Seeing through cells: Rapid measurement of intracellular target proteins.

We have studied a method for making microbial cells transparent by immersing them in a solution with a high refractive index (RI). When the RI of the solution was matching that of the cells, light scattering was greatly diminished (by a factor of up to about 100) and the cell suspension became transparent, facilitating the spectrophotometric determination of intracellular compounds such as hemoglobin. We investigated the properties of several compounds such as sucrose, glycerol, bovine serum albumin, FicollTM, and iodixanol (OptiprepTM), each with advantages and disadvantages. Particularly good overall properties were found for iodixanol at a concentration of around 36% (w/v) and bovine serum albumin at a concentration of about 30% (w/v). By using this RI-matching principle the production of intracellular compounds can easily be followed in near real-time during fermentation processes. For example, some conditions for producing plant hemoglobin in Escherichia coli were conveniently determined without the need of any cell disintegration or product purification.

A systematic review of the effects of increasing arachidonic acid intake on PUFA status, metabolism and health-related outcomes in humans.

We conducted a systematic review of randomised controlled trials (RCT) of increased intake of arachidonic acid (ARA) on fatty acid status and health outcomes in humans. We identified twenty-two articles from fourteen RCT. Most studies were conducted in adults. These used between 80 and 2000 mg ARA per d and were of 1-12 weeks duration. Supplementation with ARA doses as low as 80 mg/d increased the content of ARA in different blood fractions. Overall there seem to be few marked benefits for adults of increasing ARA intake from the typical usual intake of 100-200 mg/d to as much as 1000 mg/d; the few studies using higher doses (1500 or 2000 mg/d) also report little benefit. However, there may be an impact of ARA on cognitive and muscle function which could be particularly relevant in the ageing population. The studies reviewed here suggest no adverse effects in adults of increased ARA intake up to at least 1000-1500 mg/d on blood lipids, platelet aggregation and blood clotting, immune function, inflammation or urinary excretion of ARA metabolites. However, in many areas there are insufficient studies to make firm conclusions, and higher intakes of ARA are deserving of further study. Based on the RCT reviewed, there are not enough data to make any recommendations for specific health effects of ARA intake.

Development of permanent magnet MnAlC/polymer composites and flexible filament for bonding and 3D-printing technologies.

Searching for high-performance permanent magnets components with no limitation in shape and dimensions is highly desired to overcome the present design and manufacturing restrictions, which affect the efficiency of the final devices in energy, automotive and aerospace sectors. Advanced 3D-printing of composite materials and related technologies is an incipient route to achieve functional structures avoiding the limitations of traditional manufacturing. Gas-atomized MnAlC particles combined with polymer have been used in this work for fabricating scalable rare earth-free permanent magnet composites and extruded flexible filaments with continuous length exceeding 10 m. Solution casting has been used to synthesize homogeneous composites with tuned particles content, made of a polyethylene (PE) matrix embedding quasi-spherical particles of the ferromagnetic τ-MnAlC phase. A maximum filling factor of 86.5 and 72.3% has been obtained for the composite and the filament after extrusion, respectively. The magnetic measurements reveal no deterioration of the properties of the MnAlC particles after the composite synthesis and filament extrusion. The produced MnAlC/PE materials will serve as precursors for an efficient and scalable design and fabrication of end-products by different processing techniques (polymerized cold-compacted magnets and 3D-printing, respectively) in view of technological applications (from micro electromechanical systems to energy and transport applications).

Light and oxygen induce chain scission of conjugated polymers in solution.

Conjugated polymers have been widely studied as flexible, versatile semiconductors in organic electronics. However, the material stability is one of the problems limiting their applications. Thus, understanding the degradation process of conjugated polymers is crucial. In this work, we monitored the chain scission of the model polymer MEH-PPV in chloroform solutions under different conditions by assessing its molecular weight using gel permeation chromatography and optical spectral measurements. We showed that changes in the UV-VIS spectrum can be seen only when the degradation has already progressed substantially. The fluorescence spectrum was found to be almost totally insensitive to the degradation stage of the polymers. We demonstrate that chain scission in solutions happens even in the dark leading to a 15% decrease of the molecular weight after just one day of storage. If exposed to room light, the chain length decreases by about 10 times over one day of exposure. Using stronger light intensity or enriching the solution with oxygen accelerates the degradation process dramatically. The rate of the reaction follows approximately a square root dependence with light intensity and oxygen concentration. We conclude that some extent of polymer degradation is difficult to avoid in common laboratory practices since to prevent it, one needs to work in an oxygen-free atmosphere in the dark. Preparation of polymer films from partially degraded solutions might lead not only to losing the connection between the molecular weight and the opto-electronic properties but also to unintentional doping of the semiconductor by products of chain scission reactions.

Molecular Weight Determination by Counting Molecules.

Molecular weight (MW) is one of the most important characteristics of macromolecules. Sometimes, MW cannot be measured correctly by conventional methods like gel permeation chromatography (GPC) due to, for example, aggregation. We propose using single-molecule spectroscopy to measure the average MW simply by counting individual fluorescent molecules embedded in a thin matrix film at known mass concentration. We tested the method on dye molecules, a labeled protein, and the conjugated polymer MEH-PPV. We showed that GPC with polystyrene calibration overestimates the MW of large MEH-PPV molecules by 40 times due to chain aggregation and stiffness. This is a crucial observation for understanding correlations between the conjugated polymer length, photophysics and performances of devices. The method can measure the MW of fluorescent molecules, biological objects, and nanoparticles at ultimately low concentrations and does not need any reference; it is conformation-independent and has no limitations regarding the detected MW range.

Awareness and the arguments for and against the International Classification of Functioning, Disability and Health among representatives of disability organisations.

Disability organisations have not been engaged in the debate about the International Classification of Functioning, Disability and Health (ICF) in Sweden. We wanted to know representatives' attitudes about the ICF. The aim of the study was to elucidate the arguments for and against the ICF among representatives of disability organisations. The study consisted of eighteen representatives (from six disability organisations) that answered electronic questionnaires. The questionnaires involved ten open questions about the arguments for and against the ICF. The answers of the questionnaires have been categorized according to qualitative content analysis. Our results indicated four themes: awareness, arguments for and against, influence and the future. More than half of the representatives had very limited awareness of the ICF. There was an explicit criticism of individual classification but more positive comments about classification on an aggregated level. The most important issue for representatives was influencing social policy in society, not learning and spreading information about the ICF.

Field assessment of guar gum stabilized microscale zerovalent iron particles for in-situ remediation of 1,1,1-trichloroethane.

A pilot injection test with guar gum stabilized microscale zerovalent iron (mZVI) particles was performed at test site V (Belgium) where different chlorinated aliphatic hydrocarbons (CAHs) were present as pollutants in the subsurface. One hundred kilograms of 56µm-diameter mZVI (~70gL(-1)) was suspended in 1.5m(3) of guar gum (~7gL(-1)) solution and injected into the test area. In order to deliver the guar gum stabilized mZVI slurry, one direct push bottom-up injection (Geoprobe) was performed with injections at 5 depths between 10.5 and 8.5m bgs. The direct push technique was preferred above others (e.g. injection at low flow rate via screened wells) because of the limited hydraulic conductivity of the aquifer, and to the large size of the mZVI particles. A final heterogeneous distribution of the mZVI in the porous medium was observed explicable by preferential flow paths created during the high pressure injection. The maximum observed delivery distance was 2.5m. A significant decrease in 1,1,1-TCA concentrations was observed in close vicinity of spots where the highest concentration of mZVI was observed. Carbon stable isotope analysis (CSIA) yielded information on the success of the abiotic degradation of 1,1,1-TCA and indicated a heterogeneous spatio-temporal pattern of degradation. Finally, the obtained results show that mZVI slurries stabilized by guar gum can be prepared at pilot scale and directly injected into low permeable aquifers, indicating a significant removal of 1,1,1-TCA.

Impact of carbon, oxygen and sulfur content of microscale zerovalent iron particles on its reactivity towards chlorinated aliphatic hydrocarbons.

Zerovalent iron (ZVI) abiotically degrades several chlorinated aliphatic hydrocarbons (CAHs) via reductive dechlorination, which offers perspectives for in situ groundwater remediation applications. The difference in reactivity between ZVI particles is often linked with their specific surface area. However, other parameters may influence the reactivity as well. Earlier, we reported for a set of microscale zerovalent iron (mZVI) particles the disappearance kinetic of different CAHs which were collected under consistent experimental conditions. In the present study, these kinetic data were correlated with the carbon, oxygen and sulfur content of mZVI particles. It was confirmed that not only the specific surface area affects the disappearance kinetic of CAHs, but also the chemical composition of the mZVI particles. The chemical composition, in addition, influences CAHs removal mechanism inducing sorption onto mZVI particles instead of dechlorination. Generally, high disappearance kinetic of CAHs was observed for particles containing less oxygen. A high carbon content, on the other hand, induced nonreactive sorption of the contaminants on the mZVI particles. To obtain efficient remediation of CAHs by mZVI particles, this study suggested that the carbon and oxygen content should not exceed 0.5% and 1% respectively. Finally, the efficiency of the mZVI particles may be improved to some extent by enriching them with sulfur. However, the impact of sulfur content on the reactivity of mZVI particles is less pronounced than that of the carbon and oxygen content.

Reactivity screening of microscale zerovalent irons and iron sulfides towards different CAHs under standardized experimental conditions.

A standardized batch test procedure was developed and used to evaluate the reactivity of twelve newly designed microscale zerovalent iron (mZVI) particles and two biogenic iron sulfides towards a mixture of chlorinated aliphatic hydrocarbons (CAHs) and their breakdown products. For comparison, commercially available mZVIs, nanoscale zerovalent irons (nZVIs), iron sulfides (FeS) and granular zerovalent iron were also tested. Reactivity of the particles was based on observed (kobs) and mass normalized (kM) pseudo-first-order degradation rate constants, as well as specific surface area normalized reaction rate constants (kSA). Sorption characteristics of the particles were based on mass balance data. Among the new mZVIs, significant differences in reactivity were observed and the most reactive particles were identified. Based on kM data, nZVI degraded the examined contaminants one to two orders of magnitude faster than the mZVIs. kM values for biogenic iron sulfides were similar to the least reactive mZVIs. On the other hand, comparison of kSA data revealed that the reactivity of some newly designed mZVIs was similar to highly reactive nZVIs, and even up to one order of magnitude higher. kSA values for biogenic iron sulfides were one to two orders of magnitude lower than those reported for reactive mZVIs.

Molecular recognition with colloidosomes enabled by imprinted polymer nanoparticles and fluorogenic boronic acid.

Multifunctional colloidosomes are prepared from molecularly imprinted polymer nanoparticles and fluorogenic boronic acid using a Cu(i)-catalyzed click reaction. The molecular selectivity of the colloidosomes was investigated by radioligand binding analysis, which indicated that the inter-particle click reaction did not affect the molecular specificity of the MIP nanoparticles on the colloidosomes for the model template, propranolol. Besides specific molecular recognition of the MIP nanoparticles, the colloidosomes also displayed dose-dependent fluorescence response to fructose at physiological pH. Moreover, the immobilized boronic acid in the core could effectively bind isoproterenol, a template analogue containing a catecholamine moiety. The depletion of isoproterenol from solution allowed the MIP nanoparticles on the colloidosomes to bind propranolol more efficiently. The pre-designed molecular selectivity and fluorescence response of the colloidosomes are interesting for potential applications in controlled delivery, chemical sensing and bioseparation.

The ICF: International Classification of Functioning, Disability and Health (ICF)--a Swiss army knife? Accessibility and disability in a Scandinavian disability magazine (SDM)--a quantitative content analysis.

BACKGROUND: The study describes the extent to which the concept of accessibility and accessibility issues has been reported in a national Scandinavian disability magazine. In this study particular attention is paid to how the compatibility between the various domains of the international classification - International Classification of Functioning, Disability and Health (ICF) - has been covered in the magazine over a decade. Furthermore, other types of descriptions that the magazine has made of accessibility for people with physical disabilities are considered. METHODS: The study is based on a quantitative content analysis of a total of 326 articles from the Swedish disability magazine (SDM); 450 text pages published during the years 2000-2009. The magazine's coverage has been comprehensive. RESULTS AND CONCLUSIONS: More than half of the studied texts were news items about various accessibility issues. Every third article can be characterised as a news article or feature article. The most frequently reported of the ICF domains consist of two perspectives: environmental accessibility and civil rights. Public opinion articles in the form of letters to editors and editorials focused on accessibility have a low frequency. Likewise, research reports are few. The study has included a review of illustrations and photographs. The illustrations are generally of high quality, reinforcing the disability message of the article.

A novel combined thermometric and amperometric biosensor for lactose determination based on immobilised cellobiose dehydrogenase.

A novel method for lactose determination in milk is proposed. It is based on oxidation of lactose by cellobiose dehydrogenase (CDH) from the basidiomycete Phanerochaete chrysosporium, immobilised in an enzyme reactor. The reactor was prepared by cross-linking CDH onto aminopropyl-silanised controlled pore glass (CPG) beads using glutaraldehyde. The combined biosensor worked in flow injection analysis (FIA) mode and was developed for simultaneous monitoring of the thermometric signal associated with the enzymatic oxidation of lactose using p-benzoquinone as electron acceptor and the electrochemically generated current associated with the oxidation of the hydroquinone formed. A highly reproducible linear response for lactose was obtained between 0.05 mM and 30 mM. For a set of more than 500 samples an R.S.D. of less than 10% was achieved. The assay time was ca. 2 min per sample. The sensor was applied for the determination of lactose in dairy milk samples (milk with a fat content of 1.5% or 3% and also "lactose free" milk). No sample preparation except dilution with buffer was needed. The proposed method is rapid, suitable for repeated use and allows the possibility to compare results from two different detection methods, thus providing a built-in quality assurance. Some differences in the response observed between the methods indicate that the dual approach can be useful in mechanistic studies of redox enzymes. In addition, a dual system opens up interesting possibilities for studies of enzyme properties and mechanisms.

High-capacity composite adsorbents for nucleic acids.

Cytopore™ is a bead-shaped, macroporous and easily compressible cellulose-based anion-exchange material intended for cultivation of anchor-dependent animal cells. Reticulated vitreous carbon (RVC) is a strong, non-compressible, high voidage (97%) matrix material that can be cut to desired geometrical shapes. Cytopore and RVC were combined to cylindrical composites (25 mm × 10 mm) fitted inside chromatography columns. The composite combined the advantageous properties of both its constituents, making it suitable for column chromatography. The composite could withstand very high flow rates without compaction of the bed (>25 column volumes/min; 4000 cm h(-1)). Chromatography runs with tracers showed a low HETP value (0.3mm), suggesting that pore flow was in operation. The dynamic binding capacities (10% breakthrough) per gram of dry weight Cytopore were determined for several compounds including DNA and RNA and were found to be 240-370 mg/g. The composite was used to isolate pUC 18-type plasmids from a cleared alkaline lysate in a good yield. Confocal microscopy studies showed that plasmids were bound not only to the surface of the Cytopore material but also within the matrix walls, thus offering an explanation to the very high binding capacities observed. The concept of using a composite prepared from a mechanically weak, high-binding material and a strong scaffold material may be applied to other systems as well.

Intracellular ZnO Nanorods Conjugated with Protoporphyrin for Local Mediated Photochemistry and Efficient Treatment of Single Cancer Cell.

ZnO nanorods (NRs) with high surface area to volume ratio and biocompatibility is used as an efficient photosensitizer carrier system and at the same time providing intrinsic white light needed to achieve cancer cell necrosis. In this letter, ZnO nanorods used for the treatment of breast cancer cell (T47D) are presented. To adjust the sample for intracellular experiments, we have grown the ZnO nanorods on the tip of borosilicate glass capillaries (0.5 µm diameter) by aqueous chemical growth technique. The grown ZnO nanorods were conjugated using protoporphyrin dimethyl ester (PPDME), which absorbs the light emitted by the ZnO nanorods. Mechanism of cytotoxicity appears to involve the generation of singlet oxygen inside the cell. The novel findings of cell-localized toxicity indicate a potential application of PPDME-conjugated ZnO NRs in the necrosis of breast cancer cell within few minutes.

Fate of excitations in conjugated polymers: single-molecule spectroscopy reveals nonemissive "dark" regions in MEH-PPV individual chains.

Single chains of the conjugated polymer MEH-PPV (poly(2-methoxy-5-(2'-ethylhexyloxy)-1,4-phenylenevinylene)) were studied with wide-field fluorescence microscopy (dispersion in inert polymer matrices) and with fluorescence correlation spectroscopy (chloroform solution). The fluorescence yield of individual molecules in matrices was found to be 1-2 orders of magnitude lower than that in solution and it decreased substantially with increasing chain length. It suggests that isolation of MEH-PPV molecules in polymer matrices creates favorable conditions for photogeneration of nonemissive primary excited states.

Fluorescence blinking, exciton dynamics, and energy transfer domains in single conjugated polymer chains.

In order to understand exciton migration and fluorescence intensity fluctuation mechanisms in conjugated polymer single molecules, we studied fluorescence decay dynamics at "on" and "off" fluorescence intensity levels with 20 ps time resolution using MEH-PPV [poly(2-methoxy-5-(2'-ethylhexyloxy)-1,4-phenylenevinylene] dispersed in PMMA. Two types of intensity fluctuations were distinguished for single chains of conjugated polymers. Abrupt intensity fluctuations (blinking) were found to be always accompanied by corresponding changes in fluorescence lifetime. On the contrary, during "smooth" intensity fluctuations no lifetime change was observed. Time-resolved data in combination with data on fluorescence emission and excitation anisotropy lead to a picture where a single polymer molecule is seen as consisting of several energy transfer domains. Exciton migration is efficient within a domain and not efficient between domains. Each domain can have several emitting low-energy sites over which the exciton continuously migrates until it decays. Emission of individual domains is often highly polarized. Fluorescence from a domain can be strongly quenched by Forster energy transfer to a quencher (hole polaron) if the domain overlaps with the quenching sphere.

Plasmid adsorption to anion-exchange matrices: comments on plasmid recovery.

A number of anion-exchange adsorbents were constructed, employing nonporous silica fibers, and examined with the aim of describing factors that influence desorption and recovery of plasmid DNA (pDNA). The fibers were provided with ligands via adsorption of the polymeric amines poly(ethyleneimine) or chitosan, or via graft-polymerization of primary, tertiary, or quaternary amine monomers to vinyl-silanized fibers. Several adsorbents showed an almost irreversible plasmid binding. It was suggested that important factors affecting the DNA releasing ability are (i) type of amine ligand used (primary amines bind plasmids the strongest), (ii) the structure of the nucleic acid (supercoiled pDNA may bind stronger than linear genomic DNA), (iii) shift of ligand pK(a) (due to the proximity of highly charged pDNA), and (iv) the solid support itself (steric factors may lead to kinetically stable complexes). The last factor was derived from several comparisons between support-bound ligand and free soluble ligand. It was thus observed that polyelectrolyte complexes associated with a surface were much more difficult to dissociate than the equivalent soluble complexes.

Plasmid purification using non-porous anion-exchange silica fibres.

A new type of fibre-based anion-exchange material for plasmid purification was developed. The basic material consisted of non-porous silica fibres with a mean diameter of 1.5 microm and a surface area of 2.4m(2)g(-1). The fibre surface was provided with several types of ligands, either by adsorption of polymers (chitosan or poly(ethyleneimine)) or by polymerization of amine-containing acrylic monomers onto a propyl methacrylate-silanized surface. The resulting polymer layers contained primary, tertiary or quaternary amines as ion-exchange groups. The packing density could be varied considerably, 9-34% (v/v). The loose packing structure provided excellent flow properties suitable for high-speed operations. The best overall performance was shown by silica fibres provided with tertiary amine polymers, having a plasmid-binding capacity of 0.9 mg ml(-1) (pre-purified plasmid) and a plasmid recovery of 62% (performance data remained stable though several adsorption cycles). The high flow rates possible with the fibre material made it especially useful when large volumes of cleared lysate were processed. The columns could be operated with retention of their adsorption properties at speeds of up to 1800 cm h(-1), equivalent to 0.5 column volumes per minute. The binding capacity was found to be lower than anticipated from the design of the fibres. Fluorescence imaging showing individual plasmid molecules indicated the fibre population to be heterogeneous with respect to plasmid adsorption, some fibres displaying poor binding properties. Possible reasons for this heterogeneity are discussed.

Superporous agarose anion exchangers for plasmid isolation.

Superporous agarose beads have wide, connecting flow pores allowing large molecules such as plasmids to be transported into the interior of the beads by convective flow. The pore walls provide additional surface for plasmid binding thus increasing the binding capacity of the adsorbent. Novel superporous agarose anion exchangers have been prepared, differing with respect to bead diameter, superpore diameter and type of anion-exchange functional group (poly(ethyleneimine) and quaternary amine). The plasmid binding capacities were obtained from breakthrough curves and compared with the binding capacity of homogeneous agarose beads of the same particle size. Significantly, the smaller diameter superporous agarose beads were found to have four to five times higher plasmid binding capacity than the corresponding homogeneous agarose beads. The experimentally determined plasmid binding capacity was compared with the theoretically calculated surface area for each adsorbent and fair agreement was found. Confocal microscopy studies of beads with adsorbed, fluorescently labelled plasmids aided in the interpretation of the results. Superporous poly(ethyleneimine)-substituted beads with a high ion capacity (230 micromol/ml) showed a plasmid binding of 3-4 mg/ml adsorbent. Superporous quaternary amine-substituted beads had a lower ion capacity (81 micromol/ml) and showed a correspondingly lower plasmid binding capacity (1-2 mg/ml adsorbent). In spite of the lower capacity, the beads with quaternary amine ligand were preferred, due to their much better plasmid recovery (70-100% recovery). Interestingly, both capacity and recovery was improved when the plasmid adsorption step was carried out in the presence of a moderate salt concentration. The most suitable superporous bead type (45-75 microm diameter beads; 4 microm superpores; quaternary amine ligand) was chosen for the capture of plasmid DNA from a clarified alkaline lysate. Two strategies were evaluated, one with and one without enzymatic digestion of RNA. The strategy without RNase gave high plasmid recovery, quantitative removal of protein and a 70% reduction in RNA.

Purification of plasmid DNA with a new type of anion-exchange beads having a non-charged surface.

We have prepared a new type of anion exchanger, which effectively discriminates between RNA and plasmid DNA. The material is based on a Sephacryl S-500 HR matrix provided with quartenary amine anion-exchange groups. A distinguishing feature of the beads is that a thin (2-3 microm) outer layer of the beads lacks ion-exchange groups. In the synthesis of these beads the vinyl groups in the outer layer of vinylalkyl substituted Sephacryl S-500 HR beads are reacted with bromine. The resulting layer of bromoalkyl groups are hydrolysed, creating an inert outer layer of hydroxyalkyl groups. Finally, bromination and trimethylamine reactions of the inner vinyl groups provide the beads with a core of cationic groups. Large plasmid molecules will not bind to such beads since they are too large to enter the pores and therefore cannot come into contact with the charged matrix in the inner parts of the beads. RNA and protein molecules present in a cleared lysate, on the other hand, readily enter the pores and become adsorbed. A two-column strategy was developed for plasmid purification (recombinant pBluescript, 5.9 kilo base pairs, kbp). The first column was packed with the restricted access anion-exchanger beads (lid beads) and the second column with normal ion-exchange material (same ligand density as the lid beads). Diluted (3x), cleared lysate was pumped through the tandem columns. The first column was subsequently disconnected from the system and the purified plasmid adsorbed on the second column was eluted in a concentrated form (6x) and with 89% recovery. The two-column procedure removed 99.5% of the RNA and 96% of the proteins.