Methylene Blue Reduces Retinal Cell Inflammation, Apoptosis, and Oxidative Stress in a Rat Model of Diabetic Retinopathy via Sirtuin 1 Activation.

Objective: Diabetic retinopathy (DR), characterized by neuronal damage in the retina, is primarily driven by oxidative stress resulting from diabetes (DM). This study investigated the potential effects of methylene blue (MB) on streptozotocin (STZ)-induced DR. Methods: A rat model of DR was established via STZ injection, while a cell model was created using high-glucose (HG) exposure of human retinal microvascular endothelial cells. Evaluation of oxidative stress markers, pro-inflammatory cytokines, and pro-apoptotic proteins was performed based on their expression profiles in human retinal microvascular endothelial cells. Results: MB treatment significantly upregulated the expression of sirtuin 1 (SIRT1), which was found to be downregulated in the retinal tissues of STZ-treated rats and HG-exposed human retinal microvascular endothelial cells, as determined by polymerase chain reaction (PCR). Furthermore, MB therapy effectively suppressed STZ-induced oxidative stress, inflammation, and cell death. Consistent with the in vivo findings, MB activated the expression of SIRT1, thereby protecting HG-treated human retinal microvascular endothelial cells against oxidative stress, inflammation, and apoptosis. Conclusion: These results support the conclusion that MB mitigates DR by activating SIRT1, leading to a reduction of inflammation, apoptosis, and oxidative stress.

[Effect of methylene blue and its metabolite - azure I - on bioenergetic parameters of intact mice brain mitochondria]. / Vliianie metilenovogo sinego i ego metabolita ­ azura I ­ na bioénergeticheskie parametry izolirovannykh mitokhondrii mozga myshei.

Methylene blue is a phenothiazine dye that is widely used in medicine and clinical trials for the treatment of Alzheimer's disease. One of the factors of the unique therapeutic effect of methylene blue is its redox properties, allowing implementation of alternative electron transport - the dye accepts electrons from reducing equivalents in the mitochondria and transfer it them to other components of the respiratory chain or molecular oxygen. Azure I, an N-dimethylated metabolite of methylene blue, is potentially a more effective compound than methylene blue, but its ability for alternative electron transport has not been studied. We have shown that azure I, unlike methylene blue, is unable to restore the membrane potential in isolated mouse brain mitochondria, inhibited by rotenone and, therefore, is unable to perform bypass of the respiratory chain Complex I. Moreover, the addition of azure I does not affect the rate of mitochondrial respiration in contrast to methylene blue, which increases the rate of non-phosphorylation respiration. At the same time, both dyes stimulate an increase in H2O2 production. As a consequence, only methylene blue is capable of alternative electron transport, while azure I does not produce complex I bypass. This limits its therapeutic application only as a mitochondrial-targeted drug, but not as a substance with a potentially powerful antidepressant effect.

Methylene blue active substances in plaque of Bacillus subtilis subsp. subtilis and enrichment by supplemental calcium in culture media.

A series of experiments was conducted to identify the molecular species responsible for surface active emulsification (surfactant) bioactivity in Bacillus subtilis subsp. subtilis strain ATCC PTA-125135, and to describe culture conditions to support the enriched production of said bioactivity in cultured plaque of the strain. The assay for methylene blue active substances (MBAS) was found to be suitable for describing surfactant activity, where a solvent-extracted molecular fraction from the biofilm was found to retain surfactant activity and positively quantified as MBAS. Furthermore, an HPLC-refined protein fraction was found to quantify as MBAS with approximately 1·36-fold or greater surfactant activity per mol than sodium dodecyl sulphate, and a proteomic analysis of solvent extracted residues confirmed that biofilm surface layer protein BslA was a primary constituent of extracted residues. Surfactant bioactivity, quantified as MBAS, was enriched in cultured plaque by the supplementation of culture media with calcium chloride or calcium nitrate.

Flexible, dense and porous chitosan and alginate membranes containing the standardized extract of Arrabidaea chica Verlot for the treatment of skin lesions.

The combination of chitosan (C) with alginate (A) has been explored for the production of dressings due to the positive results on wound healing. CA films can show a dense or porous flexible structure, with characteristics tunable for different applications. Porosity and flexibility can be achieved, respectively, by the addition of surfactants such as Kolliphor® P188 (P) and silicone-based compounds as Silpuran® 2130 A/B (S). Furthermore, composite matrices of these polysaccharides have potential applications as devices for releasing bioactive compounds to skin lesions. The purpose of this study was to evaluate the physicochemical and biological characteristics of flexible dense and porous CA membranes incorporating the standardized extract of Arrabidaea chica Verlot (A. chica), and also to analyze the release mechanism of the extract from different membrane formulations. The results show that the inclusion of P in the formulation allows obtaining porous matrices, promotes greater homogeneity of the mixture of the silicone gel with the suspension of polysaccharides, and increases the swelling of the polymer matrix. All formulations presented high stability, reaching a maximum mass loss of 18% after seven days. The formulations with S showed the best performance in terms of flexibility and strain at break. The presence of A. chica standardized extract did not affect negatively the characteristics of the membranes. Incorporation efficiencies of the bioactive compound above 87% were achieved, and the addition of P and S to the membrane formulation changed the release of the A. chica extract kinetics. In addition, the developed formulations did not significantly affect Vero cells proliferation.

Antibacterial, antibiofilm, and photocatalytic activities of metals-substituted spinel cobalt ferrite nanoparticles.

This study reports the photocatalytic degradation of Methylene Blue (MB) dye (a class of dyestuffs that are resistant to biodegradation) under the influence of UV-light irradiation. Antibacterial and antibiofilm activities of ferrite nanoparticles (FO NPs) were examined against some pathogenic bacteria isolated from the medical operating room surfaces. In the same context, metals-substituted spinel cobalt ferrite nanoparticles with nominal composition [MxCo1-xFe2O4 NPs; (M = Zn, Cu, Mn; x = 0.0, 0.25, 0.5 and 0.75)] were synthesized by citrate sol-gel method. Also, the structures of the synthesized FO NPs were characterized by X-ray diffraction, and Williamson-Hall (WH) method was used to determine the crystallite size. The estimated specific surface area is found in the range from 37.99 to 107.05 m2/g, between the synthesized ferrites, Zn0.5Co0.5Fe2O4 NPs have average pore radius 1.84 nm and the pore volume was 0.136 ml/g. SEM images revealed that, the synthesized FO NPs have an unique pores and uniformly distribution, while EDX spectra shows the elemental composition for the synthesized FO NPs. The elastic properties of FO NPs have been estimated using FTIR data, whereas (M - H) hysteresis loops revealed that, by replacing cobalt ions with Zn, Cu, and Mn ions the magnetic behaviour changed from ferromagnetic to paramagnetic. Results obtained from the photocatalysis indicated that Mn0.75Co0.25Fe2O4 NPs (30.0 mg) were a promising photocatalyst achieving 96.0% removal of MB after 100 min of UV-light exposure in the alkaline solution. Antibacterial results showed that the most effective combination was Zn0.75Co0.25Fe2O4 NPs (20.0 ppm) displaying activity against Staphylococcus aureus, Enterococcus columbae, and Aerococcus viridians by 15.0, 13.0, and 12.0 mm ZOI, respectively. Additionally, Zn0.75Co0.25Fe2O4 NPs were active as antibiofilm factors producing activity by 63.7, 57.9, and 45.5% towards S. aureus, A. viridians, and E. columbae, respectively. Accordingly, Zn0.75Co0.25Fe2O4 and Mn0.75Co0.25Fe2O4 NPs can be utilized in industrial, biological and medical applications.

Biological and catalytic applications of green synthesized fluorescent N-doped carbon dots using Hylocereus undatus.

In this work, a simple hydrothermal route for the synthesis of fluorescent nitrogen doped carbon dots (N-CDs) is reported. The Hylocereus undatus (H. undatus) extract and aqueous ammonia are used as carbon and nitrogen source, respectively. The optical properties of synthesized N-CDs are analyzed using UV-Visible (UV-Vis) and fluorescence spectroscopy. The surface morphology, elemental composition, crystallinity and functional groups present in the N-CDs are examined using high resolution transmission electron microscopy (HR-TEM) with energy dispersive spectroscopy (EDS), selected area electron diffraction (SAED), X-ray diffraction (XRD) and Fourier transform infrared (FT-IR) spectroscopy, respectively. The synthesized N-CDs emit strong blue fluorescence at 400nm under the excitation of 320nm. Further, the excitation dependent emission properties are also observed from the fluorescence of synthesized N-CDs. The HR-TEM results reveal that synthesized N-CDs are in spherical shape with average diameter of 2.5nm. The XRD pattern exhibits, the graphitic nature of synthesized N-CDs. The doping of nitrogen is confirmed from the EDS and FT-IR studies. The cytotoxicity and biocompatibility of N-CDs are evaluated through MTT assay on L-929 (Lymphoblastoid-929) and MCF-7 (Michigan Cancer Foundation-7) cells. The results indicate that the fluorescent N-CDs show less cytotoxicity and good biocompatibility on both L-929 and MCF-7 cells. Moreover, the N-CDs show excellent catalytic activity towards the reduction of methylene blue by sodium borohydride.

Biosorption of methylene blue from wastewater by an extraction residue of Salvia miltiorrhiza Bge.

Efforts were made in this study to using an extraction residue of Salvia mitiorrziza Bge (SM), a widely utilized traditional Chinese medicine, as an effective biosorbent of methylene blue from polluted water. Batch experiments were carried out with original and chemical modification, particle size, dosage, solution pH, contact time, and initial concentration of the dye. Experimental data fit Langmuir isotherm and pseudo-second order kinetic best compared to other models applied in the study. Characterization of adsorption was determined by FT-IR, SEM and particle surface area measurement. The maximum monolayer biosorption capacity of raw SM is 100.0mg·g(-1). The citric acid and Na2CO3 modification can significantly enhance this value up to 161.29 and 178.57mg·g(-1), respectively. This investigation provides a novel approach for reutilizing the enormous quantity of Chinese herbal medicine wastes, which is significant since these dregs have brought out big environmental and heathy problems in the present China.

Sunlight mediated synthesis of silver nanoparticles using redox phytoprotein and their application in catalysis and colorimetric mercury sensing.

Owing to the benign nature, plant extracts mediated green synthesis of metal nanoparticles (NPs) is rapidly expanding. In this study, we demonstrated the successful green synthesis of silver nanoparticles (AgNPs) by utilizing natural sunlight and redox protein complex composed of ferredoxin-NADP(+) reductase (FNR) and ferredoxin (FD). The capping and stabilization of the AgNPs by the redox protein was confirmed by Fourier transform infrared spectroscopy. Light and redox protein is the prerequisite factor for the formation of AgNPs. The obtained result shows that the photo generated free radicals by the redox protein is responsible for the reduction of Ag(+) to Ag(0). Transmission electron microscopy revealed the formation of spherical AgNPs with size ranging from 10 to 15 nm. As-prepared AgNPs exhibit excellent catalytic activity toward the degradation of hazardous organic dyes, such as methylene blue, methyl orange and methyl red. These bio-inspired AgNPs is highly sensitive and selective in sensing hazardous mercury ions in the water at micromolar concentration. In addition, FNR/FD extract stabilized AgNPs showed good antimicrobial activity against gram positive and gram negative bacteria.

Coalesced chitosan activated carbon composite for batch and fixed-bed adsorption of cationic and anionic dyes.

A renewable waste tea activated carbon (WTAC) was coalesced with chitosan to form composite adsorbent used for waste water treatment. Adsorptive capacities of crosslinked chitosan beads (CCB) and its composite (WTAC-CCB) for Methylene blue dye (MB) and Acid blue 29 (AB29) were evaluated through batch and fixed-bed studies. Langmuir, Freundlich and Temkin adsorption isotherms were tested for the adsorption process and the experimental data were best fitted by Langmuir model and least by Freundlich model; the suitability of fitness was adjudged by the Chi-square (χ(2)) and Marquadt's percent standard deviation error functions. Judging by the values of χ(2), pseudo-second-order reaction model best described the adsorption process than pseudo-first-order kinetic model for MB/AB29 on both adsorbents. After five cycles of adsorbents desorption test, more than 50% WTAC-CCB adsorption efficiency was retained while CCB had <20% adsorption efficiency. The results of this study revealed that WTAC-CCB composite is a promising adsorbent for treatment of anionic and cationic dyes in effluent wastewaters.

A newly isolated Paecilomyces sp. WSH-L07 for laccase production: isolation, identification, and production enhancement by complex inducement.

Laccase can catalyze the oxidation of a wide range of organic and inorganic substrates. In this study, an easily detectable method was employed for screening laccase-producing microorganisms by using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as laccase-secretion indicator. A novel laccase-producing strain was isolated and identified as Paecilomyces sp. WSH-L07 according to the morphological characteristics and the comparison of internal transcribed spacer (ITS) ribosomal DNA (rDNA) gene sequences. In further investigation, the production of laccase by Paecilomyces sp. WSH-L07 was greatly enhanced by the nontoxic inducers of copper sulphate and methylene blue. Under the induction of 50 microM copper sulphate and 20 microM methylene blue, the maximum laccase production was obtained. When these inducers were added into cultivation medium at 24 h and 12 h, respectively, an increment of about 100 times of laccase activity compared with that of in inducer-free medium and about two times of that of in single copper-supplemented medium was observed. Compared with other Paecilomyces species, Paecilomyces sp. WSH-L07 exhibit the better laccase-producing characteristics with an activity of 1,650 U/l on the eighth day, suggesting its potential ability for industrial application.

Photocatalytic degradation of spill oils on TiO(2) nanotube thin films.

The nitrogen-doped TiO(2) nanotube (N-TNT) thin films were synthesized using ZnO nanorods as the template and doped with urea at 623K. Under ultraviolet (UV) and visible light irradiation, the efficiencies for photocatalytic degradation of methylene blue is as high as 30%. About 10% of toluene (representing aromatics in the spill oils) in sea water can be photocatalytically degraded under visible light radiation for 120 min. The aliphatic model compound (1-hexadecene) has, on the contrary, a less efficiency (8%) on the N-TNT photocatalyst. On the average, under visible light radiation, the effectnesses of the N-TNT for photocatalytic degradation of model compounds in the spill oils in sea water are 0.38 mg toluene/gN-TNTh and 0.25 mg 1-hexadecene/gN-TNTh. It is expected that spill oils in the harbors or seashores can be adsorbed and photocatalytically degraded on the N-TNT thin films that are coated onto levee at the sea water surface level.

Pre-clinical investigation of the efficacy of an artificial tear solution containing hydroxypropyl-guar as a gelling agent.

PURPOSE: Pre-clinical studies of a new artificial tear product (Systane Lubricating Eye Drops Alcon Laboratories, Inc., Fort Worth, TX) containing the novel gelling agent hydroxypropyl-guar (HP-guar) and two demulcents, polyethylene glycol 400 (PEG) and propylene glycol (PG) were conducted to determine the ability of the product to protect ocular surface epithelial cells from desiccation in vivo and in vitro, and to promote recovery of the damaged corneal epithelial barrier in vivo. Other leading artificial tear products were also evaluated as comparators to determine the relative effectiveness of different polymer systems. METHODS: Damage due to desiccation was assessed by measuring corneal uptake of methylene blue compared to untreated corneas. Corneas of anesthetized rabbits were treated with the new artificial tear product and subjected to desiccation by holding the eyelids open for 2 hours with a speculum. Control eyes were subjected to desiccation without application of a tear formulation. To measure recovery of the corneal epithelium from damage, corneas of anethesthetized rabbits were exposed to 0.01% benzalkonium chloride (BAC) for 5 minutes to increase epithelial permeability. The corneas were exposed to the new artificial tear for 1.5 hours followed by measurement of uptake of 5,6 carboxyfluorescein (CF). In the desiccation and CF uptake experiments, the new tear product was also compared to a tear product formulation without HP-guar and to a commercially available artificial tear containing carboxymethyl cellulose (CMC) and Purite. In a third set of experiments, immortalized human corneal epithelial cells and Chang conjunctival cells in culture were exposed to the PEG/PG/HP-guar tear product, the control formulation without HP-guar, a tear formulation preserved with BAC, or the artificial tear containing CMC/Purite for 15min. The tear formulation was removed and the cells were exposed to desiccating conditions in a laboratory safety hood for 10 or 30min. Cell viability was determined using the MTT assay. RESULTS: The in vivo desiccation model, showed that the new tear product, Systane, offered complete protection of the cornea from desiccation (methylene blue uptake not different than naïve control). Following exposure to 0.01% BAC, the new artificial tear product also provided an environment in which the corneal epithelium recovered completely from damage (CF uptake not different than normal, untreated cornea). This level of protection was not observed when corneas were treated with other formulations. Results from the in vitro desiccation procedure indicated that viability of corneal epithelial and Chang cells treated with the PEG/PG/HP-guar product was significantly greater than viability of cells treated with the tear product without HP-guar or the tear products containing BAC or CMC/Purite. CONCLUSIONS: The tear product containing HP-guar, PEG 400 and propylene glycol satisfies several pre-clinical criteria for an appropriate artificial tear formulation. It gives long-term desiccation protection of the intact cornea and also epithelial cells in culture and has no apparent deleterious affects on cells. It also provides conditions in which a damaged corneal epithelium can recover normal barrier function. The combination of ingredients in the formulation appears to provide an effective mucomimetic artificial tear product. These pre-clinical data suggest that the product will be effective in providing superior relief for the dry eye sufferer.

Detection of a novel ATP-dependent cross-linked protein at the 5' splice site-U1 small nuclear RNA duplex by methylene blue-mediated photo-cross-linking.

Assembly of spliceosomes involves a number of sequential steps in which small nuclear ribonucleoprotein particles (snRNPs) and some non-snRNP proteins recognize the splice site sequences and undergo various conformational rearrangements. A number of important intermolecular RNA-RNA duplexes are formed transiently during the process of splice site recognition. Various steps in the assembly pathway are dependent upon ATP hydrolysis, either for protein phosphorylation or for the activity of helicases, which may modulate the RNA structures. Major efforts have been made to identify proteins that interact with specific regions of the pre-mRNA during the stages of spliceosome assembly and catalysis by site-specific UV cross-linking. However, UV cross-linking is often inefficient for the detection of proteins that interact with base-paired RNA. Here we have used the complementary approach of methylene blue-mediated photo-cross-linking to detect specifically proteins that interact with the duplexes formed between pre-mRNA and small nuclear RNA (snRNA). We have detected a novel cross-link between a 65-kDa protein (p65) and the 5' splice site. A range of data suggest that p65 cross-links to the transient duplex formed by U1 snRNA and the 5' splice site. Moreover, although p65 cross-linking requires only a 5' splice site within the pre-mRNA, it also requires ATP hydrolysis, suggesting that its detection reflects a very early ATP-dependent event during splicing.

Uptake and photoeffectiveness of two thiazines in HeLa cells.

A comparative study of the uptake and photoinactivation of HeLa cells treated with methylene blue (MB) and toluidine blue (TB) has been performed. Cell toxicity induced by different concentrations of either MB or TB showed that 10(-5) M was the concentration at which dark damage was not observed, while an elevated photoinactivation could be detected with both thiazines. The uptake studies showed that the penetration kinetics of 10(-5) M MB into HeLa cells is faster than that of TB, used at the same concentration, reaching saturation (approximately 1.8 micrograms/10(6) cells) after 6 or 12 h of incubation, respectively. For both sensitizers, the survival of HeLa cells was dependent on the incubation time, as well as the light dose, for a given concentration (10(-5) M). Moreover, cell photoinactivation produced by MB was higher than that produced by TB. The differences between MB and TB obtained under our experimental conditions are discussed.

Pectin methylesterase, metal ions and plant cell-wall extension. Hydrolysis of pectin by plant cell-wall pectin methylesterase.

The hydrolysis of p-nitrophenyl acetate catalysed by pectin methylesterase is competitively inhibited by pectin and does not require metal ions to occur. The results suggest that the activastion by metal ions may be explained by assuming that they interact with the substrate rather than with the enzyme. With pectin used as substrate, metal ions are required in order to allow the hydrolysis to occur in the presence of pectin methylesterase. This is explained by the existence of 'blocks' of carboxy groups on pectin that may trap enzyme molecules and thus prevent the enzyme reaction occurring. Metal ions may interact with these negatively charged groups, thus allowing the enzyme to interact with the ester bonds to be cleaved. At high concentrations, however, metal ions inhibit the enzyme reaction. This is again understandable on the basis of the view that some carboxy groups must be adjacent to the ester bond to be cleaved in order to allow the reaction to proceed. Indeed, if these groups are blocked by metal ions, the enzyme reaction cannot occur, and this is the reason for the apparent inhibition of the reaction by high concentrations of metal ions. Methylene Blue, which may be bound to pectin, may replace metal ions in the 'activation' and 'inhibition' of the enzyme reaction. A kinetic model based on these results has been proposed and fits the kinetic data very well. All the available results favour the view that metal ions do not affect the reaction through a direct interaction with enzyme, but rather with pectin.

Use of methylene blue in suspected colovesical fistula.

Positive urine and blood assays of methylene and leukomethylene blue were obtained from 9 volunteers with normal colonic and renal function after a 100 ml, methylene blue enema (50 mg.). The study conclusively demonstrates that methylene blue is absorbed by rectal mucosa and excreted by the kidneys as a colored dye in the urine. Thus, intrarectal methylene blue cannot be used to confirm the diagnosis of a colovesical fistula.

Altered diaphorase activity in optochin-resistant pneumococci.

Methylene blue (MB) reductase activities of washed cell suspensions and of cell-free extracts prepared from optochin-resistant mutant pneumococci were two times greater than and four to eight times more resistant to optochin inhibition than those of similar preparations from the optochin-sensitive parent strains. With whole cells, optochin hydrochloride was approximately four times more potent than quinine hydrochloride in inhibiting MB reductase activity. However, with cell-free extracts, both drugs had similar inhibitory activities. Glucose uptake was not affected by optochin hydrochloride, and both optochin-sensitive and optochin-resistant pneumococci had similar glucose uptake patterns. Diaphorase activities of cell-free extracts prepared from optochin-resistant pneumococci were two times greater than and four to eight times more resistant to optochin inhibition than those of cell-free extracts prepared from the optochin-sensitive parent strains. Flavin concentrations of cell-free extracts prepared from optochin-sensitive and optochin-resistant strains were similar.