306 research outputs found
Interference-free coherence dynamics of gas-phase molecules using spectral focusing
Spectral focusing using broadband femtosecond pulses to achieve highly selective measurements has been employed for numerous applications in spectroscopy and microspectroscopy. In this work we highlight the use of spectral focusing for selective excitation and detection of gas-phase species. Furthermore, we demonstrate that spectral focusing, coupled with time-resolved measurements based upon probe delay, allows the observation of interference-free coherence dynamics of multiple molecules and gas-phase temperature making this technique ideal for gas-phase measurements of reacting flows and combustion processes
Effect of polycrystallinity on the optical properties of highly oriented ZnO grown by pulsed laser deposition
We report the results of photoluminescence and reflectance measurements on highly c-axis oriented polycrystalline ZnO grown by pulsed laser deposition. The samples measured were grown under identical conditions and were annealed in-situ at various temperatures for 10-15 min. The band-edge photoluminescence spectra of the material altered considerably with an increase in grain size, with increased free exciton emission and observable excitonic structure in the reflectance spectra. The green band emission also increased with increasing grain size. A deformation potential analysis of the effect of strain on the exciton energy positions of the A- and B-excitons demonstrated that the experimental exciton energies could not be explained solely in terms of sample strain. We propose that electric fields in the samples due to charge trapping at grain boundaries are responsible for the additional perturbation of the excitons. This interpretation is supported by theoretical estimates of the exciton energy perturbation due to electric fields. The behaviour of the green band in the samples provides additional evidence in favour of our model
The Cultural Divide: Exponential Growth in Classical 2D and Metabolic Equilibrium in 3D Environments
INTRODUCTION: Cellular metabolism can be considered to have two extremes: one is characterized by exponential growth (in 2D cultures) and the other by a dynamic equilibrium (in 3D cultures). We have analyzed the proteome and cellular architecture at these two extremes and found that they are dramatically different. RESULTS: Structurally, actin organization is changed, microtubules are increased and keratins 8 and 18 decreased. Metabolically, glycolysis, fatty acid metabolism and the pentose phosphate shunt are increased while TCA cycle and oxidative phosphorylation is unchanged. Enzymes involved in cholesterol and urea synthesis are increased consistent with the attainment of cholesterol and urea production rates seen in vivo. DNA repair enzymes are increased even though cells are predominantly in Go. Transport around the cell--along the microtubules, through the nuclear pore and in various types of vesicles has been prioritized. There are numerous coherent changes in transcription, splicing, translation, protein folding and degradation. The amount of individual proteins within complexes is shown to be highly coordinated. Typically subunits which initiate a particular function are present in increased amounts compared to other subunits of the same complex. SUMMARY: We have previously demonstrated that cells at dynamic equilibrium can match the physiological performance of cells in tissues in vivo. Here we describe the multitude of protein changes necessary to achieve this performance
Studies of viomycin, an anti-tuberculosis antibiotic: Copper(II) coordination, DNA degradation and the impact on delta ribozyme cleavage activity
Viomycin is a basic peptide antibiotic, which is among the most effective agents against multidrug-resistant tuberculosis. In this paper we provide the characteristics of its acid base properties, coordination preferences towards the Cu(II) ions, as well as the reactivity of the resulting complexes against plasmid DNA and HDV ribozyme. Careful coordination studies throughout the wide pH range allow for the characterisation of all the Cu(II)-viomycin complex species. The assignment of proton chemical shifts was achieved by NMR experiments, while the DTF level of theory was applied to support molecular structures of the studied complexes. The experiments with the plasmid DNA reveal that at the physiological levels of hydrogen peroxide the Cu(II)-viomycin complex is more aggressive against DNA than uncomplexed metal ions. Moreover, the degradation of DNA by viomycin can be carried out without the presence of transition metal ions. In the studies of antigenomic delta ribozyme catalytic activity, viomycin and its complex are shown to modulate the ribozyme functioning. The molecular modelling approach allows the indication of two different locations of viomycin binding sites to the ribozyme
Effective charge of the [pi]h11/2 orbital and the electric field gradient of Hg from the yrast structure of 206Hg
The γ-ray decay of excited states of the two-proton hole nucleus, 206Hg, has been identified using Gammasphere and 208Pb+238U collisions. The yrast states found include a T1/2 = 92(8)ns 10+ isomer located above the known 5- isomer. The B(E2;10+→8+) strength is used to derive the quadrupole polarization charge induced by the h11/2 proton hole. Also, the implied quadrupole moment has been used to provide an absolute scale for the electric field gradient of Hg in Hg metal
Removal of homeostatic cytokine sinks by lymphodepletion enhances the efficacy of adoptively transferred tumor-specific CD8(+) T cells
Depletion of immune elements before adoptive cell transfer (ACT) can dramatically improve the antitumor efficacy of transferred CD8(+) T cells, but the specific mechanisms that contribute to this enhanced immunity remain poorly defined. Elimination of CD4(+)CD25(+) regulatory T (T reg) cells has been proposed as a key mechanism by which lymphodepletion augments ACT-based immunotherapy. We found that even in the genetic absence of T reg cells, a nonmyeloablative regimen substantially augmented CD8(+) T cell reactivity to self-tissue and tumor. Surprisingly, enhanced antitumor efficacy and autoimmunity was caused by increased function rather than increased numbers of tumor-reactive T cells, as would be expected by homeostatic mechanisms. The γ (C) cytokines IL-7 and IL-15 were required for augmenting T cell functionality and antitumor activity. Removal of γ (C) cytokine–responsive endogenous cells using antibody or genetic means resulted in the enhanced antitumor responses similar to those seen after nonmyeloablative conditioning. These data indicate that lymphodepletion removes endogenous cellular elements that act as sinks for cytokines that are capable of augmenting the activity of self/tumor-reactive CD8(+) T cells. Thus, the restricted availability of homeostatic cytokines can be a contributing factor to peripheral tolerance, as well as a limiting resource for the effectiveness of tumor-specific T cells
Determination of Drug Toxicity Using 3D Spheroids Constructed From an Immortal Human Hepatocyte Cell Line
Numerous publications have documented that the immortal cells grown in three-dimensional (3D) cultures possess physiological behavior, which is more reminiscent of their parental organ than when the same cells are cultivated using classical two-dimensional (2D) culture techniques. The goal of this study was to investigate whether this observation could be extended to the determination of LD50 values and whether 3D data could be correlated to in vivo observations. We developed a noninvasive means to estimate the amount of protein present in a 3D spheroid from it is planar area (± 21%) so that a precise dose can be provided in a manner similar to in vivo studies. This avoided correction of the actual dose given based on a protein determination after treatment (when some cells may have lysed). Conversion of published in vitro LC50 data (mM) for six common drugs (acetaminophen, amiodarone, diclofenac, metformin, phenformin, and valproic acid) to LD50 data (mg compound/mg cellular protein) showed that the variation in LD50 values was generally less than that suggested by the original LC50 data. Toxicological analysis of these six compounds in 3D spheroid culture (either published or presented here) demonstrated similar LD50 values. Although in vitro 2D HepG2 data showed a poor correlation, the primary hepatocyte and 3D spheroid data resulted in a much higher degree of correlation with in vivo lethal blood plasma levels. These results corroborate that 3D hepatocyte cultures are significantly different from 2D cultures and are more representative of the liver in vivo
Evidence of non-extractable florfenicol residues: The development and validation of a confirmatory method for total florfenicol content in kidney by UPLC-MS/MS
Publication history: Accepted - 27 March 2016; Published online - 20 May 2016.The parent compound florfenicol (FF) is a broad-spectrum antibacterial compound licensed in the UK for use in cattle, pigs and the aquaculture industry. The analysis of porcine tissues in this study demonstrates that significant amounts of solvent non-extractable FF-related residues are present in incurred tissues (kidney and muscle) from treated animals. The results indicate that methods based on solvent extraction alone may carry a significant risk of reporting false-negative results. The use of a strong acid hydrolysis step prior to solvent extraction of tissue samples is necessary for an accurate estimate of the total tissue FF content. A robust and sensitive method for the determination of total FF residue content in kidney samples by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) has been developed and validated. This method covers the synthetic amphenicol drug FF and its metabolites, measured as the marker residue florfenicol amine (FFA) as per Commission Regulation (EU) No. 37/2010. Non-extractable and intermediate metabolites are converted to the hydrolysis product FFA, and then partitioned into ethyl acetate. Extracts are solvent exchanged prior to a dispersive solid-phase extraction step, then analysed using an alkaline reverse-phase gradient separation by UPLC-MS/MS. The method was validated around the maximum residue levels (MRLs) set out in Regulation (EU) No. 37/2010 for bovine kidney in accordance with Commission Decision No. 2002/657/EC. The following method performance characteristics were assessed during a single laboratory validation study: selectivity, specificity, sensitivity, linearity, matrix effects, accuracy and precision (decision limit (CCα) and detection capability (CCβ) were determined)
Characterization of the Trans Watson-Crick GU Base Pair Located in the Catalytic Core of the Antigenomic HDV Ribozyme
The HDV ribozyme’s folding pathway is, by far, the most complex folding
pathway elucidated to date for a small ribozyme. It includes 6 different steps
that have been shown to occur before the chemical cleavage. It is likely that
other steps remain to be discovered. One of the most critical of these unknown
steps is the formation of the trans Watson-Crick GU base
pair within loop III. The U23 and G28 nucleotides that
form this base pair are perfectly conserved in all natural variants of the
HDV ribozyme, and therefore are considered as being part of the signature
of HDV-like ribozymes. Both the formation and the transformation of this base
pair have been studied mainly by crystal structure and by molecular dynamic
simulations. In order to obtain physical support for the formation of this
base pair in solution, a set of experiments, including direct mutagenesis,
the site-specific substitution of chemical groups, kinetic studies, chemical
probing and magnesium-induced cleavage, were performed with the specific goal
of characterizing this trans Watson-Crick GU base pair in
an antigenomic HDV ribozyme. Both U23 and G28 can be
substituted for nucleotides that likely preserve some of the H-bond interactions
present before and after the cleavage step. The formation of the more stable trans
Watson-Crick base pair is shown to be a post-cleavage event, while a possibly
weaker trans Watson-Crick/Hoogsteen interaction seems to
form before the cleavage step. The formation of this unusually stable post-cleavage
base pair may act as a driving force on the chemical cleavage by favouring
the formation of a more stable ground state of the product-ribozyme complex.
To our knowledge, this represents the first demonstration of a potential stabilising
role of a post-cleavage conformational switch event in a ribozyme-catalyzed
reaction
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