Isolation of Novel Extreme-Tolerant Cyanobacteria from a Rock-Dwelling Microbial Community by Using Exposure to Low Earth Orbit

Many cyanobacteria are known to tolerate environmental extremes. Motivated by an interest in selecting cyanobacteria for applications in space, we launched rocks from a limestone cliff in Beer, Devon, United Kingdom, containing an epilithic and endolithic rock-dwelling community of cyanobacteria into low Earth orbit (LEO) at a height of approximately 300 kilometers. The community was exposed for 10 days to isolate cyanobacteria that can survive exposure to the extreme radiation and desiccating conditions associated with space. Culture-independent (16S rRNA) and culture-dependent methods showed that the cyanobacterial community was composed of Pleurocapsales, Oscillatoriales, and Chroococcales. A single cyanobacterium, a previously uncharacterized extremophile, was isolated after exposure to LEO. We were able to isolate the cyanobacterium from the limestone cliff after exposing the rock-dwelling community to desiccation and vacuum (0.7 × 10⁻³ kPa) in the laboratory. The ability of the organism to survive the conditions in space may be linked to the formation of dense colonies. These experiments show how extreme environmental conditions, including space, can be used to select for novel microorganisms. Furthermore, it improves our knowledge of environmental tolerances of extremophilic rock-dwelling cyanobacteria.The surface and interior of rocks is a ubiquitous environment for microorganisms. Comprehensive culturing and culture-independent analyses of endolithic (interior of rocks) and epilithic (on the surface of rocks) microbial communities have been conducted. The primary producers in these environments are phototrophs, such as cyanobacteria, that are either free living or endosymbionts in lichens (16).Epilithic microorganisms are often an important part of rock-dwelling communities. The characterization of the epilithic cyanobacteria from natural environments, such as beach rock and caves, and from human-made environments, such as hypogea and buildings, has identified a variety of cyanobacteria. This includes both unicellular and filamentous forms, for example, Lyngbya-related species and Chroococcidiopsis (5, 14, 37, 47).Many microorganisms also inhabit the interior of rocks as endoliths. The endolithic environment provides protection from environmental stresses such as desiccation, extreme temperature, UV radiation, and high photosynthetically active radiation (400 to 700 nm) (6, 16, 25, 32). Endolithic communities are often the dominant form of life in extreme environments such as hot and cold deserts (15-17), savannahs, and semideserts (3, 6, 15, 48). In these extreme environments, the endolithic cyanobacteria are mainly unicellular cyanobacteria, for example, Chroococcidiopsis, Myxosarcina, and Gloeocapsa species (11, 46, 50). Conversely, in nondesert environments, such as dolomitic rocks in Switzerland (41), the limestone of the Niagara Escarpment (19, 20), and travertine deposits in Yellowstone National Park, the endolithic communities are more diverse and include both filamentous and unicellular types of cyanobacteria, such as Leptolyngbya, Nostoc, and Synechocystis (34).Although rock-dwelling cyanobacteria communities are diverse, there has been limited, if any, use of artificial extreme conditions to select for novel extremophilic cyanobacteria from these environments. Such an investigation could have implications for understanding the physiological requirements of life in extreme environments.The work described in this paper was motivated by an interest in understanding the physiological tolerance of cyanobacteria to space conditions and their potential use in space applications, such as oxygen and feedstock provision, which are crucial for extraterrestrial settlements (23, 29). In this work, we exposed samples of a coastal limestone cliff in Beer, Devon, United Kingdom, which is inhabited by a diverse cyanobacterial community, to low Earth orbit (LEO) to isolate novel extreme-tolerant cyanobacteria.     

Arsenic in the Evolution of Earth and Extraterrestrial Ecosystems

If you were asked to speculate about the form extra-terrestrial life on Mars might take, which geomicrobial phenomenon might you select as a model system, assuming that life on Mars would be ‘primitive’? Give your reasons.     

Persistence of Biomarker ATP and ATP-Generating Capability in Bacterial Cells and Spores Contaminating Spacecraft Materials under Earth Conditions and in a Simulated Martian Environment

Most planetary protection research has concentrated on characterizing viable bioloads on spacecraft surfaces, developing techniques for bioload reduction prior to launch, and studying the effects of simulated martian environments on microbial survival. Little research has examined the persistence of biogenic signature molecules on spacecraft materials under simulated martian surface conditions. This study examined how endogenous adenosine-5′-triphosphate (ATP) would persist on aluminum coupons under simulated martian conditions of 7.1 mbar, full-spectrum simulated martian radiation calibrated to 4 W m⁻² of UV-C (200 to 280 nm), −10°C, and a Mars gas mix of CO₂ (95.54%), N₂ (2.7%), Ar (1.6%), O₂ (0.13%), and H₂O (0.03%). Cell or spore viabilities of Acinetobacter radioresistens, Bacillus pumilus, and B. subtilis were measured in minutes to hours, while high levels of endogenous ATP were recovered after exposures of up to 21 days. The dominant factor responsible for temporal reductions in viability and loss of ATP was the simulated Mars surface radiation; low pressure, low temperature, and the Mars gas composition exhibited only slight effects. The normal burst of endogenous ATP detected during spore germination in B. pumilus and B. subtilis was reduced by 1 or 2 orders of magnitude following, respectively, 8- or 30-min exposures to simulated martian conditions. The results support the conclusion that endogenous ATP will persist for time periods that are likely to extend beyond the nominal lengths of most surface missions on Mars, and planetary protection protocols prior to launch may require additional rigor to further reduce the presence and abundance of biosignature molecules on spacecraft surfaces.     

Survival of Methanogenic Archaea from Siberian Permafrost under Simulated Martian Thermal Conditions

Methanogenic archaea from Siberian permafrost complementary to the already well-studied methanogens from non-permafrost habitats were exposed to simulated Martian conditions. After 22 days of exposure to thermo-physical conditions at Martian low- and mid-latitudes up to 90% of methanogenic archaea from Siberian permafrost survived in pure cultures as well as in environmental samples. In contrast, only 0.3%–5.8% of reference organisms from non-permafrost habitats survived at these conditions. This suggests that methanogens from terrestrial permafrost seem to be remarkably resistant to Martian conditions. Our data also suggest that in scenario of subsurface lithoautotrophic life on Mars, methanogenic archaea from Siberian permafrost could be used as appropriate candidates for the microbial life on Mars.     

Effects of Cyanobacteria on Survival and Reproduction of the Littoral Crustacean Gammarus zaddachi (Amphipoda)

Toxic cyanobacteria can have harmful or fatal impacts on aquatic organisms. In the archipelagos of the northern Baltic Sea, the open sea blooms often drift into littoral areas, where they decompose and release toxins and other chemical compounds in the water. However, the effects of cyanobacteria on the littoral organisms have not previously been investigated. We studied the effects of three cyanobacteria species (toxic Nodularia spumigena, non-toxic N. sphaerocarpa and non-toxic Aphanizomenon flos-aquae) and purified dissolved nodularin (produced by N. spumigena) on a common littoral amphipod Gammarus zaddachi. Nodularin was transferred to eggs, juveniles and adults of G. zaddachi, but no significant negative effects of dissolved nodularin were detected on adults, eggs or juveniles. However, survival of adults decreased by the exposure to toxic N. spumigena cells. The egg hatching rate and juvenile survival were not affected when exposed to the three cyanobacteria species. In contrast, a weak decrease in the egg production and an increased abortion of embryos from the brood pouch of females was observed, the later indicating a failure in parental care. Further, a decrease in grazing rate on the filamentous green alga Enteromorpha intestinalis was observed. The results suggest that toxic cyanobacteria blooms are not extremely fatal, but may have, in high concentrations, negative effects on the adult survival, fecundity, and feeding behaviour of gammarids inhabiting the littoral zone.     

Factors Affecting the Germination of Akinetes of Nodularia spumigena (Cyanobacteriaceae)

Nutritional and physical factors which influence the germination of akinetes of Nodularia spumigena (Cyanobacteriaceae) were examined. Low concentrations of phosphorus (<0.9 μM) were required for germination. Nitrate had no effect, but ammonia, at concentrations of >45 μM, inhibited germination. Salinities of >20‰ were inhibitory to germination. Optimum temperatures were 22°C or greater. Germination did not take place in the dark, but only very low light intensities (0.5 microeinstein m⁻² s⁻¹) were necessary to initiate germination. Red light (620 to 665 nm) was required. More than 24 h of continuous exposure to light was necessary for any significant germination to occur. The conditions for germination corresponded with conditions in the Peel-Harvey Estuary, Western Australia, 2 to 3 weeks before large summer Nodularia blooms.     

Effects of Simulated Mars Conditions on the Survival and Growth of Escherichia coli and Serratia liquefaciens

Escherichia coli and Serratia liquefaciens, two bacterial spacecraft contaminants known to replicate under low atmospheric pressures of 2.5 kPa, were tested for growth and survival under simulated Mars conditions. Environmental stresses of high salinity, low temperature, and low pressure were screened alone and in combination for effects on bacterial survival and replication, and then cells were tested in Mars analog soils under simulated Mars conditions. Survival and replication of E. coli and S. liquefaciens cells in liquid medium were evaluated for 7 days under low temperatures (5, 10, 20, or 30°C) with increasing concentrations (0, 5, 10, or 20%) of three salts (MgCl₂, MgSO₄, NaCl) reported to be present on the surface of Mars. Moderate to high growth rates were observed for E. coli and S. liquefaciens at 30 or 20°C and in solutions with 0 or 5% salts. In contrast, cell densities of both species generally did not increase above initial inoculum levels under the highest salt concentrations (10 and 20%) and the four temperatures tested, with the exception that moderately higher cell densities were observed for both species at 10% MgSO₄ maintained at 20 or 30°C. Growth rates of E. coli and S. liquefaciens in low salt concentrations were robust under all pressures (2.5, 10, or 101.3 kPa), exhibiting a general increase of up to 2.5 orders of magnitude above the initial inoculum levels of the assays. Vegetative E. coli cells were maintained in a Mars analog soil for 7 days under simulated Mars conditions that included temperatures between 20 and −50°C for a day/night diurnal period, UVC irradiation (200 to 280 nm) at 3.6 W m⁻² for daytime operations (8 h), pressures held at a constant 0.71 kPa, and a gas composition that included the top five gases found in the martian atmosphere. Cell densities of E. coli failed to increase under simulated Mars conditions, and survival was reduced 1 to 2 orders of magnitude by the interactive effects of desiccation, UV irradiation, high salinity, and low pressure (in decreasing order of importance). Results suggest that E. coli may be able to survive, but not grow, in surficial soils on Mars.The search for extant life on Mars remains a stated goal of NASA''s Mars Exploration Program and Astrobiology Institutes (13, 17). Intrinsic within such a life detection strategy is a requirement to understand how terrestrial life might survive, replicate, and proliferate on Mars. To mitigate the risks of the forward contamination of Mars, the bioloads on spacecrafts targeted for landing must be reduced to low density and diversity (4, 7). Planetary protection guidelines are designed to prevent both the forward contamination of the martian surface and to ensure the scientific integrity of any deployed life detection experiments. To date, 12 spacecraft have landed or crashed onto the Mars surface as a result of U.S., Russian, and European space program missions, but it is currently unknown if terrestrial microorganisms typically found on spacecraft surfaces can grow and replicate under conditions encountered on the surface (44, 45, 48).Despite cleaning and sterilization measures taken to significantly reduce microbial bioloads on spacecraft (26, 56), diverse microbial communities remain at the time of launch (7, 31, 32, 44). The diversity of microorganisms found on spacecraft surfaces are generally characteristic of the clean rooms within which the spacecraft are processed. Spacecraft assembly facilities are oligotrophic extreme environments in which only the most resilient species survive the high-desiccation, low-nutrient conditions, controlled air circulation, and the rigors of bioburden reduction (56, 57). The biological inventory of microorganisms on spacecraft has mostly been limited to isolation and identification using standard culture-based microbiological assays (44, 48, 53). However, culture-based microbiological assays likely underestimate the biological diversity present on spacecraft, as traditional culture techniques fail to capture more than 99.9% of present phylotypes (7). Recently, the simultaneous use of culture-dependent and culture-independent techniques (e.g., Limulus amoebocyte lysate assay [LAL], ATP bioluminescence assay, lipopolysaccharide-based microbial detection, and DNA-based PCR) have identified many nonculturable species (31, 32, 57). Known culturable bacteria recovered from spacecraft surfaces include, but are not limited to, species of Acinetobacter, Bacillus, Corynebacterium, Escherichia, Flavobacterium, Micrococcus, Pseudomonas, Serratia, Staphylococcus, and Streptococcus (44, 53, 57).After launch, spacecraft are exposed to interplanetary conditions of ultralow pressure (3 × 10⁻¹⁰ kPa), extreme desiccating conditions, fluctuating temperatures, solar UV irradiation, and ionizing radiation (22, 44). Furthermore, upon landing, the conditions on the surface of Mars are not much improved over interplanetary space. Diverse biocidal or inhibitory conditions on Mars have been identified in a number of recent publications (8, 21, 22, 35, 36, 38, 44, 48, 59) and include the following (not in order of priority): solar UVC irradiation, low pressure, extreme desiccating conditions, extreme diurnal temperature fluctuations, solar particle events, galactic cosmic rays, UV glow discharge from blowing dust, solar UV-induced volatile oxidants (e.g., O₂⁻, O⁻, H₂O₂, NO_x, O₃), globally distributed oxidizing soils, extremely high salt levels (e.g., MgCl₂, NaCl, FeSO₄, and MgSO₄) in surficial soils at some sites on Mars, high concentrations of heavy metals in martian soils, acidic conditions in martian regolith, high CO₂ concentrations in the global atmosphere, and presence of perchlorates in some regoliths. UV irradiation, especially UVC photons (200 to 280 nm), may be the most biocidal of all factors to microbial survival on the martian surface (34, 37, 39, 47, 50, 52). Microorganisms found on sun-exposed surfaces of spacecraft are killed off within a few tens of minutes of exposure; but if covered by as little as a few hundred micrometers of martian soil, significant protection is provided (11, 34, 47). It is currently unknown if terrestrial microorganisms typically found on spacecraft surfaces can grow and replicate under conditions encountered on the surface of Mars (44, 48).In the studies cited above, most research focused on the survival of dormant spores or vegetative cells under Mars conditions. In contrast, only a few papers have explored the possibility of growth and replication of terrestrial microorganisms under environmental conditions that approach those found in surficial soils of Mars (5, 25, 45, 48). Of these four, 2.5 kPa is the lowest pressure at which replication was observed for a few bacterial species (5, 45, 48).The primary objective of the current study was to expose two non-spore-forming species to environmental stresses present on the surface of Mars to characterize the potential response of the bacteria to martian temperatures, salinities, and pressures. Two bacterial species, Escherichia coli and Serratia liquefaciens, were selected from over 30 prokaryotic species tested in preliminary experiments (5, 45). Their selection was based on their common association with humans, recovery from robotic spacecraft and space-based human life support systems (44, 53), and demonstrated replication at 2.5 kPa of total atmospheric pressure (5, 45). Experiments were conducted on cell suspensions in liquid medium at combinations of low pressure, high salt concentrations, and low temperatures, and then with cells mixed into soils and exposed to simulated Mars conditions. It was predicted for cell suspensions that (i) low temperatures would dramatically retard cell proliferation, (ii) high concentrations of salts would be biocidal on cell suspensions, and (iii) low pressure would have weak to moderate inhibitory effects on cell growth of both species. For cells in soils, growth was not expected under Mars simulations which exposed vegetative cells to low pressure, low temperatures, anaerobic gas composition, and high UVC irradiation similar to the martian surface. Although replication was not predicted, bacterial survival in analog Mars soils under simulated Mars conditions was anticipated.     

RNAi-Mediated Knockdown of Catalase Causes Cell Cycle Arrest in SL-1 Cells and Results in Low Survival Rate of Spodoptera litura (Fabricius)

Deregulated reactive oxygen species (ROS) production can lead to the disruption of structural and functional integrity of cells as a consequence of reactive interaction between ROS and various biological components. Catalase (CAT) is a common enzyme existing in nearly all organisms exposed to oxygen, which decomposes harmful hydrogen peroxide, into water and oxygen. In this study, the full length sequence that encodes CAT-like protein from Spodoptera litura named siltCAT (GenBank accession number: JQ_663444) was cloned and characterized. Amino acid sequence alignment showed siltCAT shared relatively high conservation with other insect, especially the conserved residues which defined heme and NADPH orientation. Expression pattern analysis showed that siltCAT mRNA was mainly expressed in the fat body, midgut, cuticle and malpighian tube, and as well as over last instar larvae, pupa and adult stages. RNA interference was used to silence CAT gene in SL-1 cells and the fourth-instar stage of S. litura larvae respectively. Our results provided evidence that CAT knockdown induced ROS generation, cell cycle arrest and apoptosis in SL-1 cells. It also confirmed the decrease in survival rate because of increased ROS production in experimental groups injected with double-stranded RNA of CAT (dsCAT). This study implied that ROS scavenging by CAT is important for S. litura survival.     

First report on the allelopathic effect of Tychonema bourrellyi (Cyanobacteria) against Microcystis aeruginosa (Cyanobacteria)

The allelopathic effect of the cyanobacterium Tychonema bourrellyi against the cyanobacterium Microcystis aeruginosa is reported for the first time in this paper. The filtrate of T. bourrellyi CHAB663 culture showed strong inhibitory effect on M. aeruginosa NIES-843, but the inhibitory effect was weakened by shaking culture, and such results implied that the allelopathic effect was probably mediated by the volatile substances secreted by T. bourrellyi. β-Ionone was identified as a major ingredient in the volatile substances in the cultures of T. bourrellyi, and it may act as an important allelochemical responsible for this allelopathic activity. The filtrates of T. bourrellyi culture were shown to decrease the maximum electron transport rate (ETR_max) and elevate the reactive oxygen species (ROS) levels in the cells of M. aeruginosa NIES-843.     

Investigation of fungal biomolecules after Low Earth Orbit exposure: a testbed for the next Moon missions

The Moon is characterized by extremely harsh conditions due to ultraviolet irradiation, wide temperature extremes, vacuum resulting from the absence of an atmosphere and high ionizing radiation. Therefore, its surface may provide a unique platform to investigate the effects of such conditions. For lunar exploration with the Lunar Gateway platform, exposure experiments in Low Earth Orbit are useful testbeds to prepare for lunar space experiments and to understand how and if potential biomarkers are influenced by extra-terrestrial conditions. During the BIOMEX (BIOlogy and Mars EXperiment) project, dried colonies of the fungus Cryomyces antarcticus grown on Lunar Regolith Analogue (LRA) were exposed to space conditions for 16 months aboard the EXPOSE-R2 payload outside the International Space Station. In this study, we investigated the stability/degradation of fungal biomarkers in LRA after exposure to (i) simulated space and (ii) real space conditions, using Raman spectroscopy, gas chromatography–mass spectrometry and DNA amplification. The results demonstrated that fungal biomarkers were detectable after 16 months of real space exposure. This work will contribute to the interpretation of data from future biological experiments in the Cislunar orbit with the Lunar Gateway platform and/or on the lunar surface, in preparation for the next step of human exploration.     

Biotransformation of Hg(II) by Cyanobacteria

The biotransformation of Hg(II) by cyanobacteria was investigated under aerobic and pH-controlled culture conditions. Mercury was supplied as HgCl₂ in amounts emulating those found under heavily impacted environmental conditions where bioremediation would be appropriate. The analytical procedures used to measure mercury within the culture solution, including that in the cyanobacterial cells, used reduction under both acid and alkaline conditions in the presence of SnCl₂. Acid reduction detected free Hg(II) ions and its complexes, whereas alkaline reduction revealed that meta-cinnabar (β-HgS) constituted the major biotransformed and cellularly associated mercury pool. This was true for all investigated species of cyanobacteria: Limnothrix planctonica (Lemm.), Synechococcus leopoldiensis (Racib.) Komarek, and Phormidium limnetica (Lemm.). From the outset of mercury exposure, there was rapid synthesis of β-HgS and Hg(0); however, the production rate for the latter decreased quickly. Inhibitory studies using dimethylfumarate and iodoacetamide to modify intra- and extracellular thiols, respectively, revealed that the former thiol pool was required for the conversion of Hg(II) into β-HgS. In addition, increasing the temperature enhanced the amount of β-HgS produced, with a concomitant decrease in Hg(0) volatilization. These findings suggest that in the environment, cyanobacteria at the air-water interface could act to convert substantial amounts of Hg(II) into β-HgS. Furthermore, the efficiency of conversion into β-HgS by cyanobacteria may lead to the development of applications in the bioremediation of mercury.     

Survival and Reproduction Potential of Philodina roseola (Ehrenberg) (Rotatoria,Bdelloida) under Various Temperature Conditions

Survival data, in addition to data on growth and reproduction rate, are necessary to estimate rotifer productivity under various thermal conditions. A method of individual culturing of Philodina roseola helped the authors to obtain data concerning length of life span and realisation of reproduction potential at different temperatures, ranging from 9 to 35 °C. The method also was helpful in revealing the causes of individual deaths. It is shown that at a temperature of 20 °C net reproduction rate (R₀) of Ph. roseola is maximum and is equal to reproduction potential of the individual. The peculiarity of Philodina ontogenetic reaction to temperature beyond optimum is the impairment of reproduction process.     

Extraterrestrial Flux of Potentially Prebiotic C, N, and P to the Early Earth

With growing evidence for a heavy bombardment period ending 4–3.8 billion years ago, meteorites and comets may have been an important source of prebiotic carbon, nitrogen, and phosphorus on the early Earth. Life may have originated shortly after the late-heavy bombardment, when concentrations of organic compounds and reactive phosphorus were enough to “kick life into gear”. This work quantifies the sources of potentially prebiotic, extraterrestrial C, N, and P and correlates these fluxes with a comparison to total Ir fluxes, and estimates the effect of atmosphere on the survival of material. We find (1) that carbonaceous chondrites were not a good source of organic compounds, but interplanetary dust particles provided a constant, steady flux of organic compounds to the surface of the Earth, (2) extraterrestrial metallic material was much more abundant on the early Earth, and delivered reactive P in the form of phosphide minerals to the Earth’s surface, and (3) large impacts provided substantial local enrichments of potentially prebiotic reagents. These results help elucidate the potential role of extraterrestrial matter in the origin of life.     

Hydrogen Peroxide Inhibits Photosynthetic Electron Transport in Cells of Cyanobacteria

The effect of H₂O₂ on photosynthetic O₂ evolution and photosynthetic electron transfer in cells of cyanobacteria Anabaena variabilis and Anacystis nidulans was studied. The following experiments were performed: 1) directly testing the effect of exogenous H₂O₂; 2) testing the effect of intracellular H₂O₂ generated with the use of methyl viologen (MV); 3) testing the effect of inhibiting intracellular H₂O₂ decomposition by salicylic acid (SA) and 3-amino-1,2,4-triazole (AT). H₂O₂ inhibited photosynthetic O₂ evolution and light-induced reduction of p-benzoquinone (BQ) + ferricyanide (FeCy) in the Hill reaction. The I₅₀ value for H₂O₂ was 0.75 mM. Photosynthetic electron transfer in the cells treated with H₂O₂ was not maintained by H₂O₂, NH₂OH, 1,5-diphenylcarbazide, tetraphenylboron, or butylated hydroxytoluene added as artificial electron donors for Photosystem (PS) II. The H₂O CO₂, H₂O MV (involving PSII and PSI) and H₂O BQ + FeCy (chiefly dependent on PSII) electron transfer reactions were inhibited upon incubation of the cells with MV, SA, or AT. The N,N,N",N"-tetramethyl-p-phenylenediamine MV (chiefly dependent on PSI) electron transfer was inhibited by SA and AT but was resistant to MV. The results show that H₂O₂ inhibits photosynthetic electron transfer. It is unlikely that H₂O₂ could be a physiological electron donor in oxygenic photosynthesis.     

Growth-limiting quantities and accumulation of molybdenum in Anabaena oscillarioides (Cyanobacteria)

Molybdenum deficiency and accumulation was examined in Anabaena oscillarioides (Cyanobacteria). Molybdenum deficiency was induced by: 1) culturing the cyanobacterium on modified Chu-10 (-N) medium containing 4–5 ng Mo · l⁻¹, or 2) adding tungsten to reversibly inactivate dinitrogenase. Stimulation of dinitrogenase activity, while heterocyst frequencies were decreasing, occurred in the range of 5–40 ng · l⁻¹ of added Mo. Molybdenum deficient A. oscillarioides was able to deplete Mo in the medium. This ability was rapidly lost at higher concentrations of added Mo when this cyanobacterium started to accumulate Mo. These results are of potential use in predicting potential Mo limitation in natural environments.     

Effects of Low Doses of Pioglitazone on Resting-State Functional Connectivity in Conscious Rat Brain

Pioglitazone (PIO) is a peroxisome proliferator-activated receptor-γ (PPARγ) agonist in clinical use for treatment of type 2 diabetes (T2DM). Accumulating evidence suggests PPARγ agonists may be useful for treating or delaying the onset of Alzheimer’s disease (AD), possibly via actions on mitochondria, and that dose strengths lower than those clinically used for T2DM may be efficacious. Our major objective was to determine if low doses of pioglitazone, administered orally, impacted brain activity. We measured blood-oxygenation-level dependent (BOLD) low-frequency fluctuations in conscious rats to map changes in brain resting-state functional connectivity due to daily, oral dosing with low-dose PIO. The connectivity in two neural circuits exhibited significant changes compared with vehicle after two days of treatment with PIO at 0.08 mg/kg/day. After 7 days of treatment with a range of PIO dose-strengths, connections between 17 pairs of brain regions were significantly affected. Functional connectivity with the CA1 region of the hippocampus, a region that is involved in memory and is affected early in the progression of AD, was specifically investigated in a seed-based analysis. This approach revealed that the spatial pattern of CA1 connectivity was consistent among all dose groups at baseline, prior to treatment with PIO, and in the control group imaged on day 7. Compared to baseline and controls, increased connectivity to CA1 was observed regionally in the hypothalamus and ventral thalamus in all PIO-treated groups, but was least pronounced in the group treated with the highest dose of PIO. These data support our hypothesis that PIO modulates neuronal and/or cerebrovascular function at dose strengths significantly lower than those used to treat T2DM and therefore may be a useful therapy for neurodegenerative diseases including AD.     

Unraveling Cyanobacteria Ecology in Wastewater Treatment Plants (WWTP)

Cyanobacteria may be important components of wastewater treatment plants’ (WWTP) biological treatment, reaching levels of 100% of the total phytoplankton density in some systems. The occurrence of cyanobacteria and their associated toxins in these systems present a risk to the aquatic environments and to public health, changing drastically the ecology of microbial communities and associated organisms. Many studies reveal that cyanotoxins, namely microcystins may not act as antibacterial compounds but they might have negative impacts on protozoans, inhibiting their growing and respiration rates and leading to changes in cellular morphology, decreasing consequently the treatment efficacy in WWTP. On the other side, flagellates and ciliates may ingest some cyanobacteria species while the formation of colonies by these prokaryotes may be seen as a defense mechanism against predation. Problems regarding the occurrence of cyanobacteria in WWTP are not limited to toxin production. Other cyanobacterial secondary metabolites may act as antibacterial compounds leading to the disruption of bacterial communities that biologically convert organic materials in WWTP being fundamental to the efficacy of the process. Studies reveal that the potential antibacterial capacity differs according to cyanobacteria specie and it seems to be more effective in Gram (+) bacteria. Thus, to understand the effects of cyanobacterial communities in the efficiency of the waste water treatment it will be necessary to unravel the complex interactions between cyanobacterial populations, bacteria, and protozoa in WWTP in situ studies.     

Discovery and Function of B-Cell IgD Low (BDL) B Cells in Immune Tolerance

It is now appreciated that in addition to their role in humoral immunity, B cells also exert regulatory mechanisms that lead to attenuation of inflammatory responses. The concept of B-cell regulation became well recognized when mice deficient in B cells due to genetic disruption were shown to be refractory to recovery from the signs of experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis. This seminal study spurred the search for B-cell regulatory phenotypes and mechanisms of action. Our approach was to utilize differential B-cell depletion with anti-CD20 to retain B cells whose presence were required to achieve EAE recovery. Utilizing flow cytometry, adoptive cell therapy and genetic approaches, we discovered a new B-cell subset that, upon adoptive transfer into B cell-deficient mice, was sufficient to promote EAE recovery. This B-cell subset is IgM⁺, but due to low/negative IgD cell surface expression, it was named B-cell IgD low (BD_L). Mechanistically, we found that in the absence of BD_L, the absolute cell number of CD4⁺ Foxp3⁺ T regulatory cells (Treg), essential for immune tolerance, was significantly reduced. Furthermore, we found that BD_L expression of glucocorticoid-induced tumor necrosis factor ligand (GITRL) was essential for induction of Treg proliferation and maintenance of their homeostasis. Thus, we have identified a new B-cell subset that is critical for immunological tolerance through interactions with Treg.