DNA prime-protein boost based vaccination with a conserved region of leptospiral immunoglobulin-like A and B proteins enhances protection against leptospirosis

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of theLeptospira genus. Vaccination with bacterins has severe limitations. Here, we evaluated the N-terminal region of the leptospiral immunoglobulin-like B protein (LigBrep) as a vaccine candidate against leptospirosis using immunisation strategies based on DNA prime-protein boost, DNA vaccine, and subunit vaccine. Upon challenge with a virulent strain ofLeptospira interrogans, the prime-boost and DNA vaccine approaches induced significant protection in hamsters, as well as a specific IgG antibody response and sterilising immunity. Although vaccination with recombinant fragment of LigBrep also produced a strong antibody response, it was not immunoprotective. These results highlight the potential of LigBrep as a candidate antigen for an effective vaccine against leptospirosis and emphasise the use of the DNA prime-protein boost as an important strategy for vaccine development.     

Photodynamic effect of light-emitting diode light on cell growth inhibition induced by methylene blue

The aim of this study was to propose the use of red light-emitting diode (LED) as an alternative light source for methylene blue (MB) photosensitizing effect in photodynamic therapy (PDT). Its effectiveness was tested against Staphylococcus aureus (ATCC 26923), Escherichia coli (ATCC 26922), Candida albicans (ATCC 90028) and Artemia salina. The maximum absorption of the LED lamps was at a wavelength of 663 nm, at intensities of 2,4,6 and 12 J.cm-2 for 10, 20, 30 and 60 min of exposure, respectively. Assays with and without LED exposure were carried out in plates containing MB at concentrations of 7 to 140.8 (micro) M for microorganisms and 13.35 to 668.5 (micro) M for microorganisms or microcrustaceans.The LED exposure induced more than 93.05%, 93.7% and 93.33% of growth inhibition for concentrations of 42.2 (micro)M for S.aureus (D-value=12.05 min) and 35.2 (micro)M for E.coli (D-value=11.51 min) and C.albicans (D-value=12.18 min), respectively after 20 min of exposure. LED exposure for 1 h increased the cytotoxic effect of MB against A.salina from 27% to 75%.Red LED is a promising light device for PDT that can effectively inhibit bacteria, yeast and microcrustacean growth.     

A specific effect of copper on methylene blue sensitized photodegradation of nucleic acid derivatives

Among several metal ions tested, Cu²⁺ was unique in slowing down methylene blue sensitized photodynamic breakdown of some nucleic acid bases and nucleosides. The t_1/2 values were increased in the case of xanthine and uric acid by Cu²⁺, but without any alteration in the nature or amounts of photoproducts formed. Xanthine was degraded quantitatively to allantoin and urea. The breakdown of the sugar moiety of nucleosides was more drastically retarded than that of the purine ring. The decomposition rate and its magnitude was dependent on the concentration of Cu²⁺ as well as the nucleoside. The most profound increase in t_1/2 values was found with xanthosine—7 min for the purine ring and 65 min for the ribose moiety, at 0.6 mM Cu²⁺ Hg²⁺ in the case of xanthine, and some paramagnetic metal ions in the case of the nucleosides, slowed down the photobreakdown to a small extent; however, differential effects were not observed unlike with Cu²⁺. None of the other metal ions tested significantly influenced the process.     

Degradation of methylene blue using co-dopants Mg and Se in an hydroxyapatite composite

In this work, a comparative study was made of different magnesium ion content incorporated into hydroxyapatite (HAP) and modified with selenite ions, with the aim to develop the degradation performance of methylene blue. Although the dopant metal (Mg²⁺) was present at a relatively low ratio, it induced a change in the microstructure, morphology, surface area, external surface charge, particle size, and degradation performance. The effect of magnesium and selenium binary doping on microstructure and degradation of methylene blue was evaluated. The external surface charge measured by zeta potential clarified that the highest negativity was −11.8 mV and this was accomplished in 1.0 Mg/Se-HAP. Furthermore, the roughness average increased from 36.8 nm, reaching 59.2 nm upon the addition of Mg(II). Moreover, transmission electron microscope micrographs showed that compositions were formed as rod shapes. The process of degradation was optimized, showing effectiveness in methylene blue degradation of 62.4% after 150 min of exposure to visible light. Electrostatic attraction and H-bonding, and coordination played vital roles in the adsorption process. The recyclability of the as-prepared compositions demonstrated that the effectiveness had been reduced to ~54.2% after five times of re-use.     

亚甲蓝与多聚阴离子化合物结合反应的研究

对光谱探针亚甲蓝(MB)分别与硫酸化茯苓多糖(SP)、硫酸软骨素(CS)、透明质酸(HA)等多聚阴离子化合物相互作用的吸收光谱进行了比较研究,探讨了CS等多聚阴离子化合物与MB相互作用机理及其与MB结合的功能基团,计算了MB与SP、CS最大结合数分别为54和73。研究结果表明MB与多聚阴离子化合物的磺酸基发生明显的结合反应,与羧基不能发生明显的结合反应,多聚阴离子化合物与MB结合的功能基团是磺酸基。     

Joint interaction of ethidium bromide and methylene blue with DNA. The effect of ionic strength on binding thermodynamic parameters

Large amount of data of experimental and theoretical studies have shown that ethidium bromide (EtBr) and methylene blue (MB) may bind to nucleic acids via three modes: intercalation between two adjacent base pairs, insertion into the plane between neighboring bases in the same strand (semi-intercalation), and outside binding with negatively charged backbone phosphate groups. The aim of the given research is to examine the behavior of these two ligands at both separate and joint DNA binding. The obtained experimental data show that the effect of simultaneous binding of EtBr and MB on double-stranded DNA has a non-additive effect of separate binding. The analyses of the melting thermodynamic parameters of DNA complexes with two bound ligands suggest competitive mechanism of interaction.     

Structural and functional characterization of the interaction of the photosensitizing probe methylene blue with Torpedo californica acetylcholinesterase

The photosensitizer, methylene blue (MB), generates singlet oxygen that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark, it inhibits reversibly. Binding is accompanied by a bathochromic absorption shift, used to demonstrate displacement by other acetylcholinesterase inhibitors interacting with the catalytic "anionic" subsite (CAS), the peripheral "anionic" subsite (PAS), or bridging them. MB is a noncompetitive inhibitor of TcAChE, competing with reversible inhibitors directed at both "anionic" subsites, but a single site is involved in inhibition. MB also quenches TcAChE's intrinsic fluorescence. It binds to TcAChE covalently inhibited by a small organophosphate (OP), but not an OP containing a bulky pyrene. Differential scanning calorimetry shows an ~8° increase in the denaturation temperature of the MB/TcAChE complex relative to native TcAChE, and a less than twofold increase in cooperativity of the transition. The crystal structure reveals a single MB stacked against Trp279 in the PAS, oriented down the gorge toward the CAS; it is plausible that irreversible inhibition is associated with photooxidation of this residue and others within the active-site gorge. The kinetic and spectroscopic data showing that inhibitors binding at the CAS can impede binding of MB are reconciled by docking studies showing that the conformation adopted by Phe330, midway down the gorge, in the MB/TcAChE crystal structure, precludes simultaneous binding of a second MB at the CAS. Conversely, binding of ligands at the CAS dislodges MB from its preferred locus at the PAS. The data presented demonstrate that TcAChE is a valuable model for understanding the molecular basis of local photooxidative damage.     

Prozone effects in microscopic agglutination tests for leptospirosis in the sera of mice infected with the pathogenic Leptospira interrogans serovar Canicola

Mice experimentally infected with a pathogenic strain of Leptospira interrogans serovar Canicola produced false negative results (prozone effect) in a microscopic agglutination test (MAT). This prozone effect occurred in several serum samples collected at different post-infection times, but it was more prominent in samples collected from seven-42 days post-infection and for 1:50 and 1:100 sample dilutions. This phenomenon was correlated with increased antibody titres in the early post-infection phase. While prozone effects are often observed in serological agglutination assays for the diagnosis of animal brucellosis and human syphilis, they are not widely reported in leptospirosis MATs.     

Induction of base-pair substitution and prameshift mutations in wild-type and repair-deficient strains of Salmonella typhimurium by the photodynamic action of methylene blue

Induction of back mutations to prototrophy by methylene blue (MB)-sensitized photodynamic (PD) treatment has been studied in wild-type and repair-deficient strains of Salmonella typhimurium carrying either the base-pair substitution mutation hisG46 or the frameshift mutation hisD3052. We found that reversion of the hisG46 mutation was increased in a strain carrying a uvrB deletion and decreased in a strain carrying a recA-type mutation. Reversion of the hisD3052 (frameshift) mutation, on the other hand, was decreased in both uvrB deletion and recA-type strains. The former results are consistent with the hypothesis that the majority of MB-sensitized PD-induced base-pair substitution mutations arise by a mechanism similar to that currently believed to be involved in UV mutagenesis. The latter results suggest that PD-induced frameshift mutations may arise in some other way, and two possible mechanisms involving sequential action of the excision repair and recombinational repair pathways are considered.     

In vitro evaluation of photodynamic activity of methylene blue against Trichophyton verrucosum azole-susceptible and -resistant strains

The intense search for the “Holy Grail” of antifungal therapy can be observed today. The searches are not limited only to discovery of potential antifungal drugs, but also new therapeutic strategies involving the use of chemosensitizers to achieve synergistic effect or physicochemical factors inducing stress conditions in fungal cells. In this study was examined in vitro effectiveness of photodynamic antifungal strategy with methylene blue using a light beam with a wavelength equal to 635 nm toward the Trichophyton verrucosum susceptible and itraconazole- and/or fluconazole-resistant strains. Methylene blue used at concentration equal to 5 μg/mL and in the presence of 40 J/cm² of light energy showed fungicidal effect toward the susceptible strains. However, for azole-resistant isolates, only the energy dose equal to 60 J/cm² at 5 μg/mL of methylene blue allowed to kill the pathogen. This study confirms that methylene blue induced by red light has a definite inhibitory effect on zoophilic dermatophytes.     

The effect of detergent treatment on methylene blue sensitized cytochrome b photoreduction in fractions from corn coleoptiles

It was previously demonstrated that photoexcited methylene blue can act as electron donor in red light induced reduction of a particulate b-type cytochrome in fraction from etiolated corn coleoptiles (Zea mays L. WF 9 × Bear 38). It was postulated that the same cytochrome as the one active in blue light photoprocesses was involved. This study describes the effect of detergents upon such red light induced reductions in corn coleoptile preparations fractionated after differential centrifugation into 9 KP, 21 KP and 50 KP (500–9000 g pellet, 9000–21,000 g pellet and 21,000–50,000 g pellet, spun for 20, 20, and 45 min, respectively). Both Triton X-100 (more effective) and deoxycholate (less effective and somewhat destructive) could be used as solubilizers if concentrations above the critical micellar concentration were chosen. Tween 40 was ineffective and dodecyl sulphate affected the cytochrome so that it lost its accessibility to electrons from photoexcited methylene blue. The recovery, measured as the ratio between light induced absorbance change (LIAC) in the Soret region after and before solubilization, was highest in 9 KP (70%) and lower in 21 and 50 KP fractions (50% and 43% respectively). The band in the Soret region in light minus dark spectra had its peak at longer wavelengths compared to the dithionite reduced minus no addition absorption difference band, whether the sample was solubilized or not. Similar results were obtained when the material was separated on a discontinuous sucrose gradient (15/28/33/45% w/w sucrose). In such a separation, the distribution of LIAC between fractions (collected at the interfaces) was about the same after solubilization as before (solubilization brought about a slight shift towards heavier fractions). The ratios of LIAC to the dithionite reduced minus no addition absorbance difference decreased upon detergent treatment. The LIAC still had its peak at longer wavelengths compared to the peak obtained upon dithionite reduction. The usefulness of detergents in the purification of the particulate b-type cytochrome is discussed.     

Fruit extract capped colloidal silver nanoparticles and their application in reduction of methylene blue dye

Green synthesis of silver nanoparticles (AgNPs) has become a promising environmentally benign synthetic route in nanoscience and nanotechnology during recent years. In the present work, we have developed an environment-friendly and low-cost method for synthesis of silver nanoparticles from silver nitrate using aqueous fruit extract of Dillenia indica. The as-synthesized nanoparticles were characterized by UV-Vis spectrophotometer, transmission electron microscopy (TEM) and X-ray diffraction (XRD). FTIR study was performed to know the interaction of bio-molecules present in the fruit extract with AgNPs. The catalytic application of the as-synthesized AgNPs was demonstrated against degradation of methylene blue (MB) in aqueous system. The absorption spectra of colloidal suspension of AgNPs showed characteristic surface plasmon resonance (SPR) band centred at a wavelength of 416?nm. TEM image showed that the AgNPs were almost spherical in shape having an average diameter of 10.78?±?.48?nm. XRD pattern and selected area electron diffraction (SAED) pattern with bright spots signify the crystalline nature of nanoparticles. The fruit extract-capped AgNPs was highly stable and have showed the effective catalytic activity in reduction of MB dye.     

Methylthioninium chloride (methylene blue) induces autophagy and attenuates tauopathy in vitro and in vivo

More than 30 neurodegenerative diseases including Alzheimer disease (AD), frontotemporal lobe dementia (FTD), and some forms of Parkinson disease (PD) are characterized by the accumulation of an aggregated form of the microtubule-binding protein tau in neurites and as intracellular lesions called neurofibrillary tangles. Diseases with abnormal tau as part of the pathology are collectively known as the tauopathies. Methylthioninium chloride, also known as methylene blue (MB), has been shown to reduce tau levels in vitro and in vivo and several different mechanisms of action have been proposed. Herein we demonstrate that autophagy is a novel mechanism by which MB can reduce tau levels. Incubation with nanomolar concentrations of MB was sufficient to significantly reduce levels of tau both in organotypic brain slice cultures from a mouse model of FTD, and in cell models. Concomitantly, MB treatment altered the levels of LC3-II, cathepsin D, BECN1, and p62 suggesting that it was a potent inducer of autophagy. Further analysis of the signaling pathways induced by MB suggested a mode of action similar to rapamycin. Results were recapitulated in a transgenic mouse model of tauopathy administered MB orally at three different doses for two weeks. These data support the use of this drug as a therapeutic agent in neurodegenerative diseases.     

Methylthioninium chloride (methylene blue) induces autophagy and attenuates tauopathy in vitro and in vivo

More than 30 neurodegenerative diseases including Alzheimer disease (AD), frontotemporal lobe dementia (FTD), and some forms of Parkinson disease (PD) are characterized by the accumulation of an aggregated form of the microtubule-binding protein tau in neurites and as intracellular lesions called neurofibrillary tangles. Diseases with abnormal tau as part of the pathology are collectively known as the tauopathies. Methylthioninium chloride, also known as methylene blue (MB), has been shown to reduce tau levels in vitro and in vivo and several different mechanisms of action have been proposed. Herein we demonstrate that autophagy is a novel mechanism by which MB can reduce tau levels. Incubation with nanomolar concentrations of MB was sufficient to significantly reduce levels of tau both in organotypic brain slice cultures from a mouse model of FTD, and in cell models. Concomitantly, MB treatment altered the levels of LC3-II, cathepsin D, BECN1, and p62 suggesting that it was a potent inducer of autophagy. Further analysis of the signaling pathways induced by MB suggested a mode of action similar to rapamycin. Results were recapitulated in a transgenic mouse model of tauopathy administered MB orally at three different doses for two weeks. These data support the use of this drug as a therapeutic agent in neurodegenerative diseases.     

Removal of methylene blue dye from aqueous solutions by natural clinoptilolite and clinoptilolite modified by iron oxide nanoparticles

Adsorption techniques are widely used to remove industrial wastewater contaminants, especially non-biodegradable colourants. In this study, Iranian zeolite clinoptilolite was synthesised using magnetic iron oxide as an inexpensive and efficient adsorbent. The results showed that using natural zeolite, the removal efficiency of 26.8.6% at pH?=?3 reached 48% at pH?=?9. However, the adsorption capacity of the modified clinoptilolite did not change by increasing pH; it ranged from 96.4% to 98.6%. Moreover, increase in the initial concentration of the dye did not have any effects on the removal efficacy of the modified clinoptilolite. Using natural zeolite, on the other hand, the adsorption capacity showed a significant decrease and reached less than 10% at the 200?mg/l dye concentration. At the optimal contact time of 45?min, the dye removal rate by the modified zeolite was more than 98% at the optimal dose of 0.5?g. Indeed, the adsorption isotherm complied with Freundlich equation. Overall, the results showed that in comparison to the natural zeolite, the adsorption capacity of the clinoptilolite modified by iron nanoparticles increased significantly due to the uniformity of the cavities and increase in the surface of the adsorbent.     

Enhancement of the binding affinity of methylene blue to site I in human serum albumin by cupric and ferric ions

In this work, the binding characteristics of methylene blue (MB) to human serum albumin (HSA) and the influence of Cu²⁺ and Fe³⁺ on the binding affinity of MB to HSA were investigated using fluorescence, absorption, circular dichroism (CD) spectroscopy and molecular modelling. The results of competitive binding experiments using the site probes ketoprofen and ibuprofen as specific markers suggested that MB was located in site I within sub‐domain IIA of HSA. The molecular modelling results agreed with the results of competitive site marker experiments and the results of CD spectra indicated that the interaction between MB and HSA caused the conformational changes in HSA. The binding affinity of MB to HSA was enhanced but to a different extent in the presence of Cu²⁺ and Fe³⁺, respectively, which indicated that the influence of different metal ions varied. Enhancement of the binding affinity of MB to HSA in the presence of Cu²⁺ is due to the formation of Cu²⁺–HSA complex leading to the conformational changes in HSA, whereas in the presence of Fe³⁺, enhancement of the binding affinity is due to the greater stability of the Fe³⁺–HSA–MB complex compared with the MB–HSA complex. Copyright © 2015 John Wiley & Sons, Ltd.     

The impact of crystallization conditions on structure‐based drug design: A case study on the methylene blue/acetylcholinesterase complex

Structure‐based drug design utilizes apoprotein or complex structures retrieved from the PDB. >57% of crystallographic PDB entries were obtained with polyethylene glycols (PEGs) as precipitant and/or as cryoprotectant, but <6% of these report presence of individual ethyleneglycol oligomers. We report a case in which ethyleneglycol oligomers' presence in a crystal structure markedly affected the bound ligand's position. Specifically, we compared the positions of methylene blue and decamethonium in acetylcholinesterase complexes obtained using isomorphous crystals precipitated with PEG200 or ammonium sulfate. The ligands' positions within the active‐site gorge in complexes obtained using PEG200 are influenced by presence of ethyleneglycol oligomers in both cases bound to W84 at the gorge's bottom, preventing interaction of the ligand's proximal quaternary group with its indole. Consequently, both ligands are ～3.0Å further up the gorge than in complexes obtained using crystals precipitated with ammonium sulfate, in which the quaternary groups make direct π‐cation interactions with the indole. These findings have implications for structure‐based drug design, since data for ligand‐protein complexes with polyethylene glycol as precipitant may not reflect the ligand's position in its absence, and could result in selecting incorrect drug discovery leads. Docking methylene blue into the structure obtained with PEG200, but omitting the ethyleneglycols, yields results agreeing poorly with the crystal structure; excellent agreement is obtained if they are included. Many proteins display features in which precipitants might lodge. It will be important to investigate presence of precipitants in published crystal structures, and whether it has resulted in misinterpreting electron density maps, adversely affecting drug design.     

A staining procedure for micronucleus test using new methylene blue and acridine orange: specimens that are supravitally stained with possible long-term storage

The micronucleus test has been widely used as an in vivo cytogenetic test. It employs two different kinds of supravital staining methods which use either new methylene blue (N) and Giemsa (G) or acridine orange (AO). We have developed a new staining procedure for the preparation of specimens supravitally stained with possible long-term storage, using both N and AO. This N/AO-staining method involves three steps; (1) combination of the target tissue or target cells with an equivalent volume of 0.5% solution of new methylene blue (N-staining step), (2) immediate smear of the mixture, followed by treatment with methanol for 10 min for fixation and removal of N and drying (referred to as fixed-decolorized specimens), and (3) staining with 0.007% solution of AO for 3 min, followed by washing with Sörensen's buffer (pH 6.8) and covering of specimens before observation (AO-staining step). To examine whether the N/AO-staining method is useful for the micronucleus test, comparisons were made between N-, N/AO-, and AO-stained specimens prepared supravitally from peripheral blood of rats with and without treatment of cyclophosphamide. The results indicate that N/AO-stained specimens can be supravitally observed after long-term storage with the same coloration and comparable frequencies of micronucleated reticulocytes with a positive response as AO-stained specimens, if the staining process is temporarily stopped before AO-staining (as fixed-decolorized specimens), or if the AO-staining step is repeated. The results also showed that separated reticulocyte types are supravitally stained in a similar fashion to N-stained specimens but not to AO-stained specimens, indicative of the preservation of the supravital feature of N-staining. Taken together these results suggest that the N/AO-staining procedure could offer an additional useful staining tool for the micronucleus test.     

Influence of breeding site availability on the oviposition behaviour of Aedes aegypti

Despite the importance of the mosquito Aedes aegypti in the transmission of arboviruses, such as yellow fever, Chikungunya fever and dengue fever, some aspects of their behaviour remain unknown. In the present study, the oviposition behaviour of Ae. aegypti females that were exposed to different densities of breeding sites (2, 4, 8 and 16) was evaluated in laboratory and semi-field conditions. The number of breeding sites that were used was proportional to the number available, but tended towards stabilisation. Females used four-six breeding sites on average, with a maximum of 11. A high percentage of eggs was observed in the water, along with the presence of a breeding site termed “favourite”, which received at least 40% of the eggs. The results are discussed in ecological, evolutionary and epidemiological approaches.