Energy landscape of the intact and destabilized FMO antennas from C. tepidum and the L122Q mutant: Low temperature spectroscopy and modeling study

We discuss the excitonic energy landscape of the typically studied wild-type (WT) Fenna-Matthews-Olson (FMO) antenna protein from the green sulfur bacterium Chlorobaculum tepidum (referred to as WT_M), which is described as a mixture of intact (WT_I) and destabilized (WT_D) complexes. Optical spectra of WT_M and the L122Q mutant (where leucine 122 near BChl 8 is replaced with glutamine) are compared to WT_I FMO. We show that WT_M and L122Q samples are mixtures of two subpopulations of proteins, most likely induced by protein conformational changes during the isolation/purification procedures. Absorption, emission, and HB spectra of WT_M and L122Q mutant are very similar, in which the low-energy trap (revealed by the nonresonant HB spectra) shifts to higher energies as a function of fluence, supporting a mixture model. No fluence-dependent shift is observed in the WT_I FMO trimers. New Hamiltonians are provided for WT_I and WT_D proteins. Resonant HB spectra show that the internal energy relaxation times in the WT_M and L122Q mutant are similar, and depend on excitation frequency. Fast average relaxation times (excited state lifetimes) are observed for burning into the main broad absorption band near 805 nm. Burning at longer wavelengths reveals slower total dephasing times. No resonant bleach is observed at λ_B ≤ 803 nm, implying much faster (femtosecond) energy relaxation in this spectral range in agreement with 2D electronic spectroscopy frequency maps.     

Probiotic Effect of <Emphasis Type="Italic">Bacillus licheniformis fb11</Emphasis> on the Digestive Efficiency and Growth Performance in Juvenile <Emphasis Type="Italic">Chitala chitala</Emphasis> (Hamilton, 1822)

Five isocaloric (430 kcal 100 g^?1), isonitrogenous (40% CP) experimental diets were formulated with different concentrations of Bacillus licheniformis fb11 probionts (isolated from the gut of Chitala chitala) viz. Control (without probionts), 5 × 10⁴ CFU g^?1 (D₁), 5 × 10⁵ CFU g^?1 (D₂), 5 × 10⁶ CFU g^?1 (D₃), 5 × 10⁷ CFU g^?1 (D₄), 5 × 10⁸ CFU g^?1 (D₅) to evaluate its efficiency in C. chitala juvenile. The best growth performance, feed utilisation, specific α-amylase, total protease and lipase activity were observed with the diet D₃ (P < 0.05). The lowest Presumptive Pseudomonas Count, Motile Aeromonad Count, Total Coliform Count was observed for D₃ (P < 0.05) on 90th day of trial. Two uppermost values were achieved in case of crude protein for D₃ and D₂ (P > 0.05). The highest lipid content (12.12 ± 0.4 g 100 g^?1) was found for D₅ (P < 0.05). The highest gross energy (18.75 ± 0.21 MJ 100 g^?1) of carcass was recorded for D₃. Thus B. licheniformis fb11 at the concentration 5 × 10⁶ CFU g^?1 as probiotic supplement promoted growth, digestion in C. chitala juvenile significantly by modulating intestinal microflora.     

Optimisation of biomass and lipid production by adjusting the interspecific competition mode of Dunaliella salina and Nannochloropsis gaditana in mixed culture

The high cost of algal cultivation has been a barrier associated with the commercialisation of algal biodiesel. Therefore, this study aimed to enhance lipid production by optimising the nutrient supply to benefit the coexistence of Dunaliella salina and Nannochloropsis gaditana. The effects on biomass and lipid production of using different proportions of D. salina and N. gaditana, urea and NaHCO₃ were optimised by response surface method with a 17-run Box–Behnken design. The optimal conditions for the algal growth are 58 % of D. salina in the mixture at OD₆₈₀, 150 μL day^?1 urea (0.0044 g day^?1) and no addition of NaHCO₃. The biomass concentration and lipid production reached 1.00 and 0.383 g L^?1, respectively, which are exceeded by the amount before optimisation, indicating the efficiency of the model obtained by response surface method.     

Evaluation of an improved strain of Porphyra yezoensis Ueda (Bangiales, Rhodophyta) with high-temperature tolerance

The red algae Porphyra yezoensis has high commercial and nutritional value; however, its cultivation and product quality are jeopardized by global warming. Screening of high-temperature-tolerant strains would greatly reduce cultivation risks and benefit the Porphyra industry. Single somatic cells isolated enzymatically from the wild-type (WT) blades irradiated by ⁶⁰Co-?? rays were screened at 25°C; one strain, T-17, displayed significant high-temperature resistance. Further culture studies showed that conchospore germlings of T-17 showed 76.2% survival at 23°C and 65.7% survival at 24°C, while the WT conchospore germlings survived at significantly lower percentages of 16.9% and 11.5%, respectively, over a period of 16 days. Furthermore, T-17 conchospore germlings divided at higher percentages of 100% at both 23 and 24°C compared to the WT conchospores with 99.8% and 81.8%, respectively. When the 50-day-old F₁ gametophytic blades were transferred from the optimal temperature of 18°C to high temperatures of 23 and 24°C, the T-17 blades sustained growth over a 45-day period without rot, and their mean lengths increased by 20 and 4.2 times, respectively. The mean lengths of WT blades only increased by a factor of 0.6 and 0.4 times and were severely decayed after being cultured for 15 days at high temperatures. The mean wet weights of T-17 blades increased by 418.9 and 195.3 times, but only 7.8 and 4.6 times in WT at 23 and 24°C, respectively. These results indicate that T-17 is superior to WT in growth and high-temperature tolerance, which may offer a new cultivar for the nori industry as a high-temperature-resistant strain to counteract the effects of global warming.     

Kinetic regulation of the binding of prothrombin to phospholipid membranes

A wide range of equilibrium and kinetic constants exist for the interaction of prothrombin and other coagulation factors with various model membranes from a variety of techniques. We have investigated the interaction of prothrombin with pure dioleoylphosphatidylcholine (DOPC) membranes and dioleoylphosphatidlyserine (DOPS)-containing membranes (DOPC:DOPS, 3:1) using surface plasmon resonance (SPR, with four different model membrane presentations) in addition to isotheral titration calorimetry (ITC, with suspensions of phospholipid vesicles) and ELISA methods. Using ITC, we found a simple low-affinity interaction with DOPC:DOPS membranes with a K _D = 5.1 μM. However, ELISA methods using phospholipid bound to microtitre plates indicated a complex interaction with both DOPC:DOPS and DOPC membranes with K _D values of 20 and 58 nM, respectively. An explanation for these discrepant results was developed from SPR studies. Using SPR with low levels of immobilised DOPC:DOPS, a high-affinity interaction with a K _D of 18 nM was obtained. However, as phospholipid and prothrombin concentrations were increased, two distinct interactions could be discerned: (i) a kinetically slow, high-affinity interaction with K _D in the 10^?8 M range and (ii) a kinetically rapid, low-affinity interaction with K _D in the 10^?6 M range. This low affinity, rapidly equilibrating, interaction dominated in the presence of DOPS. Detailed SPR studies supported a heterogeneous binding model in agreement with ELISA data. The binding of prothrombin with phospholipid membranes is complex and the techniques used to measure binding will report K _D values reflecting the mixture of complexes detected. Existing data suggest that the weaker rapid interaction between prothrombin and membranes is the most important in vivo when considering the activation of prothrombin at the cell surface.     

Succinic acid-producing biofilms of Actinobacillus succinogenes: reproducibility,stability and productivity

Continuous anaerobic fermentations were performed in a biofilm reactor packed with Poraver® beads. Dilution rates (D) varied between 0.054 and 0.72 h^?1, and d-glucose and CO₂ gas were used as carbon substrates. Steady-state conditions were shown to be repeatable and independent of the operational history. Production stability was achieved over periods exceeding 80 h at values of D below 0.32 h^?1. In these situations, steady-state variation (expressed as fluctuations in NaOH neutralisation flow rates) exhibited a standard deviation of less than 5 % while no indication of biofilm deactivation was detected. The total biomass amount was found to be independent of the dilution rate with an average dry concentration of 23.8?±?2.9 g L^?1 obtained for all runs. This suggests that the attachment area controls the extent of biofilm accumulation. Specific succinic acid (SA) productivities, based on the total biomass amount, exhibited a substantial decrease with decreasing D. An SA volumetric productivity of 10.8 g L^?1 h^?1 was obtained at D?=?0.7 h^?1—the highest value reported to date in Actinobacillus succinogenes fermentations. SA yields on glucose increased with decreasing D, with a yield of 0.90?±?0.01 g g^?1 obtained at a D of 0.054 h^?1. Production of formic acid approached zero with decreasing D, while the succinic to acetic acid ratio increased with decreasing D, resulting in an increasing SA yield on glucose.     

Whole cell bioconversion of vitamin D<Subscript>3</Subscript> to calcitriol using <Emphasis Type="Italic">Pseudonocardia</Emphasis> sp. KCTC 1029BP

Calcitriol is an important drug used for treating osteoporosis, which can be produced from vitamin D₃. The current method of producing calcitriol from vitamin D₃ during cultivation of microbial cells results in low yields of calcitriol and high purification costs. Therefore, in this study, the steps of cell cultivation and bioconversion of vitamin D₃ to calcitriol were separated. Cells of Pseudonocardia sp. KCTC 1029BP were utilized as a whole cell catalyst to produce a high level and yield of calcitriol from vitamin D₃. In addition, the effects of bioconversion buffers, cyclodextrins, and metal salts on the production of calcitriol were comparatively examined and selected for incorporation in the bioconversion medium, and their compositions were statistically optimized. The optimal bioconversion medium was determined as consisting of 15 mM Trizma base, 25 mM sodium succinate, 2 mM MgSO₄, 0.08 % β-cyclodextrin, 0.1 % NaCl, 0.2 % K₂HPO₄, and 0.03 % MnCl₂. Using this optimal bioconversion medium, 61.87 mg/L of calcitriol, corresponding to a 30.94 % mass yield from vitamin D₃, was produced in a 75-L fermentor after 9 days. This calcitriol yield was 3.6 times higher than that obtained using a bioconversion medium lacking β-cyclodextrin, NaCl, K₂HPO₄, and MnCl₂. In conclusion, utilizing whole cells of Pseudonocardia sp. KCTC 1029BP together with the optimal bioconversion medium markedly enhanced the production of calcitriol from vitamin D₃.     

Theoretical investigations on the structure, density, thermodynamic and performance properties of amino-, methyl-, nitroso- and nitrotriazolones

We have studied herein the effect of position and the number of -NO, -NO₂, -NH₂ and -CH₃ groups on the structure, stability, impact sensitivity, density, thermodynamic and detonation properties of triazolones by performing density functional theory calculations at the B3LYP/aug-cc-pVDZ level. The optimized structures, vibrational frequencies and thermodynamic values for triazolones have been obtained in their ground state. Kamlet-Jacob equations were used to calculate the detonation velocity and detonation pressure of model compounds. The detonation properties of NNTO (D 8.75 to 9.10 km/s, P 34.0 to 37.57 GPa), DNTO (D 8.80 to 9.05 km/s, P 35.55 to 38.27 GPa), ADNTO (D 9.01 to 9.42 km/s and P 37.81 to 41.10 GPa) and ANNTO (D 8.58 to 9.0 km/s, P 30.81 to 36.25 GPa) are compared with those of 1,3,5-trinitro-1,3,5-triazine (RDX) (D 8.75 km/s, P 34.70 Gpa) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) (D 8.96 km/s, P 35.96 GPa). The designed compounds satisfy the criteria of high energy materials.     

Association of the SNP rs1800925(C/T) in the Interleukin-13 Gene Promoter with Pulmonary Function in Chinese Han Patients with Idiopathic Pulmonary Fibrosis

The present report studied potential association of the rs1800925(C/T) single nucleotide polymorphism (SNP) of the Interleukin (IL)-13 gene promoter with idiopathic pulmonary fibrosis (IPF) in patients of Chinese Han ethnicity. Seventy patients with IPF were enrolled and divided into three subgroups: group A (61–79 % pred. D_LCO; n = 22), group B (51–60 % pred. D_LCO; n = 20), and group C (≤50 % pred. D_LCO; n = 28). Control group consisted of 80 healthy individuals of Chinese Han ethnicity. The SNP rs1800925(C/T) was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP) analysis. The IL-13 CC genotype was present in 28/70 (40.0 %), homozygous TT in 6/70 (8.6 %) and heterozygous CT in 36/70 (51.4 %) patients with IPF. In control group, these genotypes were present in 30/80 (37.5 %), 11/80 (13.75 %), 39/80 (48.75 %), respectively, indicating that the distribution of the above three genotypes was not significantly different between patients with IPF and healthy controls. When the patients were stratified according to their D_LCO and D_LCO/VA, the frequencies of genotypes CT and TT in the groups A, B, and C were, respectively, 40.9 % (9/22), 50 % (10/20), and 82.1 % (23/28). Thus, significant differences in the distribution of alleles at ?1112 region of IL-13 gene were observed among the study groups A, B, and C, with the highest frequency in group C (p < 0.05). In conclusion, the rs1800925 T allele of the IL-13 gene is associated with worse pulmonary function in patients with IPF of Chinese Han ethnicity.     

Biodegradation of Methyl Orange by alginate-immobilized Aeromonas sp. in a packed bed reactor: external mass transfer modeling

Azo dyes are recalcitrant and xenobiotic nature makes these compounds a challenging task for continuous biodegradation up to satisfactorily levels in large-scale. In the present report, the biodegradation efficiency of alginate immobilized indigenous Aeromonas sp. MNK1 on Methyl Orange (MO) in a packed bed reactor was explored. The experimental results were used to determine the external mass transfer model. Complete MO degradation and COD removal were observed at 0.20 cm bead size and 120 ml/h flow rate at 300 mg/l of initial dye concentration. The degradation of MO decreased with increasing bead sizes and flow rates, which may be attributed to the decrease in surface of the beads and higher flux of MO, respectively. The experimental rate constants (k _ps) for various beads sizes and flow rates were calculated and compared with theoretically obtained rate constants using external film diffusion models. From the experimental data, the external mass transfer effect was correlated with a model J _D = K Re ^?(1 ? n). The model was tested with K value (5.7) and the Colburn factor correlation model for 0.20, 0.40 and 0.60 bead sizes were J _D = 5.7 Re ^?0.15, J _D = 5.7 Re ^?0.36 and J _D = 5.7 Re ^?0.48, respectively. Based on the results, the Colburn factor correlation models were found to predict the experimental data accurately. The proposed model was constructive to design and direct industrial applications in packed bed reactors within acceptable limits.     

The kinetics of light-induced changes of C-550, cytochrome b559 and fluorescence yield in chloroplasts at low temperature

The kinetics of the photoreduction of C-550, the photooxidation of cytochrome b₅₅₉ and the fluorescence yield changes during irradiation of chloroplasts at ?196 °C were measured and compared. The photoreduction of C-550 proceeded more rapidly than the photooxidation of cytochrome b₅₅₉ and the fluorescence yield increase followed the cytochrome b₅₅₉ oxidation. These results suggest that fluorescence yield under these conditions indicates the dark reduction of the primary electron donor to Photosystem II, P680⁺, by cytochrome b₅₅₉ rather than the photoreduction of the primary electron acceptor.The photoreduction of C-550 showed little if any temperature dependence over the range of ?196 to ?100 °C. The amount of cytochrome b₅₅₉ photooxidized was sensitive to temperature decreasing from the maximal change at temperatures between ?196 to ?160 °C to no change at ?100 °C. To the extent that the reaction occurred at temperatures between ?160 and ?100 °C the rate was largely independent of temperature. The rate of the fluorescence increase was dependent on temperature over this range being 3–4 times more rapid at ?100 than at ?160 °C. At ?100 °C the light-induced fluorescence increase and the photoreduction of C-550 show similar kinetics. The temperature dependence of the fluorescence induction curve is attributed to the temperature dependence of the dark reduction of P680⁺.The intensity dependence of the photoreduction of C-550 and of the photooxidation of cytochrome b₅₅₉ are linear at low intensities (below 200 μW/cm²) but fall off at higher intensities. The failure of reciprocity in the photoreduction of C-550 at the higher intensities is not explained by the simple model proposed for the Photosystem II reaction centers.     

Dosimetry of the left anterior descending coronary artery in left breast cancer patients treated with postoperative external radiotherapy

Aim

To evaluate the dose distribution to the left anterior descending (LAD) coronary artery in patients treated with postoperative three-dimensional conformal radiotherapy (3DCRT).

Co-Expression of DevR and DevRN-Aph Proteins Is Associated with Hypoxic Adaptation Defect and Virulence Attenuation of Mycobacterium tuberculosis

Background

The DevR response regulator is implicated in both hypoxic adaptation and virulence of Mycobacterium tuberculosis (M. tb). DevR regulon genes are powerfully induced in vivo implicating them in bacterial adaptation to host control strategies. A better understanding of DevR function will illumine the way for new strategies to control and treat tuberculosis.

Methodology/Principal Findings

Towards this objective, we used a combination of genetic, microbiological, biochemical, cell biological tools and a guinea pig virulence assay to compare the hypoxic adaptation and virulence properties of two novel M. tb strains, namely, a devR disruption mutant, Mut1, that expresses C-terminal truncated N-terminal domain of DevR (DevR_NTD) as a fusion protein with AphI (DevR_N-Kan), and its complemented strain, Comp1, that expresses intact DevR along with DevR_N-Kan. Comp1 bacteria exhibit a defect in DevR-mediated phosphosignalling, hypoxic induction of HspX and also hypoxic survival. In addition, we find that Comp1 is attenuated in virulence in guinea pigs and shows decreased infectivity of THP-1 cells. While Mut1 bacilli are also defective in hypoxic adaptation and early growth in spleen, they exhibit an overall virulence comparable to that of wild-type bacteria.

Conclusions/Significance

The hypoxic defect of Comp1 is associated to a defect in DevR expression level. The demonstrated repression of DevR function by DevR_N-Kan suggests that such a knockdown approach could be useful for evaluating the activity of DevRS and other two-component signaling pathways. Further investigation is necessary to elucidate the mechanism underlying Comp1 attenuation.     

Engineering chimeric thermostable GH7 cellobiohydrolases in Saccharomyces cerevisiae

We report here the effect of adding different types of carbohydrate-binding modules (CBM) to a single-module GH7 family cellobiohydrolase Cel7A from a thermophilic fungus Talaromyces emersonii (TeCel7A). Both bacterial and fungal CBMs derived from families 1, 2 and 3, all reported to bind to crystalline cellulose, were used. Chimeric cellobiohydrolases with an additional S–S bridge in the catalytic module of TeCel7A were also made. All the fusion proteins were secreted in active form and in good yields by Saccharomyces cerevisiae. The purified chimeric enzymes bound to cellulose clearly better than the catalytic module alone and demonstrated high thermal stability, having unfolding temperatures (T _m) ranging from 72 °C to 77 °C. The highest activity enhancement on microcrystalline cellulose could be gained by a fusion with a bacterial CBM3 derived from Clostridium thermocellum cellulosomal-scaffolding protein CipA. The two CBM3 fusion enzymes tested were more active than the reference enzyme Trichoderma reesei Cel7A both at moderate (45 °C and 55 °C) and at high temperatures (60 °C and 65 °C), the hydrolysis yields being two- to three-fold better at 60 °C, and six- to seven-fold better at 65 °C. The best enzyme variant was also tested on a lignocellulosic feedstock hydrolysis, which demonstrated its potency in biomass hydrolysis even at 70 °C.     

Influence of carbon-dioxide on the growth of Spirulina sp. (MCRC-A0003) isolated from Muttukadu backwaters,South India

Growth of Spirulina sp. (MCRC-A0003), a cyanobacterium, was evaluated under different concentrations of carbon-dioxide (CO₂) (4–50 %) in a closed glass photobioreactor. Although significant CO₂ utilization by the cyanobacterial strain was observed up to 50 % concentration, complete utilization was observed only at 4, 10 and 20 % concentrations on 3rd, 6th and 8th day respectively. However, considerable reduction was witnessed in reactors containing 30–50 % CO₂ only between 6th and 9th day. A corresponding increase in the biomass and primary metabolites like chlorophyll-a, carbohydrate and protein were observed. Biomass productivity of Spirulina in reactors sparged with 4, 10 and 20 % CO₂ were 13.7, 43 and 44 % more than that in control reactor without CO₂. While CO₂ increased the levels of primary metabolites in the cyanobacterial cells, it was quite prominent in 10 % CO₂ concentration with the chlorophyll-a, carbohydrate and protein contents were 64, 183 and 626 mg g^?1 respectively. While 10 and 6.6 % increase were noticed in chlorophyll-a and protein, 17 % increase in carbohydrate levels was observed in Spirulina cells, which could be attributed to the conversion of CO₂ to carbohydrate by the cyanobacterium.     

Influence of elevation on canopy transpiration of temperate deciduous forests in a complex mountainous terrain of South Korea

Background and aims

Variations in microclimate and soil characteristics on mountain slopes influence forest structure and function. Precipitation, incoming solar radiation and relative humidity change along a mountain slope. Equally, soil depth and the amount of stored soil moisture vary. The objective of this study was to examine the impacts of these factors on forest water use in mountainous terrains.

Methods

Transpiration of four temperate deciduous forest stands located at different elevations in South Korea was monitored with a sap flow technique throughout the growing season in 2010. The study sites were located on the north slope at 450 m (450 N), 650 m (650 N), and 950 m (950 N). To examine the effect of aspect, an additional site with a southern aspect was studied at 650 m (650S). All the sites were dominated by Quercus species, with leaf area index (L) ranging between 5 ? 6 m² m^?2.

Results

Rainfall increased, while air temperature (T _A ) and daytime vapor pressure deficit (D) decreased with increasing elevation. We did not observe any gradients in solar radiation (R _S ), soil moisture and sap flux density of the individual trees (J _st ) with an elevational gradient. Sapwood area (A _S ), i.e., hydro-active xylem area, and daily maximum tree water use (max TWU) increased non-linearly with increasing diameter at breast height (DBH). Neither A _S nor max TWU varied among tree species or along the elevation. The total annual canopy transpiration (E _C ) was 175, 115, 110, and 90 mm for 450 N, 650 N, 650S, and 950 N, respectively. E _C declined with increasing elevation as a result of decreasing length of the growing season, D, and T _A along the elevation. Significantly (P < 0.001) higher stomatal sensitivity to changes in D was found at the 950 N, leading to lower annual E _C and lower water use efficiency (WUE) at this elevation.

Conclusions

We conclude that differences in E _C exist along the mountain slope studied, corresponding to changing T _A , D, length of the growing season, and stomatal sensitivity to D, which should be considered when establishing forest catchment water balances.