Development of a high temperature treatment device for spent nuclear fuel

Novel reprocessing schemes and techniques are the focus of the Euratom FP7 project “Actinide Recycling for Separation and Transmutation” (ACSEPT), where the Paul Scherrer Institute (PSI) is represented in the pyrochemical domain. The subject of investigation is the selective separation of fission products (FPs) from spent nuclear fuel as a head-end step to either classical hydro based or pyro processes which are not yet applied on a large scale. The selective removal of FPs that are major contributors to the overall radiation dose or bear great potentials in terms of radiotoxicity (i.e. cesium or iodine), is advantageous for further processes. At PSI a device was developed to release volatile FPs by means of inductive heating. The heating up to 2,300 °C promotes the release of material that is further transported by a carrier gas stream into an inductively coupled plasma mass spectrometer for online detection. The carrier gas can be either inert (Ar) or can contain reducing or oxidizing components like hydrogen or oxygen, respectively. The development of the device by computer aided engineering approaches, the commissioning and evaluation of the device and data from first release experiments on a simulated fuel matrix are discussed.     

A computational model for predicting fusion peptide of retroviruses

As a pivotal domain within envelope protein, fusion peptide (FP) plays a crucial role in pathogenicity and therapeutic intervention. Taken into account the limited FP annotations in NCBI database and absence of FP prediction software, it is urgent and desirable to develop a bioinformatics tool to predict new putative FPs (np-FPs) in retroviruses. In this work, a sequence-based FP model was proposed by combining Hidden Markov Method with similarity comparison. The classification accuracies are 91.97% and 92.31% corresponding to 10-fold and leave-one-out cross-validation. After scanning sequences without FP annotations, this model discovered 53,946 np-FPs. The statistical results on FPs or np-FPs reveal that FP is a conserved and hydrophobic domain. The FP software programmed for windows environment is available at https://sourceforge.net/projects/fptool/files/?source=navbar.     

Interpenetrating blends of rigid aromatic fractal polyamides and nylon-6

Blends of nylon-6 and up to 20% rigid aromatic fractal polyamides (FPs) were prepared by precipitation from a mutual solvent and by two melt-processing procedures. In general, no grafting of the flexible linear nylon chains onto the rigid FPs took place, but in several instances of melt-blending of nylon with FPs whose amine end-groups were exposed, a low level of grafting occurred. The glass transition temperature and the tensile modulus and yield strength of the blends were greatly elevated as function of the FP concentration in the blends. This was demonstrated to be caused by the openness and rigidity of the FPs, and the connectivity of the FP segments through rigid branchpoints. The great porosity of the FPs allows the chains of the amorphous fraction of the nylon to interpenetrate and pass through the FPs, and the stiff segments of the FPs to suppress the chain motions of the nylon, which accounts for the enhanced glass transition temperature (T_g) and tensile properties. When non-porous amorphous silica particles or stiff linear or essentially unbranched zigzag polyamides were blended with the nylon, the T_g of the nylon either did not change at all or changed only very little. Several analytical procedures were used to verify that the nylon chains occupied most of the free space in the pervaded volumes of the FPs in the as-prepared blends and filled this space completely when these blends were compression-molded. The point where the FPs filled all the volume of the amorphous fraction of the nylon-6 was reached between 5 and 7.5% FP concentration. Below this, traces of the original nylon-6 T_g could be occasionally detected. Above it, only the high T_g of the nylon chains interpenetrated in the FPs was detected.     

Water-Soluble Polymeric Methanofullerene and Fulleropyrrolidine Derivatives

Water-soluble methanofullerene and fulleropyrrolidine derivatives were synthesized by the reactions of methanofullerenes (MFs) and fulleropyrrolidines (FPs) with poly(N-vinylpyrrolidone) (PVP) bearing one terminal amino group. The products contain MF or FP covalently bound to the polymer, corresponding polymeric complexes, and unreacted PVP. Large aggregates of the polymeric MF and FP derivatives are found in aqueous solutions. The electrooptical properties of the products differ considerably from those of the initial polymer. The phosphorus-containing MF and FP derivatives are capable of cleaving the molecule of plasmid DNA.     

Combinatorial preferences affect molecular similarity/diversity calculations using binary fingerprints and Tanimoto coefficients

A combinatorial method was developed to calculate complete distributions of the Tanimoto coefficient (Tc) for binary fingerprint (FP) representations of specified length, regardless of the chemical parameters they reflect. Theoretical Tc distributions were calculated for FPs consisting of up to 67 bit positions which revealed significant statistical preferences of certain Tc values. Calculation of Tc distributions in a large compound database using different FPs mirrored the effects identified by our general analysis. On the basis of these findings, an average Tc is biased by statistically preferred values.     

Frontal polymerizations carried out in Deep-Eutectic mixtures providing both the monomers and the polymerization medium

Deep Eutectic Solvents (DESs) based upon mixtures of Acrylic Acid (AA) or Methacrylic Acid (MAA) and Choline Chloride (CCl) demonstrated superior performance than regular organic solvents and even ionic liquids for frontal polymerizations (FPs). Full recovering of CCl after FP provided an interesting green character to the process.     

Deep eutectic solvents as both active fillers and monomers for frontal polymerization

The deep eutectic solvents (DESs) based on the mixtures of a variety of ammonium salts and hydrogen bond donors containing acrylic acids and acrylamides are capable of sustaining frontal polymerization (FP). The selection of ammonium salt affects the reactivity and allows FP at relatively low temperature but with full conversion. Also, full conversion allows us to use these polymers for biomedical applications (e.g., drug delivery systems) as the unreactive ammonium salts can be released from the resulting polymer without by‐products. We call these components “active fillers,” which can be ammonium salts with biological or pharmaceutical importance. For instance, we prepared poly(acrylic acid) loaded with lidocaine hydrochloride (a common anesthetic), the release of which was found to occur in a controlled fashion. The ammonium salts also create a sufficiently high viscosity to suppress buoyancy‐driven convection without additional materials. The DES here described played an all‐in‐one role, providing the monomer, the active filler, and the polymerization medium for FPs. © 2013 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2013     

DNAzyme-Mediated Genetically Encoded Sensors for Ratiometric Imaging of Metal Ions in Living Cells

Genetically encoded fluorescent proteins (FPs) have been used for metal ion detection. However, their applications are restricted to a limited number of metal ions owing to the lack of available metal-binding proteins or peptides that can be fused to FPs and the difficulty in transforming the binding of metal ions into a change of fluorescent signal. We report herein the use of Mg²⁺-specific 10–23 or Zn²⁺-specific 8–17 RNA-cleaving DNAzymes to regulate the expression of FPs as a new class of ratiometric fluorescent sensors for metal ions. Specifically, we demonstrate the use of DNAzymes to suppress the expression of Clover2, a variant of the green FP (GFP), by cleaving the mRNA of Clover2, while the expression of Ruby2, a mutant of the red FP (RFP), is not affected. The Mg²⁺ or Zn²⁺ in HeLa cells can be detected using both confocal imaging and flow cytometry. Since a wide variety of metal-specific DNAzymes can be obtained, this method can likely be applied to imaging many other metal ions, expanding the range of the current genetically encoded fluorescent protein-based sensors.     

DNAzyme‐Mediated Genetically Encoded Sensors for Ratiometric Imaging of Metal Ions in Living Cells

Genetically encoded fluorescent proteins (FPs) have been used for metal ion detection. However, their applications are restricted to a limited number of metal ions owing to the lack of available metal‐binding proteins or peptides that can be fused to FPs and the difficulty in transforming the binding of metal ions into a change of fluorescent signal. We report herein the use of Mg²⁺‐specific 10–23 or Zn²⁺‐specific 8–17 RNA‐cleaving DNAzymes to regulate the expression of FPs as a new class of ratiometric fluorescent sensors for metal ions. Specifically, we demonstrate the use of DNAzymes to suppress the expression of Clover2, a variant of the green FP (GFP), by cleaving the mRNA of Clover2, while the expression of Ruby2, a mutant of the red FP (RFP), is not affected. The Mg²⁺ or Zn²⁺ in HeLa cells can be detected using both confocal imaging and flow cytometry. Since a wide variety of metal‐specific DNAzymes can be obtained, this method can likely be applied to imaging many other metal ions, expanding the range of the current genetically encoded fluorescent protein‐based sensors.     

Bond selection in the photoisomerization reaction of anionic green fluorescent protein and kindling fluorescent protein chromophore models

The chromophores of the most widely known fluorescent proteins (FPs) are derivatives of a core p-hydroxybenzylidene-imidazolinon-5-one (HBI) motif, which usually occurs as a phenolate anion. Double bond photoisomerization of the exocyclic bridge of HBI is widely held to be an important internal conversion mechanism for FP chromophores. Herein we describe the ground and excited-state electronic structures and potential energy surfaces of two model chromophores: 4- p-hydroxybenzylidiene-1,2-dimethyl-imidazolin-5-one anion (HBDI), representing green FPs (GFPs), and 2-acetyl-4-hydroxybenylidene-1-methyl-imidazolin-5-one anion (AHBMI), representing kindling FPs (KFPs). These chromophores differ by a single substitution, but we observe qualitative differences in the potential energy surfaces which indicate inversion of bond selection in the photoisomerization reaction. Bond selection is also modulated by whether the reaction proceeds from a Z or an E conformation. These configurations correspond to fluorescent and nonfluorescent states of structurally characterized FPs, including some which can be reversibly switched by specific illumination regimes. We explain the difference in bond selectivity via substituent stabilization effects on a common set of charge-localized chemical structures. Different combinations of these structures give rise to both optically active (planar) and twisted intramolecular charge-transfer (TICT) states of the molecules. We offer a prediction of the gas-phase absorption of AHBMI, which has not yet been measured. We offer a hypothesis to explain the unusual fluorescence of AHBMI in DMF solution, as well as an experimental proposal to test our hypothesis.     

An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging

Multicolor imaging based on genetically encoded fluorescent proteins (FPs) is a powerful approach to study several dynamic processes in a live cell. We report a monomeric orange FP with a large Stokes shift (LSS), called LSSmOrange (excitation/emission at 437/572 nm), which fills up an existing spectral gap between the green-yellow and red LSSFPs. Brightness of LSSmOrange is five-fold larger than that of the brightest red LSSFP and similar to the green-yellow LSSFPs. LSSmOrange allows numerous multicolor applications using a single-excitation wavelength that was not possible before. Using LSSmOrange we developed four-color single-laser fluorescence cross-correlation spectroscopy, solely based on FPs. The quadruple cross-correlation combined with photon counting histogram techniques allowed quantitative single-molecule analysis of particles labeled with four FPs. LSSmOrange was further applied to simultaneously image two F?rster resonance energy transfer pairs, one of which is the commonly used CFP-YFP pair, with a single-excitation laser line. The combination of LSSmOrange-mKate2 and CFP-YFP biosensors enabled imaging of apoptotic activity and calcium fluctuations in real time. The LSSmOrange mutagenesis, low-temperature, and isotope effect studies revealed a proton relay for the excited-state proton transfer responsible for the LSS phenotype.     

FRET in a Synthetic Flavin‐ and Bilin‐binding Protein

The last decade has seen development and application of a large number of novel fluorescence‐based techniques that have revolutionized fluorescence microscopy in life sciences. Preferred tags for such applications are genetically encoded fluorescent proteins (FP), mostly derivatives of the green fluorescent protein (GFP). Combinations of FPs with wavelength‐separated absorption/fluorescence properties serve as excellent tools for molecular interaction studies, for example, protein–protein complexes or enzyme–substrate interactions, based on the FRET phenomenon (Förster resonance energy transfer). However, alternatives are requested for experimental conditions where FP proteins or FP couples are not or less efficiently applicable. We here report as a “proof of principle” a specially designed, non‐naturally occurring protein (LG1) carrying a combination of a flavin‐binding LOV‐ and a photochromic bilin‐binding GAF domain and demonstrate a FRET process between both chromophores.     

Mitochondria from cultured cells derived from normal and thiamine-responsive megaloblastic anemia individuals efficiently import thiamine diphosphate

Background

Within the family of green fluorescent protein (GFP) homologs, one can mark two main groups, specifically, fluorescent proteins (FPs) and non-fluorescent or chromoproteins (CPs). Structural background of differences between FPs and CPs are poorly understood to date.

Results

Here, we applied site-directed and random mutagenesis in order to to transform CP into FP and vice versa. A purple chromoprotein asCP (asFP595) from Anemonia sulcata and a red fluorescent protein DsRed from Discosoma sp. were selected as representatives of CPs and FPs, respectively. For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP) were found to dramatically increase quantum yield of red fluorescence. For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs. Finally, a practically non-fluorescent mutant DsRed-NF was generated. This mutant carried four amino acid substitutions, specifically, S148C, I165N, K167M, and S203A. DsRed-NF possessed a high extinction coefficient and an extremely low quantum yield (< 0.001). These spectral characteristics allow one to regard DsRed-NF as a true chromoprotein.

Conclusions

We located a novel point in asCP sequence (position 165) mutations at which can result in red fluorescence appearance. Probably, this finding could be applied onto other CPs to generate red and far-red fluorescent mutants. A possibility to transform an FP into CP was demonstrated. Key role of residues adjacent to chromophore's phenolic ring in fluorescent/non-fluorescent states determination was revealed.     

几个荧光蛋白发色团双光子吸收性质的理论研究

实验测得的荧光蛋白的单、双光子吸收光谱在低频和高频区域都表现出明显不同的特征。为了揭示这些不同点的起源和研究荧光蛋白的构–效关系,我们详细研究了三种荧光蛋白发色团(一种增强型蓝绿色荧光蛋白的中性发色团和两种红色荧光蛋白的阴离子发色团)的单、双光子吸收特性,分别计算了纯的和振动分辨的电子谱。计算结果表明：光谱线形与计算采用的交换相关密度泛函及谱截面计算所采用的近似关系密切;如果在计算光谱截面时,我们利用长程修正的交换相关泛函CAM-B3LYP来计算几何和电子结构参数,然后把Franck-Condon (FC)效应和包含Herzberg-Teller (HT)效果的电-声耦合效应都考虑进去,理论计算的光谱与实验测定的光谱可以很好地符合;对于两种离子态的发色团,HT电-声耦合效应使得对应于基态到第一激发态跃迁的双光子吸收最强峰相对于单光子吸收的最强峰发生了蓝移,但HT电-声耦合效应对高频的双光子吸收谱没有太大的影响;分子内电荷转移是导致高频区的双光子吸收明显强于单光子吸收的主要原因。     

A simple accurate model for prediction of flash point temperature of pure compounds

In this study, a simple three-parameter linear model is presented for estimation of flash point (FP) of pure compounds. The parameters of the model contain experimental normal boiling point of the compound and two chemical structure-based parameters. A comprehensive database of FPs containing 1472 pure compounds of various chemical structures was used to develop the model. The squared correlation coefficient and average absolute error of the model calculation results for all of the compounds presented in the database are evaluated to be 0.982 and 7.2?K, respectively.     

Synthesis of 6-formylpterin nucleoside analogs and their ROS generation activities in the presence of NADH in the dark

We demonstrated previously that 3-position-modified 6-formylpterin (6FP) derivatives produce reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O(2)) from oxygen in the presence of NADH in the dark. It has been shown that 6FP derivatives markedly generate ROS, which gives rise to their particular physiological activities, such as induction of apoptosis in cellular and living systems, suggesting that such compounds provide a hint for the design of a ROS controlling agent in vivo. However, it is not well understood why such unique activities appear on chemical modification. In the present study, in order to see the effect on ROS generation activity in the dark by the modification of the 1-position in 6FP, we have developed a new synthetic procedure for nucleoside analogs of 6FP and prepared 1-(beta-d-ribofuranosyl)-2-(N,N-diethylaminomethyleneamino)-6-formylpteridin-4-one (RDEF) and 1-(beta-d-ribofuranosyl)-2-(piperidine-1-ylmethyleneamino)-6-formylpteridin-4-one (RPIF) in which the 1-position of 6FP is glycosylated. At pH 7.4, NADH was spontaneously oxidized to NAD(+) in the presence of RDEF in the dark. Using electron paramagnetic resonance analysis coupled with the spin trapping technique, we show that O(2) was converted to H(2)O(2)via superoxide anion radical ( O(2)(-)) during this reaction. The modification of the 1-position of 6FP did not cancel ROS generation activities, which were demonstrated in 3-position-modified 6FPs. Since the 6FP derivatives developed in the present study have a ribose moiety, these compounds can be subjected to further derivatization, such as incorporation into oligonucleotides, oligosaccharides, proteins, or any other compounds that recognize and interact with specific biomolecules, and therefore would be useful in pharmaceutical investigations that need generation of appropriate and controllable amounts of ROS in vivo.     

Calculation of transition dipole moment in fluorescent proteins--towards efficient energy transfer

F?rster Resonance Energy Transfer (FRET) between fluorescent proteins (FPs) is widely used to construct fluorescent sensor proteins, to study intracellular protein-protein interactions and to monitor conformational changes in multidomain proteins. Although FRET depends strongly on the orientation of the transition dipole moments (TDMs) of the donor and acceptor fluorophores, this orientation dependence is currently not taken into account in FRET sensor design. Similarly, studies that use FRET to derive structural constrains typically assume a κ(2) of 2/3 or use the TDM of green fluorescent protein, as this is the only FP for which the TDM has been determined experimentally. Here we used time-dependent density functional theory (TD-DFT) methods to calculate the TDM for a comprehensive list of commonly used fluorescent proteins. The method was validated against higher levels of calculation. Validation with model compounds and the experimentally determined TDM of GFP shows that the TDM is mostly determined by the structure of the π-conjugated fluorophore and is insensitive to non-conjugated side chains or the protein surrounding. Our calculations not only provide TDM for most of the currently used FPs, but also suggest an empirical rule that can be used to obtain the TDMs for newly developed fluorescent proteins in the future.     

Iron cross-linked carboxymethyl cellulose—gelatin complex coacervate beads for sustained drug delivery

The formation and smooth recovery of ibuprofen encapsulated in microcapsules using gelatin and carboxymethyl cellulose (CMC) complex coacervation without glutaraldehyde were the objectives of this investigation. The microcapsules were recovered as ionically cross-linked beads using aqueous ferric chloride in 50 vol.% of 2-propanol. A physical mixture of CMC/gelatin (FP1) and CMC alone (FP2) beads was also prepared for comparison. The drug-entrapment efficiency of complex coacervate beads (FP3-FP5) was dependent on the drug-to-polymer ratio and was in the range of 86–92 mass %. Beads prepared with the highest ratio of the drug (FP5) exhibited the lowest entrapment. FP1 and FP2 beads exhibited an entrapment efficiency of 98.5 mass % and 91.3 mass %, respectively. Infrared spectroscopy (FTIR) revealed different functional groups in complex coacervate, physical mixture and FP2 beads. Optical and scanning electron microscopy revealed the distinct appearance and surface morphology of the various beads. The stable and crystalline nature of ibuprofen in the beads was confirmed by FTIR and differential scanning calorimetry (DSC), respectively. Ibuprofen release from FP1 and FP2 beads was very slow and unsuitable for oral delivery. The bead prepared by complex coacervation (FP5) showed a better release profile over 48 h and could be developed as a sustained drug delivery system.     

Stalling chromophore synthesis of the fluorescent protein Venus reveals the molecular basis of the final oxidation step

Fluorescent proteins (FPs) have revolutionised the life sciences, but the mechanism of chromophore maturation is still not fully understood. Here we show that incorporation of a photo-responsive non-canonical amino acid within the chromophore stalls maturation of Venus, a yellow FP, at an intermediate stage; a crystal structure indicates the presence of O₂ located above a dehydrated enolate form of the imidazolone ring, close to the strictly conserved Gly67 that occupies a twisted conformation. His148 adopts an “open” conformation so forming a channel that allows O₂ access to the immature chromophore. Absorbance spectroscopy supported by QM/MM simulations suggests that the first oxidation step involves formation of a hydroperoxyl intermediate in conjunction with dehydrogenation of the methylene bridge. A fully conjugated mature chromophore is formed through release of H₂O₂, both in vitro and in vivo. The possibility of interrupting and photochemically restarting chromophore maturation and the mechanistic insights open up new approaches for engineering optically controlled fluorescent proteins.

Fluorescent proteins (FPs) have revolutionised the life sciences, but the chromophore maturation mechanism is still not fully understood. Here we photochemically trap maturation at a crucial stage and structurally characterise the intermediate.