Comparative investigation into the anticancer activity of analogs of marine coelenterazine and coelenteramine.

Cancer is still one of the most challenging diseases to treat, making the pursuit for novel molecules with potential anticancer activity an important research topic. Herein, we have performed a comparative investigation into the anticancer activity of analogs of marine coelenterazine and coelenteramine. The former is a well-known bioluminescent substrate, while the latter is a metabolic product of the resulting bioluminescent reaction. While both types of analogs showed anticancer activity toward lung and gastric cancer cell lines, we have obtained data that highlight relevant differences between the activity of these two types of compounds. More specifically, we observed relevant differences in structure-activity relationships between these types of compounds. Also, coelenteramine analogs showed time-dependent activity, while coelenterazine-based compounds usually present time-independent activity. Coelenterazine analogs also appear to be relatively safer toward noncancer cells than coelenteramine analogs. There was also seen a correlation between the activity of the coelenterazine-based compounds and their light-emission properties. Thus, these results further indicate the potential of the marine coelenterazine chemi-/bioluminescent system as a source of new molecules with anticancer activity, while providing more insight into their modes of action.

Copper(II)-Doped Carbon Dots as Catalyst for Ozone Degradation of Textile Dyes.

A catalytic ozonation advanced oxidation process (AOP) with a copper(II)-doped carbon dot as catalyst, Cu-CD (using L-cysteine and polyethylene glycol (PEG) as precursors and passivation agents), was developed for textile wastewater treatment (T = 25 °C and pH = 7). Four dyes were analyzed­Methyl Orange (MO), Orange II sodium salt (O-II), Reactive Black 5 (RB-5) and Remazol Brilliant Blue R (RBB-R), as well as a real effluent from the dying and printing industry. The Cu-CD, with marked catalytic ozonation properties, was successfully synthesized by one-pot hydrothermal procedure with a size of 4.0 nm, a charge of −3.7 mV and a fluorescent quantum yield of 31%. The discoloration of the aqueous dye solutions followed an apparent first-order kinetics with the following rate constants (kap in min−1): MO, 0.210; O-II, 0.133; RB-5, 0.177; RBB-R, 0.086. In the presence of Cu-CD, the following apparent first-order rate constants were obtained (kapc in min−1) with the corresponding increase in the rate constant without catalyst (%Inc): MO, 1.184 (464%); O-II, 1.002 (653%); RB-5, 0.709 (301%); RBB-R, 0.230 (167%). The presence of sodium chloride (at a concentration of 50 g/L) resulted in a marked increase of the discoloration rate of the dye solution due to generation of other radicals, such as chlorine and chlorine oxide, resulting from the reaction of ozone and chloride. Taking into consideration that the real textile effluent under research has a high carbonate concentration (>356 mg/L), which inhibits ozone decomposition, the discoloration first-order rate constants without and with Cu-CD (kap = 0.0097 min−1 and kapc = 0.012 min−1 (%Inc = 24%), respectively) were relatively small. Apparently, the Cu-CD, the surface of which is covered by a soft and highly hydrated caramelized PEG coating, accelerates the ozone decomposition and dye adsorption, increasing its degradation.

Chemical Composition, Bioactive Compounds, and Antioxidant Activity of Two Wild Edible Mushrooms Armillaria mellea and Macrolepiota procera from Two Countries (Morocco and Portugal).

The present study aimed to investigate the chemical composition, bioactive compounds, and antioxidant activity of two wild edible mushrooms, the honey fungus (Armillaria mellea) and the parasol mushroom (Macrolepiota procera), collected from Northern Morocco (MA) and Portugal (PT). Those species were chosen due to their edibility, nutraceutical, and medicinal properties. Bioactive compounds (ascorbic acid, tannin, total phenolic, total flavonoid, ß-carotene, and lycopene) and their antioxidant activity were determined by spectrophotometric methods. Herein, the fruiting body of the samples revealed a significantly higher amount of bioactive compounds, and values varied between the Moroccan and the Portuguese ones. Methanolic extracts shown a strong antioxidant capacity: Using DPPH free radical-scavenging activity radicals (IC50 1.06-1.32 mg/mL); inhibition of ß-carotene bleaching radicals (IC50 0.09-0.53 mg/mL); and, reducing power radicals (IC50 0.52-1.11 mg/mL). The mushroom species with the highest antioxidant capacity was A. mellea from MA. Chemical composition was analyzed by GC-MS and LC-MS methodologies. GC-MS analysis showed that the most abundant biomolecules group was sugar compositions in the four samples (62.90%, 48.93%, 59.00%, and 53.71%) and the main components were galactitol 16.74%, petroselinic acid 19.83%, d-galactose 38.43%, and glycerol 24.43% in A. mellea (MA), A. mellea (PT), M. procera (MA), and M. procera (PT), respectively. LC-MS analysis of individual phenolic compounds revealed that vanillic acid (198.40 ± 2.82 µg/g dry weight (dw) and cinnamic acid (155.20 ± 0.97 µg/g dw) were the main compounds detected in A. mellea, while protocatechuic acid (92.52 ± 0.45 and 125.50 ± 0.89 µg/g dw) was predominated in M. procera for MA and PT samples, respectively. In general, the results of this comparative study demonstrate that the geographic and climatic conditions of the collection site can influence biomolecule compounds and antioxidant properties of wild mushrooms. This study contributes to the elaboration of nutritional, nutraceutical, and pharmaceutical databases of the worldwide consumed mushrooms.

Hypochlorite fluorescence sensing by phenylboronic acid-alizarin adduct based carbon dots.

The selective fluorescence sensing of hypochlorite (ClO-) was achieved at pH 7.4 by a simple analytical procedure through the fluorescence quenching of autoclave synthesized carbon dots (CDs), which used as precursor an adduct formed between 3-aminophenylboronic acid (APBA) and alizarin red S (ARS). The use of this adduct allowed the preparation of CDs with a red shifted emission (560 nm) and excitation in the visible range (490 nm). Quantification of hypochlorite was achieved at physiological pH (pH 7.4) in aqueous solutions by fluorescence quenching with a linearity range of 0-200 µM (limit of detection of 4.47 µM, and limit of quantification of 13.41 µM). The selectivity of hypochlorite sensing was confirmed by comparison with other potential analytes, such as glucose, fructose and hydrogen peroxide. Finally, the validity of the proposed assay was further demonstrated by performing recovery assays in different matrices. Thus, this CDs allows the fluorescent sensing of ClO- with spectral properties more suitable for in vitro/in vivo applications.

Mechanistic Insight into the Chemiluminescent Decomposition of Cypridina Dioxetanone and the Chemiluminescent, Fluorescent Properties of the Light Emitter of Cypridina Bioluminescence.

Cypridina bioluminescence has been increasingly used in bioimaging, bioanalysis, and biomedicine, due to high quantum yield and high signal-to-noise ratio. However, there is still no consensus regarding different aspects of the chemiluminescent mechanism of this system, which impairs the development of new applications. Herein, we have used a theoretical DFT and TD-DFT approach to (i) determine the identity of the dioxetanone species responsible for efficient chemiexcitation and (ii) identify the bioluminescent emitter and determine if light-emission occurs from the fluorescent or chemiluminescent state. Our results demonstrate that upon oxygenation of the imidazopyrazinone scaffold, a dioxetanone with a neutral amide group and a cationic guanidinopropyl group is formed. This species is efficiently chemiexcited (with no obvious charge transfer step) to the corresponding oxyluciferin with a neutral amide and cationic guanidinopropyl groups. After the "dark" chemiluminescent state, this oxyluciferin species is converted into a bright blue-emitting fluorescent state.

3-Hydroxyphenylboronic Acid-Based Carbon Dot Sensors for Fructose Sensing.

The selective fluorescence sensing of fructose was achieved by fluorescence quenching of the emission of hydrothermal-synthesized carbon quantum dots prepared by 3-hydroxyphenylboronic acid. Quantification of fructose was possible in aqueous solutions with pH of 9 (Limit of Detection LOD and Limit of Quantification LOQ of 2.04 and 6.12 mM), by quenching of the emission at 376 nm and excitation ~380 nm with a linearity range of 0-150 mM. A Stern-Volmer constant (KSV) of 2.11 × 10-2 mM-1 was obtained, while a fluorescent quantum yield of 31% was calculated. The sensitivity of this assay towards fructose was confirmed by comparison with other sugars (such as glucose, sucrose and lactose). Finally, the validity of the proposed assays was further demonstrated by performing recovery assays in different matrixes. Graphical Abstract.

Theoretical analysis of the binding of potential inhibitors to protein kinases MK2 and MK3.

MK2 (or MAPKAPK2) was already known for its role in the inflammatory response, however recent studies indicate the involvement of this protein kinase in the DNA damage response mechanism. Within its kinase family the enzyme MK3 shows the highest identity with MK2. Here we report a theoretical study on the binding of two molecules, 05B and P4O, to the proteins MK2 and MK3. The data here obtained may shed light on the contribution of individual residues and binding site water molecules for the binding of potential inhibitors to these two kinases.

A theoretical study of the UV absorption of 4-methylbenzylidene camphor: from the UVB to the UVA region.

In this study, a theoretical approach was used to study the UV absorption of the UVB filter, 4-methylbenzylidene camphor. The main objective of this work was to design new UVA filters based on this rather photo-stable compound, so that photo-degradation in this UV region can be avoided without the use of other molecules. This objective was achieved by the simultaneous addition of two appropriate substituents, which led to red-shifts of up to 0.69 eV while maintaining appreciable oscillator strength. Also, useful structure-energy relationships were derived, which allow for the development of more UVA filters based on 4-methylbenzylidene camphor.

Oxyluciferin photoacidity: the missing element for solving the keto-enol mystery?

The oxyluciferin family of fluorophores has been receiving much attention from the research community and several systematic studies have been performed in order to gain more insight regarding their photophysical properties and photoprotolytic cycles. In this minireview, we summarize the knowledge obtained so far and define several possible lines for future research. More importantly, we analyze the impact of the discoveries on the firefly bioluminescence phenomenon made so far and explain how they re-open again the discussion regarding the identity (keto or enol species) of the bioluminophore.

Study on the effects of intermolecular interactions on firefly multicolor bioluminescence.

Firefly luciferase exhibits a color-tuning mechanism based on pH-induced changes in the structure of the active site. These changes increase the polarity of the active site, and thus modulate the intermolecular interactions between the light emitter and active site molecules. In this study, the effects exerted by adenosine monophosphate (AMP), water molecules, and amino acids of Luciola cruciata luciferase active site on the emission wavelength of oxyluciferin were assessed by TD-DFT calculations. The redshift results mainly from decreased interaction of oxyluciferin with AMP and increased interaction of the emitter with a water molecule and Phe249. Breaking of a hydrogen bond between the benzothiazole oxygen atom with formation of a similar bond to the thiazolone oxygen atom is also instrumental.

Theoretical modulation of the color of light emitted by firefly oxyluciferin.

One of the major mysteries regarding firefly bioluminescence is its pH-dependent multicolor variation. At basic pH, the emission is on the yellow-green region, whereas at acid pH, the light emission is observed on the red region of the visible spectrum. Theoretical calculations using density functional theory, molecular mechanics, and semiempirical methods were made to investigate the effect exerted by intermolecular forces on light emission, and their modulation by polarity, and the differences in the conformation of the active site at basic and acid pH. Red emission is achieved by the weakening of the interactions of the emitter with ionic and hydrophobic molecules, by the polarization of the benzothiazole microenvironment, by ionization of the enzyme-emitter complex and by changes of the hydrogen bond network. Arg220, Glu346, Ala350, Leu344 and adenosine-5'-monophosphate have blue-shifting effects, while His247, Phe249, Gly341, Thr253, and Ile288 exert a redshifting one.

Kinetics of inhibition of firefly luciferase by dehydroluciferyl-coenzyme A, dehydroluciferin and L-luciferin.

The inhibition mechanisms of the firefly luciferase (Luc) by three of the most important inhibitors of the reactions catalysed by Luc, dehydroluciferyl-coenzyme A (L-CoA), dehydroluciferin (L) and L-luciferin (L-LH(2)) were investigated. Light production in the presence and absence of these inhibitors (0.5 to 2 µM) has been measured in 50 mM Hepes buffer (pH = 7.5), 10 nM Luc, 250 µM ATP and D-luciferin (D-LH(2), from 3.75 up to 120 µM). Nonlinear regression analysis with the appropriate kinetic models (Henri-Michaelis-Menten and William-Morrison equations) reveals that L-CoA is a non-competitive inhibitor of Luc (K(i) = 0.88 ± 0.03 µM), L is a tight-binding uncompetitive inhibitor (K(i) = 0.00490 ± 0.00009 µM) and L-LH(2) acts as a mixed-type non-competitive-uncompetitive inhibitor (K(i) = 0.68 ± 0.14 µM and αK(i) = 0.34 ± 0.16 µM). The K(m) values obtained for L-CoA, L and L-LH(2) were 16.1 ± 1.0, 16.6 ± 2.3 and 14.4 ± 0.96 µM, respectively. L and L-LH(2) are strong inhibitors of Luc, which may indicate an important role for these compounds in Luc characteristic flash profile. L-CoA K(i) supports the conclusion that CoA can stimulate the light emission reaction by provoking the formation of a weaker inhibitor.

Computational Studies of the Luciferase Light-Emitting Product: Oxyluciferin.

Firefly luciferase is the most studied bioluminescence system, and its catalyzed reactions have been relatively well characterized. However, the color tuning mechanism that leads to firefly multicolor bioluminescence is still unknown, nor is consensual which is the yellow-green and red emitters. Computational studies have been essential in the study of oxyluciferin (OxyLH2) chemi- and bioluminescence and are responsible for most of our knowledge of this natural phenomenon. The objective of this manuscript is the analysis of the benefits and the conclusions derived from the theoretical studies of the light emitter, OxyLH2, and its applications on bioluminescence research.