Incorporation of Pectin into Vaterite Microparticles Prevented Effects of Adsorbed Mucin on Neutrophil Activation.

The application of vaterite microparticles for mucosal delivery depends on their interaction with mucin and immune cells. As we have shown previously, the binding of mucin onto particles enhances the generation of reactive oxygen species by neutrophils. The attenuation of the pro-oxidant effect of the bound mucin through the modification of vaterite could improve its biocompatibility. Hybrid microparticles composed of vaterite and pectin (CCP) were prepared using co-precipitation. In comparison with vaterite (CC), they had a smaller diameter and pores, a greater surface area, and a negative zeta-potential. We aimed to study the cytotoxicity and mucin-dependent neutrophil-activating effect of CCP microparticles. The incorporated pectin did not influence the neutrophil damage according to a lactate dehydrogenase test. The difference in the CC- and CCP-elicited luminol or lucigenin chemiluminescence of neutrophils was insignificant, with no direct pro- or antioxidant effects from the incorporated pectin. Unlike soluble pectin, the CCP particles were ineffective at scavenging radicals in an ABAP-luminol test. The fluorescence of SYTOX Green demonstrated a CCP-stimulated formation of neutrophil extracellular traps (NETs). The pre-treatment of CC and CCP with mucin resulted in a 2.5-times-higher CL response of neutrophils to the CC-mucin than to the CCP-mucin. Thus, the incorporation of pectin into vaterite microspheres enabled an antioxidant effect to be reached when the neutrophils were activated by mucin-treated microparticles, presumably via exposed ligands.

A Quantitative Luminol-Based Assay for ROS Burst Detection in Potato Leaves in Response to Biotic Stimuli.

Reactive oxygen species (ROS) are important signaling agents in plants and animals. They are involved in diverse processes, including activation of immune responses to pathogen infection. Biphasic detection of ROS in response to pathogen perception is becoming more popular even in important crops like potato as means of screening different germ plasms and mutants generated by for example CRISPR-Cas9 as well as identifying signaling pathways. Here we describe a detailed protocol for quantifying ROS bursts induced in potato leaf discs in response to a bacterial elicitor and Phytophthora infestans.

G-quadruplex-forming aptamer enhances the peroxidase activity of myoglobin against luminol.

Aptamers can control the biological functions of enzymes, thereby facilitating the development of novel biosensors. While aptamers that inhibit catalytic reactions of enzymes were found and used as signal transducers to sense target molecules in biosensors, no aptamers that amplify enzymatic activity have been identified. In this study, we report G-quadruplex (G4)-forming DNA aptamers that upregulate the peroxidase activity in myoglobin specifically for luminol. Using in vitro selection, one G4-forming aptamer that enhanced chemiluminescence from luminol by myoglobin's peroxidase activity was discovered. Through our strategy-in silico maturation, which is a genetic algorithm-aided sequence manipulation method, the enhancing activity of the aptamer was improved by introducing mutations to the aptamer sequences. The best aptamer conserved the parallel G4 property with over 300-times higher luminol chemiluminescence from peroxidase activity more than myoglobin alone at an optimal pH of 5.0. Furthermore, using hemin and hemin-binding aptamers, we demonstrated that the binding property of the G4 aptamers to heme in myoglobin might be necessary to exert the enhancing effect. Structure determination for one of the aptamers revealed a parallel-type G4 structure with propeller-like loops, which might be useful for a rational design of aptasensors utilizing the G4 aptamer-myoglobin pair.

Measurement of Reactive Oxygen Species in Semen Samples Using Chemiluminescence.

Reactive oxygen species (ROS) are continuously produced in semen and are essential for important spermatozoa functions that allow fertilization. However, an excessive amount of ROS is associated with poor sperm quality, which can compromise male fertility potential. This chemiluminescence assay is based on the production of light through the reaction between luminol and ROS. The emitted light is converted to an electrical signal by a luminometer, and the ROS levels in the sample are calculated as relative light units (RLU) per second per million spermatozoa per milliliter (RLU/s/million sperm/mL).

In Situ Formed Catalytic Interface for Boosting Chemiluminescence.

Designing the catalytic interface that preferentially attracts reactants is highly desirable for amplifying chemiluminescence (CL) emission. Herein, to boost the generation of reactive oxygen species (ROS) from dissolved O2 molecule, flower-like cobalt hydroxide (f-Co(OH)2) based catalytic interface with hierarchical and porous architecture were in situ created in the coexistence of BSA and Co2+. Benefiting from the oxidase-like catalysis capability and the unique microstructure of f-Co(OH)2, ROS was efficiently produced. Meanwhile, the capping ligands of BSA endowed the interface with the capability of enriching functionality through the interaction between BSA and luminol. 100-fold CL enhancement was achieved using the as-prepared catalytic interface compared with the classical luminol-Co2+ or luminol-BSA system. Moreover, the proposed catalytic amplification mechanism could be extended to the different proteins such as lysozyme, protamine, thrombin, papain. Based on the quenching effect on CL, a sensitive sensing platform was constructed for the determination of ascorbic acid with satisfied results. Our finding provided a novel "all-in-one" route to design the catalytic interface for amplifying CL emission.

Using Naturally Occurring Bioluminescent Enzymes to Track Specific Cell Populations.

Luminol-based bioluminescence imaging allows noninvasive tracking of oxidatively active cells such as neutrophils. Luminol is given intravenously or intraperitoneally, followed by bioluminescence imaging at 425 nm. Here we describe a method for tracking neutrophil extravasation into an inflammatory site, especially focusing on mammary carcinoma.

The horseradish smile: Demonstrating characteristic reactions of peroxidase in a visually appealing way.

Easy and visually appealing demonstrations are precious tools for introducing students to the study of enzymes. However, they most often involve purified enzymes and dedicated techniques. Here, we propose a set of demonstrations, which require only fresh horseradish root and consumer chemicals, to help support biochemistry and enzymology courses from high school to the undergraduate level. Horseradish root is a naturally rich source of horseradish peroxidase, an enzyme with many relevant practical applications. Slices of horseradish root are used to demonstrate the characteristic reaction of horseradish peroxidase with hydrogen peroxide, the selective inhibition of this enzyme by dilute hydrochloric acid, its chromogenic reaction with N,N'-diethyl-p-phenylenediamine, and its ability to trigger the chemiluminescent reaction of luminol. © 2019 International Union of Biochemistry and Molecular Biology, 48(1):38-43, 2020.

A covalent triazine framework as an oxidase mimetic in the luminol chemiluminescence system: application to the determination of the antioxidant rutin.

It is found that a covalent triazine framework (CTF-1) (that was prepared from 1,4-dicyanobenzene) exhibits oxidase-like activity toward the oxidation of luminol with dissolved oxygen in alkaline condition to produce intense blue chemiluminescence (CL). The reaction follows Michaelis-Menten kinetics and shows strong specificity for luminol. Reactive oxygen species including 1O2, •OH and O2•- are testified to be involved in the reaction and responsible for the CL. The reaction was applied to the determination of the radical-scavenging activity of antioxidants, with rutin, kaempferol and ferulic acid serving as model scavengers. A sensitive CL method was developed for the determination of rutin based on its inhibitory effect on the reaction. The CL system gave a linear response to the concentration of rutin in the range of 0.03-0.25 µmol·L-1 with a limit of detection of 0.015 µmol·L-1. The practicability of the method was demonstrated by successful determination of rutin in tablets and in Flos Sophorae Immaturus. Graphical Abstract.

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana.

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure the strength of immune activation in Arabidopsis thaliana following treatment with elicitor molecules. Presented first is a method for capturing the rapidly-induced and dynamic oxidative burst, which can be monitored using a luminol-based assay. Presented second is a method describing how to measure immune-induced inhibition of seedling growth. These protocols are fast and reliable, do not require specialized training or equipment, and are widely used to understand the genetic basis of plant immunity.

Imaging Intestinal ROS in Homeostatic Conditions Using L-012.

Reactive oxygen species (ROS) are critical redox regulators of cellular dynamics controlling homeostasis. Although numerous fluorescent probes are currently available to measure ROS in cell-based assays, the short-lived nature of these molecules renders their detection challenging in more complex biological systems, such as the gastrointestinal tract in vivo. However, in the past decade, significant progress has been made in the development of novel imaging technologies and probes, facilitating ROS quantification with high sensitivity, selectivity, and temporal resolution. The IVIS Spectrum (PerkinElmer) is an optical imaging system for small animal imaging allowing precise and noninvasive visualization of fluorescent or bioluminescent signals. Here, we describe a reproducible and comprehensive method for the measurement of physiological intestinal NADPH oxidase-derived ROS by using the chemiluminescent probe L-012. Using transgenic mice deficient in Nox isoforms expressed in the intestinal mucosa, we delineate the contribution of gut epithelial versus immune cell NADPH oxidase activity in homeostatic conditions. We also discuss L-012 probe specificity and potential alternatives for in vivo studies.

Antioxidant Activity and Toxicity of Fullerenols via Bioluminescence Signaling: Role of Oxygen Substituents.

Fullerenols are nanosized water-soluble polyhydroxylated derivatives of fullerenes, a specific allotropic form of carbon, bioactive compounds, and perspective basis for drug development. Our paper analyzes the antioxidant activity and toxicity of a series of fullerenols with different number of oxygen substituents. Two groups of fullerenols were under investigation: (1) C60Oy(OH)x, C60,70Oy(OH)x, where x + y = 24-28 and (2) C60,70Oy(OH)x, Fe0,5C60Oy(OH)x, Gd@C82Oy(OH)x, where x + y = 40-42. Bioluminescent cellular and enzymatic assays (luminous marine bacteria and their enzymatic reactions, respectively) were applied to monitor toxicity in the model fullerenol solutions and bioluminescence was applied as a signaling physiological parameter. The inhibiting concentrations of the fullerenols were determined, revealing the fullerenols' toxic effects. Antioxidant fullerenol' ability was studied in solutions of model oxidizer, 1,4-benzoquinone, and detoxification coefficients of general and oxidative types (DGT and DOxT) were calculated. All fullerenols produced toxic effect at high concentrations (>0.01 g L-1), while their antioxidant activity was demonstrated at low and ultralow concentrations (<0.001 g L-1). Quantitative toxic and antioxidant characteristics of the fullerenols (effective concentrations, concentration ranges, DGT, and DOxT) were found to depend on the number of oxygen substituents. Lower toxicity and higher antioxidant activity were determined in solutions of fullerenols with fewer oxygen substituents (x + y = 24-28). The differences in fullerenol properties were attributed to their catalytic activity due to reversible electron acceptance, radical trapping, and balance of reactive oxygen species in aqueous solutions. The results provide pharmaceutical sciences with a basis for selection of carbon nanoparticles with appropriate toxic and antioxidant characteristics. Based on the results, we recommend, to reduce the toxicity of prospective endohedral gadolinium-fullerenol preparations Gd@C82Oy(OH)x, decreasing the number of oxygen groups to x + y = 24-28. The potential of bioluminescence methods to compare toxic and antioxidant characteristics of carbon nanostructures were demonstrated.

Minocycline prevents peroxidative permeabilization of cardiolipin-containing bilayer lipid membranes mediated by cytochrome c.

Peroxidase activity of cytochrome c stimulated by interaction of the protein with cardiolipin is considered to be involved in the induction of mitochondrial apoptosis associated with cytochrome c release from mitochondria. In model systems, this activity has been previously shown to cause permeabilization of cardiolipin-containing membranes. Here, we found that the antibiotic minocycline, known to have neuroprotective properties, inhibited both the binding of cyt c to cardiolipin-containing membranes and the cyt c/H2O2-induced liposome permeabilization. The results could be relevant to inhibition of cyt c release from mitochondria exerted by minocycline.

Effect of hydroxyl group position in flavonoids on inducing single­stranded DNA damage mediated by cupric ions.

Quercetin has been demonstrated to produce DNA damage in the presence of metal ions. In the present study, 7 natural and 5 semi­synthetic glycosylated flavonoids were utilized to investigate the cupric ion (Cu2+)­dependent DNA damage in vitro. The reaction mixture, containing single­stranded DNA, different concentrations of flavonoids and cupric ion in the buffer, was incubated at three different temperatures. DNA damage was then assessed by gel electrophoresis followed by densitometric analysis. The reaction mixture with quercetin at 4, 20 and 54˚C induced DNA damage in a concentration­ and temperature­dependent manner. Furthermore, only the reaction at 54˚C resulted in DNA damage in flavonoids with glucosyl substitution of the hydroxyl group at the 3­position on the C ring in quercetin. By contrast, loss of the hydroxyl group at the 3­position on the C ring, or at the 3'­ or 4'­position on the B ring of quercetin, did not portray DNA damage formation at the investigated experimental temperatures. In addition, the experimental results suggested that the hydroxyl group at the 3­position on the C ring produced the strongest capability to induce DNA damage in the presence of cupric ions. Furthermore, hydroxyl groups at the 3'­ or 4'­position on the B ring were only able to induce DNA damage at higher temperatures, and were less efficient in comparison with the hydroxyl group at the 3­position on the C ring. Cupric ion chelating capacity was also assessed with spectroscopic analysis, and quercetin presented the largest chelating capacity among the tested flavonoids. Hydroxyl radical formation was assessed with a luminol reaction, and quercetin presented faster consumption of luminol. These results suggest that the 3­position hydroxyl group of the C ring is required to induce DNA damage at low temperatures. Furthermore, the results of the present study also indicated that the presence of cupric ions will decrease the activity of the glycosylated quercetins, in terms of their ability to induce DNA damage.

Improvement of mimetic peroxidase activity of gold nanoclusters on the luminol chemiluminescence reaction by surface modification with ethanediamine.

Peroxidase is a commonly used catalyst in luminol-H2 O2 chemiluminescence (CL) reactions. Natural peroxidase has a sophisticated separation process, short shelf life and unstable activity, therefore it is important to develop peroxidases that have both high catalytic activity and good stability as alternatives to the natural enzyme. Gold nanoclusters (Au NCs) are an alternative peroxidase with catalytic activity in the luminol-H2 O2 CL reaction. In the present study, ethanediamine was modified on the surface of Au NCs forming cationic Au NCs. The zeta potential of the cationic Au NCs maintained its positive charge when the pH of the solution was between 4 and 9. The cationic Au NCs showed higher catalytic activity in the luminol-H2 O2 CL reaction than did unmodified Au NCs. A mechanism study showed that the better performance of cationic Au NCs may be attributed to the generation of 1 O2 on the surface of cationic Au NCs and a positive surface charge, for better affinity to luminol. Cationic Au NC, acting as a peroxidase mimic, has much better stability than horseradish peroxidase over a wide range of temperatures. We believe that cationic Au NCs may be useful as an artificial peroxidase for a wide range of potential applications in CL and bioanalysis.

Production and evaluation of parathyroid hormone receptor1 ligands with intrinsic or assembled peroxidase domains.

Parathyroid hormone (PTH) can be C-terminally extended without significant affinity loss for the PTH1 receptor (PTHR1). We developed fusion protein ligands with enzymatic activity to probe PTHR1s at the cell surface. Two fusion proteins were generated by linking PTH to the N-terminus of either horseradish peroxidase (PTH-HRP) or the genetically modified soybean peroxidase APEX2 (PTH-APEX2). Alternatively, myc-tagged PTH (PTH-myc) was combined with antibodies, some of which HRP-conjugated, in the extracellular fluid. The three PTH-fusion proteins were produced as conditioned mediums (CM) by transfected producer HEK 293a cells. Binding of receptor-bound enzymatic ligands was revealed using widely available substrate/co-substrate systems. The stimulation of recipient HEK 293a expressing PTHR1s with the PTH-myc/antibodies combination or with PTH-APEX2 supported the histochemical or luminescent detection of recombinant PTHR1s (TrueBlueTM or luminol-based reagent). The PTH-HRP construction was the most sensitive and supported all tested peroxidase co-substrates (TrueBlueTM, tetramethylbenzidine (TMB), luminol, biotin-phenol with streptavidin-Qdots); the 3 latter schemes identified endogenous PTHR1 in the osteoblastic HOS cell line. The specificity of the fusion protein binding to PTHR1 was determined by its competition with an excess of PTH1-34. Bifunctional ligands possessing enzymatic activity detect intact receptors with various possible applications, including the screening of drugs that compete for receptor binding.

Rapid repeatable in vivo detection of retinal reactive oxygen species.

Oxidative injuries, such as those related to reactive oxygen species (ROS), have been implicated in various retinal and optic nerve disorders. Many ROS detection methods have been developed. Although widely utilized, many of these methods are useful only in post mortem tissues, or require relatively expensive equipment, or involve intraocular injection. In the present study, we demonstrated and characterized a chemiluminescent probe L-012 as a noninvasive, in vivo ROS detection agent in the mouse retina. Using optic nerve crush (ONC) and retinal ischemia/reperfusion (I/R) as injury models, we show that L-012 produced intensive luminescent signals specifically in the injured eyes. Histological examination showed that L-012 administration was safe to the retina. Additionally, compounds that reduce tissue superoxide levels, apocynin and TEMPOL, decreased injury-induced L-012 chemiluminescence. The decrease in L-012 signals correlated with their protective effects against retinal I/R-induced morphological and functional changes in the retina. Together, these data demonstrate the feasibility of a fast, simple, reproducible, and non-invasive detection method to monitor in vivo ROS in the retina. Furthermore, the results also show that reduction of ROS is a potential therapeutic approach for protection from these retinal injuries.

Attempts to Image the Early Inflammatory Response during Infection with the Lymphatic Filarial Nematode Brugia pahangi in a Mouse Model.

Helminth parasites remain a major constraint upon human health and well-being in many parts of the world. Treatment of these infections relies upon a very small number of therapeutics, most of which were originally developed for use in animal health. A lack of high throughput screening systems, together with limitations of available animal models, has restricted the development of novel chemotherapeutics. This is particularly so for filarial nematodes, which are long-lived parasites with a complex cycle of development. In this paper, we describe attempts to visualise the immune response elicited by filarial parasites in infected mice using a non-invasive bioluminescence imaging reagent, luminol, our aim being to determine whether such a model could be developed to discriminate between live and dead worms for in vivo compound screening. We show that while imaging can detect the immune response elicited by early stages of infection with L3, it was unable to detect the presence of adult worms or, indeed, later stages of infection with L3, despite the presence of worms within the lymphatic system of infected animals. In the future, more specific reagents that detect secreted products of adult worms may be required for developing screens based upon live imaging of infected animals.

Alteration of Neutrophil Reactive Oxygen Species Production by Extracts of Devil's Claw (Harpagophytum).

Harpagophytum, Devil's Claw, is a genus of tuberiferous xerophytic plants native to southern Africa. Some of the taxa are appreciated for their medicinal effects and have been traditionally used to relieve symptoms of inflammation. The objectives of this pilot study were to investigate the antioxidant capacity and the content of total phenols, verbascoside, isoverbascoside, and selected iridoids, as well as to investigate the capacity of various Harpagophytum taxa in suppressing respiratory burst in terms of reactive oxygen species produced by human neutrophils challenged with phorbol myristate acetate (PMA), opsonised Staphylococcus aureus, and Fusobacterium nucleatum. Harpagophytum plants were classified into different taxa according to morphology, and DNA analysis was used to confirm the classification. A putative new variety of H. procumbens showed the highest degree of antioxidative capacity. Using PMA, three Harpagophytum taxa showed anti-inflammatory effects with regard to the PBS control. A putative hybrid between H. procumbens and H. zeyheri in contrast showed proinflammatory effect on the response of neutrophils to F. nucleatum in comparison with treatment with vehicle control. Harpagophytum taxa were biochemically very variable and the response in suppressing respiratory burst differed. Further studies with larger number of subjects are needed to corroborate anti-inflammatory effects of different taxa of Harpagophytum.

Chemiluminescence and Bioluminescence as an Excitation Source in the Photodynamic Therapy of Cancer: A Critical Review.

Photodynamic therapy (PDT) of cancer is known for its limited number of side effects, and requires light, oxygen and photosensitizer. However, PDT is limited by poor penetration of light into deeply localized tissues, and the use of external light sources is required. Thus, researchers have been studying ways to improve the effectiveness of this phototherapy and expand it for the treatment of the deepest cancers, by using chemiluminescent or bioluminescent formulations to excite the photosensitizer by intracellular generation of light. The aim of this Minireview is to give a précis of the most important general chemi-/bioluminescence mechanisms and to analyze several studies that apply them for PDT. These studies have demonstrated the potential of utilizing chemi-/bioluminescence as excitation source in the PDT of cancer, besides combining new approaches to overcome the limitations of this mode of treatment.

Analysis of the lmmunity-Related Oxidative Bursts by a Luminol-Based Assay.

The rapid production of reactive oxygen species (ROS) in response to biotic and abiotic cues is a conserved hallmark of plant responses. The detection and quantification of ROS generation during immune responses is an excellent readout to analyze signaling triggered by the perception of pathogens. The assay described here is easy to employ and versatile, allowing its use in a multitude of variations. For example, ROS production can be analyzed using different tissues including whole seedlings, roots, leaves, protoplasts, and cultured cells, which can originate from different ecotypes or mutants. Samples can be tested in combination with any ROS-inducing elicitors, such as the FLS2-activating peptide flg22, but also lipids or even abiotic stresses. Furthermore, early (PAMP-triggered) and late (effector-triggered) ROS production induced by virulent and avirulent bacteria, respectively, can also be assayed.