Carbon Dot Functionalized Papers for the Selective Detection of 2,4,6-Trinitrophenol in Aqueous Solutions

The development of probes for the testing of the carcinogenic pollutant 2,4,6-trinitrophenol (TNP) is of great importance for environmental protection and human health. In this paper, a new rapid and sensitive fluorescence detection method based on carbon dots (CDs) was designed for the detection of TNP. The CDs were synthesized by simple pyrolysis using citric acid as raw material and characterized by various advanced techniques. The addition of TNP caused a significant turn off in the fluorescence of the CDs. The fluorescence quench intensity and TNP concentration exhibited a good linear correlation in the range of 0–80 μM with a minimum detection limit of 0.48 μM and a related coefficient of 0.994. The analytical method was applied to the determination of trace TNP in river water and tap water with recoveries in the range of 98%–110% and relative standard deviations less than 5%. Importantly, carbon dots functionalized papers (CDFPs) were prepared using the synthesized CDs and successfully applied to the determination of TNP in aqueous solutions, demonstrating the promising application of the method.

Carvacrol Promotes Cisplatin-induced Apoptosis of Triple-negative Breast Cancer Through TRPM7 Channels

Triple-negative breast cancer (TNBC) is the most dangerous type of breast cancer. Cisplatin is a chemotherapy agent for solid tumors treatments, but it is subjected to drug resistance. A natural compound, carvacrol, showed anti-cancer potential for TNBC and can be a cisplatin sensitizer. Carvacrol inhibits the transient receptor potential melastatin-like 7 channel (TRPM7), which plays a critical role in cancer apoptosis. In this study, we proposed that carvacrol can promote cisplatin-induced apoptosis of TNBC through TRPM7 channels. We tested this hypothesis using five TNBC cell lines. Cell viability and apoptosis were determined using the MTT and Bax (Bcl-2-associated X protein) ELISA respectively. 4T1 /BALB/c mice model was used to test the effect of carvacrol on cisplatin treatment. TRPM7 and Bax expression in the tumors were determined using western blotting. We also tested the effect of TRPM7 knockdown and TRPM7 inhibitor, 2-APB, on carvacrol actions in MDA-MB-231. The TRPM7 channel inhibition by carvacrol was confirmed using patch-clamp and Fura-2-based fluorescence quench assay. Results showed that carvacrol enhanced the suppression of cisplatin and cisplatin-induced apoptosis in all five TNBC cancer cell lines. Carvacrol improved cisplatin treatment in mice model by promoting cisplatin-induced apoptosis in tumors, but did not affect TRPM7 in tumors. Both knockdown and 2-APB inhibition of TRPM7 channels eliminated the effects of carvacrol and 2-APB showed a similar effect as carvacrol. Our study demonstrated that carvacrol is a cisplatin sensitizer in TNBC chemotherapy and TRPM7 channels might play a role in the synergistic effect of carvacrol on cisplatin.

An arrestin-1 surface opposite of its interface with photoactivated rhodopsin engages with enolase-1

Arrestin-1 is the arrestin family member responsible for inactivation of the G protein–coupled receptor rhodopsin in photoreceptors. Arrestin-1 is also well-known to interact with additional protein partners and to affect other signaling cascades beyond phototransduction. In this study, we investigated one of these alternative arrestin-1 binding partners, the glycolysis enzyme enolase-1, to map the molecular contact sites between these two proteins and investigate how the binding of arrestin-1 affects the catalytic activity of enolase-1. Using fluorescence quench protection of strategically placed fluorophores on the arrestin-1 surface, we observed that arrestin-1 primarily engages enolase-1 along a surface that is opposite of the side of arrestin-1 that binds photoactivated rhodopsin. Using this information, we developed a molecular model of the arrestin-1–enolase-1 complex, which was validated by targeted substitutions of charge-pair interactions. Finally, we identified the likely source of arrestin's modulation of enolase-1 catalysis, showing that selective substitution of two amino acids in arrestin-1 can completely remove its effect on enolase-1 activity while still remaining bound to enolase-1. These findings open up opportunities for examining the functional effects of arrestin-1 on enolase-1 activity in photoreceptors and their surrounding cells.

G-quadruplex ligands exhibit differential G-tetrad selectivity

Site-specific fluorescence quench assay allows for targeted G-quadruplex equilibrium binding measurements to investigate G-tetrad selective ligands.

Charge Transfer and Energy Transfer between CdSe Semiconductor Nano Crystals and Polyaniline Molecule

CdSe semiconductor nano crystals (NCs) and Polyaniline (PAni) are mixed uniformly to prepare CdSe NCs/PAni complex. PAni can quench the fluorescent signal of CdSe NCs. The fluorescent intensity of CdSe NCs/PAni complex is related to the size of CdSe NCs and concentration of PAni. Ultraviolet visual (UV-Vis) absorption spectra and fluorescence spectra are employed to analysis the quenching phenomenon. The mechanism of fluorescence quench is dependent on two factors: on one hand, the FÖrster resonance energy transfer conducts from CdSe to PAni; on the other hand, PAni can intercept the electron charge of CdSe and lead to the interruption of radiative recombination.

Mechanisms of resveratrol-induced changes in cytosolic free calcium ion concentrations and cell viability in OC2 human oral cancer cells

Resveratrol is a natural compound that affects cellular calcium (Ca2+) homeostasis and viability in different cells. This study examined the effect of resveratrol on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability in OC2 human oral cancer cells. The Ca2+-sensitive fluorescent dye fura-2 was used to measure [Ca2+]i, and water-soluble tetrazolium-1 was used to measure viability. Resveratrol evoked concentration-dependent increase in [Ca2+]i. The response was reduced by removing extracellular Ca2+. Resveratrol also caused manganese-induced fura-2 fluorescence quench. Resveratrol-evoked Ca2+ entry was inhibited by nifedipine and the protein kinase C (PKC) inhibitor GF109203X but was not altered by econazole, SKF96365, and the PKC activator phorbol 12-myristate 13 acetate. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor 2,5-di- tert-butylhydroquinone (BHQ) abolished resveratrol-evoked [Ca2+]i rise. Conversely, treatment with resveratrol inhibited BHQ-evoked [Ca2+]i rise. Inhibition of phospholipase C (PLC) with U73122 abolished resveratrol-evoked [Ca2+]i rise. At 20–100 μM, resveratrol decreased cell viability, which was not affected by chelating cytosolic Ca2+with 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-acetoxymethyl ester. Annexin V-fluorescein isothiocyanate staining data suggest that resveratrol at 20–40 μM induced apoptosis in a concentration-dependent manner. Collectively, in OC2 cells, resveratrol induced [Ca2+]i rise by evoking PLC-dependent Ca2+ release from the endoplasmic reticulum and by causing Ca2+ entry via nifedipine-sensitive, PKC-regulated mechanisms. Resveratrol also caused Ca2+-independent apoptosis.

Development and Optimization of a High-Throughput Bioassay for TRPM7 Ion Channel Inhibitors

TRPM7 is a ubiquitously expressed and constitutively active divalent cation channel essential for cell survival and proliferation because it provides a mechanism for Mg2+ entry. This makes the channel an attractive target for proliferative diseases. In keeping with its role in Mg2+ homeostasis, TRPM7 is inhibited by intracellular Mg2+ and Mg-ATP. TRPM7 has been implicated in anoxia-mediated cell death following brain ischemia. Despite its critical role in ischemic cell death and cell proliferation, there are no reports of selective inhibitors of TRPM7. The authors developed and optimized a fluorescent dye-based bioassay measuring the fluorescence quench of fura-2 by TRPM7-mediated Mn2+ influx in HEK293 cells that stably overexpress TRPM7. The following bioassay conditions were evaluated: (a) cell density, (b) dye loading conditions, (c) bioassay temperature, (d) concentration of the fura-2 quenching agent Mn2+, and (e) concentration of vehicle solvent. The bioassay was validated by measuring the effects of the known (nonselective) inhibitor 2-APB and La3+ on Mn2+ influx, and furthermore, the performance of the assay was evaluated by screening a subset of a marine bacteria-derived extract library. The quality of the bioassay window is excellent based on an established statistical parameter used to evaluate high-throughput screening window quality (Z and Z′ factors ≥0.5).

A Robust Screen for Inhibitors and Enhancers of Phosphoinositide-3 Kinase (PI3K) Activities by Ratiometric Fluorescence Superquenching

Aberrant regulation of phosphoinositide 3-kinase (PI3K) activity is implicated in various diseases such as cancer and diabetes. Thus, high-throughput screening (HTS) of small-molecule inhibitors for PI3 kinases is an appealing strategy for drug development. Despite the attractiveness of lipid kinases as drug targets, screening for inhibitors for PI3K activities has been hampered by limited assay formats adaptable for HTS. The authors describe a homogeneous, direct, and nonradioactive assay for highly sensitive detection of PI3Kα, β, δ, and γ activities, which is suitable for HTS. The assay is based on fluorescence superquenching of a conjugated polymer upon metal-ion-mediated association of phosphorylated and dye-labeled substrates. As a result of phosphorylation, quencher and polymer are brought into proximity, and fluorescent energy transfer occurs. This event can be monitored as either fluorescence quench of the polymer or as enhanced emission from the quencher. Ratiometric analysis of the wavelengths eliminates interferences from autofluorescing compounds, which are present in HTS libraries. The platform has been adapted for the 384-well microplate format and delivers Z factors of > 0.6 at substrate conversions as low as 7%. Using this assay platform, several unreported inhibitors and activators of PI3Ks were identified in an 84- compound screen.