Terms Of Constituents Research Articles

Heterologous Expression of Spinosyn Biosynthetic Gene Cluster in Streptomyces Species Is Dependent on the Expression of Rhamnose Biosynthesis Genes

Spinosyns are a group of macrolide insecticides produced by Saccharopolyspora spinosa. Although S. spinosa can be used for industrial-scale production of spinosyns, this might suffer from several limitations, mainly related to its long growth cycle, low fermentation biomass, and inefficient utilization of starch. It is crucial to generate a robust strain for further spinosyn production and the development of spinosyn derivatives. A BAC vector, containing the whole biosynthetic gene cluster for spinosyn (74 kb) and the elements required for conjugal transfer and site-specific integration, was introduced into different Streptomyces hosts in order to obtain heterologous spinosyn-producing strains. The exconjugants of different Streptomyces strains did not show spinosyn production unless the rhamnose biosynthesis genes from S. spinosa genomic DNA were present and expressed under the control of a strong constitutive ermE*p promoter. Using this heterologous expression system resulted in yields of 1 μg/mL and 1.5 μg/mL spinosyns in Streptomyces coelicolor and Streptomyces lividans, respectively. This report demonstrates spinosyn production in 2 Streptomyces strains and stresses the essential role of rhamnose in this process. This work also provides a potential alternative route for producing spinosyn analogs by means of genetic manipulation in the heterologous hosts.

Dyeing, fastness, and cytotoxic properties, and phenolic constituents of Anthemis tinctoria var. tinctoria (Asteraceae)

The present study focused on dyeing, fastness, cytotoxic properties, and phenolic constituents of various types of fabrics including viscose, cotton, wool, and polyester-viscose blended fabrics with different parts of Anthemis tinctoria var. tinctoria. Comparative study was carried out between ethanol and aqueous extracts of stem, flower, and root of the plant in terms of phenolic constituents, cytotoxic activity, and dyeing properties. It was found that the quantity of phenolic constituents of ethanol and aqueous extracts was quite different. All parts of the plant extract show 0% cytotoxicity except ethanol extract of the root. All the extracts exhibit better cytotoxic activity than the standard cancer drug 5-Florouracil. Ethanol and water extracts of A. tinctoria var. tinctoria plant were used to dye fabrics, but only the water extract displayed dyeing properties. Best color strength value (K/S = 9.19) was obtained with aqueous extract of the stem in the presence of alum mordant for cotton fabrics.

Taming sovereignty: constituent power in nineteenth-century French political thought

ABSTRACTPolitical theorists recently focused their attention on the history of the idea of constituent power. This, they claim, shows that the notion of pouvoir constituant expressed the radical and absolute power of the sovereign people. In other words, constituent power pointed at the democratic and irresistible core of popular sovereignty. In this paper, I argue that the analysis of nineteenth-century French political thought offers a different account of constituent power’s history. Relying upon archival resources, I show that in the aftermath of the French Revolution politicians and legal scholars used constituent power to tame the very idea of sovereignty and the powers from it derived. First, during the Restoration constituent power was used to pose a limit to the power of the monarch. Second, throughout the July Monarchy scholars resorted to constituent power to oppose the Parliament's claim to be the sovereign power and the only legitimate author of the constitution. Moreover, they also used it to claim that claim that, even if the people was sovereign, its power was restricted to authorizing the constitution. Third, during the Second Republic, jurists and politicians addressed the people’s sovereign power in terms of constituent and constituted power. While the first was meant to disappear after the constitution’s approval, the second was a second-order power limited by the hierarchy of norms and the rigidity of the constitution.

Anti-neuropathic effects of Rosmarinus officinalis L. terpenoid fraction: relevance of nicotinic receptors.

Traditional uses and current results highlight the neuroprotective properties of Rosmarinus officinalis L. The compelling need for novel strategies able to relieve neuropathic pain encouraged us to analyze different rosemary leaf extracts in rats following chronic constriction injury (CCI) of sciatic nerve. Ethanol, acetone, and the innovative ultrasound-hexane extractive methods were used to obtain: EE, AE, and for hexane extracts UREprel and URE. Extracts were characterized in terms of typical constituents and repeatedly administered to CCI-rats (13-days treatment, from the day of surgery). URE showed the best efficacy and potency in reducing hypersensitivity to noxious- and non-noxious stimuli and spontaneous pain. URE contained the higher quantity of the terpenoid carnosic acid (CA) and its efficacy was compared to pure CA. Histological analysis of the sciatic nerve revealed that URE prevented axon and myelin derangement, edema and inflammatory infiltrate. In the dorsal horn of the spinal cord, URE did not reduce astrocyte activation. Both the pain reliever and the neuroconservative effects of URE were significantly prevented by the nicotinic receptor (nAChR) antagonist mecamylamine. In conclusion, the hexane-ultrasound rosemary extract is able to reduce neuropathic hypersensitivity and protect nervous tissues. Effectiveness is mainly related to the terpenoid fraction by mechanisms involving nAChRs.

A Discussion on the Interpretation of the Darcy Equation in Case of Open-Cell Metal Foam Based on Numerical Simulations.

It is long known that for high-velocity fluid flow in porous media, the relation between the pressure drop and the superficial velocity is not linear. Indeed, the classical Darcy law for shear stress dominated flow needs to be extended with a quadratic term, resulting in the empirical Darcy–Forchheimer model. Another approach is to simulate the foam numerically through the volume averaging technique. This leads to a natural separation of the total drag force into the contribution of the shear forces and the contribution of the pressure forces. Both representations of the total drag lead to the same result. The physical correspondence between both approaches is investigated in this work. The contribution of the viscous and pressure forces on the total drag is investigated using direct numerical simulations. Special attention is paid to the dependency on the velocity of these forces. The separation of the drag into its constituent terms on experimental grounds and for the volume average approach is unified. It is shown that the common approach to identify the linear term with the viscous forces and the quadratic term with the pressure forces is not correct.

Metamaterial control of stimulated Brillouin scattering.

Using full opto-acoustic numerical simulations, we demonstrate enhancement and suppression of the SBS gain in a metamaterial comprising a subwavelength cubic array of dielectric spheres suspended in a dielectric background material. We develop a general theoretical framework and present several numerical examples using technologically important materials. For As2S3 spheres in silicon, we achieve a gain enhancement of more than an order of magnitude compared to pure silicon and for GaAs spheres in silicon, full suppression is obtained. The gain for As2S3 glass can also be strongly suppressed by embedding silica spheres. The constituent terms of the gain coefficient are shown to depend in a complex way on the filling fraction. We find that electrostriction is the dominant effect behind the control of SBS in bulk media.

Development of a Synthetic Oxytetracycline-Inducible Expression System for Streptomycetes Using de Novo Characterized Genetic Parts.

Precise control of gene expression using exogenous factors is of great significance. To develop ideal inducible expression systems for streptomycetes, new genetic parts, oxytetracycline responsive repressor OtrR, operator otrO, and promoter otrBp from Streptomyces rimosus, were selected de novo and characterized in vivo and in vitro. OtrR showed strong affinity to otrO (KD = 1.7 × 10(-10) M) and oxytetracycline induced dissociation of the OtrR/DNA complex in a concentration-dependent manner. On the basis of these genetic parts, a synthetic inducible expression system Potr* was optimized. Induction of Potr* with 0.01-4 μM of oxytetracycline triggered a wide-range expression level of gfp reporter gene in different Streptomyces species. Benchmarking Potr* against the widely used constitutive promoters ermE* and kasOp* revealed greatly enhanced levels of expression when Potr* was fully induced. Finally, Potr* was used as a tool to activate and optimize the expression of the silent jadomycin biosynthetic gene cluster in Streptomyces venezuelae. Altogether, the synthetic Potr* presents a new versatile tool for fine-tuning gene expression in streptomycetes.

Explicit, parallel Poisson integration of point vortices on the sphere

Point vortex models are frequently encountered in conceptual studies in geophysical fluid dynamics, but also in practical applications, for instance, in aeronautics. In spherical geometry, the motion of vortex centres is governed by a dynamical system with a known Poisson structure. We construct Poisson integration methods for these dynamics by splitting the Hamiltonian into its constituent vortex pair terms. From backward error analysis, the method is formally known to provide solutions to a modified Poisson system with the correct bracket, but with a modified Hamiltonian function. Different orderings of the pairwise interactions are considered and also used for the construction of higher order methods. The energy and momentum conservation of the splitting schemes is demonstrated for several test cases. For particular orderings of the pairwise interactions, the schemes allow scalable parallelization. This results in a linear–as opposed to quadratic–scaling of computation time with system size when scaling the number of processors accordingly.

Are complexity and uncertainty distinct concepts in project management? A taxonomical examination from literature

Project management research is characterized by dominance of determinism, decision-theoretic approaches, and weak theories. The growth of research interest in non-deterministic paradigms through the lenses of complexity and uncertainty is recent, and could provide stronger theoretic explanations. However, analysis of select project management literature reveals that the constructs of complexity and uncertainty are yet to be grounded in terms of definitions and constituent variables. We argue that definitional clarity is necessary for the non-deterministic research to move forward. In this paper, we propose taxonomy of constituent terms of complexity and uncertainty based on semantic analysis of select literature and show that the two constructs are broadly confounded in their constituent terms. While our finding may appear to align with complexity theoretic concept of strong interrelationship between complexity and uncertainty, we argue that such confounding represents intermingling of varying ontological and epistemological preferences within the community of project management scholars rather than a broad adherence to the complexity theory. The paper contributes to project management literature by facilitating further research toward stronger construct definitions and theory-building efforts. The paper also contributes to research methods by offering a novel methodology to elicit taxonomy of terms and to illuminate the confounding and separating terms across multiple constructs.

Effects of Chromosomal Integration of the Vitreoscilla Hemoglobin Gene (vgb) and S-Adenosylmethionine Synthetase Gene (metK) on ε-Poly-L-Lysine Synthesis in Streptomyces albulus NK660.

ε-Poly-L-lysine (ε-PL) is a widely used natural food preservative. To test the effects of the Vitreoscilla hemoglobin (VHb) and S-adenosylmethionine (SAM) on ε-PL synthesis in Streptomyces albulus NK660, the heterologous VHb gene (vgb) and SAM synthetase gene (metK) were inserted into the S. albulus NK660 chromosome under the control of the constitutive ermE* promoter. CO-difference spectrum analysis showed S. albulus NK660-VHb strain could express functional VHb. S. albulus NK660-VHb produced 26.67% higher ε-PL and 14.57% higher biomass than the wild-type control, respectively. Reversed-phase high-pressure liquid chromatography (RP-HPLC) results showed the overexpression of the metK gene resulted in increased intracellular SAM synthesis in S. albulus NK660-SAM, which caused increases of biomass as well as the transcription level of ε-PL synthetase gene (pls). Results indicated that the expression of vgb and metK gene improved on ε-PL synthesis and biomass for S. albulus NK660, respectively.

Seasonal Chemical Characteristics of Atmospheric Aerosol Particles and its Light Extinction Coefficients over Pune, India

ABSTRACTThe present study has been conducted to characterize atmospheric aerosol particles in terms of carbonaceous species and ionic constituents for a yearlong period at Pune, India. This study provides the evidence for the ionic chemistry, secondary aerosols formation, temporal variability and its climatic effect in the atmosphere. The average concentrations of PM2.5 and PM10 were 109.6 ± 23.2 and 166.9 ± 4 µg m–3, respectively, by far exceeding National Ambient Air Quality (NAAQ) and World Health Organization (WHO) standards. Seasonal analyses indicated that PM2.5 and PM10 mass concentrations were higher in the post-monsoon followed by the winter season and lower during the monsoon period. The average concentrations of organic carbon (OC) and elemental carbon (EC) were 31.3 ± 7.4 and 4.2 ± 2.4 µg m–3 for PM2.5, while, 34.2 ± 6.2 and 5.0 ± 2.3 µg m–3 for PM10, respectively. OC and EC data splits into seasons and their mass loadings were in the order of post-monsoon > monsoon > winter > summer for OC and for EC, it was as winter > post-monsoon > summer > monsoon. The overall chemical analysis revealed that particulate matter (PM) consist higher concentrations of OC followed by cations and the lowest one is EC. The ionic composition analysis indicated that cations were the abundant parts of PM in comparison to anions and Na+ and SO42– were at a higher concentration amongst all the ionic species. The estimated light extinction coefficient (bext) of the aerosol particle was 291.2 ± 55.3 Mm–1 during the study period. Further apportionment of particle extinction coefficient was estimated and the contributions of light scattering coefficient by particles (bsp) were OC (45%), (NH4)2SO4 (17%), NH4NO3 (8%) and coarse mass (12%), while, the contribution of light absorption coefficient by particle (bap) was 18% (EC). This indicates that in the present study the abundance of aerosol particles are more scattering in nature in comparison to absorption. The average value of Aerosol optical depth (AOD) was 0.46 and their positive correlation with anions and relative humidity (RH) showing same properties, while, in the case of EC, it showed contrast nature with respect to climate effect. Trajectory analysis indicated that the air masses appear as a result of long-range transportation during summer and monsoon period while during the winter and post-monsoon seasons local manmade activities showed dominant influence.

Improvement of chloramphenicol production in Streptomyces venezuelae ATCC 10712 by overexpression of the aroB and aroK genes catalysing steps in the shikimate pathway.

Streptomyces venezuelae ATCC 10712 produces chloramphenicol in small amounts. To enhance chloramphenicol production, two genes, aroB and aroK, encoding rate-limiting enzymes of the shikimate pathway were overexpressed using the expression vector pIJ86 under the control of the strong constitutive ermE* promoter. The recombinant strains, S. venezuelae/pIJ86-aroB and S. venezuelae/pIJ86-aroK, produced 2.5- and 4.3-fold greater amounts respectively of chloramphenicol than wild type at early stationary phase of growth. High transcriptional levels of aroB and aroK genes were detected at the early exponential growth of both recombinant strains and consistent with the enhanced expression of pabB gene encoding an early enzyme in chloramphenicol biosynthesis. The results suggested that the increment of carbon flux was directed towards intermediates in the shikimate pathway required for the production of chorismic acid, and consequently resulted in the enhancement of chloramphenicol production. This work is the first report of a convenient genetic approach to manipulate primary metabolite genes in S. venezuelae in order to increase chloramphenicol production.

Bio-Hydrocarbons through Catalytic Pyrolysis of Used Cooking Oils: Towards Sustainable Jet and Road Fuels

Vegetable Oil (VO) is today the most used feedstock for transport biofuel production by transesterification to biodiesel. Other commercial technologies for renewable fuels production are mainly based either on Fischer-Tropsch (FT) synthesis from coal, natural gas and possibly biomass, or hydro treating of vegetable oil (Hydrotreated Vegetable Oil, HVO): this also includes Hydrotreated Renewable Jet fuel, HRJ, Used Cooking Oil (UCO) is a highly sustainable feedstock (based on EC-RED scheme): it is therefore considered as a possible alternative to VOs for greening of air transport and, under proper circumstances, for reducing the feedstock cost component. However, the use of UCO is not trivial in reactors, as catalysts are sensitive to impurities and contaminations, which are typical of waste oils. Moreover, the chemical composition of UCO is variable regionally as well as seasonally, because the type of base-vegetable oils vary with Country and period of the year. In the framework of the ITAKA EU FP7 project, (catalytic) thermochemical conversion of UCO has been considered to obtain an intermediate biofuel suitable for upgrading by hydrotreating. The catalytic conversion of UCO and Fatty Acids were investigated in a 1.5kg/h pilot unit. UCO, properly filtered and conditioned, was characterized, and then converted in bio-oil by means of thermal and catalytic reactionsunder controlled conditions. The type of catalyst and the reaction conditions, including several parameters such as temperature, reactor geometry, heating rate and residence time, were evaluated, and selected combinations were tested. The bio-oil was characterized in terms of main constituents and hydrocarbons content, and GC-MS and GC-FID analyses were used to qualitatively and quantitatively assess the composition of the fuel.

Streptocollin, a Type IV Lanthipeptide Produced by Streptomyces collinus Tü 365.

Lanthipeptides are ribosomally synthesized and post-translationally modified microbial secondary metabolites. Here, we report the identification and isolation of streptocollin from Streptomyces collinus Tü 365, a new member of class IV lanthipeptides. Insertion of the constitutive ermE* promoter upstream of the lanthipeptide synthetase gene stcL resulted in peptide production. The streptocollin gene cluster was heterologously expressed in S. coelicolor M1146 and M1152 with 3.5- and 5.5-fold increased yields, respectively. The structure and ring topology of streptocollin were determined by high resolution MS/MS analysis. Streptocollin contains four macrocyclic rings, with one lanthionine and three methyllanthionine residues. To the best of our knowledge, this is the first report on the isolation of a class IV lanthipeptide in preparative amounts, and on the successful heterologous expression of a class IV lanthipeptide gene cluster.

A Streptomyces coelicolor host for the heterologous expression of Type III polyketide synthase genes

BackgroundRecent advances in genome sequencing, combined with bioinformatic analysis, has led to the identification of numerous novel natural product gene clusters, particularly in actinomycetes of terrestrial and marine origin. Many of these gene clusters encode uncharacterised Type III polyketide synthases. To facilitate the study of these genes and their potentially novel products, we set out to construct an actinomycete expression host specifically designed for the heterologous expression of Type III PKS genes and their gene clusters.ResultsA derivative of Streptomyces coelicolor A3(2) designed for the expression of Type III polyketide synthase (PKS) genes was constructed from the previously engineered expression strain S. coelicolor M1152 [Δact Δred Δcpk Δcda rpoB(C1298T)] by removal of all three of the endogenous Type III PKS genes (gcs,srsA,rppA) by PCR targeting. The resulting septuple deletion mutant, M1317, proved to be an effective surrogate host for the expression of actinobacterial Type III PKS genes: expression of the reintroduced gcs gene from S. coelicolor and of the heterologous rppA gene from Streptomyces venezuelae under the control of the constitutive ermE* promoter resulted in copious production of germicidin and flaviolin, respectively.ConclusionsThe newly constructed expression host S. coelicolor M1317 should be particularly useful for the discovery and analysis of new Type III polyketide metabolites.

The surface-forming energy release rate based fracture criterion for elastic–plastic crack propagation

The J-integral based criterion is widely used in elastic–plastic fracture mechanics. However, it is not rigorously applicable when plastic unloading appears during crack propagation. One difficulty is that the energy density with plastic unloading in the J-integral cannot be defined unambiguously. In this paper, we alternatively start from the analysis on the power balance, and propose a surface-forming energy release rate (ERR), which represents the energy available for separating the crack surfaces during the crack propagation and excludes the loading-mode-dependent plastic dissipation. Therefore the surface-forming ERR based fracture criterion has wider applicability, including elastic–plastic crack propagation problems. Several formulae are derived for calculating the surface-forming ERR. From the most concise formula, it is interesting to note that the surface-forming ERR can be computed using only the stress and deformation of the current moment, and the definition of the energy density or work density is avoided. When an infinitesimal contour is chosen, the expression can be further simplified. For any fracture behaviors, the surface-forming ERR is proven to be path-independent, and the path-independence of its constituent term, so-called Js-integral, is also investigated. The physical meanings and applicability of the proposed surface-forming ERR, traditional ERR, Js-integral and J-integral are compared and discussed. Besides, we give an interpretation of Rice paradox by comparing the cohesive fracture model and the surface-forming ERR based fracture criterion.

A fast numerical method for trimaran wave resistance prediction

The optimisation of a trimaran configuration is not trivial due to the influence of longitudinal and transverse clearance between the hulls. This sensitivity is illustrated in the predictions presented.The proposed calculation procedure is based on an extension of the slender ship theory of Noblesse. In particular the highly oscillatory terms within the associated Kochin function are expanded as series of products of Bessel functions and Legendre polynomials to facilitate faster computation and to separate out specific parameter dependencies. These objectives allow calculation of constituent terms of the Kochin function as a function of hull clearance parameters, which may be stored externally to the resistance calculation software.The rapid calculation procedure allows identification of optimal trimaran configurations. Comparison between the numerical predictions and experimental measurement based on the authors’ earlier experimental investigations and those of other researchers (Degiuli et al., 2003) has shown good agreement.The wave resistance differences for symmetric and asymmetric outer hull forms for three distinct cases are presented and discussed.

Heterologous coexpression of Vitreoscilla hemoglobin and Bacillus megaterium glucanase in Streptomyces lydicus A02 enhanced its production of antifungal metabolites

Streptomyces lydicus A02 is a novel producer of commercially important polyene macrocyclic antibiotic natamycin and a potential biocontrol agent to several plant fungal diseases, including wilt caused by Fusarium oxysporum f. spp. To improve the natamycin production and the antifungal activity of S. lydicus A02, we coexpressed gene vgb encoding Vitreoscilla hemoglobin (VHb) and bglC encoding Bacillus megaterium L103 glucanase, both under the control of the strong constitutive ermE* promoter, in S. lydicus A02. Our results showed that coexpressing VHb and glucanase improved cell growth, and the engineered strain produced 26.90% more biomass than the wild-type strain after 72h fermentation in YSG medium. In addition, coexpressing genes encoding VHb and glucanase led to increased natamycin production, higher endogenous chitinase activity and exogenous glucanase activity, as well as enhanced antifungal activity in the engineered S. lydicus AVG02 and AGV02, regardless of the position of the two genes on the plasmids. Compared with model strains, few reports have successfully coexpressed VHb and other foreign proteins in industrial strains. Our results illustrated an effective approach for improving antifungal activity in an industrial strain by the rational engineering of combined favorable factors.

Calcium Orthophosphate-Containing Biocomposites and Hybrid Biomaterials for Biomedical Applications.

The state-of-the-art on calcium orthophosphate (CaPO4)-containing biocomposites and hybrid biomaterials suitable for biomedical applications is presented. Since these types of biomaterials offer many significant and exciting possibilities for hard tissue regeneration, this subject belongs to a rapidly expanding area of biomedical research. Through the successful combinations of the desired properties of matrix materials with those of fillers (in such systems, CaPO4 might play either role), innovative bone graft biomaterials can be designed. Various types of CaPO4-based biocomposites and hybrid biomaterials those are either already in use or being investigated for biomedical applications are extensively discussed. Many different formulations in terms of the material constituents, fabrication technologies, structural and bioactive properties, as well as both in vitro and in vivo characteristics have been already proposed. Among the others, the nano-structurally controlled biocomposites, those containing nanodimensional compounds, biomimetically fabricated formulations with collagen, chitin and/or gelatin, as well as various functionally graded structures seem to be the most promising candidates for clinical applications. The specific advantages of using CaPO4-based biocomposites and hybrid biomaterials in the selected applications are highlighted. As the way from a laboratory to a hospital is a long one and the prospective biomedical candidates have to meet many different necessities, the critical issues and scientific challenges that require further research and development are also examined.

Metabolite Profiling and Classification of DNA-Authenticated Licorice Botanicals.

Raw licorice roots represent heterogeneous materials obtained from mainly three Glycyrrhiza species. G. glabra, G. uralensis, and G. inflata exhibit marked metabolite differences in terms of flavanones (Fs), chalcones (Cs), and other phenolic constituents. The principal objective of this work was to develop complementary chemometric models for the metabolite profiling, classification, and quality control of authenticated licorice. A total of 51 commercial and macroscopically verified samples were DNA authenticated. Principal component analysis and canonical discriminant analysis were performed on (1)H NMR spectra and area under the curve values obtained from UHPLC-UV chromatograms, respectively. The developed chemometric models enable the identification and classification of Glycyrrhiza species according to their composition in major Fs, Cs, and species specific phenolic compounds. Further key outcomes demonstrated that DNA authentication combined with chemometric analyses enabled the characterization of mixtures, hybrids, and species outliers. This study provides a new foundation for the botanical and chemical authentication, classification, and metabolomic characterization of crude licorice botanicals and derived materials. Collectively, the proposed methods offer a comprehensive approach for the quality control of licorice as one of the most widely used botanical dietary supplements.