ALBERT

Description: 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.

Description: 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.

Description: 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 .

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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 .

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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 .

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: 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 \(\hbox {CO}_2\) .

Description: 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.

Description: 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.

Description: 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.

Description: 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.

Description: Cyclonic windstorms are one of the most important natural hazards for Europe, but robust climate projections of the position and the strength of the North Atlantic storm track are not yet possible, bearing significant risks to European societies and the (re)insurance industry. Previous studies addressing the problem of climate model uncertainty through statistical comparisons of simulations of the current climate with (re-)analysis data show large disagreement between different climate models, different ensemble members of the same model and observed climatologies of intense cyclones. One weakness of such evaluations lies in the difficulty to separate influences of the climate model’s basic state from the influence of fast processes on the development of the most intense storms, which could create compensating effects and therefore suggest higher reliability than there really is. This work aims to shed new light into this problem through a cost-effective “seamless” approach of hindcasting 20 historical severe storms with the two global climate models, ECHAM6 and GA4 configuration of the Met Office Unified Model, run in a numerical weather prediction mode using different lead times, and horizontal and vertical resolutions. These runs are then compared to re-analysis data. The main conclusions from this work are: (a) objectively identified cyclone tracks are represented satisfactorily by most hindcasts; (b) sensitivity to vertical resolution is low; (c) cyclone depth is systematically under-predicted for a coarse resolution of T63 by both climate models; (d) no systematic bias is found for the higher resolution of T127 out to about three days, demonstrating that climate models are in fact able to represent the complex dynamics of explosively deepening cyclones well, if given the correct initial conditions; (e) an analysis using a recently developed diagnostic tool based on the surface pressure tendency equation points to too weak diabatic processes, mainly latent heating, as the main source for the under-prediction in the coarse-resolution runs. Finally, an interesting implication of these results is that the too low number of deep cyclones in many free-running climate simulations may therefore be related to an insufficient number of storm-prone initial conditions. This question will be addressed in future work.

Description: The effect of chitosan with different molecular weights and other natural substances on dextransucrase (DSase) activity from a representative oral pathogen Streptococcus mutans was elucidated. Among other bioactive substances, amino-monosaccharides such as glucosamine, mannosamine, and galactosamine exerted the enzyme inhibitory activity over 95 % of DSase. The specified hydrolysates derived from the hydrolysis of high molecular weight chitosan (HMWC) designated to CTSN, CTSN-P, CTSN-B, and CTSN-S with different molecular weights ranging from 3 to 8 kDa showed the similar inhibitory activity toward DSase. Also, the hyaluronic acid (MW 8.9 kDa), sulfated chitin, and amino-monosaccharides demonstrated the significant activity, CTSN, CTSN-P, CTSN-B, and CTSN-S are of potent bioactive substances that can be prepared in the cheapest way compared with other molecules tested available for antibacterial agent useful for human oral health.

Description: Rifamycin is a broad-spectrum antimicrobial drug produced commercially by submerged fermentation where the yields are far less in comparison to its demand in human drug therapy. Addressing the need, sequential mutational strain improvement was carried using UV and EtBr that resulted in improved strain yielding rifamycin SV up to 4.32 g/L. Further optimization of six important fermentation factors was followed which include temperature, agitation, inoculum level, period of fermentation, inorganic nitrogen source and amino acids. For the first time, we report a maximum yield of 5.32 g/L of rifamycin SV. Among the amino acids, proline known for its slowest assimilation by Amycolatopsis mediterranei produced the highest improvement in antibiotic yields. Following mutational strain improvement and process optimization, a total of 3.8-fold increase in antibiotic titre was achieved. Following a conventional procedure of mutational strain improvement, highest yield of rifamycin SV was reported by optimizing submerged fermentation process.

Description: A new molecularly imprinted polymer (MIP), prepared by hispidin as the template molecule, was synthesized and applied as an adsorbent phase for solid phase extraction (SPE) to isolate and enrich hispidin from eight species of mushrooms. The optimization of synthesis and the adsorption behaviors of the MIPs were investigated in detail. In comparison with C 18 -SPE, MIP-SPE displayed high selectivity and good affinity for hispidin for extract of Phellinus igniarius. The antioxidant activity of the extracts after using the MIPs was evaluated by free radical scavenging activity, and inhibition of erythrocyte hemolysis, and lipid peroxidation. This developed method provided a rapid, selective, and effective approach for separation and enrichment of active compounds from the natural products.

Description: Vinyl chloride (VC), a known human carcinogen, is a common and persistent groundwater pollutant at many chlorinated solvent contaminated sites. The remediation of such sites is challenging because of the lack of knowledge on the microorganisms responsible for in situ VC degradation. To address this, the microorganisms involved in carbon assimilation from VC were investigated in a culture enriched from contaminated site groundwater using stable isotope probing (SIP) and high-throughput sequencing. The mixed culture was added to aerobic media, and these were amended with labeled ( 13 C-VC) or unlabeled VC ( 12 C-VC). The cultures were sacrificed on days 15, 32, and 45 for DNA extraction. DNA extracts and SIP ultracentrifugation fractions were subject to sequencing as well as quantitative PCR (qPCR) for a functional gene linked to VC-assimilation ( etnE ). The gene etnE encodes for epoxyalkane coenzyme M transferase, a critical enzyme in the pathway for VC degradation. The relative abundance of phylotypes was compared across ultracentrifugation fractions obtained from the 13 C-VC- and 12 C-VC-amended cultures. Four phylotypes were more abundant in the heavy fractions (those of greater buoyant density) from the 13 C-VC-amended cultures compared to those from the 12 C-VC-amended cultures, including Nocardioides , Brevundimonas , Tissierella , and Rhodoferax. Therefore, both a previously identified VC-assimilating genus ( Nocardioides ) and novel microorganisms were responsible for carbon uptake. Enrichment of etnE with time was observed in the heavy fractions, and etnE sequences illustrated that VC-assimilators harbor similar Nocardioides- like etnE . This research provides novel data on the microorganisms able to assimilate carbon from VC.

Description: As antibiotic resistance continues to spread globally, there is growing interest in the potential to limit the spread of antibiotic resistance genes (ARGs) from wastewater sources. In particular, operational conditions during sludge digestion may serve to discourage selection of resistant bacteria, reduce horizontal transfer of ARGs, and aid in hydrolysis of DNA. This study applied metagenomic analysis to examine the removal efficiency of ARGs through thermophilic and mesophilic anaerobic digestion using bench-scale reactors. Although the relative abundance of various ARGs shifted from influent to effluent sludge, there was no measureable change in the abundance of total ARGs or their diversity in either the thermophilic or mesophilic treatment. Among the 35 major ARG subtypes detected in feed sludge, substantial reductions (removal efficiency 〉90 %) of 8 and 13 ARGs were achieved by thermophilic and mesophilic digestion, respectively. However, resistance genes of aad A, mac B, and sul 1 were enriched during the thermophilic anaerobic digestion, while resistance genes of erythromycin esterase type I, sul 1, and tet M were enriched during the mesophilic anaerobic digestion. Efflux pump remained to be the major antibiotic resistance mechanism in sludge samples, but the portion of ARGs encoding resistance via target modification increased in the anaerobically digested sludge relative to the feed. Metagenomic analysis provided insight into the potential for anaerobic digestion to mitigate a broad array of ARGs.

Description: Yersinia pestis , an infectious bacterium that is a causative agent of plague, a disease which has been shown to be one of the most feared in history and which has caused millions of deaths. The capsule-like fraction 1 (F1) antigen expressed by Y. pestis is a known specific marker for the identification of the bacteria; therefore, the detection of F1 is important for Y. pestis recognition. In this study, a rapid, sensitive, and specific technique, the lateral flow assay (LFA), was successfully developed to detect Y. pestis by the recombinant F1 antigen. The assay that utilized an anti-F1 polyclonal antibody (Pab) to identify the bacteria was based on a double-antibody sandwich format on a nitrocellulose membrane. With the LFA method, 50 ng/ml of recombinant F1 protein and 10 5 CFU/mL of Y. pestis could be detected in less than 10 min. This assay also showed no cross-reaction with other Yersinia spp. or with some selected capsule-producing Enterobacteriaceae strains. Furthermore, detection of Y. pestis in simulated samples has been evaluated. The detection sensitivity of Y. pestis in various matrices was 10 5 CFU/mL, which was identical to that in PBS buffer. The results obtained suggest that LFA is an excellent tool for detection of Y. pestis contamination in an environment and hence can be used to monitor plague diseases when they emerge.

Description: Microbial production of biodiesel from renewable feedstock has attracted intensive attention. Biodiesel is known to be produced from short-chain alcohols and fatty acyl-CoAs through the expression of wax ester synthase/fatty acyl-CoA: diacylglycerol acyltransferase that catalyzes the esterification of short-chain alcohols and fatty acyl-CoAs. Here, we engineered Escherichia coli to produce various fatty alcohol acetate esters, which depend on the expression of Saccharomyces cerevisiae alcohol acetyltransferase ATF1 that catalyzes the esterification of fatty alcohols and acetyl-CoA. The fatty acid biosynthetic pathways generate fatty acyl-ACPs, fatty acyl-CoAs, or fatty acids, which can be converted to fatty alcohols by fatty acyl-CoA reductase, fatty acyl-ACP reductase, or carboxylic acid reductase, respectively. This study showed the biosynthesis of biodiesel from three fatty acid biosynthetic pathway intermediates.

Description: Extreme precipitation and flood episodes in the Himalayas are oftentimes traced to synoptic situations involving connections between equatorward advancing upper level extratropical circulations and moisture-laden tropical monsoon circulation. While previous studies have documented precipitation characteristics in the Himalayan region during severe storm cases, a comprehensive understanding of circulation dynamics of extreme precipitation mechanisms is still warranted. In this study, a detailed analysis is performed using rainfall observations and reanalysis circulation products to understand the evolution of monsoon-extratropical circulation features and their interactions based on 34 extreme precipitation events which occurred in the Western Himalayas (WEH) during the period 1979–2013. Our results provide evidence for a common large-scale circulation pattern connecting the extratropics and the South Asian monsoon region, which is favorable for extreme precipitation occurrences in the WEH region. This background upper level large-scale circulation pattern consists of a deep southward penetrating midlatitude westerly trough, a blocking high over western Eurasia and an intensifying Tibetan anticyclone. It is further seen from our analysis that the key elements of monsoon-midlatitude interactions, responsible for extreme precipitation events over the WEH region, are: (1) midlatitude Rossby wave breaking, (2) west-northwest propagation of monsoon low-pressure system from the Bay of Bengal across the Indian subcontinent, (3) eddy shedding of the Tibetan anticyclone, (4) ageostrophic motions and transverse circulation across the Himalayas, and (5) strong moist convection over the Himalayan foothills. Furthermore, high-resolution numerical simulations indicate that diabatic heating and mesoscale ageostrophic effects can additionally amplify the convective motions and precipitation in the WEH region.

Description: This paper examines the difference in the Atlantic Meridional Overturning Circulation (AMOC) mean state between free and assimilative simulations of a common ocean model using a common interannual atmospheric forcing. In the assimilative simulation, the reproduction of cold cores in the Nordic Seas, which is absent in the free simulation, enhances the overflow to the North Atlantic and improves AMOC with enhanced transport of the deeper part of the southward return flow. This improvement also induces an enhanced supply of North Atlantic Deep Water (NADW) and causes better representation of the Atlantic deep layer despite the fact that correction by the data assimilation is applied only to temperature and salinity above a depth of 1750 m. It also affects Circumpolar Deep Water in the Southern Ocean. Although the earliest influence of the improvement propagated by coastal waves reaches the Southern Ocean in 10–15 years, substantial influence associated with the arrival of the renewed NADW propagates across the Atlantic Basin in several decades. Although the result demonstrates that data assimilation is able to improve the deep ocean state even if there is no data there, it also indicates that long-term integration is required to reproduce variability in the deep ocean originating from variations in the upper ocean. This study thus provides insights on the reliability of AMOC and the ocean state in the Atlantic deep layer reproduced by data assimilation systems.

Description: Epidermal growth factor (EGF) ameliorates stress and prevents incomplete gastrointestinal development in early-weaned piglets in commercial swine farming. This study aimed to further analyze the biological activities of intracellularly expressed EGF (IE-EGF), extracellularly expressed EGF (EE-EGF), and tagged EGF (T-EGF) from Saccharomyces cerevisiae in early-weaned pigs. In this study, we assigned 24 pigs to each of 5 groups that were provided a basic diet (the control group) or a diet supplemented with empty vector-expressing S. cerevisia e [the INVSc1(EV) group], T-EGF-expressing S. cerevisiae [the INVSc1-TE(−) group], EE-EGF-expressing S. cerevisiae [the INVSc1-EE(+) group], or IE-EGF-expressing S. cerevisiae [the INVSc1-IE(+) group]. All treatments were delivered at a dose of 60 μg EGF/kg body weight (BW) everyday. All the piglets were sacrificed after 21 day to determine their physio-biochemical indexes, immune functions, and intestinal development. In the piglet experiments, recombinant S. cerevisiae survived throughout the intestinal tract. The BW and intestinal development (e.g., mean villous height, crypt depth, villous height:crypt depth ratio (IVR), and total protein, DNA, and RNA contents) of the piglets were significantly enhanced in the INVSc1-IE(+) group compared with the animals in the INVSc1-EE(+) and INVSc1-TE(−) groups ( P  〈 0.05). In addition, increased proliferating cell nuclear antigen (PCNA) staining was observed in the piglets that received the INVSc1-IE(+) treatment (approximately 80 %) compared with those that received the INVSc1-TE(−) (approximately 70 %) and INVSc1-EE(+) treatments (approximately 70 %). The levels of lactate dehydrogenase (LDH), creatine kinase (CK), alkaline phosphatase (ALP), immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin G (IgG) were also significantly increased in the INVSc1-IE(+) group compared with the INVSc1-EE(+) and INVSc1-TE(−) groups ( P  〈 0.05). Furthermore, the proliferation of piglet enterocytes was also significantly stimulated by both IE-EGF and EE-EGF compared with T-EGF in vitro ( P  〈 0.05). Our data further demonstrate the previously reported hypothesis that IE-EGF is more suitable than EE-EGF or T-EGF for applications in early-weaned pigs.

Description: With the rapid development of molecular diagnostic techniques, there is a growing need for quality controls and standards with favorable properties to monitor the entire detection process. In this study, we describe a novel method to produce armored hepatitis B virus (HBV) and human papillomavirus (HPV) DNA for use in nucleic acid tests, which was confirmed to be stable, homogeneous, noninfectious, nuclease resistant, and safe for shipping. We demonstrated that MS2 bacteriophage could successfully package double-stranded DNA of 1.3-, 3-, 3.5-, and 6.5-kb length into viral capsids with high reassembly efficiency. This is the first application of RNA bacteriophage MS2 as a platform to encapsulate double-stranded DNA, forming virus-like particles (VLPs) which were indistinguishable from native MS2 capsids in size and morphology. Moreover, by analyzing the interaction mechanism of pac site and the MS2 coat protein (CP), we found that in addition to the recognized initiation signal TR-RNA, TR-DNA can also trigger spontaneous reassembly of CP dimers, providing a more convenient and feasible method of assembly. In conclusion, this straightforward and reliable manufacturing approach makes armored DNA an ideal control and standard for use in clinical laboratory tests and diagnostics, possessing prospects for broad application, especially providing a new platform for the production of quality controls for DNA viruses.

Description: Lipids are naturally derived products that offer an attractive, renewable alternative to petroleum-based hydrocarbons. While naturally produced long-chain fatty acids can replace some petroleum analogs, medium-chain fatty acid would more closely match the desired physical and chemical properties of currently employed petroleum products. In this study, we engineered Yarrowia lipolytica , an oleaginous yeast that naturally produces lipids at high titers, to produce medium-chain fatty acids. Five different acyl-acyl carrier protein (ACP) thioesterases with specificity for medium-chain acyl-ACP molecules were expressed in Y. lipolytica , resulting in formation of either decanoic or octanoic acid. These novel fatty acid products were found to comprise up to 40 % of the total cell lipids. Furthermore, the reduction in chain length resulted in a twofold increase in specific lipid productivity in these engineered strains. The medium-chain fatty acids were found to be incorporated into all lipid classes.

Description: Long-term stable cell growth and production of vindoline, catharanthine, and ajmalicine of cambial meristematic cells (CMCs) from Catharanthus roseus were observed after 2 years of culture. C. roseus CMCs were treated with β-cyclodextrin (β-CD) and methyl jasmonate (MeJA) individually or in combination and were cultured both in conventional Erlenmeyer flasks (100, 250, and 500 mL) and in a 5-L stirred hybrid airlift bioreactor. CMCs of C. roseus cultured in the bioreactor showed higher yields of vindoline, catharanthine, and ajmalicine than those cultured in flasks. CMCs of C. roseus cultured in the bioreactor and treated with 10 mM β-CD and 150 μM MeJA gave the highest yields of vindoline (7.45 mg/L), catharanthine (1.76 mg/L), and ajmalicine (58.98 mg/L), concentrations that were 799, 654, and 426 % higher, respectively, than yields of CMCs cultured in 100-mL flasks without elicitors. Quantitative reverse transcription (RT)-PCR showed that β-CD and MeJA upregulated transcription levels of genes related to the biosynthesis of terpenoid indole alkaloids (TIAs). This is the first study to report that β-CD induced the generation of NO, which plays an important role in mediating the production of TIAs in C. roseus CMCs. These results suggest that β-CD and MeJA can enhance the production of TIAs in CMCs of C. roseus , and thus, CMCs of C. roseus have significant potential to be an industrial platform for production of bioactive alkaloids.

Description: Mechanisms of glutathione (GSH) over-accumulation in mutant Saccharomyces cerevisiae Y518 screened by ultraviolet and nitrosoguanidine-induced random mutagenesis were studied. Y518 accumulated higher levels of GSH and l -cysteine than its wild-type strain. RNA-Seq and pathway enrichment analysis indicated a difference in the expression of key genes involved in cysteine production, the GSH biosynthesis pathway, and antioxidation processes. GSH1 , MET17 , CYS4 , GPX2 , CTT1 , TRX2 , and SOD1 and the transcriptional activators SKN7 and YAP1 were up-regulated in the mutant. Moreover, Y518 showed a dysfunctional respiratory chain resulting from dramatically weakened activity of complex III and significant elevation of intracellular reactive oxygen species (ROS) levels. The supplementation of antimycin A in the culture of the parent strain showed equivalent changes of ROS and GSH level. This study indicates that defective complex III prompts abundant endogenic ROS generation, which triggers an oxidative stress response and upregulation of gene expression associated with GSH biosynthesis. This finding may be helpful for developing new strategies for GSH fermentation process optimization or metabolic engineering.

Description: Prenyltransferases of the dimethylallyltryptophan synthase (DMATS) superfamily are involved in the biosynthesis of secondary metabolites and contribute as modification enzymes significantly to structural diversity of natural products. They show usually broad specificity toward their aromatic substrates with regiospecific prenylations on aromatic rings. However, most members of this superfamily exhibit a high specificity toward their prenyl donors and usually accept exclusively dimethylallyl diphosphate (DMAPP). Recently, several indole prenyltransferases from this family were also demonstrated to accept unnatural DMAPP analogs such as methylallyl, 2-pentenyl and benzyl diphosphate for alkylation, or benzylation of the indole ring. Partial or complete shift of the substitution position was observed for these enzymes. In this study, we report the acceptance of these DMAPP analogs by two tyrosine O -prenyltransferases TyrPT from Aspergillus niger and SirD from Leptosphaeria maculans for alkylation or benzylation of tyrosine and derivatives. NMR and mass spectrometry (MS) analyses of nine isolated enzyme products confirmed the regiospecific O- or N -alkylation or benzylation at position C-4 of the aromatic ring, which is the same prenylation position of these enzymes in the presence of DMAPP.

Description: The southeast region of Brazil experienced in austral summer 2014 a major drought event leading to a number of impacts in water availability for human consumption, agricultural irrigation and hydropower production. This study aims to perform a diagnostic analysis of the observed climate conditions during this event, including an inspection of the occurred precipitation anomalies in the context of previous years, and an investigation of possible relationships with sea surface temperatures and atmospheric circulation patterns. The sea surface temperature analysis revealed that the southwestern South Atlantic Ocean region near the coast of southeast Brazil showed strong negative association with precipitation over southeast Brazil, indicating that increased sea temperatures in this ocean region are consistent with reduced precipitation as observed in summer 2014. The circulation analysis revealed prevailing anti-cyclonic anomalies at lower levels (850 hPa) with northerly anomalies to the west of southeast Brazil, channeling moisture from the Amazon towards Paraguay, northern Argentina and southern Brazil, and drier than normal air from the South Atlantic Ocean towards the southeast region of Brazil. This circulation pattern was found to be part of a large-scale teleconnection wave train linked with the subsidence branch of the Walker circulation in the tropical east Pacific, which in turn was generated by an anomalous tropical heat source in north/northeastern Australia. A regional Hadley circulation with an ascending branch to the south of the subsidence branch of the Walker circulation in the tropical east Pacific was identified as an important component connecting the tropical and extratropical circulation. The ascending branch of this Hadley circulation in the south Pacific coincided with an identified Rossby wave source region, which contributed to establishing the extratropical component of the large-scale wave train connecting the south Pacific and the Atlantic region surrounding southeast Brazil. This connection between the Pacific and the Atlantic was confirmed with Rossby ray tracing analyses. The local circulation response was associated to downward air motion (subsidence) over Southeast Brazil, contributing to the expressive negative precipitation anomalies observed during summer 2014, and leading to a major drought event in the historical context. The analysis of atmospheric and oceanic patterns of this event helped defining a schematic framework leading to the observed drought conditions in southeast Brazil, including the involved teleconnections, blocking high pressure, radiative and humidity transport effects.

Description: The existence of cut-off lows (COLs) over South Pacific and South America is often associated with adverse weather events such as intense precipitation over the central region of South America, frost episodes in southern Brazil and the development of Andes lee cyclones and intense cyclones over the southern coast of Brazil. Despite this importance, the formation and maintenance mechanisms of the COLs are not well understood. To detail the significant variability in terms of the eddy kinetic energy equation for fifty cases of COLs that formed over the southeastern Pacific Ocean is the aim of this study. Only the cases of COLs that formed over the ocean and remained there during most of their life were chosen. The main terms of the equation [ageostrophic flux convergence (AFC), baroclinic conversion (BRC) and barotropic conversion (BRT)] were calculated using the 6-hourly gridded data from the National Centers for Environmental Prediction/Department of Energy reanalysis. The formation mechanism of the COLs was associated with BRC and AFC. During the midlife period, the BRC term converted eddy kinetic energy to eddy potential energy and the AFC had a positive contribution until 6 h after the midlife point. In the dissipation phase, the BRC term remained positive and AFC became negative. The BRT extracted kinetic energy from the COL during the entire life cycle. The AFC term was the most important in all phases of the cut-off lifetime, and it was the responsible for extending the cut-off lifetime while the others terms were negatives.

Description: The present study examines the ability of high resolution (T382) National Centers for Environmental Prediction coupled atmosphere–ocean climate forecast system version 2 (CFS T382) in simulating the salient spatio-temporal characteristics of the boreal summertime mean climate and the intraseasonal variability. The shortcomings of the model are identified based on the observation and compared with earlier reported biases of the coarser resolution of CFS (CFS T126). It is found that the CFS T382 reasonably mimics the observed features of basic state climate during boreal summer. But some prominent biases are noted in simulating the precipitation, tropospheric temperature (TT) and sea surface temperature (SST) over the global tropics. Although CFS T382 primarily reproduces the observed distribution of the intraseasonal variability over the Indian summer monsoon region, some difficulty remains in simulating the boreal summer intraseasonal oscillation (BSISO) characteristics. The simulated eastward propagation of BSISO decays rapidly across the Maritime Continent, while the northward propagation appears to be slightly slower than observation. However, the northward propagating BSISO convection propagates smoothly from the equatorial region to the northern latitudes with observed magnitude. Moreover, the observed northwest-southeast tilted rain band is not well reproduced in CFS T382. The warm mean SST bias and inadequate simulation of high frequency modes appear to be responsible for the weak simulation of eastward propagating BSISO. Unlike CFS T126, the simulated mean SST and TT exhibit warm biases, although the mean precipitation and simulated BSISO characteristics are largely similar in both the resolutions of CFS. Further analysis of the convectively coupled equatorial waves (CCEWs) indicates that model overestimates the gravest equatorial Rossby waves and underestimates the Kelvin and mixed Rossby-gravity waves. Based on analysis of CCEWs, the study further explains the possible reasons behind the realistic simulation of northward propagating BSISO in CFS T382, even though the model shows substantial biases in simulating mean state and other BSISO modes.

Description: Isolates of Aspergillus species are able to produce a large number of secondary metabolites. The profiles of biosynthetic families of secondary metabolites are species specific, whereas individual secondary metabolite families can occur in other species, even those phylogenetically and ecologically unrelated to Aspergillus . Furthermore, there is a high degree of chemo-consistency from isolate to isolate in a species even though certain metabolite gene clusters are silenced in some isolates. Genome sequencing projects have shown that the diversity of secondary metabolites is much larger in each species than previously thought. The potential of finding even further new bioactive drug candidates in Aspergillus is evident, despite the fact that many secondary metabolites have already been structure elucidated and chemotaxonomic studies have shown that many new secondary metabolites have yet to be characterized. The genus Aspergillus is cladistically holophyletic but phenotypically polythetic and very diverse and is associated to quite different sexual states. Following the one fungus one name system, the genus Aspergillus is restricted to a holophyletic clade that include the morphologically different genera Aspergillus , Dichotomomyces , Phialosimplex , Polypaecilum and Cristaspora . Secondary metabolites common between the subgenera and sections of Aspergillus are surprisingly few, but many metabolites are common to a majority of species within the sections. We call small molecule extrolites in the same biosynthetic family isoextrolites. However, it appears that secondary metabolites from one Aspergillus section have analogous metabolites in other sections (here also called heteroisoextrolites). In this review, we give a genus-wide overview of secondary metabolite production in Aspergillus species. Extrolites appear to have evolved because of ecological challenges rather than being inherited from ancestral species, at least when comparing the species in the different sections of Aspergillus . Within the Aspergillus sections, secondary metabolite pathways seem to inherit from ancestral species, but the profiles of these secondary metabolites are shaped by the biotic and abiotic environment. We hypothesize that many new and unique section-specific small molecule extrolites in each of the Aspergillus will be discovered.

Description: Gene amplification using dihydrofolate reductase gene ( dhfr ) and methotrexate (MTX) is widely used for recombinant protein production in mammalian cells and is typically conducted in DHFR-deficient Chinese hamster ovary (CHO) cell lines. Generation of DHFR-deficient cells can be achieved by an expression vector incorporating short hairpin RNA (shRNA) that targets the 3′-untranslated region (UTR) of endogenous dhfr . Thus, shRNAs were designed to target the 3′-UTR of endogenous dhfr , and shRNA-2 efficiently down-regulated dhfr expression in CHO-K1 cells. A single gene copy of shRNA-2 also decreased the translational level of DHFR by 80 % in Flp-In CHO cells. shRNA-2 was then incorporated into a plasmid vector expressing human erythropoietin (EPO) and an exogenous DHFR to develop EPO-producing cells in the Flp-In system. The specific EPO productivity ( q EPO ) was enhanced by stepwise increments of MTX concentration, and differences in the amplification rate were observed in Flp-In CHO cells that expressed shRNA-2. In addition, the q EPO increased by more than 2.5-fold in the presence of 500 nM MTX. The mRNA expression level and gene copy numbers of dhfr were correlated with increased productivity in the cells, which is influenced by inhibition of endogenous dhfr . This study reveals that an expression vector including shRNA that targets the 3′-UTR of endogenous dhfr can enhance the transgene amplification rate and productivity by generating DHFR-deficient cells. This approach may be applied for amplifying the foreign gene in wild-type cell lines as a versatile single-plasmid vector.

Description: The TAO/TRITON array is the cornerstone of the tropical Pacific and ENSO observing system. Motivated by the recent rapid decline of the TAO/TRITON array, the potential utility of TAO/TRITON was assessed for ENSO monitoring and prediction. The analysis focused on the period when observations from Argo floats were also available. We coordinated observing system experiments (OSEs) using the global ocean data assimilation system (GODAS) from the National Centers for Environmental Prediction and the ensemble coupled data assimilation (ECDA) from the Geophysical Fluid Dynamics Laboratory for the period 2004–2011. Four OSE simulations were conducted with inclusion of different subsets of in situ profiles: all profiles (XBT, moorings, Argo), all except the moorings, all except the Argo and no profiles. For evaluation of the OSE simulations, we examined the mean bias, standard deviation difference, root-mean-square difference (RMSD) and anomaly correlation against observations and objective analyses. Without assimilation of in situ observations, both GODAS and ECDA had large mean biases and RMSD in all variables. Assimilation of all in situ data significantly reduced mean biases and RMSD in all variables except zonal current at the equator. For GODAS, the mooring data is critical in constraining temperature in the eastern and northwestern tropical Pacific, while for ECDA both the mooring and Argo data is needed in constraining temperature in the western tropical Pacific. The Argo data is critical in constraining temperature in off-equatorial regions for both GODAS and ECDA. For constraining salinity, sea surface height and surface current analysis, the influence of Argo data was more pronounced. In addition, the salinity data from the TRITON buoys played an important role in constraining salinity in the western Pacific. GODAS was more sensitive to withholding Argo data in off-equatorial regions than ECDA because it relied on local observations to correct model biases and there were few XBT profiles in those regions. The results suggest that multiple ocean data assimilation systems should be used to assess sensitivity of ocean analyses to changes in the distribution of ocean observations to get more robust results that can guide the design of future tropical Pacific observing systems.

Description: Biological activities of medicinal mushrooms have been attributed to β-(1→3),(1→6)-glucans that are present in the cell wall of fungi and some plants. Antitumor, immunomodulatory, antimicrobial, antinociception, antiinflammatory, prebiotic, antioxidant, and antidiabetic are some of different properties already described for β-(1→3),(1→6)-glucans. Immune activation systems, including specific β-glucan receptors like Dectin-1, complement (CR3), and Toll (TLR), have been identified to clarify these biological effects. The β-(1→3)-glucans are synthesized by β-(1→3)-glucan synthase (GLS), an enzyme belonging to the glucosyltransferase group, which has a catalytic unit (FKS) and another regulatory (RHO). The mechanisms for adding β-(1→6) branches to the non-reducing ends of the β-(1→3)-glucan chains are unclear until now. Due to the biological importance of β-(1→3),(1→6)-glucan, it is necessary to understand the biochemical and molecular mechanisms of its synthesis, both to optimize the production of bioactive compounds and to develop antifungal drugs that interrupt this process. Therefore, the aim of this review is to gather information about the potential of β-(1→3),(1→6)-glucans, their methods of isolation, purification, and chemical characterization, as well as how these biomolecules are synthesized by fungi and what studies involving biotechnology or molecular biology have contributed to this subject.

Description: Bluetongue virus (BTV) is the causative agent of bluetongue (BT), an important sheep disease that caused great economic loss to the sheep industry. There are 26 BTV serotypes based on the outer protein VP2. However, the serotypes BTV-1 and BTV-16 are the two most prevalent serotypes in China. Vaccination is the most effective method of preventing viral infections. Therefore, the need for an effective vaccine against BTV is urgent. In this study, DNA vaccines and recombinant fowlpox virus (rFPV) vaccines expressing VP2 alone or VP2 in combination with VP5 or co-expressing the VP2 and VP5 proteins of BTV-1 were evaluated in both mice and sheep. Several strategies were tested in mice, including DNA vaccine prime and boost, rFPV vaccine prime and boost, and DNA vaccine prime and rFPV vaccine boost. We then determined the best vaccine strategy in sheep. Our results indicated that a strategy combining a DNA vaccine prime (co-expressing VP2 and VP5) followed by an rFPV vaccine boost (co-expressing VP2 and VP5) induced a high titer of neutralizing antibodies in sheep. Therefore, our data suggest that a DNA vaccine consisting of a pCAG-(VP2+VP5) prime and an rFPV-(VP2+VP5) boost is an important candidate for the design of a novel vaccine against BTV-1.

Description: Numerous species of wild-grown mushrooms are among the most vulnerable organisms for contamination with radiocesium released from a radioactive fallout. A comparison was made on radiocesium as well as the natural gamma ray-emitting radionuclide ( 40 K) activity concentrations in the fruiting bodies of several valued edible Boletus mushrooms collected from the region of Europe and Yunnan Province in China. Data available for the first time for Boletus edulis collected in Yunnan, China, showed a very weak contamination with 137 Cs. Radiocesium concentration activity of B. edulis samples that were collected between 2011 and 2014 in Yunnan ranged from 5.2 ± 1.7 to 10 ± 1 Bq kg −1 dry matter for caps and from 4.7 ± 1.3 to 5.5 ± 1.0 Bq kg −1 dry matter for stipes. The mushrooms Boletus badius , B. edulis , Boletus impolitus , Boletus luridus , Boletus pinophilus , and Boletus reticulatus collected from the European locations between 1995 and 2010 showed two to four orders of magnitude greater radioactivity from 137 Cs compared to B. edulis from Yunnan. The nuclide 40 K in B. badius was equally distributed between the caps and stipes, while for B. edulis , B. impolitus , B. luridus , B. pinophilus , and B. reticulatus , the caps were richer, and for each mushroom, activity concentration seemed to be more or less species-specific.

Description: A novel TaqMan-based multiplex real-time PCR method combined with propidium monoazide (PMA) treatment was firstly developed for the simultaneous quantification of viable Vibrio parahaemolyticus and Listeria monocytogenes in raw shrimp. The optimization of PMA concentration showed that 100 μM was considered optimal to effectively inhibit 10 8  CFU/mL dead cells of both bacteria. The high specificity of this method was confirmed on tests using 96 target and non-target strains. The optimized assay could detect as low as 10 1 –10 2  CFU/g of each strain on the artificially contaminated shrimp, and its amplification efficiencies were up to 100 and 106 % for V. parahaemolyticus and L. monocytogenes , respectively. Furthermore, this assay has been successfully applied to describe the behavior of these two pathogens in raw shrimps stored at 4 °C. In conclusion, this PMA TaqMan-based multiplex real-time PCR technique, where the whole procedure takes less than 5 h, provides an effective and rapid tool for monitoring contamination of viable V. parahaemolyticus and L. monocytogenes in seafood, improving seafood safety and protecting public health.

Description: A hybrid airlift reactor was adopted to retain aerobic granules in the reactor successfully for continuous operation. It was found that aerobic granules maintained excellent physical structure stability in the continuous-flow reactor with reactor performance as good as batch operation. However, flocs appeared after batch operation was switched to continuous operation, and chemical oxygen demand (COD) in the wastewater was thus removed by co-existed granules and flocs in the reactor. Furthermore, excessive precipitation of CaCO 3 as needled shaped aragonite in the continuous aerobic granular sludge reactor was observed, which led to the further enhancement of settling ability of granules with sludge volume index (SVI) reduction from 32 to 2 ml g −1 but specific oxygen utilization rate (SOUR) decrease from 61 to 23 mg O 2  g −1 MLVSS h −1 . Thus, apart from the physical structure stability, bioactivity stability of granules should be also considered as an important parameter to evaluate the continuous operation of aerobic granular sludge. Furthermore, the decrease in granule polysaccharide content implied that protein was more important for aragonite precipitation. The excessive aragonite precipitation in the continuous-flow reactor could be due to the competition between flocs and granules. In addition, the degradation of polysaccharide in aerobic granules under a continuous-flow mode may also contribute to excessive aragonite precipitation.

Description: Recent numerical studies in stratospheric dynamics and its variability as well as climate, have highlighted the need of more observational analyses to improve simulation of the West African monsoon (WAM). In this paper, activity and spectral characteristics of short-scale vertical waves (wavelengths 〈4 km) are analysed in equatorial coastal and tropical lower stratosphere during the WAM. A first detailed description of such waves over West Africa is derived from high-resolution vertical profiles of temperature and horizontal wind obtained during Intensive Observation Period of the African Monsoon Multidisciplinary Analyses (AMMA) Campaign 2006. Monthly variation of wave energy density is revealed to trace the progression of the inter-tropical convergence zone (ITCZ) over West Africa. Mesoscale inertia gravity-waves structures with vertical and horizontal wavelengths of 1.5–2.5 and 400–1100 km respectively and intrinsic frequencies of 1.1–2.2 f or periods 〈2 days are observed in the tropical LS with intense activity during July and August when the WAM is installed over the tropical West Africa. Over equatorial region, gravity waves with intrinsic frequencies of 1.4–4 f or periods 〈5.2 days, vertical wavelength of 2.1 km and long horizontal wavelengths of 1300 km are intense during the WAM coastal phase. From July to October, gravity waves with intrinsic frequencies of 1.2–3.8 f or periods 〈6 days, vertical wavelength of 2.1 km and horizontal wavelengths of 1650 km are less intense during the WAM Sahelian phase of the WAM, March–June. Unlike potential energy density, kinetic energy density is observed to be a good proxy for the activity of short-scale vertical waves during the WAM because quasi-inertial waves are dominant. Long-term wave activity variation from January 2001 to December 2009, highlights strong year-to-year variation superimposed on convective activity and quasi-biennial oscillation-like variations especially above tropical stations.

Description: Satellite based top-of-atmosphere (TOA) and surface radiation budget observations are combined with mass corrected vertically integrated atmospheric energy divergence and tendency from reanalysis to infer the regional distribution of the TOA, atmospheric and surface energy budget terms over the globe. Hemispheric contrasts in the energy budget terms are used to determine the radiative and combined sensible and latent heat contributions to the cross-equatorial heat transports in the atmosphere (AHT EQ ) and ocean (OHT EQ ). The contrast in net atmospheric radiation implies an AHT EQ from the northern hemisphere (NH) to the southern hemisphere (SH) (0.75 PW), while the hemispheric difference in sensible and latent heat implies an AHT EQ in the opposite direction (0.51 PW), resulting in a net NH to SH AHT EQ (0.24 PW). At the surface, the hemispheric contrast in the radiative component (0.95 PW) dominates, implying a 0.44 PW SH to NH OHT EQ . Coupled model intercomparison project phase 5 (CMIP5) models with excessive net downward surface radiation and surface-to-atmosphere sensible and latent heat transport in the SH relative to the NH exhibit anomalous northward AHT EQ and overestimate SH tropical precipitation. The hemispheric bias in net surface radiative flux is due to too much longwave surface radiative cooling in the NH tropics in both clear and all-sky conditions and excessive shortwave surface radiation in the SH subtropics and extratropics due to an underestimation in reflection by clouds.

Description: Venom, the mucus layer covering the body surface, ink glands, mammary glands, milk, and various animal secretory functions as both a physical and chemical defense barrier against bacteria and virus infections. Previously, several studies reported that l -amino acid oxidases (LAAOs) present in animal secretary fluids have strong antimicrobial activities and selective cytotoxic activities against Gram-positive and Gram-negative bacteria, various pathogenic bacteria, viruses, and parasite species. These LAAOs catalyze oxidative deamination of an l -amino acid substrate with the generation of hydrogen peroxide. The antibacterial activity of LAAOs is completely inhibited by catalase; thus, LAAOs kill bacteria by the hydrogen peroxide generated from the oxidation of l -amino acid substrates. This review focuses on the selective, specific, and local antibacterial actions of various LAAOs that may be used as novel therapeutic agents against infectious diseases. LAAOs that are suitable leads for combating multidrug-resistant bacterial infections are also studied.

Description: Experiments were designed to validate the two common DNA extraction protocols (CTAB-based method and DNeasy Blood & Tissue Kit) used to effectively recover actinobacterial DNA from sponge samples in order to study the sponge-associated actinobacterial diversity. This was done by artificially spiking sponge samples with actinobacteria (spores, mycelia and a combination of the two). Our results demonstrated that both DNA extraction methods were effective in obtaining DNA from the sponge samples as well as the sponge samples spiked with different amounts of actinobacteria. However, it was noted that in the presence of the sponge, the bacterial 16S rRNA gene could not be amplified unless the combined DNA template was diluted. To test the hypothesis that the extracted sponge DNA contained inhibitors, dilutions of the DNA extracts were tested for six sponge species representing five orders. The results suggested that the inhibitors were co-extracted with the sponge DNA, and a high dilution of this DNA was required for the successful PCR amplification for most of the samples. The optimized PCR conditions, including primer selection, PCR reaction system and program optimization, further improved the PCR performance. However, no single PCR condition was found to be suitable for the diverse sponge samples using various primer sets. These results highlight for the first time that the DNA extraction methods used are effective in obtaining actinobacterial DNA and that the presence of inhibitors in the sponge DNA requires high dilution coupled with fine tuning of the PCR conditions to achieve success in the study of sponge-associated actinobacterial diversity.

Description: Mineral dust aerosols are an essential component of climate over West Africa, however, little work has been performed to investigate their contributions to potential climate change. A set of regional climate model experiments with and without mineral dust processes and land cover changes is performed to evaluate their climatic effects under the Representative Concentration Pathway 8.5 for two global climate models. Results suggest surface warming to be in the range of 4–8 °C by the end of the century (2081–2100) over West Africa with respect to the present day (1981–2000). The presence of mineral dusts dampens the warming by 0.1–1 °C in all seasons. Accounting for changes in land cover enhances the warming over the north of Sahel and dampens it to the south in spring and summer; however, the magnitudes are smaller than those resulting from dusts. Overall dust loadings are projected to increase, with the greatest increase occurring over the Sahara and Sahel in summer. Accounting for land cover changes tends to reduce dust loadings over the southern Sahel. Future precipitation is projected to decrease by 5–40 % in the western Sahara and Sahel and increase by 10–150 % over the eastern Sahel and Guinea Coast in JJA. A dipole pattern of future precipitation changes is attributed to dust effects, with decrease in the north by 5–20 % and increase by 5–20 % in the south. Future changes in land cover result in a noisy non-significant response with a tendency for slight wetting in MAM, JJA, and SON and drying in DJF.

Description: Bacterial cell envelope is generally accepted as the primary target for a photo-induced oxidative stress. It is plausible that DNA damage occurs during the antimicrobial photoinactivation. Here we investigate the correlation between DNA damage and photoinactivation by evaluating the level of RecA-based DNA repair system in Staphylococcus aureus . By using exogenous photosensitizers (new methylene blue (NMB), toluidine blue O (TBO), 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra( p -toluenesulfonate) (TMPyP), zinc phthalocyanine (ZnPc), Rose Bengal (RB)) and ALA-induced endogenous porphyrin-dependent blue light (405 nm), several outcomes were observed: (i) an increase of DNA damage (from gel electrophoresis in DNA damage assay), (ii) an increase of recA expression (luminescence assay in recA - lux strain), and (iii) an increase of RecA protein level (Western blotting). When recA expression was repressed by novobiocin, or abolished by deleting the gene, S . aureus susceptibility towards photoinactivation was increased at approximately a hundred-fold. The absence of RecA increases DNA damage to yield bactericidal effect. In novobiocin-resistant mutant ( gyrB ), as opposed to wild type, neither RecA protein level nor cell’s susceptibility was affected by photoinactivation (when novobiocin is present). This is to suggest that GyrB-dependent inhibition mediated recA repression. Therefore, we have established the role of RecA in DNA damage during photoinactivation. With the use of rifampicin mutation frequency and Ames tests, we demonstrated that photoinactivation did not increase S . aureus mutagenesis and potentially is not mutagenic toward eukaryotic cells. The results suggest that the treatment is considered safe. In conclusion, we provide an evidence that recA inhibitor may serve as therapeutic adjuvant for antimicrobial photoinactivation. Clinical relevance of our findings warrants further investigations.

Description: Calcitonin participates in controlling homeostasis of calcium and phosphorus and plays an important role in bone metabolism. The aim of this study was to endow an industrial strain of Saccharomyces cerevisiae with the ability to express chimeric human/salmon calcitonin (hsCT) without the use of antibiotics. To do so, a homologous recombination plasmid pUC18- rDNA2 - ura 3- P pgk - 5hsCT - rDNA1 was constructed, which contains two segments of ribosomal DNA of 1.1 kb ( rDNA1 ) and 1.4 kb ( rDNA2 ), to integrate the heterologous gene into host rDNA. A DNA fragment containing five copies of a chimeric human/salmon calcitonin gene ( 5hsCT ) under the control of the promoter for phosphoglycerate kinase ( P pgk ) was constructed to express 5hsCT in S. cerevisiae using ura 3 as a selectable auxotrophic marker gene. After digestion by restriction endonuclease Hpa I, a linear fragment, rDNA2 - ura 3- P pgk - 5hsCT - rDNA1 , was obtained and transformed into the △ ura3 mutant of S. cerevisiae by the lithium acetate method. The ura 3- P pgk - 5hsCT sequence was introduced into the genome at rDNA sites by homologous recombination, and the recombinant strain YS- 5hsCT was obtained. Southern blot analysis revealed that the 5hsCT had been integrated successfully into the genome of S. cerevisiae . The results of Western blot and ELISA confirmed that the 5hsCT protein had been expressed in the recombinant strain YS- 5hsCT . The expression level reached 2.04 % of total proteins. S. cerevisiae YS- 5hsCT decreased serum calcium in mice by oral administration and even 0.01 g lyophilized S. cerevisiae YS- 5hsCT /kg decreased serum calcium by 0.498 mM. This work has produced a commercial yeast strain potentially useful for the treatment of osteoporosis.

Description: Since total petroleum hydrocarbons (TPH) are toxic and persistent in environments, studying the impact of oil contamination on microbial communities in different soils is vital to oil production engineering, effective soil management and pollution control. This study analyzed the impact of oil contamination on the structure, activity and function in carbon metabolism of microbial communities of Chernozem soil from Daqing oil field and Cinnamon soil from Huabei oil field through both culture-dependent techniques and a culture-independent technique—pyrosequencing. Results revealed that pristine microbial communities in these two soils presented disparate patterns, where Cinnamon soil showed higher abundance of alkane, (polycyclic aromatic hydrocarbons) PAHs and TPH degraders, number of cultivable microbes, bacterial richness, bacterial biodiversity, and stronger microbial activity and function in carbon metabolism than Chernozem soil. It suggested that complicated properties of microbes and soils resulted in the difference in soil microbial patterns. However, the changes of microbial communities caused by oil contamination were similar in respect of two dominant phenomena. Firstly, the microbial community structures were greatly changed, with higher abundance, higher bacterial biodiversity, occurrence of Candidate_division_BRC1 and TAO6 , disappearance of BD1-5 and Candidate_division_OD1 , dominance of Streptomyces , higher percentage of hydrocarbon-degrading groups, and lower percentage of nitrogen-transforming groups. Secondly, microbial activity and function in carbon metabolism were significantly enhanced. Based on the characteristics of microbial communities in the two soils, appropriate strategy for in situ bioremediation was provided for each oil field. This research underscored the usefulness of combination of culture-dependent techniques and next-generation sequencing techniques both to unravel the microbial patterns and understand the ecological impact of contamination.