39 research outputs found
Role of the flat-designed surface in improving the cyclic fatigue resistance of endodontic NiTi rotary instruments
The aim of this study was to investigate the role of the flat-designed surface in improving the resistance to cyclic fatigue by comparing heat-treated F-One (Fanta Dental, Shanghai, China) nickel-titanium (NiTi) rotary instruments and similar prototypes, differing only by the absence of the flat side. The null hypothesis was that there were no differences between the two tested instruments in terms of cyclic fatigue lifespan. A total of 40 new NiTi instruments (20 F-One and 20 prototypes) were tested in the present study. The instruments were rotated with the same speed (500 rpm) and torque (2 N) using an endodontic motor (Elements Motor, Kerr, Orange, CA, USA) in the same stainless steel, artificial canal (90° angle of curvature and 5 mm radius). A Wilcoxon-Mann-Whitney test was performed to assess the differences in terms of time to fracture and the length of the fractured segment between the flat- and non-flat-sided instruments. Significance was set at p = 0.05. The differences in terms of time to fracture between non-flat and flat were statistically significant (p < 0.001). In addition, the differences in terms of fractured segment length were statistically significant (p = 0.034). The results of this study highlight the importance of flat-sided design in increasing the cyclic fatigue lifespan of NiTi rotary instruments
BLUF Domain Function Does Not Require a Metastable Radical Intermediate State
BLUF
(blue light using flavin) domain proteins are an important
family of blue light-sensing proteins which control a wide variety
of functions in cells. The primary light-activated step in the BLUF
domain is not yet established. A number of experimental and theoretical
studies points to a role for photoinduced electron transfer (PET)
between a highly conserved tyrosine and the flavin chromophore to
form a radical intermediate state. Here we investigate the role of
PET in three different BLUF proteins, using ultrafast broadband transient
infrared spectroscopy. We characterize and identify infrared active
marker modes for excited and ground state species and use them to
record photochemical dynamics in the proteins. We also generate mutants
which unambiguously show PET and, through isotope labeling of the
protein and the chromophore, are able to assign modes characteristic
of both flavin and protein radical states. We find that these radical
intermediates are not observed in two of the three BLUF domains studied,
casting doubt on the importance of the formation of a population of
radical intermediates in the BLUF photocycle. Further, unnatural amino
acid mutagenesis is used to replace the conserved tyrosine with fluorotyrosines,
thus modifying the driving force for the proposed electron transfer
reaction; the rate changes observed are also not consistent with a
PET mechanism. Thus, while intermediates of PET reactions can be observed
in BLUF proteins they are not correlated with photoactivity, suggesting
that radical intermediates are not central to their operation. Alternative
nonradical pathways including a keto–enol tautomerization induced
by electronic excitation of the flavin ring are considered
Photoinduced Electron Transfer Facilitates Tautomerization of the Conserved Signaling Glutamine Side Chain in BLUF Protein Light Sensors
The BLUF domain (sensor of blue light using flavin adenine
dinucleotide)
from a bacterial photoreceptor protein AppA undergoes a cascade of
chemical transformations, including hydrogen bond rearrangements around
the flavin adenine dinucleotide (FAD) chromophore, in response to
light illumination. These transformations are initiated by photoinduced
electron and proton transfer from a tyrosine residue to the photoexcited
flavin which is assisted by a glutamine residue. According to the
recent studies, the proton-coupled electron transfer leads to formation
of a radical-pair intermediate Tyr•···FADH•
and a tautomeric EE form of glutamine in the ground electronic state.
This intermediate is a precursor of the light-induced state of the
BLUF photoreceptor implicated in biological signaling. In order to
describe evolution of the radical pair, we computed reaction pathways
on the ground state potential energy surface employing quantum-chemical
calculations in the DFT PBE0/cc-pVDZ approximation for a molecular
cluster mimicking the chromophore containing pocket of the AppA BLUF
protein. We found a minimum-energy pathway comprised of the following
consecutive reaction steps: (1) rotation of the imidic group of the
EE glutamine side chain around the Cγ–Cδ bond;
(2) flip of the OεH group and formation of the ZE form of the
glutamine side chain; and (3) biradical recombination via coupled
proton and electron transfer, leading to the ZZ form of the glutamine
side chain. The potential-energy barriers for stages 1–3 do
not exceed 9 kcal/mol. Energy barrier 3 describing the ZE to ZZ glutamine
tautomerization is significantly smaller in the BLUF model than in
isolated glutamine, since tautomerization in BLUF is facilitated by
electron transfer and radical recombination. Thus, our study shows
that tautomerization of the conserved glutamine is coupled to the
light-induced electron transfer process in BLUF and, thus, is a viable
candidate for the photoactivation mechanism which at present is very
much debated
Computational Characterization of Reaction Intermediates in the Photocycle of the Sensory Domain of the AppA Blue Light Photoreceptor
The AppA protein with the BLUF (blue light using flavin adenine dinucleotide) domain is a blue light photoreceptor that cycle between dark−adapted and light−induced functional states. We characterized possible reaction intermediates in the photocycle of AppA BLUF. Molecular dynamics (MD), quantum chemical and quantum mechanical−molecular mechanical (QM/MM) calculations were carried out to describe several stable structures of a molecular system modeling the protein. The coordinates of heavy atoms from the crystal structure (PDB code 2IYG) of the protein in the dark state served as starting point for 10 ns MD simulations. Representative MD frames were used in QM(B3LYP/cc−pVDZ)/MM(AMBER) calculations to locate minimum energy configurations of the model system. Vertical electronic excitation energies were estimated for the molecular clusters comprising the quantum subsystems of the QM/MM optimized structures using the SOS−CIS(D) quantum chemistry method. Computational results support the occurrence of photoreaction intermediates that are characterized by spectral absorption bands between those of the dark and light states. They agree with crystal structures of reaction intermediates (PDB code 2IYI) observed in the AppA BLUF domain. Transformations of the Gln63 side chain stimulated by photo−excitation and performed with the assistance of the chromophore and the Met106 side chain are responsible for these intermediate
Sonic vs Ultrasonic activation of sodium hypoclorite for root canal treatments. In vitro assessment of debris removal from main and lateral canals
Aim: Aim of this study is to evaluate the efficacy of two different sonic and ultrasonic devices in the elimination of debris from artificial main and accessory canals. Methodology: Two different irrigant activator devices were tested: The sonic handpiece Endo- Activator (Dentsply Maillefer, Baillagues, Switzerland) and the ultrasonic handpiece Ultra X (Eighteeth, Changzhou Sifary Medical Technology Co., Ltd, Changzhou City, China). Two groups of 18 artificial root canals were analyzed (n=36): Main and lateral canals were embedded in a transparent resin model. Canals were filled with organic paste to simulate the necrotic pulp tissues. With both devices, irrigation was performed using 5% sodium hypoclorite and two activation times of 30 seconds each. Sodium hypochlorite was replaced every 30 seconds. After a photographic exam, debris removal was evaluated by a software and assessed in terms of percentage of cleaned canal. Means and standard deviations were calculated and data were statistically analyzed with the Anova test. Results: Under the same experimental conditions (same canal, time and irrigant), both sonic and ultrasonic devices completely cleaned the main canal. On the contrary, a statistically significant difference was noted in the debridement of lateral canals, with ultrasonic device removing more debris than the sonic one (p<0.05). No tested device was able to remove all debris from accessory canals. Conclusions: The cordless ultrasonic handpiece Ultra X used with maximum power showed significantly greater efficacy in cleaning accessory canals when compared to the sonic EndoActivator
Genetically Encoded FRET-Sensor Based on Terbium Chelate and Red Fluorescent Protein for Detection of Caspase-3 Activity
This article describes the genetically encoded caspase-3 FRET-sensor based on the terbium-binding peptide, cleavable linker with caspase-3 recognition site, and red fluorescent protein TagRFP. The engineered construction performs two induction-resonance energy transfer processes: from tryptophan of the terbium-binding peptide to Tb3+ and from sensitized Tb3+ to acceptor—the chromophore of TagRFP. Long-lived terbium-sensitized emission (microseconds), pulse excitation source, and time-resolved detection were utilized to eliminate directly excited TagRFP fluorescence and background cellular autofluorescence, which lasts a fraction of nanosecond, and thus to improve sensitivity of analyses. Furthermore the technique facilitates selective detection of fluorescence, induced by uncleaved acceptor emission. For the first time it was shown that fluorescence resonance energy transfer between sensitized terbium and TagRFP in the engineered construction can be studied via detection of microsecond TagRFP fluorescence intensities. The lifetime and distance distribution between donor and acceptor were calculated using molecular dynamics simulation. Using this data, quantum yield of terbium ions with binding peptide was estimated