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High-resolution four-dimensional carbon-correlated 1H-1H ROESY experiments employing isotags and the filter diagonalization method for effective assignment of glycosidic linkages in oligosaccharides.

Four-dimensional nuclear magnetic resonance spectroscopy of oligosaccharides that correlates 1H-1H ROESY cross peaks to two additional 13C frequency dimensions is reported. The 13C frequencies were introduced by derivatization of all free hydroxyl groups with doubly 13C-labeled acetyl isotags. Pulse sequences were optimized for processing with the filter diagonalization method. The extensive overlap typically observed in 2D ROESY 1H-1H planes was alleviated by resolution of ROESY cross peaks in the two added dimensions associated with the carbon frequencies of the isotags. This enabled the interresidue 1H-1H ROESY cross peaks to be unambiguously assigned hence spatially proximate sugar spin systems across glycosidic bonds could be effectively ascertained. An experiment that selectively amplifies interresidue ROESY 1H-1H cross peaks is also reported. It moves the magnetization of an intraresidue proton normally correlated to a sugar H-1 signal orthogonally along the z axis prior to a Tr-ROESY mixing sequence. This virtually eliminates the incoherent intraresidue ROESY transfer, suppresses coherent TOCSY transfer, and markedly enhances the intensity of interresidue ROESY cross peaks.

Carbon Isotopes↗

Optimization of HPLC chromatographic conditions for determination of Transkarbam 12 and its degradation products.

This paper deals with searching of HPLC chromatographic conditions for determination and separation of Transkarbam 12 (T 12) and its two main degradation products (omega-aminocaproic acid and dodecylalcohol). T 12 is a new substance which belongs to the group of accelerators of transdermal penetration. Chromatographic separation was achieved using Separon SGX C18 analytical column (150 mm x 3 mm i.d.; 5 microm). Mobile phase contained acetonitrile and sodium acetate buffer pH 4.5 at the flow rate of 1 ml/min. Separation was carried out under the conditions of gradient elution. After the modification of the structure by derivatization reagent (3,5-dinitrobenzoyl chloride) detection at wavelength 230 nm was realized. The aim of this study was not only the optimization of the separation of derivatization reagent and derivatized T 12, Ak and D but also optimal derivatization processes for all three substances.

Aminocaproates↗

Role of soil microbes in the rhizospheres of plants growing on trace metal contaminated soils in phytoremediation.

This article reviews recent developments in in situ bioremediation of trace metal contaminated soils, with particular reference to the microbial dynamics in the rhizospheres of plants growing on such soils and their significance in phytoremediation. In non-agricultural conditions, the natural role of plant growth promoting rhizobacteria (PGPR), P-solubilizing bacteria, mycorrhizal-helping bacteria (MHB) and arbuscular mycorrhizal fungi (AMF) in maintaining soil fertility is more important than in conventional agriculture, horticulture, and forestry where higher use of agrochemicals minimize their significance. These microbes initiate a concerted action when a particular population density is achieved, i.e. quorum sensing. AMF also recognize their host by signals released by host roots, allowing a functional symbiosis. AM fungi produce an insoluble glycoprotein, glomalin, which sequester trace elements and it should be considered for biostabilization leading to remediation of contaminated soils. Conclusions drawn from studies of metal uptake kinetics in solution cultures may not be valid for more complex field conditions and use of some combination of glasshouse and field experiments with organisms that occur within the same plant community is suggested. Phytoextraction strategies, such as inoculation of plants to be used for phytoremediation with appropriate heavy metal adapted rhizobial microflora, co-cropping system involving a non-mycorrhizal hyperaccumulator plant and a non-accumulator but mycorrhizal with appropriate AMF, or pre-cropping with mycotrophic crop systems to optimize phytoremediation processes, merit further field level investigations. There is also a need to improve our understanding of the mechanisms involved in transfer and mobilization of trace elements by rhizosphere microbiota and to conduct research on selection of microbial isolates from rhizosphere of plants growing on heavy metal contaminated soils for specific restoration programmes. This is necessary if we are to improve the chances of successful phytoremediation.

Metals↗

Metabolic engineering of Escherichia coli and Corynebacterium glutamicum for biotechnological production of organic acids and amino acids.

Industrial microorganisms have been developed as biocatalysts to provide new or to optimize existing processes for the biotechnological production of chemicals from renewable plant biomass. Rational strain development by metabolic engineering is crucial to successful processes, and is based on efficient genetic tools and detailed knowledge of metabolic pathways and their regulation. This review summarizes recent advances in metabolic engineering of the industrial model bacteria Escherichia coli and Corynebacterium glutamicum that led to efficient recombinant biocatalysts for the production of acetate, pyruvate, ethanol, d- and l-lactate, succinate, l-lysine and l-serine.

Amino Acids↗

A novel lysozyme from Xanthomonas oryzae phage varphiXo411 active against Xanthomonas and Stenotrophomonas.

In this study, a bacteriophage of Xanthomonas oryzae pv. oryzae designated as varphiXo411 was isolated. Random sequencing of its genome revealed that it is closely related to another X. oryzae phage, Xp10. A cloned fragment carries the lysozyme gene, lys411. The deduced protein, Lys411, shares 92% identity with Xp10 lysozyme, which contains an extra 46 aa at the N-terminus. Lys411 shows over 40% identities to several other phage lysozymes. The His-tagged protein, Lys411H, expressed in Escherichia coli largely formed as inclusion bodies. The insoluble protein was solubilized in urea and purified by passing through a His-bind column, and the lytic activity was then restored by a refolding process. The optimal assay conditions determined for Lys411H are in 0.1M potassium phosphate buffer, pH 6.6 containing 1 mM CuCl(2) at 25 degrees C. Lysis assays using different bacterial cells as the substrates indicate that Lys411H is the first lysozyme active against both Xanthomonas and Stenotrophomonas maltophilia. This suggests that Lys411 can be a candidate to be developed into a therapeutic agent for treating S. maltophilia infections, in addition to the potential use in control of the plant diseases caused by Xanthomonas. By analogy to the situation in Xp10, we predict that varphiXo411 has no holin, the protein required for lysozyme export, and the N-terminal signal-arrest-release sequence of Lys411 can accommodate its own export to the periplasm.

Amino Acid Sequence↗

Cloud point extraction, preconcentration and simultaneous spectrophotometric determination of nickel and cobalt in water samples.

Cloud point extraction has been used for the preconcentration and simultaneous spectrophotometric determination of nickel and cobalt after the formation of a complex with 2-amino-cyclopentene-1-dithiocarboxylic acid (ACDA), and latter analysis by spectrophotometer using Triton X-114 as surfactant. The parameters affecting the separation phase and detection process were optimized. Under the optimum experimental conditions (i.e. pH=5, 0.07 mM ACDA, Triton X-114=0.25% (w/v)), calibration graphs were linear in the range of 20-500 and 20-200 microg l(-1) with detection limits of 10 and 7.5 microg l(-1) for Ni and Co, respectively. The method was applied to the determination of Ni and Co in natural and waste water samples with satisfactory results.

Carboxylic Acids↗

The double regulation of endothelial nitric oxide synthase by caveolae and caveolin: a paradox solved through the study of angiogenesis.

Caveolae are plasmalemmal invaginations formed by the sequestration of cholesterol and glycosphingolipids with self-associating molecules named caveolins, resulting in a platform for the assembly of signaling complexes at the surface of the cell. The enrichment of the endothelial nitric oxide synthase in caveolae and its direct interaction with caveolin both account for the exquisite regulation of nitric oxide production in cardiovascular tissues. Dissection of the angiogenic signaling cascade downstream vascular endothelial growth factor recently led to recognition that although the former enables the compartmentation of endothelial nitric oxide synthase and optimizes the process leading to its activation, the latter maintains the enzyme in its inactivated state in the absence of stimulation. Alteration in caveolin abundance or subcellular location may lead endothelial cells or cardiac myocytes to favor one mode of regulation over the other and thereby alter the subtle equilibrium governing nitric oxide production in these cells.

Animals↗

Memory consolidation and reconsolidation: what is the role of sleep?

Memory consolidation and reconsolidation reflect molecular, cellular and systems-level processes that convert labile memory representations into more permanent ones, available for continued reactivation and recall over extended periods of time. Here, we discuss the complexities of consolidation and reconsolidation, and suggest they should be viewed not as all-or-none phenomena, but as a continuing series of biological adjustments that enhance both the efficiency and the utility of stored memories over time and in response to changing needs of the organism. As such, consolidation and reconsolidation might be better thought of as memory organization and reorganization. A rapidly growing body of evidence suggests that many of these processes are optimally engaged during sleep.

Animals↗

Optimization of microcarrier cell culture process for the inactivated enterovirus type 71 vaccine development.

Enterovirus 71 (EV71) is an enterovirus that could lead to severe neurological disorders and fatalities. The inactivated vaccine is an appropriate EV71 vaccine format for meeting current needs. Large-scale preparation of the inactivated EV71vaccine depends on a scalable cell culture system for industrial mass production. In this paper, Vero cells were found to produce higher titers of EV71 than did MRC-5 and WI-38 cells. High-density microcarrier Vero cell cultures were established using 5g/L Cytodex 1 microcarriers and found to promote the release of EV71s from infected Vero cells. For the large-scale production of the inactivated vaccine antigen, the extracellular virus titers produced in the 2L bioreactor were found to be 10 times lower than the spinner flask culture but improved by 30-folds using glucose/glutamine feedings during infection. A serum-free Vero cell microcarrier culture was also established in the bioreactor, yielding a high-titer of 5.8 x 10(7) TCID50/mL for EV71 production. The immunogenicity of the inactivated virions produced in serum-free culture elicited a slightly higher level of neutralizing antibody response in immunized mice. These results constitute valuable information on the development of a large-scale microcarrier cell culture process for producing inactivated EV71 vaccine.

Animals↗

Encapsulation of antigenic extracts of Salmonella enterica serovar. Abortusovis into polymeric systems and efficacy as vaccines in mice.

The properties of drug-vaccine delivery systems based on the use of biodegradable polymers, and its application in the control of experimental infection by Salmonella enterica serovar. Abortusovis (SAO), are described in this manuscript. Micelles of major membrane antigens from SAO (HSao extract) can be encapsulated in microparticles of poly(epsilon-caprolactone) or in nanoparticles of Gantrez polymer. The encapsulation process was optimized by the combined use of cyclodextrins. The resulting particles contained unaltered significant amounts of the antigenic complex. To establish the protective value of these subunit vaccines, particles were injected in one single dose (20 microg of HSao) subcutaneously in BALB/c mice in order to observe the protection conferred against experimental infection with the virulent strains S. Abortusovis 15/5. Control non-immunized animals resulted infected, as well as the group that received unloaded or HSao loaded into microparticles. In contrast, nanoparticles conferred a significant protection when compared to unvaccinated controls, similar to that induced by the attenuated commercial vaccine Rv6. In conclusion, protection against experimental infection in mice after one single shoot, and its potential for mucosal vaccination suggest that HSao-nanoparticles may represent a serious alternative to the conventional attenuated vaccines against S. Abortusovis.

Animals↗

Inducible flocculation in Komagataella phaffii enables enhanced biomass separation for biopharmaceutical production.

Biomass separation represents a critical bottleneck in Komagataella phaffii-based biopharmaceutical processes, as typically high cell densities of 40 - 50 % create significant operational, technical and economic challenges for harvest operations. Yeast cell aggregation (flocculation) provides a solution to accelerate cell sedimentation by increasing particle size, thus allowing to improve biomass-supernatant separation efficiency during both natural gravity settling and (continuous) centrifugation operations. This study demonstrates successful engineering of K. phaffii strains with an inducible flocculation phenotype using CRISPR/Cas9-based genome editing to integrate the Saccharomyces cerevisiae FLO1 (ScFLO1) gene under control of various regulatory elements, including methanol-inducible and derepressible promoters. Flocculation strength could be enhanced by implementing transcriptional positive feedback circuits based on the methanol-inducible AOX1 promoter. To address methanol-free production requirements, we developed alternative systems to retrofit PAOX1-based ScFLO1 expression and exploited the derepressible PDF promoter, offering broader compatibility with biopharmaceutical manufacturing facilities. Flocculating cells cultivated in a bioreactor demonstrated significantly improved sedimentation behavior, with considerably lower supernatant turbidity after short low-speed centrifugation or gravity sedimentation compared to non-flocculating controls. Crucially, cell flocculation had no negative impact on product amount and quality when expressing a multivalent NANOBODY® VHH molecule with pharmaceutical relevance. Thus, this work establishes the first genetically engineered flocculation system in K. phaffii compatible with recombinant protein production, providing the basis for an innovative approach to streamline harvest operations in biopharmaceutical processes.

Flocculation↗

Towards European urinalysis guidelines. Introduction of a project under European Confederation of Laboratory Medicine.

Improved standardized performance is needed because urinalysis continues to be one of the most frequently requested laboratory tests. Since 1997, the European Confederation of Laboratory Medicine (ECLM) has been supporting an interdisciplinary project aiming to produce European urinalysis guidelines. More than seventy clinical chemists, microbiologists and ward-based clinicians, as well as representatives of manufacturers are taking part. These guidelines aim to improve the quality and consistency of chemical urinalysis, particle counting and bacterial culture by suggesting optimal investigative processes that could be applied in Europe. The approach is based on medical needs for urinalysis. The importance of the pre-analytical stage for total quality is stressed by detailed illustrative advice for specimen collection. Attention is also given to emerging automated technology. For cost containment reasons, both optimum (ideal) procedures and minimum analytical approaches are suggested. Since urinalysis mostly lacks genuine reference methods (primary reference measurement procedures; Level 4), a novel classification of the methods is proposed: comparison measurement procedures (Level 3), quantitative routine procedures (Level 2), and ordinal scale examinations (Level 1). Stepwise strategies are suggested to save costs, applying different rules for general and specific patient populations. New analytical quality specifications have been created. After a consultation period, the final written text will be published in full as a separate document.

Europe↗

Aspects of column fabrication for packed capillary electrochromatography.

Various parameters have been evaluated to develop a process for optimization of column manufacture for packed capillary electrochromatography (CEC). Spherisorb ODS-1 was packed into 75 microm I.D. capillaries to establish a standard set of packing conditions to afford high-performance columns free of voids. Numerous silica-based packing materials including porous and non-porous reversed-phase and ion-exchange phases were employed to evaluate the applicability of the standard conditions. Success of column manufacture and performance demonstrate a relationship to the colligative properties of the packing materials under the applied conditions. Frequently encountered difficulties arising from inadequate column conditioning and void formation in the packed bed are identified and discussed.

Electrophoresis, Capillary↗

Purification of lentil lectins using preparative electrophoresis.

Electrophoresis presents an interesting alternative to chromatography for the purification of biological compounds. To document the performance of three preparative electrophoresis apparatus currently available, they were applied to the purification of lectins from lentil seeds which contain two isolectins usually purified by chromatography. Purification by electrophoresis consists of first isolating a mixture of the two isolectins and then separating them. For the first step, either of two free-flow electrophoresis apparatus were employed: the Elphor VaP 22, using field step electrophoresis and the Biostream using zone electrophoresis. To optimize the process, the Biostream was modified to a recycling mode. This required repositioning one dialysis membrane which separates an electrode from the separation chamber. This allowed the lentil extract to be desalted by electrodialysis directly in the apparatus prior to fractionation. A high concentration of lectins was collected at the cathode and acidic proteins were collected at the anode. The bulk of the extract was recycled until the whole volume was processed. In a second step the isolectins were separated by recycling isoelectric focusing in the recycling isoelectric focusing apparatus. The present work clearly demonstrates that electrophoretic methods provide lectins with higher purity than chromatographically purified commercial products.

Electrophoresis↗

Determination of phthalate esters in water samples by solid-phase microextraction and gas chromatography with mass spectrometric detection.

Solid-phase microextraction (SPME) with an 85 microm polyacrylate fiber, coupled to gas chromatography-mass spectrometry was used to determine six phthalate esters and bis(2-ethylhexyl) adipate in water samples. The variables affecting the SPME absorption process were optimized and the method developed was applied to analyze both tap and commercial mineral water samples as well as water from the Ebro river and fishing and industrial ports. For real samples, the linear range in full scan acquisition mode was between 0.02 and 10 microg l(-1) for most compounds, and the limits of detection of the method were between 0.006 and 0.17 microg l(-1). Commercial water samples contained in recipients which were made from different materials were analyzed, and the influence of the material of the recipients on the concentration of phthalates was evaluated.

Esters↗

Optimal harvesting from a population in a stochastic crowded environment.

We study the (Ito) stochastic differential equation [equation: see text] as a model for population growth in a stochastic environment with finite carrying capacity K > 0. Here r and alpha are constants and Bt denotes Brownian motion. If r > or = 0, we show that this equation has a unique strong global solution for all x > 0 and we study some of its properties. Then we consider the following problem: What harvesting strategy maximizes the expected total discounted amount harvested (integrated over all future times)? We formulate this as a stochastic control problem. Then we show that there exists a constant optimal "harvest trigger value" x* epsilon (0, K) such that the optimal strategy is to do nothing if Xt < x* and to harvest Xt-x* if Xt > x*. This leads to an optimal population process Xt being reflected downward at x*. We find x* explicitly.

Environment↗

Lipoprotein-mimicking biovectorized systems for methotrexate delivery.

The present investigation reports a new family of lipid nanoparticles biomimetic of lipoproteins, lipoprotein-mimicking biovectorized systems (LMBVs), for the delivery of methotrexate. LMBVs were prepared by microemulsion congealing technique, and the composition and process were optimized. The palmitoylpolyethylene glycol 4000 (p-PEG 4000) was anchored on LMBVs as apoprotein analogue. Various formulations were prepared and characterized for size and polydispersity, zeta potential, drug entrapment efficiency, anchoring efficiency, release kinetics, pharmacokinetics and tissue distribution. The size was 70-76 nm and the polydispersity was 0.09-0.18. The zeta potential was -63.2 which was reduced to -19.3 after p-PEG 4000 coating. Entrapment efficiency varied from 22.6% to 30.2%. Anchoring efficiency was 74.0 +/- 3.9%. The drug release showed zero-order profile. Their circulation half-life was enhanced and exhibited capability to accumulate in tissues for longer periods. The composition of lipidic part of LMBVs and p-PEG 4000 anchoring impart similarity with natural lipoproteins in terms of in vivo behaviour. This new drug carrier named LMBVs, holds promise for targeting and systemic controlled release, which may prove effective in the treatment of various types of cancer.

Animals↗