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The formation of secondary Ln(III) solid phases (e.g., Nd-2(CO3)(3) and Sm-2(CO3)(3)) was studied as a function of the humic acid concentration in 0.1 mol/L NaClO4 aqueous solution in the neutral pH range (5-6.5). The solid phases under investigation were prepared by alkaline precipitation under 100% CO2 atmosphere and characterized by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), X-ray diffraction (XRD), time-resolved laser fluorescence spectroscopy (TRLFS), diffuse reflectance ultraviolet-visible (DR-UV-Vis), Raman spectroscopy, and solubility measurements. The spectroscopic data obtained indicated that Nd-2(CO3)(3) and Sm-2(CO3)(3) were stable and remained the solubility limiting solid phases even in the presence of increased humic acid concentration (0.5 g/L) in solution. Upon base addition in the Ln(III)-HA system, decomplexation of the previously formed Ln(III)-humate complexes and precipitation of two distinct phases occurred, the inorganic (Ln(2)(CO3)(3)) and the organic phase (HA), which was adsorbed on the particle surface of the former. Nevertheless, humic acid affected the particle size of the solid phases. Increasing humic acid concentration resulted in decreasing crystallite size of the Nd-2(CO3)(3) and increasing crystallite size of the Sm-2(CO3)(3) solid phase, and affected inversely the solubility of the solid phases. However, this impact on the solid phase properties was expected to be of minor relevance regarding the chemical behavior and migration of trivalent lanthanides and actinides in the geosphere.
The disease risk and age of onset of Huntington disease (HD) and nine other repeat disorders strongly depend on the expansion of CAG repeats encoding consecutive polyglutamines (polyQ) in the corresponding disease protein. PolyQ length-dependent misfolding and aggregation are the hallmarks of CAG pathologies. Despite intense effort, the overall structure of these aggregates remains poorly understood. Here, we used sensitive time-dependent fluorescent decay measurements to assess the architecture of mature fibrils of huntingtin (Htt) exon 1 implicated in HD pathology. Varying the position of the fluorescent labels in the Htt monomer with expanded 51Q (Htt51Q) and using structural models of putative fibril structures, we generated distance distributions between donors and acceptors covering all possible distances between the monomers or monomer dimensions within the polyQ amyloid fibril. Using Monte Carlo simulations, we systematically scanned all possible monomer conformations that fit the experimentally measured decay times. Monomers with four-stranded 51Q stretches organized into five-layered beta-sheets with alternating N termini of the monomers perpendicular to the fibril axis gave the best fit to our data. Alternatively, the core structure of the polyQ fibrils might also be a zipper layer with antiparallel four-stranded stretches as this structure showed the next best fit. All other remaining arrangements are clearly excluded by the data. Furthermore, the assessed dimensions of the polyQ stretch of each monomer provide structural evidence for the observed polyQ length threshold in HD pathology. Our approach can be used to validate the effect of pharmacological substances that inhibit or alter amyloid growth and structure.
The standard procedure in the lab for plasmid isolation usually involves a 2-mL, 16 h over-night cultivation in 15-mL bioreaction tubes in LB medium. This is time consuming, and not suitable for high-throughput applications. This study shows that it is possible to produce plasmid DNA (pDNA) in a 1.5-mL microcentrifuge tube with only 100 L cultivation volume in less than 7 h with a simple protocol. Compared with the standard LB cultivation for pDNA production reaching a final pDNA concentration range of 1.5-4 mu g mL(-1), a 6- to 10-fold increase in plasmid concentration (from 10 up to 25 mu g mL(-1) cultivation volume) is achieved using an optimized medium with an internal substrate delivery system (EnBase (R)). Different strains, plasmids, and the applicability of different inoculation tools (i.e. different starting ODs) were compared, demonstrating the robustness of the system. Additionally, dissolved oxygen was monitored in real time online, indicating that under optimized conditions oxygen limitation can be avoided. We developed a simple protocol with a significantly decreased procedure time, enabling simultaneous handling of more samples, while a consistent quality and a higher final pDNA concentration are ensured.
We report on a new three-color FRET system consisting of three fluorescent dyes, i.e., of a carbostyril (=quinolin-2(1H)-one)-derived donor D, a (bathophenanthroline)ruthenium complex as a relay chromophore A1, and a Cy dye as A2 (FRET=Forster resonance-energy-transfer) (cf. Fig. 1). With their widely matching spectroscopic properties (cf. Fig. 2), the combination of these dyes yielded excellent FRET efficiencies. Furthermore, fluorescence lifetime measurements revealed that the long fluorescence lifetime of the Ru complex was transferred to the Cy dye offering the possibility to measure the whole system in a time-resolved mode. The FRET system was established on double-stranded DNA (cf. Fig. 3) but it should also be generally applicable to other biomolecules.
Ultrasound (20 kHz, 29 W. cm(-2)) is employed to form three types of erbium oxide nanoparticles in the presence of multiwalled carbon nanotubes as a template material in water. The nanoparticles are (i) erbium carboxioxide nanoparticles deposited on the external walls of multiwalled carbon nanotubes and Er(2)O(3) in the bulk with (ii) hexagonal and (iii) spherical geometries. Each type of ultrasonically formed nanoparticle reveals Er(3+) photoluminescence from crystal lattice. The main advantage of the erbium carboxioxide nanoparticles on the carbon nanotubes is the electromagnetic emission in the visible region, which is new and not examined up to the present date. On the other hand, the photoluminescence of hexagonal erbium oxide nanoparticles is long-lived (mu s) and enables the higher energy transition ((4)S(3/2)-(4)I(15/2)), which is not observed for spherical nanoparticles. Our work is unique because it combines for the first time spectroscopy of Er(3+) electronic transitions in the host crystal lattices of nanoparticles with the geometry established by ultrasound in aqueous solution of carbon nanotubes employed as a template material. The work can be of great interest for "green" chemistry synthesis of photoluminescent nanoparticles in water.
In this work, the photophysical properties of two oxazine dyes (ATTO 610 and ATTO 680) covalently attached via a C6-amino linker to the 5'-end of short single-stranded as well as double-stranded DNA (ssDNA and dsDNA, respectively) of different lengths were investigated. The two oxazine dyes were chosen because of the excellent spectral overlap, the high extinction coefficients, and the high fluorescence quantum yield of ATTO 610, making them an attractive Forster resonance energy transfer (FRET) pair for bioanalytical applications in the far-red spectral range. To identify possible molecular dye-DNA interactions that cause photophysical alterations, we performed a detailed spectroscopic study, including time-resolved fluorescence anisotropy and fluorescence correlation spectroscopy measurements. As an effect of the DNA conjugation, the absorption and fluorescence maxima of both dyes were bathochromically shifted and the fluorescence decay times were increased. Moreover, the absorption of conjugated ATTO 610 was spectrally broadened, and a dual fluorescence emission was observed. Steric interactions with ssDNA as well as dsDNA were found for both dyes. The dye-DNA interactions were strengthened from ssDNA to dsDNA conjugates, pointing toward interactions with specific dsDNA domains (such as the top of the double helix). Although these interactions partially blocked the dye-linker rotation, a free (unhindered) rotational mobility of at least one dye facilitated the appropriate alignment of the transition dipole moments in doubly labeled ATTO 610/ATTO 680-dsDNA conjugates for the performance of successful FRET. Considering the high linker flexibility for the determination of the donor-acceptor distances, good accordance between theoretical and experimental FRET parameters was obtained. The considerably large Forster distance of similar to 7 nm recommends the application of this FRET pair not only for the detection of binding reactions between nucleic acids in living cells but also for monitoring interactions of larger biomolecules such as proteins.
We report on a new three-color FRET system which we were able to verify in peptides as well as in synthetic DNA. All three chromophores could be introduced by a building block approach avoiding postsynthetic labeling. Additional features are robustness, matching spectroscopic properties, high-energy transfer, and sensitivity. The system was investigated in detail on a set of peptides as well as an array of tailored oligonucleotides. The detailed analysis of the experimental data and comparison with theoretical considerations were in excellent agreement. It is shown that in the case of polypeptides specific interaction with the fluorescence probes has to be considered. In contrast with DNA, the fluorescence probes did not show any indications of such interactions. The novel three-color FRET toolbox revealed the potential for applications studying fundamental processes of three interacting molecules in life science applications.
This article gives an overview of the current status of knowledge concerning the role of nanoparticles (inorganic and organic) in deep geological host rocks and the potential influence of these nanoparticles on radionuclide migration in far-field systems. The manuscript is not intended to be a full review paper or overview paper concerning nanoparticles, here the intention is to refer to recent publications but to highlight the progress made in the 6th framework project IP FUNMIG (Fundamental processes of radionuclide migration) and the open literature over the past 5 a concerning the process understanding of nanoparticle related issues in the three host rock formations investigated, namely: claystones, crystalline rocks and salt rock overburden. The results show inter alia that the inorganic nanoparticle concentration in deep groundwaters of advection dominated systems rarely exceeds 1 mg L (1) and is expected to be in the ng L (1) range in diffusion controlled systems. For organic nanoparticles DOC concentrations up to tens of milligrams in diffusion-controlled indurated clays with molecular sizes mostly <500 Da have been found. Fulvic acid type organics have been identified in crystalline environments and plastic Clay formations (Boom Clay) with molecular sizes <= 300 kDa. Additional sources of inorganic nanoparticles from the repository near-field (compacted bentonite) were identified and the initial erosion rates were determined. The results indicate under stagnant conditions similar to 38 mg cm (2) a (1) for bi-distilled water, similar to 20 mg cm (2) a (1) for glacial melt water (Grimsel groundwater) and very low rates similar to 0.02 mg cm (2) a (1) for 5 mM CaCl2 contact water. The low critical coagulation concentration (CCC) indicative for purely diffusion controlled coagulation of 1 mM L (1) Ca2+ found in bentonite nanoparticle stability analysis matches the low nanoparticle mobilization from compacted bentonite found in these systems.
Time-resolved emission data (fluorescence decay and fluorescence depolarization) of two three-color Forster resonance energy transfer (tc-FRET) systems consisting of a carbostyril donor (D), a ruthenium complex (Ru) as relay dye, and a Cy5 derivative (Cy) or, optionally, an anthraquinone quencher (Q) were carefully analyzed using advanced distribution analysis models. Thereby, it is possible to get information on the flexibility and mobility of the chromophores which are bound to double stranded (ds) DNA. Especially the distance distribution based on the analysis of the fluorescence depolarization is an attractive approach to complement data of fluorescence decay time analysis. The distance distributions extracted from the experimental data were in excellent agreement with those determined from accessible volume (AV) simulations. Moreover, the study showed that for tc-FRET systems the combination of dyes emitting on different time scales (e.g., nanoseconds vs microseconds) is highly beneficial in the distribution analysis of time-resolved luminescence data in cases where macromolecules such as DNA are involved. Here, the short lifetimes can yield information on the rotation of the dye molecule itself and the long lifetime can give insight in the overall dynamics of the macromolecule.
The formation of secondary Ln(III) solid phases (e.g. Nd(OH)CO3 and Sm(OH)CO3) has been studied as a function of the humic acid (HA) concentration in 0.1 M NaClO4 aqueous solution and their solubility has been investigated in the neutral pH range (6.5-8) under normal atmospheric conditions. Nd(III) and Sm(III) were selected as analogues for trivalent lanthanide and actinide ions. The solid phases under investigation have been prepared by alkaline precipitation and characterized by TGA, ATR-FTIR, XRD, TRLFS, DR-UV-Vis and Raman spectroscopy, and solubility measurements. The spectroscopic data obtained indicate that Nd(OH)CO3 and Sm(OH)CO3 are stable and remain the solubility limiting solid phases even in the presence of increased HA concentration (0.5 g/L) in solution. Upon base addition in the Ln(III)-HA system decomplexation of the previously formed Ln(III)-humate complexes and precipitation of two distinct phases occurs, the inorganic (Ln(OH)CO3) and the organic phase (HA), which is adsorbed on the particle surface of the former. Nevertheless, HA affects the particle size of the solid phases. Increasing HA concentration results in decreasing crystallite size of the Nd(OH)CO3 and increasing crystallite size of the Sm(OH)CO3 solid phase, and affects inversely the solubility of the solid phases. However, this impact on the solid phase properties is expected to be of minor relevance regarding the chemical behavior and migration of trivalent lanthanides and actinides in the geosphere.