Formation of polymeric nano-emulsions by a low-energy method and their use for nanoparticle preparation

Formation of polymeric O/W nano-emulsions has been studied in the water/polyoxyethylene 4 sorbitan monolaurate/ethylcellulose solution system by the phase inversion composition (PIC) method. These nano-emulsions were used for the preparation of nanoparticles by solvent evaporation. Composition variables such as O/S ratio or final water content as well as emulsification path have been found to play a key role in the formation of stable, nanometer sized emulsions. Nano-emulsions with a constant water content of 90 wt.% and O/S ratios from 50/50 to 70/30 showed an average droplet size of about 200 nm as assessed by dynamic light scattering. Mean nanoparticle diameters, as determined by transmission electron microscopy image analysis, were of the order of 50 nm and showed a slight increase as well as a broader size distribution at increasing O/S ratios. The findings verify that the low-energy emulsification methods are not only valid for aliphatic and semipolar oils, but also for a highly polar solvent such as ethylacetate containing a preformed polymer.     

SnpFilt: A pipeline for reference-free assembly-based identification of SNPs in bacterial genomes

De novo assembly of bacterial genomes from next-generation sequencing (NGS) data allows a reference-free discovery of single nucleotide polymorphisms (SNP). However, substantial rates of errors in genomes assembled by this approach remain a major barrier for the reference-free analysis of genome variations in medically important bacteria. The aim of this report was to improve the quality of SNP identification in bacterial genomes without closely related references. We developed a bioinformatics pipeline (SnpFilt) that constructs an assembly using SPAdes and then removes unreliable regions based on the quality and coverage of re-aligned reads at neighbouring regions. The performance of the pipeline was compared against reference-based SNP calling for Illumina HiSeq, MiSeq and NextSeq reads from a range of bacterial pathogens including Salmonella, which is one of the most common causes of food-borne disease. The SnpFilt pipeline removed all false SNP in all test NGS datasets consisting of paired-end Illumina reads. We also showed that for reliable and complete SNP calls, at least 40-fold coverage is required. Analysis of bacterial isolates associated with epidemiologically confirmed outbreaks using the SnpFilt pipeline produced results consistent with previously published findings. The SnpFilt pipeline improves the quality of de-novo assembly and precision of SNP calling in bacterial genomes by removal of regions of the assembly that may potentially contain assembly errors. SnpFilt is available from https://github.com/LanLab/SnpFilt.     

Identification and characterization of a bacterial glutamic peptidase

Background  

Glutamic peptidases, from the MEROPS family G1, are a distinct group of peptidases characterized by a catalytic dyad consisting of a glutamate and a glutamine residue, optimal activity at acidic pH and insensitivity towards the microbial derived protease inhibitor, pepstatin. Previously, only glutamic peptidases derived from filamentous fungi have been characterized.     

Identification of products formed during UV irradiation of tamoxifen and their use for fluorescence detection in high-performance liquid chromatography

During the UV irradiation of tamoxifen, isomerization of the trans to the cis isomer takes place and consequently corresponding highly fluorescent phenanthrene derivatives are formed. Their formation can be used for the sensitive and selective detection of tamoxifen in high-performance liquid chromatography (HPLC). The structure of photoproducts was identified by 1H NMR spectroscopy, HPLC, gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. Owing to the variety of products formed and the higher selectivity and fluorescence response, on-line postcolumn photocyclization is preferred to the precolumn mode. A chromatographic system for the separation of isomers and photoproducts is suggested.     

Optimization of a kinetic-catalytic method by use of a numerical model and the simplex method

Optimization of a kinetic-catalytic method based on a numerical model is demonstrated for the copper(II)-catalyzed decomposition of hydrogen peroxide in the presence of pyridine. The responses are calculated as initial rates of the catalytic reaction after calculating the equilibrium distributions of all species in the reaction mixture. Simulation of response surfaces as well as simplexes showed optimal concentrations for hydrogen peroxide, pyridine and pH. Experimental verifications of the simulated reaction conditions showed that the catalytic determination of copper down to 1.5 x 10^-7 M is possible.     

Features of electronic circular dichroism and tips for its use in determining absolute configuration

This review focuses on the general features of electronic circular dichroism (ECD) as applied in determining the absolute configuration of organic compounds. The high sensitivity and straightforward spectral interpretation of the exciton chirality method makes this approach very useful, and complementary to X-ray crystallography. A brief tutorial is provided on ECD, with precautions and tips for using it, especially the exciton chirality method. The spectroscopic ECD of several examples are analyzed.     

The use of circular dichroism in the investigation of protein structure and function

Circular Dichroism (CD) relies on the differential absorption of left and right circularly polarised radiation by chromophores which either possess intrinsic chirality or are placed in chiral environments. Proteins possess a number of chromophores which can give rise to CD signals. In the far UV region (240-180 nm), which corresponds to peptide bond absorption, the CD spectrum can be analysed to give the content of regular secondary structural features such as alpha-helix and beta-sheet. The CD spectrum in the near UV region (320-260 nm) reflects the environments of the aromatic amino acid side chains and thus gives information about the tertiary structure of the protein. Other non-protein chromophores such as flavin and haem moieties can give rise to CD signals which depend on the precise environment of the chromophore concerned. Because of its relatively modest resource demands, CD has been used extensively to give useful information about protein structure, the extent and rate of structural changes and ligand binding. In the protein design field, CD is used to assess the structure and stability of the designed protein fragments. Studies of protein folding make extensive use of CD to examine the folding pathway; the technique has been especially important in characterising molten globule intermediates which may be involved in the folding process. CD is an extremely useful technique for assessing the structural integrity of membrane proteins during extraction and characterisation procedures. The interactions between chromophores can give rise to characteristic CD signals. This is well illustrated by the case of the light harvesting complex from photosynthetic bacteria, where the CD spectra can be analysed to indicate the extent of orbital overlap between the rings of bacteriochlorophyll molecules. It is therefore evident that CD is a versatile technique in structural biology, with an increasingly wide range of applications.     

Identification of critical genes in microarray experiments by a Neuro-Fuzzy approach

Gene expression profiling by microarray technology is usually difficult to interpret into a simpler pattern. One approach to resolve the complexity of gene expression profiles is the application of artificial neural networks (ANNs). A potential difficulty in this strategy, however, is that the non-linear nature of ANN makes it essentially a 'black-box' computation process. Addition of a fuzzy logic approach is useful because it can complement ANN by explicitly specifying membership function during computation. We employed a hybrid approach of neural network and fuzzy logic to further analyze a published microarray study of gene responses to eight bacteria in human macrophages. The original analysis by hierarchical clustering found common gene responses to all bacteria but did not address individual responses. Our method allowed exploration of the gene response of the host to individual bacterium. We implemented a two-layer, feed-forward neural network containing the principle of 'competitive learning' (i.e. 'winner-take-all'). The weights of the trained neural network were fed into a fuzzy logic inference system. A new measurement, called the impact rating (IR) was also introduced to explore the degree of importance of each gene. To assess the reliability of the IR value, a bootstrap re-sampling method was applied to the dataset and a confidence level for each IR was obtained. Our approach has successfully uncovered the unique features of host response to individual bacterium. Further, application of gene ontology (GO) annotation to the genes of high IR values in each response has suggested new biological pathways for individual host-pathogen interactions.     

The use of a gold-containing membrane for ion-sensitive electrodes and their application in analysing systems

Summary As an example of an automatic continuous monitoring device a chloride titrator is described. A sample stream is mixed with a varying titrant stream (AgNO₃). The flow of the titrant stream is controlled by the difference between a set point signal and an actual signal (Ag⁺-ion activity). The signal is measured with an ion-sensitive flow-through electrode. The liquid stream is forced through the electrodes by a motor driven spinning brush with its hairs implanted in a screw-like form. Any maintenance during a two weeks period was redundant. The linearity was in the range of 10–100 ppm Cl^– better than 1% rel.

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Verwendung einer goldhaltigen Membran für ionensensitive Elektroden und ihre Anwendung in analytischen SystemenIII. Überwachung von Inenkonzentrationen (z.B. Chlorid) in einem Lösungsmittelstrom durch kontinuierliche Titrationen

Zusammenfassung Als Beispiel eines kontinuierlich messenden, automatischen Geräts wird ein Chlorid-Titrator beschrieben. Hierbei wird der Probenstrom mit einem variablen Titrantstrom (AgNO₃) gemischt. Die Titrantmenge pro Sekunde wird aus der Differenz zwischen einem Sollwert und einem Signalwert (Ag⁺-Ionen-Aktivität) bestimmt, der mittels einer Durchfluß-Elektrode gemessen wird. Der Flüssigkeitsstrom wird mit Hilfe einer von einem Motor gedrehten Bürste mit schraubenförmig eingesetzten Haaren durch die Elektroden getrieben. Während einer Zeit von 2 Wochen war eine Wartung unnötig. Die Linearität war im Bereich von 10–100 ppm Cl^– besser als 1% rel.

     

Identification of family-specific residue packing motifs and their use for structure-based protein function prediction: I. Method development

Protein function prediction is one of the central problems in computational biology. We present a novel automated protein structure-based function prediction method using libraries of local residue packing patterns that are common to most proteins in a known functional family. Critical to this approach is the representation of a protein structure as a graph where residue vertices (residue name used as a vertex label) are connected by geometrical proximity edges. The approach employs two steps. First, it uses a fast subgraph mining algorithm to find all occurrences of family-specific labeled subgraphs for all well characterized protein structural and functional families. Second, it queries a new structure for occurrences of a set of motifs characteristic of a known family, using a graph index to speed up Ullman’s subgraph isomorphism algorithm. The confidence of function inference from structure depends on the number of family-specific motifs found in the query structure compared with their distribution in a large non-redundant database of proteins. This method can assign a new structure to a specific functional family in cases where sequence alignments, sequence patterns, structural superposition and active site templates fail to provide accurate annotation.     

Ionic liquids and their use for the dissolution of natural polymers

The experimental and calculated data on the structure and properties of ionic liquids are presented. The prospects for their use as the solvents for natural polymers are considered.     

Blocking and ripening of colloids in porous media and their implications for bacterial transport

A model accounting for the dynamics of colloid deposition in porous media was developed and applied to systems containing similarly charged particles and collectors. Colloid breakthrough and intracolumn retention data confirmed that blocking reduced overall colloidal adhesion to soil. The surface coverage at which blocking occurred varied for the type of colloid, as shown by changes in the clean-bed collision efficiency, _0, and the excluded area parameter, β. Excluded area parameters were relatively high due to unfavorable interactions between particles and collectors, and ranged from 11.5 for one bacterium (Pseudomonas putida KT2442) to 13.7 and 24.1 for carboxylated latex microspheres with differing degrees of charged groups on their surfaces. Differences in β values for the three colloids were correlated with electrophoretic mobility, with the most negatively charged colloid (carboxylated latex; CL microspheres) having the highest β. No correlation between hydrophobicity and ₀ or β was found. Besides using colloidal particles capable of blocking, the addition of chemical additives to the soil has been suggested as a means for reducing attachment of colloids to porous media. Dextran addition caused an order-of-magnitude reduction in the overall (for carboxylated-modified latex; CMLs). This reduction was not attributed to blocking, but to the sorption of dextran to the soil which lowered ₀. The filtration-based numerical model used to fit the ₀ and β parameters was used to demonstrate that blocking could result in significantly enhanced bacterial transport in field situations.     

Modular synthesis of diphospholipid oligosaccharide fragments of the bacterial cell wall and their use to study the mechanism of moenomycin and other antibiotics

We present a flexible, modular route to GlcNAc-MurNAc-oligosaccharides that can be readily converted into peptidoglycan (PG) fragments to serve as reagents for the study of bacterial enzymes that are targets for antibiotics. Demonstrating the utility of these synthetic PG substrates, we show that the tetrasaccharide substrate lipid IV (3), but not the disaccharide substrate lipid II (2), significantly increases the concentration of moenomycin A required to inhibit a prototypical PG-glycosyltransferase (PGT). These results imply that lipid IV and moenomycin A bind to the same site on the enzyme. We also show the moenomycin A inhibits the formation of elongated polysaccharide product but does not affect length distribution. We conclude that moenomycin A blocks PG-strand initiation rather than elongation or chain termination. Synthetic access to diphospholipid oligosaccharides will enable further studies of bacterial cell wall synthesis with the long-term goal of identifying novel antibiotics.