ALBERT

Beschreibung: Abstract Pseudomonas aeruginosa biofilms contribute heavily to chronic lung infection in cystic fibrosis patients, leading to morbidity and mortality. Nitric oxide (NO) has been shown to disperse P. aeruginosa biofilms in vitro, ex vivo and in clinical trials as a promising anti-biofilm agent. Traditional NO donors such as sodium nitroprusside (SNP) have been extensively employed in different studies. However, the dosage of SNP in different studies was not consistent, ranging from 500 nM to 500 μM. SNP is light sensitive and produces cyanide, which may lead to data misinterpretation and inaccurate predictions of dispersal responses in clinical settings. New NO donors and NO delivery methods have therefore been explored. Here we assessed 7 NO donors using P. aeruginosa PAO1 and determined that SNP and Spermine NONOate (S150) successfully reduced 〉 60% biomass within 24 and 2 h, respectively. While neither dosage posed toxicity towards bacterial cells, chemiluminescence assays showed that SNP only released NO upon light exposure in M9 media and S150 delivered much higher performance spontaneously. S150 was then tested on 13 different cystic fibrosis P. aeruginosa (CF-PA) isolates; most CF-PA biofilms were significantly dispersed by 250 μM S150. Our work therefore discovered a commercially available NO donor S150, which disperses CF-PA biofilms efficiently within a short period of time and without releasing cyanide, as an alternative of SNP in clinical trials in the future. Key points • S150 performs the best in dispersing P. aeruginosa biofilms among 7 NO donors. • SNP only releases NO in the presence of light, while S150 releases NO spontaneously. • S150 successfully disperses biofilms formed by P. aeruginosa cystic fibrosis clinical isolates.

Beschreibung: In this study, the influence of twenty different single (i.e. 19 amino acids and ammonium sulphate) and two multiple nitrogen sources (N-sources) on growth and fermentation (i.e. glucose consumption and ethanol production) performance of Saccharomyces cerevisiae and of four wine-related non- Saccharomyces yeast species ( Lachancea thermotolerans , Metschnikowia pulcherrima , Hanseniaspora uvarum and Torulaspora delbrueckii ) was investigated during alcoholic fermentation. Briefly, the N-sources with beneficial effects on all performance parameters (or for the majority of them) for each yeast species were alanine, arginine, asparagine, aspartic acid, glutamine, isoleucine, ammonium sulphate, serine, valine and mixtures of 19 amino acids and of 19 amino acids plus ammonium sulphate (for S. cerevisiae ), serine (for L. thermotolerans ), alanine (for H. uvarum ), alanine and asparagine (for M. pulcherrima ), arginine, asparagine, glutamine, isoleucine and mixture of 19 amino acids (for T. delbrueckii ). Furthermore, our results showed a clear positive effect of complex mixtures of N-sources on S. cerevisiae and on T. delbrueckii (although to a lesser extent) as to all performance parameters studied, whereas for L. thermotolerans , H. uvarum and M. pulcherrima , single amino acids affected growth and fermentation performance to the same extent as the mixtures. Moreover, we found groups of N-sources with similar effects on the growth and/or fermentation performance of two or more yeast species. Finally, the influences of N-sources observed for T. delbrueckii and H. uvarum resembled those of S. cerevisiae the most and the least, respectively. Overall, this work contributes to an improved understanding of how different N-sources affect growth, glucose consumption and ethanol production of wine-related yeast species under oxygen-limited conditions, which, in turn, may be used to, e.g. optimize growth and fermentation performance of the given yeast upon N-source supplementation during wine fermentations.

Beschreibung: Saccharopolyspora spinosa produces tetra-cyclic macrolide spinosyns, a group of highly efficient pesticidal agents. However, this species lacks efficient vectors for genetic manipulation. In this study, the circular plasmid pCM32 was newly isolated from Saccharopolyspora endophytica YIM 61095. The complete nucleotide sequence of pCM32 consists of 14,611 bp and is predicted to encode 17 open reading frames (ORFs). Interestingly, a putative int gene in pCM32 was predicted by homologous alignment to encode an integrase belonging to the tyrosine family of integrases/recombinases. Plasmid pCM238 containing this int locus derived from pCM32 could be transferred by conjugation from Escherichia coli into Sa. spinosa at a high frequency. Integration of pCM238 in the host chromosome was demonstrated as site-specific recombination (at the tRNA Ser gene) via a 56-bp core sequence within the attP / attB sites. Plasmid pCM265, a shuttle vector containing the int and attP sequences of pCM32, was constructed to introduce foreign genes into Sa. spinosa . The production of spinosad approximately doubled in Sa. spinosa NRRL18395 after introducing pCM265-derived plasmids carrying the genes for phosphofructokinase (PFK) or anthranilate synthase. These results indicate that plasmid pCM32 is an actinomycete integrative and conjugative element (AICE) and that its derived integrative vectors are useful for efficiently introducing foreign DNA into Sa. spinosa .

Beschreibung: Impacts of spectral nudging on simulations of Arctic climate in coupled simulations have been investigated in a set of simulations with a regional climate model (RCM). The dominantly circumpolar circulation in the Arctic lead to weak constraints on the lateral boundary conditions (LBCs) for the RCM, which causes large internal variability with strong deviations from the driving model. When coupled to an ocean and sea ice model, this results in sea ice concentrations that deviate from the observed spatial distribution. Here, a method of spectral nudging is applied to the atmospheric model RCA4 in order to assess the potentials for improving results for the sea ice concentrations when coupled to the RCO ocean-sea ice model. The spectral nudging applied to reanalysis driven simulations significantly improves the generated sea ice regarding its temporal evolution, extent and inter-annual trends, compared to simulations with standard LBC nesting. The method is furthermore evaluated with driving data from two CMIP5 GCM simulations for current and future conditions. The GCM biases are similar to the RCA4 biases with ERA-Interim, however, the spectral nudging still improves the surface winds enough to show improvements in the simulated sea ice. For both GCM downscalings, the spectrally nudged version retains a larger sea ice extent in September further into the future. Depending on the sea ice formulation in the GCM, the temporal evolution of the regional sea ice model can deviate strongly.

Beschreibung: The increased rate of Tropical Indian Ocean (TIO) surface warming has gained a lot of attention in the recent years mainly due to its regional climatic impacts. The processes associated with this increased surface warming is highly complex and none of the mechanisms in the past studies could comprehend the important features associated with this warming such as the negative trends in surface net heat fluxes and the decreasing temperature trends at thermocline level. In this work we studied a previously unexplored aspect, the changes in large scale surface circulation pattern modulating the surface warming pattern over TIO. We use ocean reanalysis datasets and a suit of Ocean General Circulation Model (OGCM) experiments to address this problem. Both reanalysis and OGCM reveal strengthening large scale surface circulation pattern in the recent years. The most striking feature is the intensification of cyclonic gyre circulation around the thermocline ridge in the southwestern TIO. The surface circulation change in TIO is mainly associated with the surface wind changes and the geostrophic response to sea surface height decrease in the western/southwestern TIO. The surface wind trends closely correspond to SST warming pattern. The strengthening mean westerlies over the equatorial region are conducive to convergence in the central and divergence in the western equatorial Indian Ocean (IO) resulting central warming and western cooling. The resulting east west SST gradient further enhances the equatorial westerlies. This positive feedback mechanism supports strengthening of the observed SST trends in the equatorial Indian Ocean. The cooling induced by the enhanced upwelling in the west is compensated to a large extent by warming due to reduction in mixed layer depth, thereby keeping the surface temperature trends in the west to weak positive values. The OGCM experiments showed that the wind induced circulation changes redistribute the excess heat received in the western TIO to central and east thereby enhancing warming in the central equatorial IO. The increased surface warming in central TIO increases the latent heat loss, and keeps the net heat flux trends negative. Model sensitivity experiments reveal that the subsurface cooling at thermocline level in TIO is contributed by variability in Pacific via Indonesian Through Flow whereas the surface warming trend is mainly induced by the changes in the local forcing. The long term changes in IO Rossby waves are not induced by local atmospheric forcing but are forced by Pacific. The thermocline shoaling in the west is therefore amplified by the remote influence of Pacific via wave transmission through Indonesian archipelago.

Beschreibung: In this work, Nannochloropsis salina was cultivated in a continuous-flow flat-plate photobioreactor, working at different residence times and irradiations to study the effect of the specific light supply rate on biomass productivity and photosynthetic efficiency. Changes in residence times lead to different steady-state cell concentrations and specific growth rates. We observed that cultures at steady concentration were exposed to different values of light intensity per cell. This specific light supply rate was shown to affect the photosynthetic status of the cells, monitored by fluorescence measurements. High specific light supply rate can lead to saturation and photoinhibition phenomena if the biomass concentration is not optimized for the selected operating conditions. Energy balances were applied to quantify the biomass growth yield and maintenance requirements in N. salina cells.

Beschreibung: Two classes of GRBs have been identified thus far without doubt and are prescribed to different physical scenarios—NS-NS or NS-BH mergers, and collapse of massive stars, for short and long GRBs, respectively. The existence of two distinct populations was inferred through a bimodal distribution of the observed durations \(T_{90}\) , and the commonly applied \(2~\mbox{s}\) limit between short and long GRBs was obtained by fitting a parabola between the two peaks in binned data from BATSE 1B. Herein, by means of a maximum likelihood (ML) method a mixture of two Gaussians is fitted to the datasets from BATSE, Swift , BeppoSAX , and Fermi in search for a local minimum that might serve as a new, more proper, limit for the two GRB classes. It is found that Swift and BeppoSAX distributions are unimodal, hence no local minimum is present, Fermi is consistent with the conventional limit, whereas BATSE gives the limit significantly longer (equal to \(3.38\pm 0.27~\mbox{s}\) ) than \(2~\mbox{s}\) . These new values change the fractions of short and long GRBs in the samples examined, and imply that the observed \(T_{90}\) durations are detector dependent, hence no universal limiting value may be applied to all satellites due to their different instrument specifications. Because of this, and due to the strong overlap of the two-Gaussian components, the straightforward association of short GRBs to mergers and long ones to collapsars is ambiguous.

Beschreibung: When luminosity of black hole accretion disks is greater than 0.06 Eddington luminosity ( \(L \geq0.06 L_{E} \) ), according to standard disk model, the inner region of these disks is radiation-pressure dominated (RPD) and unstable in thermal and viscous modes. Whereas this object conflicts with the observations, if \(0.01 L_{E}\leq L \leq0.5 L_{E}\) . Thus, the change of viscosity or considering relativistic corrections that are essential in realistic black hole accretion disks may solve this problem between theory and observation. An explanation by taking into account the effect of black hole spin with the presence of different viscosities on the secular and thermal instabilities in geometrically thin optically thick accretion disks is presented. In order to consider the effects of spin and general relativity, kerr space-time is used. A diffusive form of viscous stress tensor, a new form of fully relativistic shear stress tensor that is not used so far, power law, and parametric viscosity are considered. Investigations have been done in some parameters of viscosity and some values of spin. We conclude that the type of viscosity is effective in stabilization of the disk, whereas spin has no effect. Increasing stability or instability in the disk can be caused by spin, which this behavior is related to the type of viscous parameter. Thereby, spin can be effective on the temperature, accretion rate, and in the luminosity of the system.

Beschreibung: We describe a new type of NADH sensor based on a screen-printed electrode (SPE) modified with reduced graphene oxide (RGO) and poly(allylamine hydrochloride) (PAH). A mixture of graphene oxide (GO) and PAH was deposited on the surface of a carbon working electrode, and RGO was prepared in-situ by electrochemical reduction of GO. The oxidation peak of NADH was recorded at +450 mV (vs . silver pseudo-reference). Under optimized conditions, the electrode exhibits high electrocatalytic activity toward NADH oxidation, expressed by a high rate constant and a stable response up to 0.8 mM concentrations. The sensitivity is 108.6 μA·mM −1 ·cm −2 , the response time is 20 s (for 95 % of the steady state current), and the detection limit is 6.6 μM (at an S/N ratio of 3). A peak separation of about 300 mV was achieved in differential pulse voltammetric determination of NADH in the presence of ascorbic acid. This makes the new sensor a useful tool with potential analytical application in different dehydrogenase based systems. Graphical abstract ᅟ

Beschreibung: We describe an electrochemical immunosensor for the simultaneous determination of alpha-fetoprotein (AFP) and prostate specific antigen (PSA) via a modified glassy carbon electrode. Silica nanoparticles (200–300 nm i.d.) with good monodispersity and uniform shape were synthesized, and the following species were then consecutively immobilized on their surface: gold nanoparticles (AuNPs; 5–15 nm i.d.), secondary antibody (Ab 2 ) and the redox-probes Azure A or ferrocenecarboxy acid (Fc). In parallel, two types of primary antibodies (Ab 1 ) were co-immobilized on the surface of the dissolved reduced graphene oxide sheets (rGO) that were also decorated with AuNPs. In the presence of antigens (AFP or PSA), the Ab 2 /Si@AuNPs carrying Azure A and Fc are attached to the AuNP/rGO conjugate via a sandwich type immunoreaction. Differential pulse voltammetry (DPV) was employed to measure the resulting changes in the signal of Fc or Azure A. Two well-resolved oxidation peaks, one at −0.48 V (corresponding to Azure A) and other at + 0.12 V (corresponding to Fc; both vs. SCE) can be observed in the DPV curves. Under optimal conditions, AFP and PSA can be simultaneously determined in the range from 0.01 to 25 ng mL‾ 1 for AFP, and from 0.012 to 25 ng mL‾ 1 for PSA. The detection limits are 3.3 pg mL‾ 1 for AFP and 4.0 pg mL‾ 1 for PSA (at a signal-to-noise ratio of 3). The method was applied to (spiked) real sample analysis, and the recoveries are within 96.0 and 107.2 % for PSA, and within 100.9 and 105.8 % for AFP, indicating that this dual immunosensor matches the requirements of clinical analysis. Graphical abstract (A) Two types of signal labels preparation process. (B) The immunosensor preparation and detection process.

Beschreibung: An open reading frame CC1225 from the Caulobacter crescentus CB15 genome sequence belongs to the Gfo/Idh/MocA protein family and has 47 % amino acid sequence identity with the glucose-fructose oxidoreductase from Zymomonas mobilis ( Zm GFOR). We expressed the ORF CC1225 in the yeast Saccharomyces cerevisiae and used a yeast strain expressing the gene coding for Zm GFOR as a reference. Cell extracts of strains overexpressing CC1225 (renamed as Cc aaor ) showed some Zm GFOR type of activity, producing D-gluconate and D-sorbitol when a mixture of D-glucose and D-fructose was used as substrate. However, the activity in Cc aaor expressing strain was 〉100-fold lower compared to strains expressing Zm gfor . Interestingly, C. crescentus AAOR was clearly more efficient than the Zm GFOR in converting in vitro a single sugar substrate D-xylose (10 mM) to xylitol without an added cofactor, whereas this type of activity was very low with Zm GFOR. Furthermore, when cultured in the presence of D-xylose, the S. cerevisiae strain expressing Cc aaor produced nearly equal concentrations of D-xylonate and xylitol (12.5 g D-xylonate l −1 and 11.5 g D-xylitol l −1 from 26 g D-xylose l −1 ), whereas the control strain and strain expressing Zm gfor produced only D-xylitol (5 g l −1 ). Deletion of the gene encoding the major aldose reductase, Gre3p, did not affect xylitol production in the strain expressing Cc aaor , but decreased xylitol production in the strain expressing Zm gfor . In addition, expression of Cc aaor together with the D-xylonolactone lactonase encoding the gene xylC from C. crescentus slightly increased the final concentration and initial volumetric production rate of both D-xylonate and D-xylitol. These results suggest that C. crescentus AAOR is a novel type of oxidoreductase able to convert the single aldose substrate D-xylose to both its oxidized and reduced product.

Beschreibung: We have previously shown that overexpression of the human tumor suppressor protein P53 causes cell death of the yeast Saccharomyces cerevisiae . P53 overproduction led to transcriptional downregulation of some yeast genes, such as the TRX1/2 thioredoxin system, which plays a key role in cell protection against various oxidative stresses induced by reactive oxygen species (ROS). In the present work, the impact of TRX2 overexpression on apoptosis mediated by p53 overexpression in yeast is investigated. In yeast cells expressing P53 under an inducible promoter together with TRX2 under a strong constitutive promoter, we showed that Tr2p overproduction reduced the apoptotic effect exerted by P53 and increased the viability of the P53-overproducing cells. Furthermore, measurements of ROS amounts by flow cytometry and fluorescence microscopy indicated that the TRX2 protein acted probably through its increased detoxifying activity on the P53-generated ROS. The steady-state level and activity of P53 were not affected by TRX2 overexpression, as shown by western blotting and functional analysis of separated alleles in yeast (FASAY), respectively. The growth inhibitory effect of P53 was partially reversed by the antioxidant N -acetylcysteine. Our data strengthen the idea that overexpression of a single gene ( trx2 ) decreases the p53 -mediated cell death by decreasing ROS accumulation.

Beschreibung: The moving bed biofilm reactor-membrane bioreactor (MBBR-MBR) is a novel solution to conventional activated sludge processes and membrane bioreactors. In this study, a pure MBBR-MBR was studied. The pure MBBR-MBR mainly had attached biomass. The bioreactor operated with a hydraulic retention time (HRT) of 9.5 h. The kinetic parameters for heterotrophic and autotrophic biomasses, mainly nitrite-oxidizing bacteria (NOB), were evaluated. The analysis of the bacterial community structure of the ammonium-oxidizing bacteria (AOB), NOB, and denitrifying bacteria (DeNB) from the pure MBBR-MBR was carried out by means of pyrosequencing to detect and quantify the contribution of the nitrifying and denitrifying bacteria in the total bacterial community. The relative abundance of AOB, NOB, and DeNB were 5, 1, and 3 %, respectively, in the mixed liquor suspended solids (MLSS), and these percentages were 18, 5, and 2 %, respectively, in the biofilm density (BD) attached to carriers. The pure MBBR-MBR had a high efficiency of total nitrogen (TN) removal of 71.81 ± 16.04 %, which could reside in the different bacterial assemblages in the fixed biofilm on the carriers. In this regard, the kinetic parameters for autotrophic biomass had values of Y A  = 2.3465 mg O 2  mg N −1 , μ m, A  = 0.7169 h −1 , and K NH  = 2.0748 mg N L −1 .

Beschreibung: Slow sand filtration (SSF) is an effective low-tech water treatment method for pathogen and particle removal. Yet despite its application for centuries, it has been uncertain to which extent pathogenic microbes are removed by mechanical filtration or due to ecological interactions such as grazing and competition for nutrients. In this study, we quantified the removal of bacterial faecal indicators, Escherichia coli and Enterococcus faecalis , from secondary effluent of a wastewater treatment plant and analysed the microbial community composition in compartments of laboratory model SSF columns. The columns were packed with different sand grain sizes and eliminated 1.6–2.3 log units of faecal indicators, which translated into effluents of bathing water quality according to the EU directive (〈500 colony forming units of E. coli per 100 ml) for columns with small grain size. Most of that removal occurred in the upper filter area, the Schmutzdecke . Within that same zone, total bacterial numbers increased however, thus suggesting a specific elimination of the faecal indicators. The analysis of the microbial communities also revealed that some taxa were removed more from the wastewater than others. These results accentuate the contribution of biological mechanisms to water purification in SSF.

Beschreibung: Pseudoalteromonas is widespread in various marine environments, and most strains can affect invertebrate larval settlement and metamorphosis by forming biofilms. However, the impact and the molecular basis of population diversification occurring in Pseudoalteromonas biofilms are poorly understood. Here, we show that morphological diversification is prevalent in Pseudoalteromonas species during biofilm formation. Two types of genetic variants, wrinkled (frequency of 12 ± 5 %) and translucent (frequency of 5 ± 3 %), were found in Pseudoalteromonas lipolytica biofilms. The inducing activities of biofilms formed by the two variants on larval settlement and metamorphosis of the mussel Mytilus coruscus were significantly decreased, suggesting strong antifouling activities. Using whole-genome re-sequencing combined with genetic manipulation, two genes were identified to be responsible for the morphology alternations. A nonsense mutation in AT00 _ 08765 led to a wrinkled morphology due to the overproduction of cellulose, whereas a point mutation in AT00 _ 17125 led to a translucent morphology via a reduction in capsular polysaccharide production. Taken together, the results suggest that the microbial behavior on larval settlement and metamorphosis in marine environment could be affected by the self-generated variants generated during the formation of marine biofilms, thereby rendering potential application in biocontrol of marine biofouling.

Beschreibung: A nanocomposite consisting of coral-like gold nanostructures on reduced graphene oxide (RGO) was synthesized with the assistance of dimethylbiguanide (DMBG). It was then fabricated on a glassy carbon electrode, coating with cysteamine in order to enable the immobilization of acetylcholinesterase (AChE) as a model enzyme whose activity of hydrolyzing the substrate of acetylthiocholine is inhibited by the pesticide triazophos. The biosensor has response to acetylthiocholine in the 0.3 ~ 300 μM concentration range at 0.65 V (vs. SCE). The inhibition of the enzyme by triazophos can be determined in concentrations of up to 210 ppb, with a detection limit of 0.35 ppb of triazophos ( S / N  = 3). The biosensor is highly reproducible and acceptably stable. Graphical Abstract Coral-like gold nanostructures supported on reduced graphene oxide were synthesized with the assistance of dimethylbiguanide to fabricate an acetylcholinesterase (AChE) biosensor, which exhibited high reproducibility and good stability, providing a good platform for the detection of organophosphorus pesticides.

Beschreibung: This study used an artificial enrichment microbial consortium to examine the effects of different substrate conditions on microbial diversity, composition, and function (e.g., zinc leaching efficiency) through adding pyrite (SP group), chalcopyrite (SC group), or both (SPC group) in sphalerite bioleaching systems. 16S rRNA gene sequencing analysis showed that microbial community structures and compositions dramatically changed with additions of pyrite or chalcopyrite during the sphalerite bioleaching process. Shannon diversity index showed a significantly increase in the SP (1.460), SC (1.476), and SPC (1.341) groups compared with control (sphalerite group, 0.624) on day 30, meanwhile, zinc leaching efficiencies were enhanced by about 13.4, 2.9, and 13.2 %, respectively. Also, additions of pyrite or chalcopyrite could increase electric potential (ORP) and the concentrations of Fe 3+ and H + , which were the main factors shaping microbial community structures by Mantel test analysis. Linear regression analysis showed that ORP, Fe 3+ concentration, and pH were significantly correlated to zinc leaching efficiency and microbial diversity. In addition, we found that leaching efficiency showed a positive and significant relationship with microbial diversity. In conclusion, our results showed that the complicated substrates could significantly enhance microbial diversity and activity of function.

Beschreibung: A metagenomic fosmid expression library established from environmental DNA (eDNA) from the shallow hot vent sediment sample collected from the Levante Bay, Vulcano Island (Aeolian archipelago) was established in Escherichia coli . Using activity-based screening assays, we have assessed 9600 fosmid clones corresponding to approximately 350 Mbp of the cloned eDNA, for the lipases/esterases/lactamases, haloalkane and haloacid dehalogenases, and glycoside hydrolases. Thirty-four positive fosmid clones were selected from the total of 120 positive hits and sequenced to yield ca. 1360 kbp of high-quality assemblies. Fosmid inserts were attributed to the members of ten bacterial phyla, including Proteobacteria , Bacteroidetes , Acidobateria , Firmicutes , Verrucomicrobia , Chloroflexi , Spirochaetes , Thermotogae , Armatimonadetes , and Planctomycetes . Of ca. 200 proteins with high biotechnological potential identified therein, we have characterized in detail three distinct α/β-hydrolases (LIPESV12_9, LIPESV12_24, LIPESV12_26) and one new α-arabinopyranosidase (GLV12_5). All LIPESV12 enzymes revealed distinct substrate specificities tested against 43 structurally diverse esters and 4 p -nitrophenol carboxyl esters. Of 16 different glycosides tested, the GLV12_5 hydrolysed only p -nitrophenol-α-( l )-arabinopyranose with a high specific activity of about 2.7 kU/mg protein. Most of the α/β-hydrolases were thermophilic and revealed a high tolerance to, and high activities in the presence of, numerous heavy metal ions. Among them, the LIPESV12_24 was the best temperature-adapted, retaining its activity after 40 min of incubation at 90 °C. Furthermore, enzymes were active in organic solvents (e.g., 〉30 % methanol). Both LIPESV12_24 and LIPESV12_26 had the GXSXG pentapeptides and the catalytic triads Ser-Asp-His typical to the representatives of carboxylesterases of EC 3.1.1.1.

Beschreibung: Feruloyl or ferulic acid esterase (Fae, EC 3.1.1.73) catalyzes the hydrolysis of ester bonds between polysaccharides and phenolic acid compounds in xylan side chain. In this study, the thermostability of a type A feruloyl esterase (AuFaeA) from Aspergillus usamii was increased by iterative saturation mutagenesis (ISM). Two amino acids, Ser33 and Asn92, were selected for saturation mutagenesis according to the B-factors analyzed by B-FITTER software and ΔΔG values predicted by PoPMuSiC algorithm. After screening the saturation mutagenesis libraries constructed in Pichia pastoris , 15 promising variants were obtained. The best variant S33E/N92-4 (S33E/N92R) produced a T m value of 44.5 °C, the half-lives ( t 1/2 ) of 35 and 198 min at 55 and 50 °C, respectively, corresponding to a 4.7 °C, 2.33- and 3.96-fold improvement compared to the wild type. Additionally, the best S33 variant S33-6 (S33E) was thermostable at 50 °C with a t 1/2 of 82 min, which was 32 min longer than that of the wild type. All the screened S33E/N92 variants were more thermostable than the best S33 variant S33-6 (S33E). This work would contribute to the further studies on higher thermostability modification of type A feruloyl esterases, especially those from fungi. The thermostable feruloyl esterase variants were expected to be potential candidates for industrial application in prompting the enzymic degradation of plant biomass materials at elevated temperatures.

Beschreibung: Riemerella anatipestifer infection causes high mortality for ducks which results in major economic losses in the duck industry. In this study, we identified a mutant strain RA-M1 by Tn 4351 transposon mutagenesis, in which the M949_1603 gene encoding glycosyl transferase was inactivated. PCR analysis revealed that M949_1603 gene is specifically existed in R. anatipestifer serotype 1 strains. RA-M1 presented no reactivity to the anti-lipopolysaccharide (LPS) MAb in an indirect ELISA. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting demonstrated that RA-M1 LPS had a deficiency in ladder-like binding pattern to rabbit antiserum against R. anatipestifer serotype 1 strain CH3, indicating that the O-antigen structure of RA-M1 was changed. RA-M1 showed significant attenuated virulence in ducks and higher sensitivity to normal duck serum, compared with its parent strain CH3. Furthermore, cross-protection of RA-M1 for R. anatipestifer serotypes 1, 2, and 10 strains was evaluated. Ducks that received two immunizations with inactivated RA-M1 vaccine were 100 % protected from challenge with R. anatipestifer serotype 1 strain WJ4, serotype 2 strain Yb2, and serotype 10 strain HXb2. No changes were observed in the liver, heart, or spleen samples from the protected ducks during autopsy and histological examination. Furthermore, vaccination generated high antibody titers of 1:12,800 against serotypes 1, 2, and 10 strains and enhanced production of interleukin 2 (IL-2) and IL-4 in ducks. These results suggested that M949_1603 gene is associated with serotype 1 O-antigen biosynthesis, and mutant RA-M1 could be used as a novel cross-protection vaccine candidate to protect ducks against R. anatipestifer infection.

Beschreibung: Saccharopolyspora spinosa can produce spinosad as a major secondary metabolite, which is an environmentally friendly agent for insect control. Cobalamin-independent methionine synthase (MetE) is an important enzyme in methionine biosynthesis, and this enzyme is probably closely related to spinosad production. In this study, its corresponding gene metE was inactivated, which resulted in a rapid growth and glucose utilisation rate and almost loss of spinosad production. A label-free quantitative proteomics-based approach was employed to obtain insights into the mechanism by which the metabolic network adapts to the absence of MetE. A total of 1440 proteins were detected from wild-type and Δ metE mutant strains at three time points: stationary phase of Δ metE mutant strain (S1 Δ metE , 67 h), first stationary phase of wild-type strain (S1 WT , 67 h) and second stationary phase of wild-type strain (S2 WT , 100 h). Protein expression patterns were determined using an exponentially modified protein abundance index (emPAI) and analysed by comparing S1 Δ metE /S1 WT and S1 Δ metE /S2 WT . Results showed that differentially expressed enzymes were mainly involved in primary metabolism and genetic information processing. This study demonstrated that the role of MetE is not restricted to methionine biosynthesis but rather is involved in global metabolic regulation in S. spinosa .

Beschreibung: Chlorella vulgaris encapsulated in alginate beads were added into a bioreactor treating synthetic wastewater using Pseudomonas putida . A symbiotic CO 2 /O 2 gas exchange was established between the two microorganisms for photosynthetic aeration of wastewater. During batch operation, glucose removal efficiency in the bioreactor improved from 50 % in 12 h without aeration to 100 % in 6 h, when the bioreactor was aerated photosynthetically. During continuous operation, the bioreactor was operated at a low hydraulic retention time of 3.3 h at feed concentrations of 250 and 500 mg/L glucose. The removal efficiency at 500 mg/L increased from 73 % without aeration to 100 % in the presence of immobilized microalgae. The initial microalgae concentration was critical to achieve adequate aeration, and the removal rate increased with increasing microalgae concentration. The highest removal rate of 142 mg/L-h glucose was achieved at an initial microalgae concentration of 190 mg/L. Quantification of microalgae growth in the alginate beads indicated an exponential growth during symbiosis, indicating that the bioreactor performance was limited by oxygen production rates. Under symbiotic conditions, the chlorophyll content of the immobilized microalgae increased by more than 30 %. These results indicate that immobilized microalgae in symbiosis with heterotrophic bacteria are promising in wastewater aeration.

Beschreibung: We consider the effect of a partial coverage of quasar broad-line regions (QSO BLRs) by intervening \(\mbox{H}_{2}\) -bearing clouds when a part of quasar (QSO) radiation passes by a cloud not taking part in absorption-line system formation of the QSO spectrum. That leads to modification of observable absorption line profiles and consequently to a bias in physical parameters derived from standard absorption line analysis. In application to the \(\mbox{H}_{2}\) absorption systems the effect has been revealed in the analysis of \(\mbox{H}_{2}\) absorption system in the spectrum of Q 1232+082 (see Ivanchik et al. in Mon. Not. R. Astron. Soc. 404:1583, 2010 , Balashev et al. in Mon. Not. R. Astron. Soc. 418:357, 2011 ). We estimate a probability of the effect to be detected in QSO spectra. To do this we derive distribution of BLR sizes of high-z QSOs from Sloan Digital Sky Survey (SDSS) Data Release 9 (DR9) catalogue and assume different distributions of cloud sizes. We conclude that the low limit of the probability is about 11 %. The latest researches shows that about a fifth of observed \(\mbox{H}_{2}\) absorption systems can be partially covered. Accounting of the effect may allow to revise significantly physical parameters of interstellar clouds obtained by the spectral analysis.

Beschreibung: The spatially homogeneous and anisotropic Bianchi type-V universe filled with interacting Dark matter and Holographic dark energy has been studied. The exact solutions of Einstein’s field equations are obtained by (i) applying the special law of variation of Hubble parameter that yields constant values of the deceleration parameter and (ii) using a special form of deceleration parameter. It has been observed that for suitable choice of interaction between dark matter and holographic dark energy there is no coincidence problem (unlike \(\varLambda\) CDM). Also, in all the resulting models the anisotropy of expansion dies out very quickly and attains isotropy after some finite time. The physical and geometrical aspects of the models are also discussed.

Beschreibung: The properties of nonlinear dust-acoustic rogue waves in an unmagnetized, collisionless, four-component dusty plasma system consisting of electrons, nonthermal ions, hot and cold dust species have been investigated. The basic set of fluid equations is reduced to a nonlinear Schrodinger equation. The dependence of the rogue wave properties on the ion energetic population parameter is discussed. The results of the present investigation could be applicable in Saturn F-ring.

Beschreibung: In this paper, we study the reconstruction scenario of a dark energy model in the framework of modified Horava-Lifshitz \(F(R)\) gravity or \(F(\tilde{R})\) gravity. We assume generalized ghost pilgrim dark energy model in flat universe. We consider three well-known scale factors to analyze the behavior of reconstructed \(F(\tilde{R})\) model. These scale factors include bouncing and intermediate scale factors as well as scale factor representing the unification of matter and accelerated phases. The graphical representation is adopted to analyze the behavior of reconstructed model and equation of state parameter for different values of model parameter. The reconstructed model represents increasing and decreasing behavior with respect to time in all cases. The equation of state parameter represents phantom-like universe after transition for intermediate scale factor while quintessence behavior for bouncing and unified scale factors. We also found that the squared speed of sound exhibits the stability of all reconstructed models.

Beschreibung: We have performed a study on the performance of two microbial glucose sensors based on immobilized Gluconobacter oxydans ( G. oxydans ). The first one was prepared by modifying a glassy carbon paste electrode (GCPE) containing the microbial cells with graphene oxide (GO), the other one by modifying it with graphene-platinum hybrid nanoparticles (graphene-Pt NPs). The electrode was characterized by following the voltammetric signals of the oxidation of hexacyanoferrate(II) to hexacyanoferrate(III) via the oxidative enzymes contained in G. oxydans which convert glucose to gluconic acid. Optimizations were conducted with a conventional GCPE containing G. oxydans . After material optimization, the biosensors were applied to the determination of glucose. The linear and analytical ranges for GO based biosensor range from 1 to 75 μM (linear) and 1 to 100 μM (analytical), respectively, with a limit of detection (LOD) of (3 s/m) 1.06 μM (at an S/m of 3). On the other hand, the graphene-Pt hybrid nanoparticle based biosensor showed two linear ranges (from 0.3 to 1 µM and from 1 to 10 μM), a full analytical range from 1 to 50 μM, and an LOD of 0.015 μM. The graphene-Pt hybrid NP based sensors performs better and was applied to the determination of glucose in synthetically prepared plasma samples where it gave recoveries as 101.8 and 104.37 % for two different concentrations. Selectivity studies concerning fructose, galactose, L-ascorbic acid and dopamine were also conducted. Graphical Abstract The electrochemical performances of graphene oxide and graphene-platinum hybrid nanoparticle modified G. oxydans biosensors were compared.

Beschreibung: Copper nanoclusters (Cu-NCs) were fabricated by chemical reduction of Cu(II) ions using formaldehyde as the reductant and poly(vinyl pyrrolidone) as the protecting agent. The resulting Cu-NCs were characterized by TEM, FT-IR, UV–vis and XPS and fluorescence spectroscopy. The Cu-NCs display a luminescence quantum yield of about 13 %, and the emission peaks shift from 398 to 457 nm on increasing the excitation wavelength from 310 to 390 nm. The Cu-NCs possess a storage stability of at least 2 months and are stable in the presence of high concentrations of salt. Their fluorescence is strongly quenched by hypochlorite, while other common cations, anions and hydrogen peroxide have minor (or no) effects on fluorescence. On this basis, a fluorometric hypochlorite assay was developed that has a 0.1 μM detection limit and a linear range that extends from 1 to 30 μM. The method was successfully used to the determination of hypochlorite in local tap water samples, and the results agreed well with those obtained by a colorimetric method. Graphical Abstract Hypochlorite ion is found to quench the fluorescence copper nanoclusters synthesized by reduction of Cu(II) ion by formaldehyde in the presence of poly(vinyl pyrrolidone) serving as protecting agent. This finding resulted in a new fluorescence assay for hypochlorite.

Beschreibung: A nanoporous carbon derived from an aluminum-based metal-organic framework was deposited on stainless steel wires in a sol–gel matrix. The resulting fibers were applied to the solid-phase microextraction of the polycyclic aromatic hydrocarbons (PAHs) naphthalene, acenaphthene, fluorene, phenanthrene and anthracene from water and soil samples. The fiber was then directly inserted into the GC injector and the PAHs were quantified by GC-MS. The effects of salt addition, extraction temperature, extraction time, sample volume and desorption conditions on the extraction efficiency were optimized. A linear response to the analytes was observed in the 0.1 to 12 μg∙L −1 range for water samples, and in the 0.6 to 30 μg∙kg −1 for soil samples, with the correlation coefficients ranging from 0.9934 to 0.9985. The limits of detection ranged from 5.0 to 20 ng∙L −1 for water samples, and from 30 to 90 ng∙kg −1 for soil samples. The recoveries of spiked samples were between 72.4 and 108.0 %, and the precision, expressed as the relative standard deviations, is 〈12.8 %. Graphical Abstract A MOF derived nanoporous carbon coated fiber for use in solid-phase microextraction was prepared via sol–gel technology. The coated fiber has a porous, rough and wrinkled structure, and shows a high thermal stability, good extraction repeatability and long lifetime. The established HS-SPME-GC-MS method is suitable for the determination of the PAHs from water and soil samples.

Beschreibung: We describe uniform and high-temperature-stable mesoporous TiO 2 beads functionalized with gold nanoparticles (AuNPs-TiO 2 ) for use in conductometric sensing of gases and organic vapors. The size of the interconnected main mesopores of the TiO 2 beads ranges from 8 to 15 nm, and the AuNPs have diameters between 8 and 10 nm. The mesoporous TiO 2 beads are formed during calcination while the structure-directing template agent is removed. Monodispersed AuNPs are formed by reduction in-situ and are placed inside the mesoporous TiO 2 framework. This prevents aggregation of the AuNPs even at 500 °C. The materials were characterized by UV–vis spectroscopy, scanning and transmission electron microscopy, nitrogen adsorption-desorption, and X-ray diffraction. Comb-type gold electrodes were then fabricated on an alumina substrate and are shown to display excellent properties in terms of sensing ammonia, ethanol, methanol or acetone. The sensitivity (defined as the ratio of resistivities under vapor and air) of a typical AuNPs(0.5 %)-TiO 2 gas sensor for ethanol reached up to 5.65 at above 600 ppm at 75 °C. Response time and recovery times (t 90  ≤ 20 s) are faster than (or comparable to) other metal-doped TiO 2 sensors, and working temperatures are much lower. An interesting observation was made in that the changes in the conductivity of the sensor change with temperature. The sensor prepared with AuNPs(0.5 %)-TiO 2 is of the p-type (in its response to ammonia gas) at 45 °C, but becomes n-type at 20 °C. Obviously, rather slight changes in temperature lead to a complete change in the direction of the conductometric signal change. This may provide a new aspect in terms of selective and highly sensitive detection of ammonia at ambient and slightly elevated temperatures. Graphical abstract We describe uniform and high-temperature-stable mesoporous TiO 2 beads functionalized with gold nanoparticles (AuNPs-TiO 2 ) for use in conductometric sensing of gases and organic vapors. Interestingly, the changes in the conductivity of typical sensors were opposite with the increasing of temperature.

Beschreibung: Humidity and temperature sensors were fabricated from a nanocomposite consisting of CeO 2 -Co 3 O 4 hybrid nanoparticle-silicone adhesive and CeO 2 -Co 3 O 4 hybrid nanoparticle-polymer adhesive, respectively, to fix the material on a glass supported copper electrode. The impedance of the sensor decreases by a factor of 960 at a working frequency of 100 Hz, and by a factor of 800 at 1 kHz, on increasing relative humidity (RH) from 30 to 90 %. In parallel, the capacitances increase by factors of 567 and 355, respectively, under the same experimental conditions. The effect of temperature in the range from 25 to 70 °C on impedance (again at 100 Hz and 1 kHz) was also studied and found to decrease with increasing temperature. On going from 25 to 70 °C, the impedance measured at 100 Hz and 1 kHz decreases 2.22 and 1.58 times, respectively, in surface type sensors, while in sandwich type sensors this decrease is 3.0 and 2.08 times. The calculated average sensitivity to temperature is −1.02 and −0.8 % °C −1 for the surface type and −1.5 and −1.2 % °C −1 for the sandwich type sensors at frequencies of 100 Hz and 1 kHz, respectively. Graphical abstract A highly sensitive sensor with dual functionality for humidity and temperature has been fabricated by using CeO 2 -codoped Co 3 O 4 nanoparticles with silicone and polymer adhesive.

Beschreibung: Single-walled carbon nanotubes (SWCNT), multi-walled carbon nanotubes (MWCNT) and graphene have been tested as carbon allotropes for the modification of carbon screen-printed electrodes (CSPEs) to simultaneously determine melatonin (MT) and serotonin (5-HT). Two groups of CSPEs, both 4 mm in diameter, were explored: The first includes commercial SWCNT, MWCNT and graphene, the second includes SWCNT, MWCNT, graphene oxide nanoribbons and reduced nanoribbons that were drop casted on the electrodes. The carbon nanomaterials enhanced the electroactive area in the following order: CSPE 〈MWCNTs 〈SWCNTs 〈graphene. This allowed the simultaneous determination of 5-HT and MT at the working potentials of +50 mV and +390 mV (vs. Ag), respectively. The use of carbon nanomaterials, in particular of graphene oxide nanoribbons on CSPEs, represents an excellent and disposable tool for sensing the two target molecules in even small sample volumes. Figures of merit for MT and 5-HT include (a) detection limit of 1.1 and 0.4 μM for MT and 5-HT, respectively; (b) an inter-electrode reproducibility with RSD ≤ 8 %; (c) 120 s response time, and (d) recoveries (in case of spiked samples) ranging from 94 to 103 % (with an RSD 〈 1 %). Graphical Abstract Carbon nanomaterials on screen-printed electrodes: smart electrochemistry for fast, simultaneous and reliable detection of serotonin the molecule of happiness and melatonin the molecule of darkness.

Beschreibung: We describe a sensitive photoelectrochemical (PEC) sensor for the determination of the herbicide clethodim. The PEC sensor was constructed by using amino-MIL-125/TiO 2 (MIL stands for Materials from Institute Lavoisier), an amino-functionalized metal-organic framework (MOF) modified with TiO 2 . The amino-MIL-125/TiO 2 was synthesized by a simple one-step solvothermal method and placed on a glassy carbon electrode where it displays photoelectrocatalytic activity. Scanning electron microscopy (SEM), Fourier-transform infrared (FTIR) spectroscopy and X-ray diffractometry (XRD) were used to characterize the amino-MIL-125/TiO 2 . In the sensing process, amino-MIL-125/TiO 2 is illuminated by visible light to produce electrons. These excited electrons are delivered to the glassy carbon electrode, leaving positively charged holes (h + ) on the surface of the amino-MIL-125/TiO 2 . The holes react with H 2 O to generate hydroxy radicals (•OH). Clethodim rapidly attacks the hydroxy radicals and improves the efficiency of charge separation, this leading to an enhanced photocurrent. Under the optimal experimental conditions, this photoelectrochemical method enables clethodim to be quantified in the concentration range from 0.2 to 25 μmol L −1 , with a detection limit (3 S/N) of 10 nmol L −1 . The assay was applied to the determination of clethodim in soil samples, and results were in acceptable agreement with data obtained by liquid chromatography/mass spectrometry. Graphical Abstract An amino-functionalized metal-organic framework (MOF) modified with titanium dioxide was synthesized and used as a new platform for photoelectrochemical sensing of the herbicide clethodim.

Beschreibung: The microbiological production of 2,3-butanediol (2,3-BDO) has attracted considerable attention as an alternative way to produce high-value chemicals from renewable sources. Among the number of 2,3-BDO-producing microorganisms, Klebsiella pneumoniae has been studied most extensively and is known to produce large quantity of 2,3-BDO from a range of substrates. On the other hand, the pathogenic characteristics of the bacteria have limited its industrial applications. In this study, two major virulence traits, outer core LPS and fimbriae, were removed through homologous recombination from 2,3-BDO-producing K. pneumoniae 2242 to expand its uses to the industrial scale. The K. pneumoniae 2242 ∆wabG mutant strain was found to have an impaired capsule, which significantly reduced its ability to bind to the mucous layer and evade the phagocytic activity of macrophage. The association with the human ileocecal epithelial cell, HCT-8, and the bladder epithelial cell, T-24, was also reduced dramatically in the K. pneumoniae 2242 ∆fimA mutant strain that was devoid of fimbriae. However, the growth rate and production yield for 2,3-BDO were unaffected. The K. pneumoniae strains developed in this study, which are devoid of the major virulence factors, have a high potential for the efficient and sustainable production of 2,3-BDO.

Beschreibung: Various food proteins including, e.g. gluten, collagen and casein are rich in l -proline residues. Due to the cyclic structure of proline, these proteins are well protected from enzymatic degradation by typical digestive enzymes. Proline-specific peptidases (PsP) belong to different families of hydrolases acting on peptide bonds (EC 3.4.x.x). They occur in various organisms including bacteria, fungi, plants and insects. Based on their biochemical characteristics, PsP type enzymes are further grouped into different subclasses of which prolyl aminopeptidases (EC 3.4.11.5, PAP), prolyl carboxypeptidases (EC 3.4.17.16, PCP) and prolyl oligopeptidases/prolyl endopeptidases (EC 3.4.21.26, POP/PEP) are of major interest for applications in food biotechnology. This mini review summarises the biochemical assays employed for these subclasses of PsP and their structural properties and the reaction mechanisms. A special focus was set on PsP derived from fungi and insects and important industrial applications in the field of food biotechnology. The degradation of gluten and collagen as well as the hydrolysis of bitter peptides are discussed.

Beschreibung: The paper describes the preparation of new probiotic formulations based on chitosan-coated alginate microcapsules containing three different probiotic strains, Lactobacillus plantarum PBS067, Lactobacillus rhamnosus PBS070, and Bifidobacterium animalis subsp. lactis PBS075 taken individually and as a mixture of them. The effects of microencapsulation on the viability of the strains in conditions simulating the gastrointestinal tract and under industrial processes conditions were studied. In addition, an evaluation of their probiotic properties was also investigated by in vitro tests on the human intestinal cell line HT-29 to explore the effect of microencapsulation on health beneficial effect of the considered strains. Non-encapsulated cells were completely destroyed when exposed to simulated gastric juice and other stress conditions, while encapsulated cells exhibited a significantly higher resistance to artificial intestinal juice and heat and osmotic treatment. Moreover, in this study, the effect of the various microencapsulated probiotic strain formulations was compared with analogous formulations also containing the β-glucan Pleuran. The microencapsulation effectively protected the selected bacteria, as single strain and as a mixture of the three strains in both the formulations with and without Pleuran, from simulating gastrointestinal tract and industrial process conditions in delivering the viable cells without any significant adverse effect on their functionalities. The comparative study of the immunomodulatory properties of each single strain and the mixture of the three strains revealed a synergistic effect of the probiotic mixture, but no appreciable difference between the two kinds of formulations could be detected, as the effect of Pleuran is covered by the higher potential of the probiotic strains.

Beschreibung: Strategies for bioremediation of atrazine, a pesticide commonly polluting groundwater in low concentrations, were studied in two boreal nonagricultural soils. Atrazine was not mineralized in soil without bioremediation treatments. In biostimulation treatment with molasses, up to 52 % of atrazine was mineralized at 10 °C, even though the degradation gene copy numbers did not increase. Incubations with radioactively labeled atrazine followed by microautoradiographic analysis revealed that bioremediation strategies increased the relative proportion of active degraders from 0.3 up to 1.9 % of the total bacterial count. These results indicate that atrazine degradation might not solely be facilitated by atzA / trzN – atzB genes. In combined biostimulation treatment using citrate or molasses and augmentation with Pseudomonas citronellolis ADP or Arthrobacter aurescens strain TC1, up to 76 % of atrazine was mineralized at 30 °C, and the atrazine degradation gene numbers increased up to 10 7  copies g −1 soil. Clone libraries from passive samplers in groundwater monitoring wells revealed the presence of phylogenetic groups formerly shown to include atrazine degraders, and the presence of atrazine degradation genes atzA and atzB . These results show that the mineralization of low concentrations of atrazine in the groundwater zone at low temperatures is possible by bioremediation treatments.

Beschreibung: We expressed a glucansucrase, DsrI, from Leuconostoc mesenteroides that catalyzes formation of water-insoluble glucans from sucrose using a nisin-controlled gene expression system in Lactococcus lactis . These polymers have potential for production of biodegradable gels, fibers, and films. We optimized production of DsrI using several different background vectors, signal peptides, strains, induction conditions, and bioreactor parameters to increase extracellular accumulation. Optimal production of the enzyme utilized a high-copy plasmid, pMSP3535H3, which contains a nisin immunity gene, L. lactis LM0230, and bioreactors maintained at pH 6.0 to stabilize the enzyme. We were able to significantly improve growth using the lactic acid inhibitor heme and by continuous removal of lactic acid with anion exchange resins, but enzyme production was less than the controls. The recombinant enzyme under optimized conditions accumulated in the culture medium to approximately 380 mg/L, which was over 150-fold higher compared to the native L. mesenteroides strain. Methods are also included for purification of DsrI utilizing the glucan-binding domain of the enzyme.

Beschreibung: Glutathione is a valuable tripeptide widely used in the pharmaceutical, food, and cosmetic industries. In industrial fermentation, glutathione is currently produced primarily using the yeast Saccharomyces cerevisiae . Intracellular glutathione exists in two forms; the majority is present as reduced glutathione (GSH) and a small amount is present as oxidized glutathione (GSSG). However, GSSG is more stable than GSH and is a more attractive form for the storage of glutathione extracted from yeast cells after fermentation. In this study, intracellular GSSG content was improved by engineering thiol oxidization metabolism in yeast. An engineered strain producing high amounts of glutathione from over-expression of glutathione synthases and lacking glutathione reductase was used as a platform strain. Additional over-expression of thiol oxidase (1.8.3.2) genes ERV1 or ERO1 increased the GSSG content by 2.9-fold and 2.0-fold, respectively, compared with the platform strain, without decreasing cell growth. However, over-expression of thiol oxidase gene ERV2 showed almost no effect on the GSSG content. Interestingly, ERO1 over-expression did not decrease the GSH content, raising the total glutathione content of the cell, but ERV1 over-expression decreased the GSH content, balancing the increase in the GSSG content. Furthermore, the increase in the GSSG content due to ERO1 over-expression was enhanced by additional over-expression of the gene encoding Pdi1, whose reduced form activates Ero1 in the endoplasmic reticulum. These results indicate that engineering the thiol redox metabolism of S. cerevisiae improves GSSG and is critical to increasing the total productivity and stability of glutathione.

Beschreibung: Baculoviruses have a long history of safe use as specific, environmentally friendly insecticides that provide alternatives to chemical pesticides for controlling insect pests. However, their use has been limited by several factors, particularly their slow pathogenicity. In this study, we constructed a recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) and an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) that expressed an insect-specific cyto-insectotoxin (Cit1a) from the venom of the central Asian spider Lachesana tarabaevi . Cit1a is a comparatively long linear cytolytic molecule that contains a predicted α-helix structure composed of two short membrane-acting antimicrobial peptides (MAMPs) that are joined together in a “head-to-tail” shape. Cit1a fused to polyhedrin gene ( polh ) ( polh - cit1a ) was expressed in the nuclei as polyhedra in silkworm larvae, Bm5 and Sf9 cells. An early death of Bm5 and Sf9 cells by recombinant BmNPV/Polh-Cit1a and AcMNPV/Polh-Cit1a was observed compared with control viruses that lacked the toxin gene. The infected cells showed a loss of cytoplasm, membrane integrity, and structural changes, suggesting that recombinant baculovirus-infected cells were killed by the necrosis caused by Cit1a. In addition, the BmNPV/Polh-Cit1a showed a significant reduction in the median lethal time (LT 50 ) against silkworm larvae compared with those of control BmNPV that lacked the cit1a gene.

Beschreibung: Organic carbon, nitrogen, and sulfur are highly concentrated in municipal solid waste (MSW) landfill leachate, which usually frustrates conventional leachate treatment technologies from the perspective of energy costs. Therefore, the possibility of converting leachate to a new energy source via microbial fuel cell (MFC) technology has been examined recently. This paper summarizes the power output and energy recovery efficiency of the leachate-fed MFCs according to different feeding patterns, cell structures, and loading rates. Also, we assess potential energy-generating chemicals in leachate like nitrogen and sulfur compounds and propose alternative pathways, which may lift strict ratios between organic carbon and nitrogen content in conventional denitrification of leachate and are expected to achieve a higher voltage than traditional organic-oxygen based cells. Although currently power output of leachate-fed MFCs is limited, it seems well possible that dynamic characteristics of MSW leachates and microbial physiologies underlying some bio-electrochemically efficient activities (e.g., direct interspecies electron transfer) could be stimulated in MFC systems to improve the present status.

Beschreibung: An on-line, ensemble-based data assimilation (DA) method is performed to reconstruct the climate for 1750–1850 AD, and the performance is evaluated on large and small spatial scales. We use a low-resolution version of the Max Planck Institute for Meteorology MPI-ESM model and assimilate the PAGES 2K continental mean temperature reconstructions for the Northern Hemisphere (NH). The ensembles are generated sequentially for sub-periods based on the analysis of previous sub-periods. The assimilation has good skill for large-scale temperatures, but there is no agreement between the DA analysis and proxy-based reconstructions for small-scale temperature patterns within Europe or with reconstructions for the North Atlantic Oscillation (NAO) index. To explain the lack of added value in small spatial scales, a maximum covariance analysis (MCA) of links between NH temperature and sea level pressure is performed based on a control simulation with MPI-ESM. For annual values, winter and spring the Northern Annular Mode (NAM) is the pattern that is most closely linked to the NH continental temperatures, while for summer and autumn it is a wave-like pattern. This link is reproduced in the DA for winter, spring and annual means, providing potential for constraining the NAM/NAO phase and in turn regional temperature variability. It is shown that the lack of actual small-scale skill is likely due to the fact that the link might be too weak, as the NH continental mean temperatures are not the best predictors for large-scale circulation anomalies, or that the PAGES 2K temperatures include noise. Both factors can lead to circulation anomalies in the DA analysis that are substantially different from reality, leading to unrealistic representation of small-scale temperature variability. Moreover, we show that even if the true amplitudes of the leading MCA circulation patterns were known, there is still a large amount of unexplained local temperature variance. Based on these results, we argue that assimilating temperature reconstructions with a higher spatial resolution might improve the DA performance.

Beschreibung: We analyse the impact of migration and strength of Southern Hemisphere westerly winds on the ocean carbon cycle in a systematic sensitivity study with the University of Victoria Earth System Climate Model. We find that changes in the biological pump are mainly driven by changes in ocean residence times while changes in export production are negligible. Changes in the biological and physical pumps are always of opposite sign; with the physical pump being dominant for southward shifts and the biological pump being dominant for northward shifts. Furthermore, changes in the Pacific Ocean carbon budget dictate the overall changes in global marine and atmospheric carbon. Overall, atmospheric \(\hbox {CO}_2\) increases (and \(\Delta ^{14}\hbox {C}\) decreases) for northward shifts or a strengthening in wind forcing. The opposite is true for a southward shift or a weakening in wind forcing. Combining forcings (shift and intensity change) results in a combination of their impacts with the direction of the shift being the first order forcing. The terrestrial carbon reservoir absorbs (releases) 50–70 % of the net oceanic carbon loss (increase), counterbalancing the effect on atmospheric \(\hbox {CO}_2\) .

Beschreibung: Effects of wind and fresh water on the Atlantic meridional overturning circulation (AMOC) are investigated using a fully coupled climate model. The AMOC can change significantly when perturbed by either wind stress or freshwater flux in the North Atlantic. This study focuses on wind stress effect. Our model results show that the wind forcing is crucial in maintaining the AMOC. Reducing wind forcing over the ocean can cause immediately weakening of the vertical salinity diffusion and convection in the mid-high latitudes Atlantic, resulting in an enhancement of vertical salinity stratification that restrains the deep water formation there, triggering a slowdown of the thermohaline circulation. As the thermohaline circulation weakens, the sea ice expands southward and melts, providing the upper ocean with fresh water that weakens the thermohaline circulation further. The wind perturbation experiments suggest a positive feedback between sea-ice and thermohaline circulation strength, which can eventually result in a complete shutdown of the AMOC. This study also suggests that sea-ice variability may be also important to the natural AMOC variability on decadal and longer timescales.

Beschreibung: Many filamentous fungi produce only conidia for dispersal and survival in vitro or in vivo. Here, we show that the developmental regulator WetA and the velvet protein VosA are not only required for conidial maturation but indispensable for conidiation in Beauveria bassiana , a filamentous entomopathogen. Deletion of wetA or vosA resulted in more than 90 % transcriptional depression of brlA and abaA , two activator genes in the central developmental pathway, during the critical period of conidiophore development and conidiation. Consequently, Δ wetA and Δ vosA strains lost 98 % in and 88 % of their conidiation capacities under optimal culture conditions, respectively. The conidia of Δ wetA showed more defective features than those of Δ vosA , including smaller size, lesser density, lower hydrophobicity, and impaired cell walls although intracellular trehalose content decreased more in the aging culture of Δ vosA than of Δ wetA . As a result, conidial sensitivity to cell wall perturbation was elevated in Δ wetA but unaffected in Δ vosA , which produced conidia more sensitive to the oxidant menadione and the wet-heat stress at 45 °C. Both deletion mutants showed similar defects in conidial tolerance to high osmolarity or UV-B irradiation but no change in conidial sensitivity to the other oxidant H 2 O 2 or the fungicide carbendazim. Moreover, Δ wetA lost more virulence to Galleria mellonella larvae than Δ vosA . All these phenotypical changes were restored by either wetA or vosA complementation. Taken together, WetA and VosA are indispensable for asexual development and contribute differentially to conidial quality and hence the biological control potential of B. bassiana against insect pests.

Beschreibung: In this paper, we investigate the role of electromagnetic field on the stability regions of charged self-gravitating compact objects by using the concept of cracking. For this purpose, we have applied local density perturbation scheme to the hydrostatic equilibrium equation as well as on physical parameters involved in the model. In particular, we have examined the cracking of charged compact objects like PSR J1614-2230, PSR J1903+327, Vela X-1, SMC X-1 and Cen X-3 with different values of charge. We conclude that these objects exhibit cracking and stability regions decreases with the increase of charge.

Beschreibung: In this paper we have studied the behavior of static spherically symmetric relativistic objects with locally anisotropic matter distribution considering the Tolman VII form for the gravitational potential \(g_{rr}\) in curvature coordinates together with the linear relation between the energy density and the radial pressure. The interior spacetime has been matched continuously to the exterior Schwarzschild geometry. We have investigated and analyzed different physical properties of the stellar model and presented graphically.

Beschreibung: The relationship between the large-scale circulation dynamics and regional precipitation regime in the Tibetan Plateau (TP) has so far not been well understood. In this study, we classify the circulation types using the self-organizing maps based on the daily field of 500 hPa geopotential height and link them to the precipitation climatology in the eastern and central TP. By virtue of an objective determining method, 18 circulation types are quantified. The results show that the large amount of precipitation in summer is closely related to the circulation types in which the enhanced and northward shifted subtropical high (SH) over the northwest Pacific and the obvious cyclconic circulation anomaly over the Bay of Bengal are helpful for the Indian summer monsoon and East Asian summer monsoon to take abundant low-latitude moisture to the eastern and southern TP. On the contrary, the dry winter in the central and eastern Tibet corresponds to the circulation types with divergence over the central and eastern TP and the water vapor transportations of East Asian winter monsoon and mid-latitude westerly are very weak. Some circulation types are associated with some well-known circulation patterns/monsoons influencing the TP (e.g. East Atlantic Pattern, El Niño Southern Oscillation, Indian Summer Monsoon and the mid-latitude westerly), and exhibit an overall good potential for explaining the variability of regional seasonal precipitation. Moreover, the climate shift signals in the late 1970s over the eastern Pacific/North Pacific Oceans could also be reflected by both the variability of some circulation types and their correspondingly composite precipitations. This study extends our understandings for the large-scale atmospheric dynamics and their linkages with regional precipitation and is beneficial for the climate change projection and related adaptation activities in the highest and largest plateau in the world.

Beschreibung: We report on a new amplification strategy for use in an immunoassay for influenza virus subtype H7N9. Graphene sheets were first placed on a glassy carbon electrode (GCE), and gold nanoparticles were then electrodeposited as a support for a layer of alcohol dehydrogenase (ADH) in a sol–gel containing thiol groups. Protein A was used to properly orientate immobilized antibody against H7N9 on the sol–gel, and this is shown to result in strongly improved specificity of the antigen-antibody binding. Thus, a sensitive and specific immunosensor was obtained in which a quadruple signal amplification strategy is employed, viz. (a) via the use of graphene sheets, (b) via a hybridization chain reaction, (c) the use of hemin/G-quadruplex DNAzyme concatamers, and (d) the use of ADH. The hemin/G-quadruplex is a typical DNAzyme, which simultaneously acts as NADH oxidase and HRP-mimicking DNAzyme. The hybridization chain reaction-based DNAzyme concatamers assembled on multi-walled carbon nanotubes (MWCNTs) and the ADH represent a triple electrocatalytic enzyme cascade system. Sandwich immunoreactions occurred between the capture antibody on the electrode and the secondary antibody labeled with MWCNTs. Positively charged Methylene Blue (MB) was then used as an intercalator to detect the DNAzyme concatamer formed. The differential pulse voltammetric signals for MB are related to the concentration of H7N9 in the range from 8 to 60 pg · mL −1 , and the detection limit is 0.81 pg · mL −1 (at an S/N ratio of 3). This immunoassay is very sensitive, specific and robust. Graphical Abstract An electrochemical sandwich immunosensor has been developed for sensitive and specific detection of influenza virus subtype H7N9. Protein A was used to properly orientate antibody. The hybridization chain reaction based DNAzyme concatamers assembled on multi-walled carbon nanotubes (MWCNTs) and the ADH represent a triple electrocatalytic enzyme cascade system.

Beschreibung: This paper explores structure scalars of cylindrically symmetric spacetime in Brans-Dicke gravity. We construct twelve scalar factors using orthogonal splitting of the Reimann tensor and study their distinct dynamical interpretations. These structure scalars are used to derive a set of governing equations of the evolving system. It is concluded that cylindrical systems are necessarily inhomogeneous. Finally, we show that all possible static inhomogeneous cylindrical solutions can be obtained through structure scalars.

Beschreibung: In this Letter, based on Bianchi-I type cosmological model, we have studied the evolution of universe in terms of a vector field coupled to the scalar field. The symmetries of the system are calculated in the Noether approach, the scalar potential and the gauge kinetic function are also obtained following these symmetries. The dynamical equations are integrated numerically and the behaviors of cosmological evolution are presented certainly. Furthermore, the early-time and late-time scenarios are also reached asymptotically, that is, the universe is during the period of decelerating expansion in the early time while during the period of accelerating expansion in the late time. Moreover, we have also studied the expansion isotropization of the universe during evolution. There is no Kasner solution in the present case, while the universe would approach an isotropic Robertson-Walker one, concretely, a de Sitter type one in the late time.

Beschreibung: We describe a turn-off fluorescence-based strategy for the detection of ATP by making use of aptamer-triggered dsDNA concatamers. This sensitive and easily controlled method is based on consecutive hybridization induced by ATP aptamers and their sectional complementary DNAs to form dsDNA concatamers. The intercalator SYBR Green I (SGI) was employed as a fluorescent probe. In the absence of ATP, the probe produces a strong signal. However, on addition of ATP, the binding of aptamer and ATP cause the concatamers to collapse and to release SGI whose fluorescence then is quenched. The effect was exploited to design a selective ATP assay by relating the decrease in fluorescence to the ATP concentration. A lower detection limit of 6.1 μM and a linear response in the 0 to 5000 μM concentration range was accomplished. The strategy was applied to cellular ATP assays, and the results obtained by this strategy and by the gold standard method are in good agreement. The method is sensitive, simple and cost efficient, and hence is promising in terms of future applications to determine ATP in cellular and other systems. Graphical Abstract A turn-off fluorescence-based strategy for the selective detection of ATP by using aptamer-triggered dsDNA concatamers.

Beschreibung: Based on the historical and RCP8.5 experiments from 25 Coupled Model Intercomparison Project phase 5 (CMIP5) models, the impacts of sea surface temperature (SST) warming in the tropical Indian Ocean (IO) on the projected change in summer rainfall over Central Asia (CA) are investigated. The analysis is designed to answer three questions: (1) Can CMIP5 models reproduce the observed influence of the IO sea surface temperatures (SSTs) on the CA rainfall variations and the associated dynamical processes? (2) How well do the models agree on their projected rainfall changes over CA under warmed climate? (3) How much of the uncertainty in such rainfall projections is due to different impacts of IO SSTs in these models? The historical experiments show that in most models summer rainfall over CA are positively correlated to the SSTs in the IO. Furthermore, for models with higher rainfall-SSTs correlations, the dynamical processes accountable for such impacts are much closer to what have been revealed in observational data: warmer SSTs tend to favor the development of anti-cyclonic circulation patterns at low troposphere over north and northwest of the Arabian Sea and the Bay of Bengal. These anomalous circulation patterns correspond to significantly enhanced southerly flow which carries warm and moisture air mass from the IO region up to the northeast. At the same time, there is a cyclonic flow over the central and eastern part of the CA which further brings the tropical moisture into the CA and provides essential moist conditions for its rainfall generation. In the second half of twenty-first century, although all the 25 models simulate warmed SSTs, significant uncertainty exists in their projected rainfall changes over CA: half of them suggest summer rainfall increases, but the other half project rainfall decreases. However, when we select seven models out of the 25 based on their skills in capturing the dynamical processes as observed, then the model projected changes are much closer. Five out of the seven models predicted more rainfall over CA. Such a result is helpful for allowing us to attribute part of the observed upward rainfall trend in the CA region in the last several decades to the IO SST warming.