Nitroblue Tetrazolium Reduction Research Articles

Innovative engineering of superoxide dismutase for enhanced cardioprotective biocatalysis in myocardial ischemia-reperfusion injury

The present research compares the stability, catalytic activity, and cardioprotective benefits of the designed superoxide dismutase (SOD) variation SOD-M3 to those of the naturally occurring enzyme, along with additional alternatives. SOD-M3 exhibited a 51.5 % increase in expression yield, reaching 50 mg/L, compared to the wild-type's 33 mg/L. In structural biology, the average distance between atoms of stacked proteins or molecules is measured by RMSD. Testing the precision of protein-ligand docking models is a typical application. The projected structure closely resembles the reference or native structure when the RMSD is low. The enzyme's durability improved significantly, resulting in negligible aggregation in the Size Exclusion Chromatography (SEC) and root mean square deviation (RMSD) of 2.1 Å. SOD-M3 catalytically increased superoxide radical scavenging capability by 40 % and inhibited nitroblue tetrazolium (NBT) reduction by 90 % at 1 μg/mL concentration. The material exhibited increased stability, as seen by its decomposition temperature (Tm) of about 80 °C, as opposed to 65 °C in the wild-type strains. In cardiomyocyte protection experiments, SOD-M3 reduced lactate dehydrogenase (LDH) production by 55 % while decreasing reactive oxygen species (ROS) concentrations by 60 %. Since a 51.5 % increase in expression yield for SOD-M3 indicates improved production efficiency, which makes large-scale manufacturing more viable for clinical applications, it is therapeutically essential. Higher expression yields mean more cost-effective manufacture for enzyme-based therapy research and commercialization. In vivo, SOD-M3 decreased myocardial infarction diameter by 44 % while improving cardiac function, including increased ejection percentage and fractional contraction. The histopathological investigation revealed undamaged heart tissue and less necrotic areas. The results reported here demonstrate SOD-M3's superior activity of enzymes, cardioprotective perspective, and stability, making it a promising therapeutic alternative for illnesses related to cellular oxidation & ischemic cardiac diseases. An increase in enzyme concentrations can enhance the obtaining of reactive oxygen species (ROS), which build up during ischaemia episodes; therefore, this increase is essential. Improving the enzyme's solubility and stability by site-directed modifications makes SOD-M3 more stable and effective in physiological circumstances where its activity is crucial for therapeutic efficacy maximization. A further benefit of increased expression yield is that it can lower manufacturing costs, which will help get SOD-M3 into clinical settings. These modifications raise SOD-M3's cardioprotective potential, making it a strong contender for novel therapies against cardiac ischemia-reperfusion impacts.

PBA2, a novel inhibitor of the β-catenin/CBP pathway, eradicates chronic myeloid leukemia including BCR-ABL T315I mutation

BackgroundBCR-ABL is a constitutively active tyrosine kinase that stimulates multiple downstream signaling pathways to promote the survival and proliferation of chronic myeloid leukemia (CML) cells. The clinical application of specific BCR-ABL tyrosine kinase inhibitors (TKIs) has led to significantly improved prognosis and overall survival in CML patients compared to previous treatment regimens. However, direct targeting of BCR-ABL does not eradicate CML cells expressing T315I-mutated BCR-ABL. Our previous study revealed that inhibiting CREB binding protein (CBP) is efficacious in activating β-catenin/p300 signaling, promoting cell differentiation and inducing p53/p21-dependent senescence regardless of BCR-ABL mutation status. We hypothesize that the specific inhibition of CBP may represent a novel strategy to promote β-catenin/p300-mediated differentiation and suppress cancer cell proliferation for treating CML patients.MethodsThe anticancer efficacy of PBA2, a novel CBP inhibitor, in CML cells expressing wild-type or T315I-mutated BCR-ABL was investigated in vitro and in vivo. Cell differentiation was determined by the nitroblue tetrazolium (NBT) reduction assay. The extent of cellular senescence was assessed by senescence-associated β-galactosidase (SA-β-Gal) activity. Cytotoxicity was measured by MTS assay. RNA interference was performed to evaluate the cell proliferation effects of CBP knockdown. The interaction of β-catenin and CBP/p300 was examined by co-immunoprecipitation assay.ResultsPBA2 exhibited significantly higher anticancer effects than imatinib in CML cells harboring either wild-type or T315I-mutated BCR-ABL both in vitro and in vivo. Mechanistically, PBA2 reduced CBP expression and promoted β-catenin-p300 interaction to induce cell differentiation and senescence.ConclusionOur data supported the rational treatment of CML by inhibiting the β-catenin/CBP pathway regardless of BCR-ABL mutation status.

Effect of proteins from the coelomic fluid of superficially wounded holothurians on the activity of phagocytes

Holothurians are among the most capable of regenerating animals. Their phagocytes are analogues of macrophages, but the mechanisms of their participation in wound healing have not been studied. The aim of the work is to determine the effect of individual protein components isolated from the coelomic fluid of holothurians with superficial damage to the body wall on the functional activity of the two types of phagocytes (P1 and P2) in holothurian Eupentacta fraudatrix. Protein components of the coelomic fluid of holothurians with superficial wounds of the body wall were analyzed and collected by gel permeation chromatography. We used proteins whose content changed significantly during the wound healing period in preliminary experiments. Two proteins and a peptide were added to phagocytes, isolated by gradient centrifugation, simultaneously with the thermostable toxin of Yersinia pseudotuberculosis and incubated for 24 h. The production of superoxide anion radical was determined by the reduction of nitroblue tetrazolium (NBT) using a colorimetric method. To assess the specificity of the proteins, bovine serum albumin (BSA) was used as an internal control. In one-day in vivo experiments, proteins were administered to wounded animals. Two proteins, when compared with the effect of BSA, did not show a specific effect on the production of reactive oxygen species in phagocytes in vitro. However, a comparison of the effect of the peptide on the oxidative activity and viability of phagocytes with those of BSA revealed the specificity of its action. The peptide reduced the oxidative activity of P1 phagocytes and increased it in P2 phagocytes in a direct concentration-dependent manner. One of the two proteins being administered to wounded holothurians caused a multidirectional concentration-dependent effect on the oxidative activity of the two types of phagocytes, with a predominant suppression of the P1 phenotype activity. The ability of protein components to cause a shift in the ratio of oxidative activity of the two types of phagocytes towards the preferential activation of P2 phagocytes, which are considered as functional analogues of M2 macrophages, suggests that they act as anti-inflammatory agents.

Pirarucu requires taurine to maximize growth and antioxidant status when fed diets high in plant-based ingredients

Taurine (Tau) is a free conditionally essential amino acid for most fish species that plays a significant nutritional role, mainly in carnivorous fish species. However, limited studies are still available regarding the role of Tau on pirarucu nutrition, an important Amazonian fish species. Therefore, this study used diets high in plant-based ingredients to investigate the effect of taurine (Tau) supplementation on juvenile pirarucu's growth, metabolism, and health (n = 115, Weight = 504.12 ± 19 g). Fish were fed five diets containing increasing levels of Tau: 0.95, 5.1, 9.6, 12.9, and 17.2 g/kg in a completely randomized design with three replicates per treatment. After a 60-day feeding trial, growth, hematology, blood chemistry, Tau content in muscle, carcass, and liver, antioxidant status, and immunological parameters were evaluated. The control group (0.95 g/kg Tau) showed the lowest growth. According to the segmented regression model, the recommended Tau level for maximizing the development of juvenile pirarucu was 4.49 g/kg of feed. Additionally, fish fed the lowest dietary Tau level showed the highest ROS (reactive oxygen species) concentration. Dietary Tau levels did not affect hematological parameters and blood chemistry (p > 0.05). Fish fed 9.6 g/kg of Tau showed increased catalase activity, while the control group had the worst result in NBT (nitroblue tetrazolium reduction test). Fish fed with the highest dietary Tau levels (12.9 and 17.2 g/kg) showed increased total serum immunoglobulin concentration. These results suggest that the optimal inclusion level of Tau in diets for juvenile pirarucu is 4.49 g/kg of feed and that this amino acid acts as a mitigator of oxidative stress.

Role of reactive carbonyls and superoxide radicals in protein damage by cigarette smoke extracts: Comparison of Heat-not-Burn e-cigarettes to conventional cigarettes

Oxidative protein damage involving carbonylation of respiratory tract proteins typically accompanies exposure to tobacco smoke. Such damage can arise via multiple mechanisms, including direct amino acid oxidation by reactive oxygen species or protein adduction by electrophilic aldehydes. This study investigated the relative importance of these pathways during exposure of a model protein to fresh cigarette emission extracts. Briefly, protein carbonyl adducts were estimated in bovine serum albumin following incubation in buffered solutions with whole cigarette emissions extracts prepared from either a single 1R6F research cigarette or a single “Heat-not-Burn” e-cigarette. Although both extracts caused concentration-dependent protein carbonylation, conventional cigarette extracts produced higher adduct yields than e-cigarette extracts. Superoxide radical generation by conventional and e-cigarette emissions was assessed by monitoring nitro blue tetrazolium reduction and was considerably lower in extracts made from “Heat-Not-Burn” e-cigarettes. The superoxide dismutase/catalase mimic EUK-134 strongly suppressed radical production by whole smoke extracts from conventional cigarettes, however, it did not diminish protein carbonyl adduction when incubating smoke extracts with the model protein. In contrast, edaravone, a neuroprotective drug with strong carbonyl-trapping properties, strongly suppressed protein damage without inhibiting superoxide formation. Although these findings require extension to appropriate cell-based and in vivo systems, they suggest reactive aldehydes in tobacco smoke make greater contributions to oxidative protein damage than smoke phase radicals.

Ethanolic extract of Euphorbia Gypsicola induces differentiation and apoptosis in human myeloid leukemia K562 cells

IntroductionThe Euphorbiaceae family, widely distributed and long utilized in traditional medicine, has been recognized for its potential in developing anti-cancer drugs. The current study investigates the capacity of Euphorbia gypsicola extract to induce apoptosis and differentiation in the human myeloid leukemia K562 cell line. MethodsCell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptotic activity was visually evaluated through acridine orange/ethidium bromide (AO/EB) dye staining and annexin V/PI double labeling procedures. Induction of differentiation was examined using nitro blue tetrazolium (NBT) reduction and Wright-Giemsa staining assays. The expression of apoptosis-related proteins including Bax, Bcl2 and Survivin was evaluated by western blotting method. ResultsThe E. gypsicola extract exhibited potent medication properties with IC50 values of 207.59, 48.58, and 7.08 ng/mL at 24, 48, and 72 h, respectively. Fluorescence microscopy, DNA fragmentation, annexin V/PI double staining, and sub-G1 cell cycle arrest confirmed apoptosis occurrence in K562 cells treated with the E. gypsicola extract. Our findings demonstrated that differentiated cells reduced NBT yellow solution after endocytosis, forming dark crystals. Wright Giemsa staining observations indicated a decrease in nuclear volume-to-cytoplasm ratio in K562 cells treated with the crude extract, signifying Induction of differentiation. Furthermore, western blot analysis revealed down-regulation of Survivin protein expression and up-regulation of Bax protein level following treatment with the E. gypsicola extract. However, no significant decrease in Bcl2 protein expression level was observed in K562 cells. ConclusionThese findings indicate the growth inhibitory effects of the E. gypsicola extract on leukemia K562 cells, which supports the further study of this plant to discover novel therapeutic compounds for the treatment of leukemia.

Antioxidant and Anti-Inflammatory Activity of Fractionated Artemia Urmiana Extract in Human Peripheral Blood Neutrophils System

Marine organisms are great resources for discovering bioactive compounds with new antioxidant properties. The aim of this work was to purify and isolate the protein fractions of Artemia urmiana and to evaluate antioxidant activities of them on human peripheral blood neutrophils response. For these purposes, crude extracts protein was extracted from napulii of A. urmiana and sequentially fractionated by ion exchange. The antioxidant activities of the samples were determined by DPPH free radical scavenging, reducing power ability assays in comparison with ascorbic acid. In the second phase, the human peripheral blood neutrophils (HPBN) were separated and cultured. The cells were treated with increasing concentrations of crude and partially purified fractions for 24 hrs. After that, the pellet and supernatant of treated cells were separately collected and assayed for Nitro Blue Tetrazolium (NBT) reduction, the activity of Super Oxide Dismutase (SOD) and Nitric oxide content. The results showed that the IC50 values of ascorbic acid, crude extracts, partially purified protein fractions(F1-F8) were 150, 126, 141, 295, 299, 114, 164 and 132 µg/ml respectively in the DPPH radical scavenging activity. The reducing power activity at the highest concentration (200 µg/ml) based on the optical density of the sample listed, were 2.8, 1.4, 0.13, 0.14, 0.17, 0.14, 0.13, 0.66, 0.18 and 0.35 respectively. In NBT reduction and production of Nitric oxide, treated cells did not show significant difference between different concentrations of protein extract and untreated control cells. The activity of SOD was increased in presence of 50-100 µg/ml of crud extract and fractions 1, 2, 3, 4 and 5 in comparison to untreated control cells (p<0.05).

Highly efficient hydrogen production and selective CO2 reduction by the C3N5 photocatalyst using only visible light.

The production of energy sources by metal-free photocatalysts based on graphitic carbon nitride (g-C3N4) has garnered substantial attention. In this study, nitrogen-rich carbon nitride (C3N5) was successfully synthesized through the thermal polycondensation of 3-amino-1,2,4-triazole. The structural and physical characterization has suggested that a portion of the triazine rings, which constitute the structural framework of g-C3N4, may be substituted with five-membered rings in C3N5. Furthermore, the polymerization of C3N5 proceeded more extensively than that of g-C3N4 from melamine precursors. The increased nitrogen content in C3N5 resulted in a heightened number of π-electrons and a narrowed energy bandgap, with the potential of the valence band maximum being negatively shifted. Additionally, photocatalytic assessments encompassing nitro blue tetrazolium reduction, H2 production from triethanolamine aqueous solution, and CO2 reduction in the liquid phase were performed. All findings demonstrated that C3N5 exhibits significantly superior photocatalytic properties compared to g-C3N4. It is particularly noteworthy that C3N5 selectively generates methanol and H2 from oversaturated CO2 solutions under visible light irradiation, while g-C3N4 selectively generates formaldehyde. These outcomes strongly indicate that C3N5 serves as a metal-free, visible-light-responsive photocatalyst, capable of contributing to both the production of renewable energy sources and the reduction of greenhouse effect gases.

Immunomodulatory effect of Myrtenol on benzo (a) pyrene-induced lung cancer in Swiss albino mice via modulation of tumor markers, cytokines and inhibition of PCNA expression.

Lung cancer is one of the most common cancers in men. Although many diagnostic and treatment regimens have been followed in the treatment for lung cancer, increasing mortality rate due to lung cancer is depressing and hence requires alternative plant based therapeutics with with less side-effects. Myrtenol exhibits anti-inflammatory and antioxidant properties. Hence we intended to study the effect of Myrtenol on B(a)P-induced lung cancer. Our study showed that B(a)P lowered hematological count, decreased phagocyte and avidity indices, nitroblue tetrazolium (NBT) reduction, levels of immunoglubulins, antioxidant levels, whereas Myrtenol treatment restored them back to normal levels. On the other hand, xenobiotic and liver dysfunction marker enzymes and pro-inflammatory cytokines were elevated on B(a)P exposure, which retuned back to normal by Myrtenol. This study thus describes the immunomodulatory and antioxidant effects of Myrtenol on B[a]P-induced immune destruction.

Exploring the Relationship between Neutrophil Activation and Different States of Canine L. infantum Infection: Nitroblue Tetrazolium Test and IFN-γ.

This study aimed to investigate the role of neutrophils in canine leishmaniosis by assessing neutrophil activation and its relationship with different states of L. infantum infection and antibody and IFN-γ production. Dogs were categorized into five groups: healthy-seronegative (n = 25), healthy-seropositive (n = 21), LeishVet-stage I (n = 25), Leishvet-stage II (n = 41), and LeishVet-stage III-IV (n = 16). Results of the nitroblue tetrazolium reduction test (NBT) showed significantly higher neutrophil activation in stage I (median:17.17, range: [7.33-31.50]%) compared to in healthy-seronegative (4.10 [1.20-18.00]%), healthy-seropositive (7.65 [3.98-21.74]%), stage II (6.50 [1.50-28.70]%), and stage III-IV (7.50 [3.00-16.75]%) groups (p < 0.0001). Healthy-seropositive dogs also displayed higher values than all groups except stage I. Stages II and III-IV did not show significant differences compared to healthy-seronegative. Regarding IFN-γ, stage I dogs had higher concentrations (median:127.90, range: [0-3998.00] pg/mL) than healthy-seronegative (0 [0-109.50] pg/mL) (p = 0.0002), stage II (9.00 [0-5086.00] pg/mL) (p = 0.045), and stage III-IV (3.50 [80.00-548.80] pg/mL) (p = 0.02) dogs. Stage II dogs showed increased IFN-γ compared to healthy-seronegative dogs (p = 0.015), while stage III-IV dogs had no significant differences compared to healthy-seronegative dogs (p = 0.12). Healthy-seropositive dogs had elevated IFN-γ concentrations compared to healthy-seronegative dogs (p = 0.001) and dogs in stage III-IV (p = 0.03). In conclusion, neutrophil activation was higher in dogs with mild disease and healthy-seropositive dogs, and a relationship between neutrophil activation and the production of IFN-γ was found.

Effect of storage on the nitro blue tetrazolium reduction test in dog blood samples.

The nitro blue tetrazolium (NBT) reduction test (NBTT) has been used for measuring the metabolic activity of phagocytes of mammals. Activated neutrophils transform NBT into formazan in the cytoplasm. The NBTT can detect the activation of neutrophils in peripheral blood and is used to assess neutrophil function in dogs. However, the NBTT is not used frequently in the clinical setting, as samples should be processed after blood collection. The aim of this study was to evaluate the effect of storage on NBTT in dog blood samples. Residual EDTA blood samples from 22 dogs were included of different ages, breeds, and sex. The buffy coat layer was separated from the blood and incubated with 0.1% NBT. The NBTT was performed at 0, 24, 48, and 72 h after the collection of blood. Blood samples were stored at 4°C until the tests were performed. Blood smears were evaluated by light microscopy, and the NBT reduction rate was reported, which represents the percentage of activated neutrophils. The NBT reduction rate was calculated after counting 300 neutrophils in each slide. The means of NBTT in dog blood samples at 0, 24, 48, and 72 h were 8.3%, 8.5%, 8.7%, and 7.8%, respectively. No significant differences were observed between time points. This study showed that the NBTT can be performed up to 72 h after the collection of canine blood if correctly refrigerated at 4°C. This finding supports the performance of the NBTT in the clinical setting.

Oncolytic Newcastle disease virus effects on immune response - a new issue in cancer treatment.

Millions of people are diagnosed with cancer each year, and fighting it puts a heavy financial burden on communities and governments. Numerous advances have been made in the field of cancer; one of the newest methods is using oncolytic viruses. This study aimed to evaluate the effect of oncolytic Newcastle disease virus wild-type strains (NDV-WTS) on the immune system. Forty mice were divided into four groups (10 animals in each group). The control group received phosphate buffered saline, and experimental group 1 (NDV-WTS 1), experimental group 2 (NDV-WTS 2), and experimental group 3 (NDV-WTS 3) received 10-1, 10-2, and 10-3 titers of Newcastle virus on 0, 14th, and 28th days. On the 31st day, 100 µL of Newcastle virus was injected into the left footpads of animals. After 48 hours, delayed-type hypersensitivity (DTH) reactions were measured. On the 33rd day, peritoneal macrophages were isolated. Then proliferation of the cells was measured by the methyl-thiazolyl-tetrazolium (MTT) test. Neutral red uptake and respiratory burst of peritoneal macrophages were also assessed. Data were analyzed using statistical software SPSS, version 19. The results of the DTH test showed that footpad swelling in control, NDV-WTS 1, NDV-WTS 2, and NDV-WTS 3 groups were 23.5%, 23.5%, 23.6% and 23.6%. No significant differences were seen between the groups in this regard (P &gt; 0.05). A negative nitroblue tetrazolium (NBT) reduction test as an indicator of macrophage's respiratory burst, showed no significant difference between the groups (P &gt; 0.05). The neutral red uptake assay and MTT test showed no significant differences between the groups (P &gt; 0.05). The results of this study showed that NDV-WTS in doses of 10-1, 10-2, and 10-3 have no adverse effects on healthy normal cells.

EFFECT OF MODULATION OF NITROGEN OXIDE (II) SYNTHESIS UNDER CHRONIC NORMOBARIC HYPOXIA ON THE ISOENZYME SPECTRUM OF RAT EPIDIDIMIS LACTATE DEHYDROGENASE

Lactate dehydrogenase have a key role in providing energy to cells under physiological and hypoxic conditions. Changes in the activity of the enzyme can be facilitated by nitric oxide (II), regulating the expression of individual fractions of lactate dehydrogenase. Aim. To study changes in the isoenzyme spectrum of rat epididymis lactate dehydrogenase under conditions of hypoxia and modulation of nitric oxide (II) synthesis. Material and methods. Male rats (32) were divided into 4 groups (n=8): 1) chronic normobaric hypoxia; 2) control to group 1; 3) hypoxia together with modeling of nitric oxide deficiency; 4) hypoxia against the background of induction of NO synthesis. The mitochondrial fraction and non-mitochondrial cytoplasm of the head and tail of the epididymis were isolated for laboratory research. The zymogram was obtained by electrophoresis in 7% gel followed by detection by the precipitation reaction of the reduction of nitro blue tetrazolium. The overall activity of lactate dehydrogenase was assessed and the percentage of isoenzyme fractions was calculated. Fractions 1 and X of lactate dehydrogenase were analyzed, the results were con-sidered statistically significant if p 0.05 when comparing two independent samples, and p 0.0167 when comparing three independent samples. Results. Chronic normobaric hypoxia leads to a decrease in lactate dehydrogenase activity compared to the control group. At the same time, the share of the X-fraction of the enzyme and lactate dehydrogenase 1 increases. Modulation of nitric oxide (II) deficiency against the background of hy-poxia increased the activity of the enzyme in the head of the epididymis, where a decrease in the studied fractions of lactate dehydrogenase was ob-served compared to the group of animals subjected only to hypoxia. Stimulation of the synthesis of nitric oxide (II) during hypoxia led to a decrease in the X-fraction, but an increase in the 1st fraction of lactate dehydrogenase in the head of the epididymis, in the tail of the epididymis, opposite changes were observed. Conclusion. Hypoxia causes a change in the ratio of lactate dehydrogenase isoenzymes towards an increase in lactate dehydrogenase B and lactate dehydrogenase X, while a deficiency of nitric oxide (II) contributes to a decrease in lactate dehydrogenase X synthesis.

Superoxide Generation by Nicotinamide Coenzymes

Nicotinamide coenzymes can generate superoxide radicalsin alkaline environment. Their formation was registered by reduction of nitroblue tetrazolium (NBT) present in the buffer with formation of diformasan. Inhibition of diformazan formation occurs when superoxide dimutase (SOD) is added to the system, thus confirming generation of O─●. The highest superoxide generating activity was observed with NADPH. In the case of NADPH and NADH, the rate of superoxide generation was significantly lower (by approximately 50%). No O─● was detected when NAD was used under the same conditions and in the same time; however, 4 h later, diformasan was detected in the same sample. The superoxide generating activity decreased in the following order: NADPH &gt; NADH ? NADP &gt; NAD. Other compounds tested (adenosine, ADP and ATP) did not generate superoxide radicals even after prolonged incubation. In a cell, where a local changes in the pH of the environment are possible, nicotinamide coenzymes can be potential sources of O─● and thus participate in cell signaling. A change in pH can initiate this process.

MEGAMOX MEDIATES ECO-FRIENDLY SYNTHESIS OF SELENIUM NANOPARTICLES AS IMMUNOMODULATORY, ANTIOXIDANT AND ANTIMICROBIAL AGENTS

The research herein includes methodology for eco-friendly preparation of selenium nanoparticles (SeNPs) using the broad-spectrum antibiotic, Megamox (Mega.). Characterization of SeNPs was done using UV-Visible spectroscopy, FTIR, XRD, DLS and TEM imaging. Additionally, the immunomodulatory, antioxidant and antimicrobial activities of both SeNPs and Mega. were checked against different strains of bacteria and fungi. TEM and DLS images showed that SeNPs are polydisperse spheres with mean diameter of 22.4 nm. FTIR analysis indicated that the hydroxyl and nitrogen moieties in Mega. were effective for reduction plus binding manner. According to the results of the nitro blue tetrazolium reduction test, both SeNPs and Mega. presented high intracellular killing activity, which confirmed their immunostimulatory effect. Antioxidant activity of SeNPs and Mega. were 90 and 82 %, respectively. SeNPs presented great activity facing multi-drug resistant bacteria and TB. SeNPs are considered promising cost-effective and eco-friendly anti-bacterial and anti-fungal agents that can represent a new potential nano-platform in both medical and infectious diseases control.

Reprint of: Generation of Superoxide Radical during Autoxidation of Hydroxylamine and an Assay for Superoxide Dismutase

Accompanying the autoxidation of hydroxylamine at pH 10.2, nitroblue tetrazolium was reduced and nitrite was produced in the presence of EDTA. The rate of autoxidation was negligible below pH 8.0, but sharply increased with increasing pH. The reduction of nitroblue tetrazolium was inhibited by superoxide dismutase, indicating the participation of superoxide anion radical in the autoxidation. Hydrogen peroxide stimulated the autoxidation and superoxide dismutase inhibited the hydrogen peroxide-induced oxidation, results which suggest the participation of hydrogen peroxide in autoxidation and in the generation of superoxide radical. An assay for superoxide dismutase using autoxidation of hydroxylamine is described.

Single Cell Assessment of Mitochondrial Function

Cellular mitochondrial function can be assessed using high resolution respirometry that measures O2 consumption rate during various conditions that systematically alter the tricarboxylic acid (TCA) cycle or the electron transport chain (ETC). However, current high resolution respirometry does not measure O2 consumption rate at the single cell level, but actually measures average mitochondrial function across a number of cells (either isolated or in tissues). Thus, respirometry assumes physiological homogeneity across cells. However, in many tissues, mitochondrial function varies across cells and this heterogeneity is physiologically important. Therefore, a direct measurement of cellular mitochondrial function will provide valuable novel information and physiological insight. In the present study, we used a quantitative histochemical technique to measure the activity of succinate dehydrogenase (SDH), a key enzyme located in the inner mitochondrial membrane, and the only enzyme to participate in both the TCA cycle and the ETC as Complex II. SDH mediates the oxidation of succinate to fumarate in the TCA cycle, which is coupled to the reduction of ubiquinone to ubiquinol in the ETC. In this study we determined the maximum velocity of the SDH reaction (SDHmax) in isolated human airway smooth muscle (hASM) cells using 1‐methoxyphenazine methosulphate (mPMS), as an exogenous electron carrier, and azide to inhibit cytochrome oxidase. To measure SDHmax, the cells were exposed to a solution containing 80 mM succinate and 1.5 mM nitroblue tetrazolium (NBT) as the reaction indicator. hASM cells were imaged in 3D (Z optical slice of 0.5 μm) using a Nikon Eclipse A1 laser scanning confocal system with a ×60/1.4 NA oil‐immersion lens. In the quantitative histochemical procedure, changes in cell optical density (OD) due to the progressive reduction of NBT to its diformazan (peak absorbance wavelength of 570 nm) were measured every 15 s over a 10 min period. Linearity of the SDH reaction was confirmed across the 10 min period, and SDHmax was expressed as mM fumarate/liter of tissue/min. Validation of this technique included specific ETC inhibitors including oligomycin (ATP synthase inhibitor), FCCP (proton ionophore), antimycin A (Complex III inhibitor) and rotenone (Complex I inhibitor), similar to those used in high resolution respirometry. We observed that FCCP‐mediated disruption of the mitochondrial proton gradient does not affect SDHmax, while SDHmax is decreased by rotenone and antimycin A. In addition, we used MitoTracker Green to label and image mitochondria in hASM cells and determined mitochondrial volume density. The SDHmax was then normalized to mitochondrial content. Our results confirm that this quantitative technique is rigorous and reproducible, and that measurements of cellular SDHmax can serve as a reliable surrogate for the measurement of maximum mitochondrial respiration in single cells.

Determination of secondary metabolites including curcumin in Rheum ribes L. and surveying of its antioxidant and anticancer activity

Dry stem and root of Rheum ribes are used for the treatment of different diseases. In this study, the organic components in the root of R. ribes were extracted by three extraction methods (soxhlet, ultrasonic and maceration) and the efficiencies of these methods were calculated. To validate the existence of Curcumin, analysis such as UV–Vis (Ultraviolet–Visible), HPLC (High Performance Liquid Chromatography), GC (Gas Chromatography) and GC/Mass were performed and in order to separate Curcuminoids, TLC (Thin Layer Chromatography) technique was implemented on the extract which had the highest yield. Total phenolic, flavonoid and flavonol contents of three kinds of extract were measured. For studying antioxidant activity, DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging was done. Finally, for investigating anticancer activity, MTT (Methyl Thiazolyl Tetrazolium), NBT (Nitro Blue Tetrazolium) and DNA-Binding tests were done. The most yield was belonged to soxhlet extract. The results showed the presence of Curcumin in soxhlet extract has not been reported previously. Furthermore, the total phenolic, flavonoid and flavonol contents were belonged to ultrasonic extract. As well as the maximum antioxidants properties were belonged to ultrasonic and maceration extraction. The MTT assay showed the lowest IC50 in the ultrasonic extract. At NBT test, the highest NBT reduction percentage was belonged to maceration extraction. DNA-binding test revealed that all the extracts have shown some degree of fragmentation. Due to the presence of Curcumin in R. ribes and the notable features that the results showed, it can be a good source for Curcumin extraction and use in the drug industry.

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5'-Phosphate.

Riboflavin-5'-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can be generated from FMN photolysis upon irradiation with visible light. The ROS generated from FMN photolysis are harmful to microorganisms, including pathogenic bacteria such as Staphylococcus aureus (S. aureus). This article presents a protocol for deactivating S. aureus, as an example, via photochemical reactions involving FMN under visible light irradiation. The superoxide radical anion () generated during the FMN photolysis is evaluated via nitro blue tetrazolium (NBT) reduction. The microbial viability of S. aureus that is attributed to reactive species was used to determine the effectiveness of the process. The bacterial inactivation rate is proportional to FMN concentration. Violet light is more efficient in inactivating S. aureus than blue light irradiation, while the red or green light does not drive FMN photolysis. The present article demonstrates FMN photolysis as a simple and safe method for sanitary processes.

Growth Promoter, Immunostimulant and Antioxidant for Rainbow Trout (Oncorhynchus mykiss): Terebinth (Pistacia terebinthus) Extract

In this study, the effects of Pistacia terebinthus (PT) fruit extract supplemented diet on growth performance, haematology, digestive and antioxidant enzyme activities, and non-specific immune responses were evaluated in juvenile rainbow trout (Onchoryhnchus mykiss). The fish were fed diets containing three doses of Pistacia terebinthus extract (0.1, 0.5 and 1% of diet) and a control diet without extract for 63 days. Final weight, weight gain and specific growth rate were significantly improved in all the treated groups. In addition, feed conversion ratio was significantly reduced in all PT diet fed groups. Pepsin and lipase activities were significantly increased in all the treated groups. Trypsin was significantly improved in PT 0.1% and PT 1% groups. Amylase was significantly increased in PT 0.5 and 1% groups. In haematological assays, red blood cell, haemoglobin, haematocrit, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration values were not changed among all experimental groups. Superoxide dismutase, catalase, glutathione peroxidase and glucose-6-phosphate dehydrogenase activities were significantly improved in all the treatment groups. However, catalase activity decreased in PT 0.5% group at the end of 63 days. In addition, hepatic and white muscle lipid peroxidation activities were significant decreased in all the treated groups compared to the control. Non-specific immune parameters, such as nitroblue tetrazolium reduction, myeloperoxidase and lysozyme activities were increased in all the treated fish groups. These results indicated that extract of P. terebinthus can be used to improve fish health in aquaculture.