evident only when the bacteria under investigation are flagellate: clearly, motile cells have the capacity to seek out microenvironments in liquid cultures that have not been depleted of nutrients and flagellar action may in itself mix the quiescent layer around the cell. Although no definitive experiments have been undertaken to underpin this contention, studies with microgravity analogs such as clinostats and the high aspect ratio vessel (HARV), a rotating wall bioreactor described below, support the idea that mixing of microgravity-grown cultures to eliminate differences in fluid dynamics abrogates these growth kinetic effects.

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Alterations in bacterial growth kinetics in space appear to stimulate the production of secondary metabolites.

Thus, production of the antibiotic monorden by the parasitic fungus Humicola fuscoatra was greater when grown aboard Space Shuttle mission STS-77 than in ground samples, even though agar media were employed.

Similarly, the time course of elaboration of the antibiotic actinomycin D by Streptomyces plicatus in both defined and complex liquid media was altered in comparison to terrestrial cultures during flight on Shuttle STS-80, with more of the drug produced during the first 12 days in orbit. Interestingly, flight samples maintained their sporulation capacity when plated on agar medium postflight, while the residual ground controls did not sporulate.

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Some microorganisms adapt and thrive in the unique environment within spacecraft. A cloudy humidity condensate collected in January 1998 from behind a service panel on the orbiting platform Mir contained a wide range of bacteria, including Gram-negative species only infrequently associated with the contamination of shortduration missions. One sample yielded evidence of a member of the genus Legionella, bacteria that can cause lethal infections. Microbial consortia that accumulated over the 12 years since the launch of Mir included fungi of medical importance, protozoa, dust mites, and spirochetes. Some bacteria were recovered from surfaces in biofilms, suggesting a microbial strategy for increased onboard survival in comparison to less developed bacterial communities.

Pseudomonas aeruginosa PAO-1 formed biofilms more readily than in Earth-based parallel experiments when grown on surfaces or solid medium in the Biorack facility aboard Shuttle missions STS-81 and STS-95. During later missions STS-132 and STS-135, P. aeruginosa biofilms exhibited a "column and canopy" structure that has not been observed on Earth; thus, spaceflight affects not only the physiology of planktonic bacterial cultures but also their community-level behavior. A high proportion of Gram-positive isolates from the ISS were able to grow as biofilms under standard laboratory conditions, suggesting that the capacity to form complex communities on surfaces and interfaces provides competitive advantage aboard spacecraft.

Spaceflight increased the virulence of S. Typhimurium, while global gene expression profiling revealed a general downregulation of key virulence genes in this pathogen. The opportunistic pathogen P. aeruginosa responded to culture in the microgravity environment of spaceflight through differential regulation of 167 genes and 28 proteins. A significant part of the spaceflight stimulon was under the control of the RNA binding protein Hfq. Hfq is important for the virulence and stress resistance of several (opportunistic) pathogens, including P. aeruginosa PAO1, by modulating the function and stability of small regulatory RNAs (sRNAs) and interfering with their interactions with mRNAs. Interestingly, Hfq was also found to be an important regulator in the responses of (i) P. aeruginosa to microgravity analogue low-fluid-shear conditions (LSMMG, using the RWV bioreactor) and (ii) S. Typhimurium to actual spaceflight and LSMMG conditions. Hence, Hfq is the first transcriptional regulator ever shown to be commonly involved in the spaceflight and LSMMG responses of two bacterial species.

Among the P. aeruginosa genes with the highest fold inductions under spaceflight conditions were the genes encoding the lectins LecA and LecB. Lectins bind galactosides, play a role in the bacterial adhesion process to eukaryotic cells, and are thus important virulence factors in P. aeruginosa. P. aeruginosa lectins have cytotoxic effects in human peripheral lymphocytes and respiratory epithelial cells in vitro and increase alveolar barrier permeability in vivo. Lectin production in P. aeruginosa is regulated through the N-butanoyl-L-homoserine lactone (C4 -HSL) quorum-sensing system. However, the downregulation of rhlI, the gene encoding the C4 -HSL synthase, under spaceflight conditions was unexpected. Nevertheless, rhlA, which is dependent on C4 -HSL quorumsensing regulation and encodes the rhamnosyltransferase I involved in rhamnolipid surfactant biosynthesis, was induced during spaceflight culture. Rhamnolipids are glycolipidic surface-active molecules that have cytotoxic and immunomodulatory effects in eukaryotic cells. Interestingly, rhamnolipids and rhlA transcripts were also found in P. aeruginosa in larger amounts under low-fluid-shear compared to higher-fluid-shear growth conditions, using the RWV bioreactor. These data indicate that rhamnolipid production could be induced upon sensing of low fluid shear.

Gene expression profiles of P. aeruginosa grown under spaceflight conditions also revealed the differential regulation of a significant fraction of genes involved in growth under oxygen-limiting conditions. Spaceflight induced mainly genes involved in anaerobic metabolism, which was reinforced by a lower expression in spaceflight samples of CcoP2, a cytochrome with high affinity for oxygen that is typically induced under microaerophilic conditions. At the time of measurement, the most prominent way to cope with the apparent oxygen shortage under spaceflight conditions seemed to occur through denitrification and not through fermentation. Indeed, under oxygen-limiting conditions, P. aeruginosa switches to anaerobic respiration in the presence of the alternative electron acceptor nitrate or nitrite. The downregulation of ArcA, a protein involved in arginine fermentation, accentuates that fermentation was presumably not activated in spaceflightgrown bacteria.

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When comparing the gene expression profiles of P. aeruginosa grown in spaceflight and P. aeruginosa grown in LSMMG, a limited but significant overlap was found. Besides the role of Hfq and its regulon in the response of P. aeruginosa PAO1 to both spaceflight and LSMMG (see above), a significant fraction of genes involved in both microaerophilic and anaerobic metabolism were commonly induced. In contrast to P. aeruginosa grown under spaceflight conditions, LSMMGgrown P. aeruginosa induced genes involved in arginine and pyruvate fermentation, while denitrification did not appear to play a role in the LSMMG response of this bacterium. The observation that spaceflight samples were presumably more deprived of oxygen than LSMMGgrown bacteria, compared to their respective controls, could be explained by the fact that actual spaceflight conditions are characterized by even lower fluid shear levels than LSMMG conditions. Indeed, due to the absence of convection currents in microgravity, oxygen limitation will be more pronounced in space than in LSMMG.

Furthermore, the role of the experimental setup needs to be considered. As depicted in Fig. 2, cells grown in the bioreactors used for growth of P. aeruginosa in LSMMG and spaceflight have different oxygen availabilities. While the bioreactors have a gas-permeable membrane, the membrane surface-to-volume ratio of FPA bioreactors (used in spaceflight) is 12 times lower than that of the RWVs (LSMMG) [based on the formula Ď&#x20AC;r2 /(Ď&#x20AC;r2 X h) or 1/h, with r = radius and h = height]. Hence, oxygen availability overall will be higher in RWVs than in the FPA devices. It also needs to be mentioned that despite differences in aeration and fluid shear between the spaceflight and LSMMG studies, the RWV mimics only certain aspects of the spaceflight environment. Indeed, enhanced irradiation and vibration or potential direct effects of microgravity (such as effects on the cell or cellular components instead of on the extracellular environment) during spaceflight could lead to differences in gene and protein expression profiles between spaceflight and LSMMG-grown P. aeruginosa.

Accordingly, the RWV bioreactor was unable to mimic the complete repertoire of spaceflight-induced alterations in P. aeruginosa. Since the present study was conducted by growing P. aeruginosa in a liquid environment under spaceflight conditions, authors results are relevant mainly to the assessment of bacterial virulence in fluid niches of the spacecraft. Indeed, astronauts are in regular contact with water-containing sources that could be contaminated with P. aeruginosa, such as drinking water, rinseless shampoo, toothpaste, mouthwash, and water for laundry. Similarly, water-related sites in the hospital environment are most likely to harbor P. aeruginosa (e.g., faucets, showers, medication, disinfectants, mouthwash, and other hygiene products) and are at the origin of a significant number of nosocomial infections. Furthermore, P. aeruginosa is occasionally part of the normal human flora of the mouth, pharynx, anterior urethra, and lower gastrointestinal tract. In these regions of the human body, P. aeruginosa is present in a fluid environment, which will be affected by microgravity and will presumably result in the exposure of P. aeruginosa to lower-fluid-shear conditions than on Earth.

This study was the first to characterize the comprehensive transcriptional and translational responses of an opportunistic pathogen that is frequently found in the space habitat. Authors demonstrated that spaceflight conditions activated pathways in P. aeruginosa that have been shown previously to be involved in the in vivo

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Authors also identified interesting similarities and differences between P. aeruginosa grown in spaceflight and under the LSMMG conditions of the RWV. Despite the limited overlap of identical genes between spaceflightand LSMMG-grown P. aeruginosa, it was observed that different genes of the same regulon or stimulon could be induced or downregulated in spaceflight and LSMMG. The experimental setup was proposed as one of the putative factors at the origin of the oxygen-related transcriptional differences between LSMMG culture in the RWV bioreactor and spaceflight- cultured P. aeruginosa in the FPAs. These data emphasize the importance of using identical hardware for spaceflight experiments and ground simulations, especially when oxygen is a limiting factor.

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In addition, differences in fluid shear and other environmental conditions (such as irradiation) between actual microgravity and LSMMG need to be considered when comparing bacterial responses to the two test conditions.

This study represents an important step in understanding the response of bacterial opportunistic pathogens to the unique spaceflight environment. Furthermore, it allows assessment of the role that low-fluid-shear regions found in the human body play in the regulation of bacterial virulence. It remains to be determined whether the phenotype of P. aeruginosa acquired under spaceflight conditions will effectively lead to increased pathogenicity, as was observed for S. Typhimurium. This will be an important consideration and key area of future study in order to further assess the risk for infectious disease during long-term missions.

Spaceflight causes increased virulence of Serratia marcescens on a Drosophila melanogaster host Drosophila melanogaster, or the fruit fly, has long been an important organism for Earth-based research, and is now increasingly utilized as a model system to understand the biological effects of spaceflight. Studies in Drosophila melanogaster have shown altered immune responses in 3rd instar larvae and adult males following spaceflight, changes similar to those observed in astronauts. In addition, spaceflight has also been shown to affect bacterial physiology, as evidenced by studies describing altered virulence of Salmonella typhimurium following spaceflight and variation in biofilm growth patterns for the opportunistic pathogen Pseudomonas aeruginosa during flight.

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Fruit Fly Lab (Fruit Fly Lab)

The Fruit Fly Lab provides a research platform aboard the International Space Station (ISS) for longduration fruit fly (Drosophila Melanogaster) experiments in space. Such experiments will examine how microgravity and other aspects of the space environment affect these insects, providing information relevant to long-term human spaceflight, in particular the response to illness. Approximately 77% of the human disease genes have close matches in the fruit fly genome.

NASA sent Serratia marcescens Db11, a Drosophila pathogen and an opportunistic human pathogen, to the ISS on SpaceX-5 (Fruit Fly Lab-01). S. marcescens samples were stored at 4â&#x2014;Ś C for 24 days on-orbit and then allowed to grow for 120 hours at ambient station temperature before being returned to Earth. Upon return, bacteria were isolated and preserved in 50% glycerol or RNAlater. Storage, growth, and isolation for ground control samples were performed using the same procedures.

Spaceflight and ground samples stored in 50% glycerol were diluted and injected into 5-7-day-old groundborn adult D. melanogaster. Lethality was significantly greater in flies injected with the spaceflight samples compared to those injected with ground bacterial samples.

These results indicate a shift in the virulence profile of the spaceflight S. marcescens Db11 and will be further assessed with molecular biological analyses. The findings strengthen the conclusion that spaceflight impacts the virulence of bacterial pathogens on model host organisms such as the fruit fly.

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• Space flown samples of S. marcescens Db11 display increased virulence.

• This cause a significant increase in bacteria growth in w1118flies at 9hr and 12hrs post injection.

• Mutant line data show that altered bacterial growth are the primary cause for altered virulence.

• Survival of pathway mutants suggest that the imdpathway, not the toll pathway, is still important in the flies response to Ground Grey and Space Red sample injection.

Effects of Microgravity and Clinorotation on the Virulence of Klebsiella, Streptococcus, Proteus, and Pseudomonas To evaluate effects of microgravity on virulence, authors studied the ability of four common clinical pathogensKlebsiella, Streptococcus, Proteus, and Pseudomonas- to kill wild type Caenorhabditis elegans (C. elegans) nematodes at the larval and adult stages. Simultaneous studies were performed utilizing spaceflight, rotation in a 2D clinorotation device, and static ground controls. Nematodes, microbes, and growth media were separated until exposed to true or modeled microgravity, then mixed and grown for 48 hours.

Figure 3 illustrates the virulence of the four microorganisms towards nematodes in static/ground condition, clinorotation on ground, and microgravity of spaceflight. When cultured alone as growth controls, the four microorganisms grew as well as or better in spaceflight as they did in matched ground-based cultures, whereas clinorotation induced a striking increase in growth (Table 1). A comparison of worm/microorganism co-cultures with the growth controls revealed that the raw OD620 of microorganisms incubated with worms was higher than the raw OD620 of microorganisms cultured alone (Table 1). This does not reflect light absorption by the worms, as their light absorption at this wavelength is negligible. Instead, it appears the increased OD620 in the microorganism/worm mixtures reflects debris from worms that have been killed by the microorganism. Authors therefore calculated for each condition the difference in OD620 for microorganism mixed with worms minus the OD620 for microorganisms cultured alone. Authors used the delta (∆) OD620 as an index of virulence to compare the different culture conditions. Decreased virulence is indicated by a lower ∆ OD620 due to consumption of more microorganisms by the C. elegans. Table 1 provides the raw data used to calculate the ∆ OD620 and Figure 3 is based on those ∆ OD620 .

Pseudomonas was slightly numerically more virulent in spaceflight, but not statistically significantly when tested with adult worms (p = 0.07). There was no difference in virulence when assayed with larval worms (Figure 3).

Streptococcus was slightly less virulent in spaceflight when tested with adult worms (p=0.06), but showed

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no difference with larval worms. Virulence of Klebsiella was reduced by spaceflight when assayed with larval worms (p <0.01), but not adult worms. Virulence of Proteus was not different in spaceflight. When assayed under clinorotation conditions with adult or larval worms, all four organisms were significantly less virulent compared to static controls. The one exception was Pseudomonas with adult worms where the clinorotated samples were significantly less virulent than samples from spaceflight, but did not reach statistical significance when compared to static controls.

None of the wild type organisms that we have tested to date show increased virulence under either spaceflight or clinorotation (Table 3).

Spaceflight decreased the virulence of Streptococcus for adult C. elegans, which is similar to what we have previously reported with Candida, MRSA, Enterococcus, and Listeria (Table 3).

When larval C. elegans were the targets, spaceflight decreased the virulence of Klebsiella, which is what we have previously reported with Candida, Enterococcus, Listeria, and MRSA. Spaceflight had minimal effect on the virulence of Pseudomonas or Proteus, which is similar to what we have seen with Salmonella (Table 3).

Under clinorotation conditions, Klebsiella, Proteus, Pseudomonas, and Streptococcus were all less virulent with larval C. elegans, as authors have reported previously with Candida and Enterococcus (Table 3). Pseudomonas virulence for adult C. elegans was unaffected by clinorotation, just as authors have previously reported with Enterococcus, Listeria, MRSA, and Salmonella (Table 3). Overall, Pseudomonas, Klebsiella, Proteus, and Streptococcus showed far less virulence when tested in clinorotation than was observed in spaceflight (Table 2).

The current experiments found only modest changes in the virulence of Pseudomonas, Klebsiella, and Streptococcus, and no changes in the virulence of Proteus in spaceflight. This contrasts with our previous studies of Listeria, MRSA, Salmonella, and Candida albicans (C.albicans) that all showed significantly reduced virulence in spaceflight when tested with both larval and adult C. elegans.

When tested under conditions of clinorotation, the current report showed significantly reduced virulence of Pseudomonas, Klebsiella, Proteus, and Streptococcus. Authors have also previously found that Candida and Enterococcus were less virulent for larval worms, but not adult worms, when tested under clinorotation; whereas virulence of Salmonella, MRSA, and Listeria were unaffected in clinorotated tests with both adult and larval worms. Thus, using shear force to offset gravity did not consistently produce the same effect on virulence as did true spaceflight microgravity. This discordance may reflect variability in how different microorganisms are affected by the shear forces that are required to offset

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gravity in the clinorotation model.

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Effect of Spaceflight on Growth of Ulocladium chartarum Colonies on the ISS

The species Ulocladium chartarum has been chosen for this space flight experiment because it is well known to be involved in biodeterioration of organic and inorganic substrates covered with organic deposits and expected to be a possible contaminant in space stations. Ulocladium botrytis for example was isolated by Novikova et al next to Aspergillus niger and Cladosporium herbarum in samples taken from structural elements and internal surfaces of Soyuz taxi-flights and ISS between 1998-2005. Another reason to choose U. chartarum is the fact that it is also supposed to be resistant to space radiation due to its melanin content in hyphae and spores.

The objective of the 14 days spaceflight experiment was: first to investigate the effect of spaceflight on the colonies of different age and hyphae growth of U. chartarum, second to study the viability of the aerial and submerged mycelium and third to put in evidence changes at the cellular level.

Coloniesâ&#x20AC;&#x2122; Morphology, Growth and Viability The aerial mycelium was observed among the different BC and it showed differences in texture and colour, exudates size and colour, and colony surface and margin; the extension of the submerged mycelium outside the colony margin was also depending on the growth conditions (Table 1). Figure 3 shows clearly that the longer the colony could grow before launch the less pitted areas (Figure 4) and exudate drops were produced (BC#3: 5 days versus BC#2: 1 day). The irregular shape of the colony margin and the presence of pitted areas correlate with the amount of exudates, which keeps secondary metabolites, acting as toxins, near the hyphae. All these observations clearly suggest that the growth conditions have an influence on the morphology of the colonies.

The survival of hyphae, their ability to develop aerial and submerged mycelium as a consequence of branching, and finally their apical growth was evaluated by measuring the diameters of the colonies from pictures taken

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both on ISS and at the Institute of Biology (Bucharest, Romania). It was found that the growth rate of aerial and submerged mycelium were similar, therefore the overall colonyâ&#x20AC;&#x2122;s growth rate was evaluated using the diameter of the thick aerial mycelium only. As visible in Figure 5 (A, B, C), the diameter of the colonies was always larger in the flight samples than in the ground control, but smaller than in the laboratory control (optimal conditions).

Interestingly, the diameter difference between ground control and flight samples seems to be related with the time the colonies were growing before launch. In fact, the longer the colonies developed on ground (BC#3) the smaller the difference.

Not only the diameter of the colonies was larger but also the growth rate was mostly higher in flight than on ground (Figure 5 D, E, F). Only colonies in BC#3 showed a lower growth rate of the aerial mycelium in flight than on ground, though the submerged mycelium growth rate in this BC was high. Comparing the BC among each other, it is interesting to see that the highest growth rate was obtained in BC#2 during the 5 first days in space. These colonies have covered a larger surface of the substrate, compared to those from BC#1, which were two days older. Then, between FD5-FD9, the growth rate became very low, even lower than the one of BC#1. Generally, while in the laboratory control, under optimal conditions, full growth was found after five

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days of incubation, in flight and in the ground control the growth rate was decreasing over time until the aerial growth totally stopped. Growth curves in BC#2, 1 and 3 (Figure 5 A, B, C) showed that the aerial growth stopped between FD4-FD8, with a dependency on the inoculation day. A further growth of the colonies was observed at the level of the submerged mycelium, which gave rise to new colonies initially growing inside the nutrient layer (Figure 6). The formation of such microcolonies was not observed in the ground colonies of the same age. It seems that spaceflight conditions stimulate the growth of submerged mycelium. All viability tests performed on aerial mycelium and spores of all culture plates (flight and ground) revealed that hyphae from margins and sporulated aerial mycelium were not viable.

Microscopic examination of spores showed that about 10% were able to germinate but did not grow further to long and branched hyphae and, in consequence, were not able to form a colony. Submerged mycelium however was always alive and developed normal colonies. Viability tests performed on the laboratory control on the aerial mycelium and spores showed that they are viable.

Hyphal Growth and Curvature The length of the young hyphae growing at the margin of the colony was measured from the branching point to the tip. The maximal length observed between the different growth conditions was not statistically significant (data not shown), but authors observed an interesting difference in the length distribution between ground and flight (Figure 7A, C). The hyphae in the ground BCs were homogeneous and showed an average length of about 200 Âľm, determined from the fitting curve, where in the flight BCs hyphae were longer and showed a broader distribution. Especially interesting is the flight BC#2, with the colonies grown shortest before flight, i.e.1d; this sample showed the longest hyphae (about 500 Âľm) with the broadest distribution. It can be deduced, that spaceflight conditions positively influence the growth of the hyphae independently of their age. The curvature of the hyphae after branching was also investigated (Figure 7B, D). The mean value of the main population is determined from the fitting curve to be about 0.15 degrees/mm for all conditions. In the case of BC#3 (oldest colonies) a second population was observed at almost the double value 0.27 degrees/mm. In flight there was even a third peak of highly curved hyphae (<0.42 degrees/mm - multiple tip folding). Highly curved hyphae are easily damaged and are more prone to defects. This can explain the high density of defects observed in this sample (Figure 8).

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Branches close to the edge of the colony represent young hyphae, which are expected to be good "sensors" of the spaceflight effects, since growth of aerial mycelium had stopped after a certain time under flight conditions. Topologically, branches can grow equally to their parent hyphae from the point they branched, or two branches can evolve from a hyphae tip and grow simultaneously. As for the hyphae distribution, the length distribution of the branches was also much broader in flight than on ground (Figure 9A, B). Interestingly, in flight, the branch length in BC#2 was uniformly distributed up to 400 Âľm, whereas in BC#3 (longest growth before flight, i.e.

5d) the distribution is similar to the ground sample; and in BC#1 an intermediate behaviour is detected.

As for the hyphae curvature, the branch curvature did not show significant difference between flight and ground samples. The only difference to be noticed is the larger amount of branches with higher degree of curvature in the samples of BC#3 flight (Figure 10A, B). Here many hyphae did heavily fold, correlating well with the results presented in Figure 8.

Since in-flight the submerged growth was important, authors also performed depth profiles measurements on all BC, except for the ground BC#3 in which the aerial mycelium reached the edge of the culture plate producing blurred and dark images. The flight experiment produced hyphae that extended in depth over long distances (Figure 11), in some cases very close to the edge of the culture plate and always initiating new colonies (Figure 6). This was not observed for the ground samples during the timeframe of this experiment.

Microcolonies Under spaceflight conditions, microcolonies evolved from a submerged hyphae extending towards the edge of the culture plates far away from the originating initial colony (Figure 6 and 11). Normally, individual hyphae exhibit an apical dominance; the growing tip suppresses branching in its vicinity, but sub-apical cells may generate new hyphae by lateral branching. In flight, some of the submerged hyphae lost the apical dominance and a chaotic branching took place. Microcolonies started in the depth of the substrate as a very dense mycelium, and then submerged hyphae oriented themselves towards the surface where oxygen is available, as proven by

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Authors observed on pictures of BC#2 and BC#1 taken just after landing submerged mycelium looking like white dots. While, on the pictures of BC#3, the microcolonies had already reached the surface of the substrate and sporulated between FD9 and return; in BC#2 the microcolonies became visible on the surface of the substrate 3 days after return, in BC#1, 4 days after return. Similarly, microcolonies were found inside the substrate (white dots) in samples grown on the RPM (Random Positioning Machine) in the frame of ground studies (unpublished results) and in ground samples, which have been incubated for much longer time than the duration of the mission.

Microcolonies have the same characteristics as the initial colonies but are of smaller size (2-5 mm in diameter). They are constituted of aerial and submerged mycelium (Figure 12A, B). Hyphae of microcolonies are shorter and wider than those from submerged mycelium. In fact, the length of the microcolonies’ hyphae are mostly around 20-60 µm (Figure 13A) in comparison with the average of 200 µm in the main colonies (Figure 7). The degree of curvature (Figure 13B) is much larger in microcolonies (average about 1.1 degree/µm) than

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in the initial colony (0.1-0.12 degrees/Âľm). The limit between 2 microcolonies is made of healthy submerged hyphae (Figure 14A), differently between the initial and new colony folded and empty hyphae can be found (Figure 14B).

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This study clearly indicates that Ulocladium chartarum is able to grow under spaceflight conditions developing, as a response, a complex colony morphotype that has not been previously mentioned. The colony growth cycle of U. chartarum provides a useful eukaryotic system for the study of fungal growth under spaceflight conditions.

Here we prove that the growth of U. chartarum colonies is not affected by spaceflight.

Broad-Spectrum Antibiotic or G-CSF as Potential Countermeasures for Impaired Control of Bacterial Infection Associated with an SPE Exposure during Spaceflight In the study, authors investigated the efficacy of enrofloxacin (an orally bioavailable antibiotic) and Granulocyte colony stimulating factor (G-CSF) (Neulasta) on enhancing resistance to Pseudomonas aeruginosa infection in mice subjected to HS and SPE-like radiation. The results revealed that treatment with enrofloxacin or G-CSF enhanced bacterial clearance and significantly decreased morbidity and mortality in challenged mice exposed to suspension and radiation. These results establish that antibiotics, such as enrofloxacin, and G-CSF could be effective countermeasures to decrease the risk of bacterial infections after exposure to SPE radiation during extended space flight, thereby reducing both the risk to the crew and the danger of mission failure.

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