CULTURE OF THE TERRESTRIAL CYANOBACTERIUM,NOSTOC FLAGELLIFORME (CYANOPHYCEAE), UNDER AQUATIC CONDITIONS1

Both colonies and free‐living cells of the terrestrial cyanobacterium, Nostoc flagelliforme (Berk. & Curtis) Bornet & Flahault, were cultured under aquatic conditions to develop the techniques for the cultivation and restoration of this endangered resource. The colonial filaments disintegrated with their sheaths ruptured in about 2 days without any desiccating treatments. Periodic desiccation played an important role in preventing the alga from decomposing, with greater delays to sheath rupture with a higher frequency of exposure to air. The bacterial numbers in the culture treated with seven periods of desiccation per day were about 50% less compared with the cultures without the desiccation treatment. When bacteria in the culture were controlled, the colonial filaments did not disintegrate and maintained the integrity of their sheath for about 20 days even without the desiccation treatments, indicating the importance of desiccation for N. flagelliforme to prevent them from being disintegrated by bacteria. On the other hand, when free‐living cells obtained from crushed colonial filaments were cultured in liquid medium, they developed into single filaments with sheaths, within which multiple filaments were formed later on as a colony. Such colonial filaments were developed at 15, 25, and 30° C at either 20 or 60 μmol photons·m^?2·s^?1; colonies did not develop at 180 μmol photons·m^?2·s^?1, though this light level resulted in the most rapid growth of the cells. Conditions of 60 μmol photons·m^?2·s^?1 and 25° C appeared to result in the best colonial development and faster growth of the sheath‐held colonies of N. flagelliforme when cultured indoor under aquatic conditions.     

Carbon,nitrogen and O2 fluxes associated with the cyanobacterium Nodularia spumigena in the Baltic Sea

Photosynthesis, respiration, N₂ fixation and ammonium release were studied directly in Nodularia spumigena during a bloom in the Baltic Sea using a combination of microsensors, stable isotope tracer experiments combined with nanoscale secondary ion mass spectrometry (nanoSIMS) and fluorometry. Cell-specific net C- and N₂-fixation rates by N. spumigena were 81.6±6.7 and 11.4±0.9 fmol N per cell per h, respectively. During light, the net C:N fixation ratio was 8.0±0.8. During darkness, carbon fixation was not detectable, but N₂ fixation was 5.4±0.4 fmol N per cell per h. Net photosynthesis varied between 0.34 and 250 nmol O₂ h⁻¹ in colonies with diameters ranging between 0.13 and 5.0 mm, and it reached the theoretical upper limit set by diffusion of dissolved inorganic carbon to colonies (>1 mm). Dark respiration of the same colonies varied between 0.038 and 87 nmol O₂ h⁻¹, and it reached the limit set by O₂ diffusion from the surrounding water to colonies (>1 mm). N₂ fixation associated with N. spumigena colonies (>1 mm) comprised on average 18% of the total N₂ fixation in the bulk water. Net NH₄⁺ release in colonies equaled 8–33% of the estimated gross N₂ fixation during photosynthesis. NH₄⁺ concentrations within light-exposed colonies, modeled from measured net NH₄⁺ release rates, were 60-fold higher than that of the bulk. Hence, N. spumigena colonies comprise highly productive microenvironments and an attractive NH₄⁺ microenvironment to be utilized by other (micro)organisms in the Baltic Sea where dissolved inorganic nitrogen is limiting growth.     

Metabolomic approach to optimizing and evaluating antibiotic treatment in the axenic culture of cyanobacterium Nostoc flagelliforme

The application of antibiotic treatment with assistance of metabolomic approach in axenic isolation of cyanobacterium Nostoc flagelliforme was investigated. Seven antibiotics were tested at 1–100 mg L^?1, and order of tolerance of N. flagelliforme cells was obtained as kanamycin > ampicillin, tetracycline > chloromycetin, gentamicin > spectinomycin > streptomycin. Four antibiotics were selected based on differences in antibiotic sensitivity of N. flagelliforme and associated bacteria, and their effects on N. flagelliforme cells including the changes of metabolic activity with antibiotics and the metabolic recovery after removal were assessed by a metabolomic approach based on gas chromatography–mass spectrometry combined with multivariate analysis. The results showed that antibiotic treatment had affected cell metabolism as antibiotics treated cells were metabolically distinct from control cells, but the metabolic activity would be recovered via eliminating antibiotics and the sequence of metabolic recovery time needed was spectinomycin, gentamicin > ampicillin > kanamycin. The procedures of antibiotic treatment have been accordingly optimized as a consecutive treatment starting with spectinomycin, then gentamicin, ampicillin and lastly kanamycin, and proved to be highly effective in eliminating the bacteria as examined by agar plating method and light microscope examination. Our work presented a strategy to obtain axenic culture of N. flagelliforme and provided a method for evaluating and optimizing cyanobacteria purification process through diagnosing target species cellular state.     

Desiccation and rehydration dynamics in the epiphytic resurrection fern Pleopeltis polypodioides

The epiphytic resurrection—or desiccation-tolerant (DT)—fern Pleopeltis polypodioides can survive extreme desiccation and recover physiological activity within hours of rehydration. Yet, how epiphytic DT ferns coordinate between deterioration and recovery of their hydraulic and photosynthetic systems remains poorly understood. We examined the functional status of the leaf vascular system, chlorophyll fluorescence, and photosynthetic rate during desiccation and rehydration of P. polypodioides. Xylem tracheids in the stipe embolized within 3–4 h during dehydration. When the leaf and rhizome received water, tracheids refilled after ∼24 h, which occurred along with dramatic structural changes in the stele. Photosynthetic rate and chlorophyll fluorescence recovered to predesiccation values within 12 h of rehydration, regardless of whether fronds were connected to their rhizome. Our data show that the epiphytic DT fern P. polypodioides can utilize foliar water uptake to rehydrate the leaf mesophyll and recover photosynthesis despite a broken hydraulic connection to the rhizome.     

N2-fixation,ammonium release and N-transfer to the microbial and classical food web within a plankton community

We investigated the role of N₂-fixation by the colony-forming cyanobacterium, Aphanizomenon spp., for the plankton community and N-budget of the N-limited Baltic Sea during summer by using stable isotope tracers combined with novel secondary ion mass spectrometry, conventional mass spectrometry and nutrient analysis. When incubated with ¹⁵N₂, Aphanizomenon spp. showed a strong ¹⁵N-enrichment implying substantial ¹⁵N₂-fixation. Intriguingly, Aphanizomenon did not assimilate tracers of ¹⁵NH₄⁺ from the surrounding water. These findings are in line with model calculations that confirmed a negligible N-source by diffusion-limited NH₄⁺ fluxes to Aphanizomenon colonies at low bulk concentrations (<250 nm) as compared with N₂-fixation within colonies. No N₂-fixation was detected in autotrophic microorganisms <5 μm, which relied on NH₄⁺ uptake from the surrounding water. Aphanizomenon released about 50% of its newly fixed N₂ as NH₄⁺. However, NH₄⁺ did not accumulate in the water but was transferred to heterotrophic and autotrophic microorganisms as well as to diatoms (Chaetoceros sp.) and copepods with a turnover time of ~5 h. We provide direct quantitative evidence that colony-forming Aphanizomenon releases about half of its recently fixed N₂ as NH₄⁺, which is transferred to the prokaryotic and eukaryotic plankton forming the basis of the food web in the plankton community. Transfer of newly fixed nitrogen to diatoms and copepods furthermore implies a fast export to shallow sediments via fast-sinking fecal pellets and aggregates. Hence, N₂-fixing colony-forming cyanobacteria can have profound impact on ecosystem productivity and biogeochemical processes at shorter time scales (hours to days) than previously thought.     

CMR-derived TAPSE measurement: a semi-quantitative method of right ventricular function assessment in patients with hypertrophic cardiomyopathy

Aim

To compare cardiovascular magnetic resonance (CMR)-derived right ventricular fractional shortening (RVFS), tricuspid annular plane systolic excursion with a reference point within the right ventricular apex (TAPSE_in) and with one outside the ventricle (TAPSE_out) with the standard volumetric approach in patients with hypertrophic cardiomyopathy (HCM).

Methods and results

105 patients with HCM and 20 healthy subjects underwent CMR. In patients with HCM, TAPSE_in (r = 0.31, p = 0.001) and RVFS (r = 0.35, p = 0.0002) revealed a significant but weak correlation with right ventricular ejection fraction (RVEF), whereas TAPSE_out (r = 0.57, p < 0.0001) showed a moderate correlation with RVEF. The ability to predict RVEF < 45 % in HCM patients was best for TAPSE_out. In patients with hypertrophic obstructive cardiomyopathy (HOCM), RVEF showed a significant but weak correlation with TAPSE_out (r = 0.36, p = 0.02) and no correlation with TAPSE_in (r = 0.05, p = 0.07) and RVFS (r = 0.02, p = 0.2). In patients with hypertrophic non-obstructive cardiomyopathy (HNCM), there was a moderate correlation between RVEF and TAPSE_out (r = 0.57, p < 0.0001) and a weak correlation with TAPSE_in (r = 0.39, p = 0.001) and RVFS (r = 0.38, p = 0.002). In the 20 healthy controls, there was a strong correlation between RVEF and all semi-quantitative measurements.

Conclusion

CMR-derived TAPSE_in is not suitable to determine right ventricular function in HCM patients. TAPSE_out showed a good correlation with RVEF in HNCM patients but only a weak correlation in HOCM patients. TAPSE_out might be used for screening but the detection of subtle changes in RV function requires the 3D volumetric approach.     

Nitrogen loss by anaerobic oxidation of ammonium in rice rhizosphere

Anaerobic oxidation of ammonium (anammox) is recognized as an important process for nitrogen (N) cycling, yet its role in agricultural ecosystems, which are intensively fertilized, remains unclear. In this study, we investigated the presence, activity, functional gene abundance and role of anammox bacteria in rhizosphere and non-rhizosphere paddy soils using catalyzed reporter deposition–fluorescence in situ hybridization, isotope-tracing technique, quantitative PCR assay and 16S rRNA gene clone libraries. Results showed that rhizosphere anammox contributed to 31–41% N₂ production with activities of 0.33–0.64 nmol N₂ g⁻¹ soil h⁻¹, whereas the non-rhizosphere anammox bacteria contributed to only 2–3% N₂ production with lower activities of 0.08–0.26 nmol N₂ g⁻¹ soil h⁻¹. Higher anammox bacterial cells were observed (0.75–1.4 × 10⁷ copies g⁻¹ soil) in the rhizosphere, which were twofold higher compared with the non-rhizosphere soil (3.7–5.9 × 10⁶ copies g⁻¹ soil). Phylogenetic analysis of the anammox bacterial 16S rRNA genes indicated that two genera of ‘Candidatus Kuenenia'' and ‘Candidatus Brocadia'' and the family of Planctomycetaceae were identified. We suggest the rhizosphere provides a favorable niche for anammox bacteria, which are important to N cycling, but were previously largely overlooked.     

Unique WSPA protein from terrestrial macroscopic cyanobacteria can confer resistance to osmotic stress in transgenic plants

The terrestrial macroscopic cyanobacterium Nostoc commune exhibits remarkable resistance to desiccation stress. This species synthesizes abundant acidic water stress protein (WSPA) in cells upon desiccation and secretes it into the extracellular polysaccharide sheath upon rehydration. However, our knowledge about its cellular role in stress resistance is still rather limited. In this paper, we first revealed that WSPA also occurred in two other macroscopic cyanobacteria Nostoc flagelliforme and Nostoc sphaeroides, but it is more abundant in N. commune. The N. commune wspa1 gene was then heterologously expressed in Arabidopsis thaliana. Phenotypic observation found that WSPA1 conferred increased tolerance to osmotic stress in transgenic plants. The physiological indexes such as relative electrolyte leakage, malondialdehyde, proline accumulation and the maximal quantum efficiency of Photosystem II, were also improved in transgenic plants upon osmotic stress, compared to wild types. In addition, GFP fluorescence analysis of eGFP::wspa1 transgenic plant showed that WSPA1 was localized in the cytoplasm. Therefore, the role of WSPA revealed by this study mainly represented its intracellular function. In general, our research suggested that WSPA may act as a stress protein and involve cellular osmotic stress resistance.     

Diffusible Factors Secreted by Glioblastoma and Medulloblastoma Cells Induce Oxidative Stress in Bystander Neural Stem Progenitors

Harmful effects that alter the homeostasis of neural stem or progenitor cells (NSPs) can affect regenerative processes in the central nervous system. We investigated the effect of soluble factors secreted by control or ¹³⁷Cs-γ-irradiated glioblastoma or medulloblastoma cells on redox-modulated endpoints in recipient human NSPs. Growth medium harvested from the nonirradiated brain tumor cells, following 24 h of growth, induced prominent oxidative stress in recipient NSPs as judged by overall increases in mitochondrial superoxide radical levels (p < .001), activation of c-jun N-terminal kinase, and decrease in the active form of FoxO3a. The induced oxidative stress was associated with phosphorylation of p53 on serine 15, a marker of DNA damage, induction of the cyclin-cyclin dependent kinase inhibitors p21^Waf1 and p27^Kip1, and perturbations in cell cycle progression (p < .001). These changes were also associated with increased apoptosis as determined by enhanced annexin V staining (p < .001) and caspase 8 activation (p < .05) and altered expression of critical regulators of self-renewal, proliferation, and differentiation. Exposure of the tumor cells to radiation only slightly altered the induced oxidative changes in the bystander NSPs, except for medium from irradiated medulloblastoma cells that was more potent at inducing apoptosis in the NSPs than medium from nonirradiated cells (p < .001). The elucidation of such stressful bystander effects provides avenues to understand the biochemical events underlying the development or exacerbation of degenerative outcomes associated with brain cancers. It is also relevant to tissue culture protocols whereby growth medium conditioned by tumor cells is often used to support the growth of stem cells.     

Desiccation independence of terrestrialNostoc commune ecotypes (cyanobacteria)

The subspeciesNostoc commune var.flagelliforme andN. commune var.commune are found in China (Ningxia Province, Inner Mongolia) as two morphologically different ecotypes of the desiccation-independent cyanobacteriumN. commune. The first ecotype, but not the second, colonizes arid areas. Various biochemical parameters and water dependence of photosynthesis and nitrogen fixation were compared for both ecotypes. Different patterns of water stress proteins were found in the two ecotypes. Repeated desiccation resulted in an enhanced desiccation independence for photosynthesis and, in the case of the ecotypecommune, for nitrogen fixation. The different response of nitrogenase of both ecotypes towards repeated cycles of rewetting and desiccation under conditions simulating the natural environment is discussed in terms of the energy balance of the colonies that are adapted to different environmental conditions.     

Subjective and objective levels of physical activity and their association with cardiorespiratory fitness in rheumatoid arthritis patients

IntroductionThe aims of the present study were: (a) to examine the agreement between subjective (assessed via the International Physical Activity Questionnaire; IPAQ) and objective (accelerometry; GT3X) physical activity (PA) levels in patients with rheumatoid arthritis (RA), and (b) to evaluate the associations of RA patients’ subjective and objective PA to their scores on the maximal oxygen uptake test (VO2max).MethodsThe participants wore the GT3X for seven days before completing the IPAQ and VO2max test. The Bland-Altman plot was used to illustrate the agreement between the objective and subjective PA data, and the Wilcoxon test was employed to examine the differences. The association between the PA measurement and VO2max test was examined via the correlations and the magnitude was presented by the Steiger’s Z value.ResultsSixty-eight RA patients (age = 55 ± 13 years, body mass index: 27.8 ± 5.4 kg/m2, median of disease duration = 5 (2–8) yrs) were recruited. Smaller differences between the subjective and objective measures were found when PA was assessed at the moderate level. Wilcoxon tests revealed that patients reported less time spent engaged in sedentary behaviours (Z = −6.80, P < 0.01) and light PA (Z = −6.89, P < 0.01) and more moderate PA (Z = −6.26, P < 0.01) than was objectively indicated. Significant positive correlations were revealed between VO2max with all PA levels derived from accelerometry (light PA rho = .35, P < .01; moderate PA rho = .34, P = .01; moderate and vigorous PA, (MVPA) rho = .33, P = .01), and a negative association to sedentary time (ST) emerged (rho = −.27, P = .04). IPAQ-reported moderate PA and MVPA positively correlated with maxV02 (rho = .25, P = .01, rho = .27, P = .01, respectively). Differences between the magnitude of correlations between the IPAQ-VO2 max and GT3X-VO2 max were only significant for ST (Z = 3.43, P < .01).ConclusionsVia responses to the IPAQ, RA patients reported that they were less sedentary and engaged in more higher intensity PA than what was objectively assessed. Accelerometry data correlated with VO2max at all PA levels. Only subjective moderate and MPVA correlated with VO2max. Findings suggest that self-reported PA and ST should be interpreted with caution in people with RA and complemented with accelerometry when possible.

Trial registration

Trial registration: ClinicalTrials.gov ISRCTN04121489. Registered 5 September 2012.     

Dynamic expression of intra- and extra-cellular proteome and the influence of epiphytic bacteria for Nostoc flagelliforme in response to rehydration

Nostoc flagelliforme is well known for its strong ecological adaptability in inhabiting desert biological soil crusts. However, the mechanism of its recovery from quiescent to active state after prolonged dormancy remains poorly characterized. Especially how exoproteome be related to the adaptive strategies and participate in the microalgae-bacteria interaction. In the present work, we analysed the intra- and extra-cellular proteome of N. flagelliforme over a complete rehydration period both in sterilization and in natural condition for the first time. The protein expression profile for N. flagelliforme has more fluctuations during the first 1 h after wetting but been relatively steady after fully hydrated. According to the extracellular proteomic datasets, we found a dynamic secretion of various extracellular hydrolytic enzymes and membrane transport proteins, which were related to peptidoglycan digestion and nutrient exchange respectively. Two-hundred and thirteen differentially expressed proteins induced by sterilization also reflect variation in nutrient exchange and highlight symbiosis between N. flagelliforme and surrounding bacteria. We also identified 112 phosphopeptides and 217 phosphorylation site of 95 protein of hydrated N. flagelliforme. The time course datasets we present here will be a reference for understanding the molecular processes underlying N. flagelliforme resuscitation and its potential role in microbial community diversification and soil desertification control.     

Expansion of bilin-based red light sensors in the subaerial desert cyanobacterium Nostoc flagelliforme

Light is the crucial environmental signal for desiccation-tolerant cyanobacteria to activate photosynthesis and prepare for desiccation at dawn. However, the photobiological characteristics of desert cyanobacteria adaptation to one of the harshest habitats on Earth remain unresolved. In this study, we surveyed the genome of a subaerial desert cyanobacterium Nostoc flagelliforme and identified two phytochromes and seven cyanobacteriochromes (CBCRs) with one or more bilin-binding GAF (cGMP phosphodiesterase/adenylyl cyclase/FhlA) domains. Biochemical and spectroscopic analyses of 69 purified GAF-containing proteins from recombinant phycocyanobilin (PCB), biliverdin or phycoerythrobilin-producing Escherichia coli indicated that nine of these proteins bind chromophores. Further investigation revealed that 11 GAFs form covalent adducts responsive to near-UV and visible light: eight GAFs contained PCB chromophores, three GAFs contained biliverdin chromophores and one contained the PCB isomer, phycoviolobilin. Interestingly, COO91_03972 is the first-ever reported GAF-only CBCR capable of sensing five wavelengths of light. Bioinformatics and biochemical analyses revealed that residue P132 of COO91_03972 is essential for chromophore binding to dual-cysteine CBCRs. Furthermore, the complement of N. flagelliforme CBCRs is enriched in red light sensors. We hypothesize that these sensors are critical for the acclimatization of N. flagelliforme to weak light environments at dawn.     

Aerobic nitrous oxide production through N-nitrosating hybrid formation in ammonia-oxidizing archaea

Soil emissions are largely responsible for the increase of the potent greenhouse gas nitrous oxide (N₂O) in the atmosphere and are generally attributed to the activity of nitrifying and denitrifying bacteria. However, the contribution of the recently discovered ammonia-oxidizing archaea (AOA) to N₂O production from soil is unclear as is the mechanism by which they produce it. Here we investigate the potential of Nitrososphaera viennensis, the first pure culture of AOA from soil, to produce N₂O and compare its activity with that of a marine AOA and an ammonia-oxidizing bacterium (AOB) from soil. N. viennensis produced N₂O at a maximum yield of 0.09% N₂O per molecule of nitrite under oxic growth conditions. N₂O production rates of 4.6±0.6 amol N₂O cell⁻¹ h⁻¹ and nitrification rates of 2.6±0.5 fmol NO₂⁻ cell⁻¹ h⁻¹ were in the same range as those of the AOB Nitrosospira multiformis and the marine AOA Nitrosopumilus maritimus grown under comparable conditions. In contrast to AOB, however, N₂O production of the two archaeal strains did not increase when the oxygen concentration was reduced, suggesting that they are not capable of denitrification. In ¹⁵N-labeling experiments we provide evidence that both ammonium and nitrite contribute equally via hybrid N₂O formation to the N₂O produced by N. viennensis under all conditions tested. Our results suggest that archaea may contribute to N₂O production in terrestrial ecosystems, however, they are not capable of nitrifier-denitrification and thus do not produce increasing amounts of the greenhouse gas when oxygen becomes limiting.     

Superiority of zinc complex of acetylsalicylic acid to acetylsalicylic acid in preventing postischemic myocardial dysfunction

The pathophysiology of ischemic myocardial injury involves cellular events, reactive oxygen species, and an inflammatory reaction cascade. The zinc complex of acetylsalicylic acid (Zn(ASA)₂) has been found to possess higher anti-inflammatory and lower ulcerogenic activities than acetylsalicylic acid (ASA). Herein, we studied the effects of both ASA and Zn(ASA)₂ against acute myocardial ischemia. Rats were pretreated with ASA (75 mg/kg) or Zn(ASA)₂ (100 mg/kg) orally for five consecutive days. Isoproterenol (85 mg/kg, subcutaneously [s.c.]) was applied to produce myocardial infarction. After 17–22 h, animals were anesthetized with sodium pentobarbital (60 mg/kg, intraperitoneally [i.p.]) and both electrical and mechanical parameters of cardiac function were evaluated in vivo. Myocardial histological and gene expression analyses were performed. In isoproterenol-treated rats, Zn(ASA)₂ treatment normalized significantly impaired left-ventricular contractility index (E_max 2.6 ± 0.7 mmHg/µL vs. 4.6 ± 0.5 mmHg/µL, P < 0.05), increased stroke volume (30 ± 3 µL vs. 50 ± 6 µL, P < 0.05), decreased systemic vascular resistance (7.2 ± 0.7 mmHg/min/mL vs. 4.2 ± 0.5 mmHg/min/mL, P < 0.05) and reduced inflammatory infiltrate into the myocardial tissues. ECG revealed a restoration of elevated ST-segment (0.21 ± 0.03 mV vs. 0.09 ± 0.02 mV, P < 0.05) and prolonged QT-interval (79.2 ± 3.2 ms vs. 69.5 ± 2.5 ms, P < 0.05) by Zn(ASA)₂. ASA treatment did not result in an improvement of these parameters. Additionally, Zn(ASA)₂ significantly increased the mRNA-expression of superoxide dismutase 1 (+73 ± 15%), glutathione peroxidase 4 (+44 ± 12%), and transforming growth factor (TGF)-β₁ (+102 ± 22%). In conclusion, our data demonstrate that oral administration of zinc and ASA in the form of bis(aspirinato)zinc(II) complex is superior to ASA in preventing electrical, mechanical, and histological changes after acute myocardial ischemia. The induction of antioxidant enzymes and the anti-inflammatory cytokine TGF-β₁ may play a pivotal role in the mechanism of action of Zn(ASA)₂.