Uniform amplification of multiple DNAs by emulsion PCR

When several DNAs are amplified by PCR in one PCR tube, biased amplification is known to occur because amplification efficiency differs from one DNA to another. Therefore, we conducted PCR in the water in oil-emulsion (W/O emulsion) to examine whether the procedure allows the uniform amplification of several DNAs. In the amplification of a model library consisting of two clones, the emulsification of the PCR mixture successfully reduced the difference in its amplification efficiency to approximately one-seventh the value obtained without emulsification. Furthermore, we conducted repeated PCR to amplify a model library consisting of ten short hairpin RNA (shRNA) expression vectors as a model experiment for gene discovery using an shRNA expression library. Consequently, the emulsification of the PCR mixture successfully reduced PCR bias. Our results indicate that emulsion PCR is capable of uniformly amplifying libraries of shRNA, ribozyme, cDNA, and others, and is useful also for gene discovery using these libraries.     

Biomechanics of smooth adhesive pads in insects: influence of tarsal secretion on attachment performance

Many insects possess smooth adhesive pads on their legs, which adhere by thin films of a two-phasic secretion. To understand the function of such fluid-based adhesive systems, we simultaneously measured adhesion, friction and contact area in single pads of stick insects (Carausius morosus). Shear stress was largely independent of normal force and increased with velocity, seemingly consistent with the viscosity-effect of a continuous fluid film. However, measurements of the remaining force 2 min after a sliding movement show that adhesive pads can sustain considerable static friction. Repeated sliding movements and multiple consecutive pull-offs to deplete adhesive secretion showed that on a smooth surface, friction and adhesion strongly increased with decreasing amount of fluid. In contrast, pull-off forces significantly decreased on a rough substrate. Thus, the secretion does not generally increase attachment but does so only on rough substrates, where it helps to maximize contact area. When slides were repeated at one position so that secretion could accumulate, sliding shear stress decreased but static friction remained clearly present. This suggests that static friction which is biologically important to prevent sliding is based on non-Newtonian properties of the adhesive emulsion rather than on a direct contact between the cuticle and the substrate.     

Physicochemical characterization of a thermostable glycoprotein bioemulsifier from Solibacillus silvestris AM1

A novel estuarine bacterial strain, Solibacillus silvestris AM1, was found to produce an extracellular, multimeric glycoprotein bioemulsifier, termed AM1, with a MW of 200 kDa and containing 30 kDa monomeric subunits. The bioemulsifier contained 3.6% of the minor carbohydrate components galactose and ribose/xylose. LC/MS-MS of the 30 kDa subunit revealed its homology with a flagellin-like protein arranged in the form of fibers, as shown by transmission electron micrographs. This is the first report of a flagellin-like protein that exhibits bioemulsifier activity being produced from a member of the Solibacillus genus. Bioemulsifier AM1 has a high emulsification index of 62.5% with 10^?2 critical micellar dilution. It was found to be thermostable and active in the pH 5–9 and 0–5 M NaCl ranges. Moreover, AM1 formed stable emulsions with a broad range of solvents, including aliphatics, aromatic hydrocarbons and oils, performing better than the well-known bioemulsifier emulsan. Emulsions formed with trichlorobenzene and paraffin oil have a pseudoplastic non-Newtonian rheological property, as observed by particle size and shear stress analysis. AM1, an eco-friendly bioemulsifier, formed stable emulsions in varied physical conditions, and these attributes may prove to be advantageous in cosmetic, pharmaceutical and environmental applications.     

A novel fiber-optic photometer for in situ stability assessment of concentrated oil-in-water emulsions

The purpose of this research was to evaluate a novel fiberoptic photometer for its ability to monitor physical instabilities occurring in concentrated emulsions during storage. For this, the fiber-optic photometer was used to measure transmission of oil-in-water emulsions stabilized with hypromellose (HPMC) as a function of oil volume fraction and droplet size distribution (DSD). To detect physical instabilities like creaming and coalescence, the transmissivity of the samples was studied at 2 different hight levels over a certain period of time. The corresponding droplet size distributions were determined by laser diffraction with PIDS. Transmissivity was found to depend on the number of dispersed droplets and thus is sensitive to both the variation of phase volume fraction as well as the emulsions droplet size distribution. At constant DSD, light transmission decreased linearly with increasing oil content within a large interval of phase volume fractions from 0.01 to 0.3. At constant phase volume fraction, an increase in droplet size increased light transmission. Investigation of creaming on emulsions with different droplet size distributions showed changes in the initial delay times and creaming velocities. In contrast to creaming phenomenon coalescence can be identified by height independent changes of the transmissivity. In conclusion, transmissivity of oil-in-water emulsions observed by the novel fiber-optic photometer is sensitive to phase volume fraction, droplet size distribution, and thus can be used as a tool for stability studies on concentrated emulsions. Published: August 31, 2007     

Modulation of pH Sensitivity of Surface Charge and Aggregation Stability of Protein-Coated Lipid Droplets by Chitosan Addition

The impact of a cationic polyelectrolyte on the pH sensitivity of the electrical charge and aggregation stability of protein-coated lipid droplets was examined. One percent (w/w) corn oil-in-water emulsions containing lipid droplets coated by β-lactoglobulin [0.05% (w/w) β-Lg, 10 mM acetate buffer, pH 3] were prepared in the absence (“primary” emulsions) and presence (“secondary” emulsions) of chitosan (0 to 0.05 wt%). The pH (3 to 8) of these emulsions was adjusted, and the particle charge, particle size, creaming stability, and microstructure were measured. Chitosan adsorbed to the β-Lg-coated droplets from pH 4.5 to 7.5, which was attributed to electrostatic attraction between the cationic polyelectrolyte and anionic patches on the droplet surfaces. Droplets coated by β-Lg–chitosan had better stability to flocculation than those coated by β-Lg alone around the isoelectric point of the adsorbed proteins (pH 4.5 to 5.5), which was attributed to increased electrostatic and steric repulsion between the droplets. We have shown that chitosan may be used to modulate the electrical characteristics and stability of protein-coated lipid droplets, which may be useful in the design and formation of delivery systems for use in the food, pharmaceutical, and other industries.     

生物破乳菌的筛选及发酵条件的优化

生物破乳剂的开发可以降低油田乳状液对石油工业和生态环境的负面影响并减少化学破乳剂的使用量。本研究建立了一套高效、便捷的破乳菌筛选方法, 并对破乳菌的特性进行研究。利用大庆油田受石油污染土壤作为菌种来源, 将Tween 60-water(0.072%, V/V)和Span 60-oil (0.028%, V/V)以6.5:3.5体积比配置出可以稳定200 h以上的O/W型乳状液, 用于破乳菌破乳效能的评价。经过分离纯化、血平板试验、排油试验和破乳试验最终筛选出2株24 h平均排油率在80%以上的优势破乳菌, 初步鉴定为芽孢杆菌属(Bascillus)。通过该破乳菌发酵条的件优化得到, 当温度为25°C, 摇床转数为160 r/min, pH值为9, 接菌量为20%时对该破乳菌生长速率最快, 积累发酵产物的量最多; 当温度为35°C, 摇床转数为120 r/min, pH值为9, 接菌量为2%时该破乳菌代谢产物的破乳活性最高。     

Comparison between waste frying oil and paraffin as carbon source in the production of biodemulsifier by Dietzia sp. S-JS-1

In order to lower the production cost, waste frying oils were used in the biosynthesis of demulsifier by Dietzia sp. S-JS-1, which was isolated from petroleum contaminated soil. After 7 days of cultivation, the biomass concentration of the most suitable waste frying oil (WFO II) culture reached 3.78 g/L, which was 2.4 times the concentration of paraffin culture. The biodemulsifier produced with WFO II culture broke the emulsions more efficiently than that produced with paraffin culture, given the same volume ratio of carbon source in the culture medium and the same cultivation conditions. It achieved 88.3% of oil separation ratio in W/O emulsion and 76.4% of water separation ratio in O/W emulsion within 5 h. With the aid of thin layer chromatography (TLC) and Fourier transform infrared (FTIR) spectrometry, biodemulsifiers produced from both paraffin and WFO II were identified as a mixture of lipopeptide homologues. The subtle variation in the distribution of these homologues and high biomass concentration of WFO II cultures may account for the afore-mentioned good demulsification performance.     

Controlling phospholipid self-assembly and film properties using highly fluorinated components--fluorinated monolayers, vesicles, emulsions and microbubbles

Use of fluorinated components instead or along with standard phospholipids in film, vesicle, bubble and emulsion engineering, can cause drastic modifications of the formation processes, structure and dynamics, and functional behavior of these systems. Perfluoroalkyl chains provide a powerful driving force for self-assembly and ordering. They allow, for example, obtainment of thermally stable vesicles from single-chain phosphocholine derivatives, tubules from non-chiral amphiphiles, faceted vesicles with fluid bilayer membranes, exceptionally stable and narrowly dispersed emulsions and microbubbles. Contact of a monolayer of DPPC with a fluorocarbon gas modifies the monolayer’s phase behavior, suppressing the liquid expanded/liquid condensed transition. Phospholipid absorption kinetics at an air/water interface can be substantially accelerated, and the equilibrium interfacial tension reduced by exposure to a fluorocarbon gas. Perfluoroalkyl chains induce nanocompartmentation in films and membranes, allowing, for example, polymerization within vesicular membranes. Vesicles involving highly fluorinated components generally exhibit stability, permeability, fusion and recognition characteristics, different from those of their hydrogenated analogues. Drastic stabilization can be gained for phospholipid-coated emulsions through a co-surfactant effect of (perfluoroalkyl)alkyl diblocks. Stable, size-controlled, narrowly dispersed populations of microbubbles have been obtained using fluorinated wall and/or internal gas components, allowing progress in the understanding of microbubble physics, and open new application perspectives.     

Stimulation of Armillaria mellea by phenolic fungicides

Phenolic fungicides, which were initially fungicidal to mycelium of Armillaria mellea on the surface of well‐colonised wood billets, eventually stimulated the growth of A. mellea. An extensive growth of rhizomorphs was produced from A. mellea inoculum, which had been exposed to phenolic chemicals for 3 months, compared to few or no rhizomorphs produced from inoculum exposed to water or a suspension of a non‐phenolic fungicide, fenpropidin. Inoculated privet plants grown either in pots or under field conditions were treated with a range of fungicides; fenpropidin, phenyl phenol, cresylic acid or water (control) was applied every 6 months over 21/2 yr. Fenpropidin caused a slightly (but significantly) lower incidence of infection than occurred in untreated plants, but the phenolic fungicides, cresylic acid and phenyl phenol, did not reduce the incidence of infection. The severity of infection (% root circumference colonised at 5 cm depth) was greater following cresylic acid treatments than the other fungicides or water‐treated controls. Use of phenolic fungicides such as cresylic acid for the control of A. mellea may therefore be counter‐productive.     

Biosurfactants produced by Pseudomonas syringae pv tabaci: A versatile mixture with interesting emulsifying properties

In recent years, worldwide awareness about environmental concerns has led to an extensive searching for products of natural origin. Biosurfactants (BS), are an attractive alternative to the chemical counterparts. In these sense, this works evaluate for the first time the emulsifying and surface activity of biosurfactants produced by P. syringae pv. tabaci. Chemical characterization revealed that the produced BS extract mostly consisted in a mixture of syringopeptins and arthrofactins. Besides, this sample was able to reduce water surface tension up to 36.89 mN/m. This activity was improved by modifying conditions of pH, temperature and salinity. For instance, the surface tension was reduced up to 29 mN/m when using NaCl 5 % w/w. The BS also exhibited a high emulsifying activity, this being comparable or even superior to that of commercial bioemulsifiers such xanthan, tara and guar gums. A notorious emulsification index, close to 90 %, was exhibited by BS in coconut oil/water mixtures. Moreover, emulsifying activity resulted to be little affected by different operational conditions, thus retaining about 80 % of its emulsificant index in presence of 20 % w/w of NaCl at 80 °C. These findings are indicative of the BS promising properties and its potential as additive in a variety of industrial applications.