Intratubular penetration of endodontic sealers depends on the fluorophore used for CLSM assessment.

Root canal filling aims at eliminating empty spaces into the root canal system using biologically compatible materials. Three-dimensional root canal obturation must prevent or minimize the reinfection caused by microorganisms' leakage. This study aimed at evaluating whether fluorophore (Rhodamine or Fluo-3) influences the CLSM images of intratubular penetration of four endodontic sealers. Eighty bovine teeth were prepared using K files up to a size #70 and irrigated with 2.5% sodium hypochlorite. All roots were divided into eight groups (n = 10) according to the sealer and fluorophore used: AH Plus/Rhodamine, AH Plus/Fluo-3, Sealer Plus/Rhodamine, Sealer Plus/Fluo-3, Sealer Plus BC/Rhodamine, Sealer Plus BC/Fluo-3, Endosequence/Rhodamine, and Endosequence/Fluo-3. All roots were filled using cold lateral compaction technique. After 7 days, the roots were transversely sectioned, and three slices, one of each canal third, were obtained. Intratubular penetration was evaluated using CLSM. Sealer Plus BC/Rhodamine and Endosequence BC/Rhodamine presented higher intratubular penetration than AH Plus/Fluo-3 and Sealer Plus/Fluo-3 (p ˂ .05). The intragroup analysis showed similar intratubular penetration, regardless of the root third, except for the apical third in AH Plus/Fluo-3 and Sealer Plus BC/Fluo-3 groups. The type of fluorophore influences the calcium silicate sealers' tubular penetration but not of epoxy resin-based ones using CLSM. Bioceramic sealers should not be used associated with Rhodamine for CLSM evaluation. RESEARCH HIGHLIGHTS: The type of fluorophore influences the calcium silicate sealers' tubular penetration but not of epoxy resin-based ones when CLSM is used for assessment. Bioceramic sealers should not be used associated with Rhodamine.

A rapid response near-infrared ratiometric fluorescent probe for the real-time tracking of peroxynitrite for pathological diagnosis and therapeutic assessment in a rheumatoid arthritis model.

Peroxynitrite (ONOO-) is a potent bio-oxidant involved in many physiological and pathological processes; however, most of the pathological effects associated with ONOO-in vivo are still ambiguous. Herein, we designed and synthesized two near-infrared ratiometric fluorescent probes, Ratio-A and Ratio-B, for the detection and biological evaluation of ONOO-. The recognition unit diene in the probes could be specifically cleaved by ONOO- with a 94-fold enhancement in the ratiometric fluorescence signal. By imaging ONOO- in immune stimulated cells and acute inflammation mice model using Ratio-A, we investigated the fluctuations of ONOO- levels in a rheumatoid arthritis (RA) model of mice. Ratio-A could be applied for the effective imaging of RA and could rapidly evaluate the response of the RA treatment with methotrexate (MTX). Thus, Ratio-A can be considered as a promising tool for pathological diagnosis and the therapeutic assessment of a wide range of diseases including RA.

Removal of rhodamine 6G from synthetic effluents using Clitoria fairchildiana pods as low-cost biosorbent.

Many organic dye pollutants have been identified in rivers and lakes around the world, and concern is growing with them as they cause serious changes in the ecological balance of aquatic environments. One of these dyes is rhodamine R6G, which is very water-soluble and has a high corrosive power. Therefore, Clitoria fairchildiana (CF) pods were used as a biosorbent to remove R6G from synthetic dye effluents. CF was characterized by infrared spectroscopy, thermogravimetric analysis, x-ray diffraction, elemental analysis, Boehm titration, and zero charge point measurements. The influence of various factors, such as solution pH, contact time, adsorbent mass, and concentration of R6G, was studied using batch equilibrium experiments. The optimum contact time to reach equilibrium was found to be 15 min, while the optimum adsorbent dose was 8 g L-1. The maximum adsorption capacity of CF (73.84 mg g-1) was observed at pH 6.4 and 298.15 K. Adsorption kinetics followed a pseudo-second-order law, and the isotherm could be best fitted with a Liu model. The obtained thermodynamic parameters indicate that the adsorption of R6G is spontaneous and enthalpy-driven. We thus conclude that CF is an efficient, green, and readily available biosorbent for dye removal from wastewater.

A fluorescent hydrazone exchange probe of pyridoxal phosphate for the assessment of vitamin B6 status.

Abnormal vitamin B6 status, marked by deficient intracellular concentrations of pyridoxal phosphate (PLP), is classified as a direct biomarker based on its biomedical significance. However, there exist no direct methods for measuring vitamin B6 status in intact cells. Here we describe the development of a fluorogenic probe, RAB6, which shows remarkable selectivity for PLP among the B6 vitamers and other cellular aldehydes.

A robust and economical pulse-chase protocol to measure the turnover of HaloTag fusion proteins.

The self-labeling protein HaloTag has been used extensively to determine the localization and turnover of proteins of interest at the single-cell level. To this end, halogen-substituted alkanes attached to diverse fluorophores are commercially available that allow specific, irreversible labeling of HaloTag fusion proteins; however, measurement of protein of interest half-life by pulse-chase of HaloTag ligands is not widely employed because residual unbound ligand continues to label newly synthesized HaloTag fusions even after extensive washing. Excess unlabeled HaloTag ligand can be used as a blocker of undesired labeling, but this is not economical. In this study, we screened several inexpensive, low-molecular-weight haloalkanes as blocking agents in pulse-chase labeling experiments with the cell-permeable tetramethylrhodamine HaloTag ligand. We identified 7-bromoheptanol as a high-affinity, low-toxicity HaloTag-blocking agent that permits protein turnover measurements at both the cell population (by blotting) and single-cell (by imaging) levels. We show that the HaloTag pulse-chase approach is a nontoxic alternative to inhibition of protein synthesis with cycloheximide and extend protein turnover assays to long-lived proteins.

Virtual Combinatorial Library Design, Synthesis and In vitro Anticancer Assessment of -2-Amino-3-Cyanopyridine Derivatives.

AIM AND OBJECTIVE: For the development of new class of anticancer agents, a series of novel 2-amino-3-cyanopyridine derivatives were designed from virtual screening with Glide program by setting Topoisomerase II as the target. MATERIALS AND METHODS: The top ranked ten molecules from the virtual screening were synthesized by microwave assisted technique and investigated for their cytotoxic activity against MCF-7 and A- 549 cell lines by using sulforhodamine B assay method. RESULTS: The most active compound 2-amino-4-(3,5-dibromo-4-hydroxyphenyl)-6-(2,4- dichlorophenyl) nicotinonitrile (CG-5) showed significant cytotoxic profile with (LC50 = 97.1, TGI = 29.9 and GI50 = <0.1 µM) in MCF-7 and (LC50= 93.0, TGI= 50.0 and GI50= <7 µM) in A-549 cell lines. A molecular docking study was performed to explore the binding interaction of CG-5with the active site of Topoisomerase II. CONCLUSION: It can be concluded that halogen substituent pyridine ring was benefit for cytotoxicity.

A low-cost method for visible fluorescence imaging.

A wide variety of crystallization solutions are screened to establish conditions that promote the growth of a diffraction-quality crystal. Screening these conditions requires the assessment of many crystallization plates for the presence of crystals. Automated systems for screening and imaging are very expensive. A simple approach to imaging trace fluorescently labeled protein crystals in crystallization plates has been devised, and can be implemented at a cost as low as $50. The proteins ß-lactoglobulin B, trypsin and purified concanavalin A (ConA) were trace fluorescently labeled using three different fluorescent probes: Cascade Yellow (CY), Carboxyrhodamine 6G (CR) and Pacific Blue (PB). A crystallization screening plate was set up using ß-lactoglobulin B labeled with CR, trypsin labeled with CY, ConA labeled with each probe, and a mixture consisting of 50% PB-labeled ConA and 50% CR-labeled ConA. The wells of these plates were imaged using a commercially available macro-imaging lens attachment for smart devices that have a camera. Several types of macro lens attachments were tested with smartphones and tablets. Images with the highest quality were obtained with an iPhone 6S and an AUKEY Ora 10× macro lens. Depending upon the fluorescent probe employed and its Stokes shift, a light-emitting diode or a laser diode was used for excitation. An emission filter was used for the imaging of protein crystals labeled with CR and crystals with two-color fluorescence. This approach can also be used with microscopy systems commonly used to observe crystallization plates.

Assessment of dually labelled PEGylated liposomes transplacental passage and placental penetration using a combination of two ex-vivo human models: the dually perfused placenta and the suspended villous explants.

Uptake and passage of nanocarriers through the placenta are critical information to develop new therapeutic approaches during pregnancy. In order to assess nanocarriers transplacental passage and penetration into the placenta, we studied and optimized two ex-vivo human models: the dually perfused placenta and the placenta explants. Doubly labelled PEGylated liposomes were used as models to provide data on the penetration and transplacental passage of drugs and liposomes. A HPLC method was set-up to quantify both carboxyfluorescein and lipid-rhodamine. Transplacental passage was then quantified using HPLC and placental penetration was assessed using spinning disk microscopy. We found a similar transplacental passage rate for both free and encapsulated carboxyfluorescein as well as a homogeneous fluorescence intensity in the outer cell layer of the placental villous, the syncytiotrophoblast, and the mesenchyma. Besides, liposome-rhodamine was not detected in the fetal circulation. The absence of transplacental passage of PEGylated liposomes is also supported by their detection in the sole syncytiotrophoblast. The combination of two ex-vivo models and the monitoring of both the drug and the carrier provided consistent and complementary information. Overall, we suggest combining the perfused human placenta and the human explants villous models to evaluate nanocarriers designed for treatments during pregnancy.

Remote in vivo stress assessment of aquatic animals with microencapsulated biomarkers for environmental monitoring.

Remote in vivo scanning of physiological parameters is a major trend in the development of new tools for the fields of medicine and animal physiology. For this purpose, a variety of implantable optical micro- and nanosensors have been designed for potential medical applications. At the same time, the important area of environmental sciences has been neglected in the development of techniques for remote physiological measurements. In the field of environmental monitoring and related research, there is a constant demand for new effective and quick techniques for the stress assessment of aquatic animals, and the development of proper methods for remote physiological measurements in vivo may significantly increase the precision and throughput of analyses in this field. In the present study, we apply pH-sensitive microencapsulated biomarkers to remotely monitor the pH of haemolymph in vivo in endemic amphipods from Lake Baikal, and we compare the suitability of this technique for stress assessment with that of common biochemical methods. For the first time, we demonstrate the possibility of remotely detecting a change in a physiological parameter in an aquatic organism under ecologically relevant stressful conditions and show the applicability of techniques using microencapsulated biomarkers for remote physiological measurements in environmental monitoring.

At-line nanofractionation with parallel mass spectrometry and bioactivity assessment for the rapid screening of thrombin and factor Xa inhibitors in snake venoms.

Snake venoms comprise complex mixtures of peptides and proteins causing modulation of diverse physiological functions upon envenomation of the prey organism. The components of snake venoms are studied as research tools and as potential drug candidates. However, the bioactivity determination with subsequent identification and purification of the bioactive compounds is a demanding and often laborious effort involving different analytical and pharmacological techniques. This study describes the development and optimization of an integrated analytical approach for activity profiling and identification of venom constituents targeting the cardiovascular system, thrombin and factor Xa enzymes in particular. The approach developed encompasses reversed-phase liquid chromatography (RPLC) analysis of a crude snake venom with parallel mass spectrometry (MS) and bioactivity analysis. The analytical and pharmacological part in this approach are linked using at-line nanofractionation. This implies that the bioactivity is assessed after high-resolution nanofractionation (6 s/well) onto high-density 384-well microtiter plates and subsequent freeze drying of the plates. The nanofractionation and bioassay conditions were optimized for maintaining LC resolution and achieving good bioassay sensitivity. The developed integrated analytical approach was successfully applied for the fast screening of snake venoms for compounds affecting thrombin and factor Xa activity. Parallel accurate MS measurements provided correlation of observed bioactivity to peptide/protein masses. This resulted in identification of a few interesting peptides with activity towards the drug target factor Xa from a screening campaign involving venoms of 39 snake species. Besides this, many positive protease activity peaks were observed in most venoms analysed. These protease fingerprint chromatograms were found to be similar for evolutionary closely related species and as such might serve as generic snake protease bioactivity fingerprints in biological studies on venoms.

Action-FRET: probing the molecular conformation of mass-selected gas-phase peptides with Förster resonance energy transfer detected by acceptor-specific fragmentation.

The use of Förster resonance energy transfer (FRET) as a probe of the structure of biological molecules through fluorescence measurements in solution is well-attested. The transposition of this technique to the gas phase is appealing since it opens the perspective of combining the structural accuracy of FRET with the specificity and selectivity of mass spectrometry (MS). Here, we report FRET results on gas-phase polyalanine ions obtained by measuring FRET efficiency through specific photofragmentation rather than fluorescence. The structural sensitivity of the method was tested using commercially available chromophores (QSY 7 and carboxyrhodamine 575) grafted on a series of small, alanine-based peptides of differing sizes. The photofragmentation of these systems was investigated through action spectroscopy, and their conformations were probed using ion mobility spectrometry (IMS) and Monte Carlo minimization (MCM) simulations. We show that specific excitation of the donor chromophore results in the observation of fragments that are specific to the electronic excitation of the acceptor chromophore. This shows that energy transfer took place between the two chromophores and hence that the action-FRET technique can be used as a new and sensitive probe of the structure of gas-phase biomolecules, which opens perspectives as a new tool in structural biology.

Time-dependent approach to spin-vibronic coupling: implementation and assessment.

In this work, we present the generalization of a time-dependent method for the calculation of intersystem crossing (ISC) rates in the Condon approximation. When ISC takes place between electronic states with the same orbital type, i.e., when the transition is forbidden according to the El-Sayed rules, it is necessary to go beyond the Condon approximation. Similar to the Herzberg-Teller expansion of the vibronic interaction, the electronic spin-orbit matrix elements are assumed to depend linearly on the nuclear coordinates. The ISC rate is then a sum of three contributions: a direct, mixed direct-vibronic, and vibronic term. The method, presented in this work, is based on the generating function formalism and the multi-mode harmonic oscillator approximation. In addition to the zero-temperature case, we implemented formulae for finite-temperature conditions assuming a Boltzmann population of vibrational levels in the initial state. Tests have been carried out for a variety of molecules for which literature data were available. We computed vibronic one-photon spectra of free-base porphyrin and free-base chlorin and calculated ISC rates for xanthone, thioxanthone, thionine, as well as free-base porphyrin and found excellent agreement with previous results. Quantitative rates for triplet formation in rhodamine A have been determined theoretically for the first time. We find the S1↝ T2 channel to be the major source of triplet rhodamine formation in the gas phase.

Integrated multiplatform method for in vitro quantitative assessment of cellular uptake for fluorescent polymer nanoparticles.

Studies of cellular internalization of nanoparticles (NPs) play a paramount role for the design of efficient drug delivery systems, but so far they lack a robust experimental technique able to quantify the NP uptake in terms of number of NPs internalized in each cell. In this work we propose a novel method which provides a quantitative evaluation of fluorescent NP uptake by combining flow cytometry and plate fluorimetry with measurements of number of cells. Single cell fluorescence signals measured by flow cytometry were associated with the number of internalized NPs, exploiting the observed linearity between average flow cytometric fluorescence and overall plate fluorimeter measures, and previous calibration of the microplate reader with serial dilutions of NPs. This precise calibration has been made possible by using biocompatible fluorescent NPs in the range of 20-300 nm with a narrow particle size distribution, functionalized with a covalently bonded dye, Rhodamine B, and synthesized via emulsion free-radical polymerization. We report the absolute number of NPs internalized in mouse mammary tumor cells (4T1) as a function of time for different NP dimensions and surface charges and at several exposure concentrations. The obtained results indicate that 4T1 cells incorporated 10(3)-10(4) polymer NPs in a short time, reaching an intracellular concentration 15 times higher than the external one.

Assessment of labile plasma iron in patients who undergo hematopoietic stem cell transplantation.

Body iron disorders have been reported after myeloablative conditioning in patients undergoing hematopoietic stem cell transplantation (HSCT). There is a concern that labile plasma iron (LPI), the redox-active form of iron, can be involved in the occurrence of toxicity and other complications commonly observed in the early post-HSCT period. In order to better understand the LPI kinetics and its determinants and implications, we undertook sequential LPI determinations before and after conditioning until engraftment in 25 auto-HSCT patients. Increased LPI was present in only 5 patients before starting conditioning. Shortly after conditioning, LPI levels were increased in 23 patients, with peak at day 0, returning to normal range upon engraftment in 21 patients. Overall, LPI levels correlated weakly with serum ferritin and more strongly with transferrin saturation; however, both parameters were apparently not applicable as surrogate markers for increased LPI. Although this was a small cohort, logistic regression suggested that baseline LPI levels could predict occurrence of grade III or IV toxicity. In conclusion, LPI kinetics is influenced by aplasia following conditioning and engraftment. Measuring LPI before starting conditioning can offer an opportunity to predict toxicity and, perhaps, the need for chelation therapy.

Membrane destabilization by monomeric hIAPP observed by imaging fluorescence correlation spectroscopy.

Monomeric hIAPP significantly destabilizes both model and live cell membranes by increasing membrane fluidity. This interaction with membranes happens via carpet formation followed by lipid extraction in a concentration dependent manner and thus we propose that hIAPP aggregation prior to membrane interaction may not be necessary for its cytotoxicity.

Fluorogenic probing of specific recognitions between sugar ligands and glycoprotein receptors on cancer cells by an economic graphene nanocomposite.

Economical nanocomposites based on π-stacking of N-acetyl glycosyl rhodamine B to graphene oxide (GO) are simply prepared. These "sweet" GO-materials are proven to be admirable for the fluorogenic recognition of specific intercellular sugar-based ligand-glycoprotein receptor interactions of interest.

Controllable synthesis of recyclable core-shell γ-Fe2O3@SnO2 hollow nanoparticles with enhanced photocatalytic and gas sensing properties.

Composite materials containing different components with well-defined structures may cooperatively enhance their performance and extend their applications. In this work, core-shell γ-Fe2O3@SnO2 hollow nanoparticles (NPs) were synthesized by a low-cost and environmentally friendly seed-mediated hydrothermal method. Firstly, the γ-Fe2O3 hollow NPs were synthesized by a template-free method. Then they were used as the cores for the growth of SnO2 shells. The thickness of the shell can be simply tailored by controlling the reaction time. Various techniques, including SEM, XRD, TEM and HRTEM, were employed to investigate the morphology, structure and formation process of the special core-shell hollow structures. The combination of magnetic semiconductor (γ-Fe2O3) and wide band-gap semiconductor (SnO2) endowed them with great potential to be used as recyclable photocatalysts. Experiments on photo-degradation of Rhodamin B (RhB) dye in the presence of the samples showed that the hybrid structures possessed higher photocatalytic activities than the monomer structures of SnO2 and γ-Fe2O3 materials indicating a strong coupling enhancement effect between the wide and narrow band-gap semiconductors. Moreover, the gas sensing tests of the γ-Fe2O3@SnO2 hollow NPs revealed that the samples exhibited fast response and recovery rates, which enable them to be promising materials for gas sensors.

NaYF4:Yb3+/Er3+ nanoparticle-based upconversion luminescence resonance energy transfer sensor for mercury(II) quantification.

Upconversion luminescence is an anti-Stokes' emission process by converting long wavelength near-infrared (NIR, 980 nm) irradiation into shorter wavelength visible light emission, which demonstrates many advantages including no autofluorescence, low damage to samples, no photobleaching, and high sensitivity. Based on the Rhodamine B thiolactone (RBT) functionalized NaYF(4):15%Yb(3+),5%Er(3+) (UCNPs@RBT) nanocomposites, an ultrasensitive, selective, and rapid upconversion luminescence resonance energy transfer (UC-LRET) sensor has been developed for the detection of mercury ions (Hg(2+)) in water. Upconverting luminescence resonance energy transfer from the UCNPs to the RBT derivates occurs after the addition of Hg(2+) ions into the UCNPs@RBT colloidal solution. This UC-LRET recognition of Hg(2+) can be finished within 1 min and other cations have no influence on the detection of mercury ions. This newly developed sensor demonstrates high selectivity toward the mercury ions and enables ultrasensitive and rapid detection of mercury ions in water in the range of 5 nM to 10 µM with a 3σ limit of detection of 3.7 nM. This sensor can be used for a naked-eye detection of Hg(2+) ions via its green upconverting luminescence response under the infrared excitation (980 nm) with the merit of no autofluorescence interference and good photostability. In addition, by dipping the hydrogel of UCNPs@RBT nanocomposites onto the filter paper, a highly selective and convenient luminescent paper sensor for Hg(2+) ions was also developed.

DHR123: an alternative probe for assessment of ROS in human spermatozoa.

The aim of this study was to assess the potential of dihydrorhodamine 123 (DHR123) to measure oxidative stress produced by human spermatozoa. The results were compared with 2', 7'-dichlorodihydrofluorescein diacetate (DCFH-DA) that is routinely used for assessment of H(2)O(2) produced by spermatozoa. Fluorescence intensity and percentage R123 and DCF positive sperm were measured by flow cytometry. The optimal condition for assessment of reactive oxygen species (ROS) produced by sperm with DHR123 was 0.05 µM for 1 million sperm per ml for 20 minutes. The results of ROS measurement by DHR123 showed a significant correlation (r= +0.818, P<0.001) with DCFH-DA staining. Immunofluorescence of sperm stained with DHR123 revealed ROS production in the sperm mid-piece. In addition a significant correlation was observed between oxidant production assessed by DHR123 and semen parameters. Therefore, DHR123 may be considered a suitable probe for estimating oxidants produced by human spermatozoa, and can present heterogeneity in oxidant production between different samples.