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Dexmedetomidine (Dex) plays protective effects on brain ischemia-reperfusion (I/R) injury, but its mechanism remains unclear. In this study, we aimed to investigate whether Dex protects neurons against I/R injury by activating SIRT3 mediated autophagy. The oxygen glucose deprivation-reperfusion (OGD/R) model was constructed in HT22 cells. Different doses of Dex (50 ng/mL, 100 ng/mL and 500 ng/mL) were treated to observe the changes of autophagy and SIRT3 expression. Further, the mimic of SIRT3 and SIRT3 inhibitor were used to analyze the effects of Dex on the SIRT3 expression in HT22 cells. Additionally, the autophagy inhibitor and AMPK inhibitor were used to analyze the effects of Dex on SIRT3 mediated autophagy. The cells viability, oxidative stress and ATP were observed using assay kits. The mitochondrial membrane potential (MMP) and death were analyzed by flow cytometry. The degree of autophagy was observed by acridine orange staining. Western blotting was used to analyze the expression of autophagy related proteins and AMPK/mTOR pathway related proteins. After Dex treatment, the OGD/R induced cell injury was significantly improved through decreasing the levels of LDH and H 2 O 2 , increasing levels of ATP and MMP. Furthermore, Dex increased the degree of autophagy and expression of SIRT3 in OGD/R injured cells. Through overexpression of SIRT3, the OGD/R induced cell injury was also clearly improved. But the SIRT3 inhibitor or autophagy inhibitor covered the roles of Dex. Additionally, AMPK inhibitor played an opposite role compared with the effects of Dex treatment. From this study, the protection mechanism of Dex on neurons I/R injury might related to the activation of SIRT3 mediated autophagy., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Dengue virus (DENV) is considered one of the most important pathogens in the world causing 390 million infections each year. Currently, the development of vaccines against DENV presents some shortcomings and there is no antiviral therapy available for its infection. An important challenge is that both treatments and vaccines must be effective against all four DENV serotypes. Nordihydroguaiaretic acid (NDGA), isolated from Larrea divaricata Cav. (Zygophyllaceae) has shown a significant inhibitory effect on a broad spectrum of viruses, including DENV serotypes 2 and 4., Purpose: We evaluated the in vitro virucidal and antiviral activity of NDGA on DENV serotype 1 (DENV1), including the study of its mechanism of action, to provide more evidence on its antiviral activity., Methods: The viability of viral particles was quantified by the plaque-forming unit reduction method. NDGA effects on DENV1 genome and viral proteins were evaluated by qPCR and immunofluorescence, respectively. Lysosomotropic activity was assayed using acridine orange and neutral red dyes., Results: NDGA showed in vitro virucidal and antiviral activity against DENV1. The antiviral effect would be effective within the first 2 h after viral internalization, when the uncoating process takes place. In addition, we determined by qPCR that NDGA decreases the amount of intracellular RNA of DENV1 and, by immunofluorescence, the number of cells infected. These results indicate that the antiviral effect of NDGA would have an intracellular mechanism of action, which is consistent with its ability to be incorporated into host cells. Considering the inhibitory activity of NDGA on the cellular lipid metabolism, we compared the antiviral effect of two inhibitors acting on two different pathways of this type of metabolism: 1) resveratrol that inhibits the sterol regulatory element of binding proteins, and 2) caffeic acid that inhibits the 5-lipoxygenase (5-LOX) enzyme. Only caffeic acid produced an inhibitory effect on DENV1 infection. We studied the lysosomotropic activity of NDGA on host cells and found, for the first time, that this compound inhibited the acidification of cell vesicles which would prevent DENV1 uncoating process., Conclusion: The present work contributes to the knowledge of NDGA activity on DENV. We describe its activity on DENV1, a serotype different to those that have been already reported. Moreover, we provide evidence on which stage/s of the viral replication cycle NDGA exerts its effects. We suggest that the mechanism of action of NDGA on DENV1 is related to its lysosomotropic effect, which inhibits the viral uncoating process., Competing Interests: Declaration of Competing Interest All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy.”, (Copyright © 2022. Published by Elsevier GmbH.)

Methamphetamine (MA) and 3,4-methylenedioxymethamphetamine (MDMA) are empathogen (entactogen) psychoactive designer drugs which are mainly used for recreational purposes. Both MA and MDMA are central nervous system stimulants which are classified as monoamine neurotransmitter reuptake inhibitors. They have strong cytotoxic effects on dopaminergic and serotonergic neurons. Neurotoxicities of MA and MDMA by glial activation have been shown. The present work has investigated and measured cytotoxic, necrotic and apoptotic, and autophagic effects of MA and MDMA on U-87 MG (glial) and B104-1-1 (neuronal) cell lines by janus green, ethidium bromide/acridine orange, and monodansylcadaverine/propidium iodide staining to evaluate and compare their effects on glial and neuronal cells, respectively. The results of the present work showed that: (1) MDMA induced more potent mitochondrial toxicity, stronger necrotic and autophagic effects than MA in both B104-1-1 (neuronal) and U-87 MG (glial) cell lines; (2) although MDMA induced stronger apoptotic effect than MA on U-87 MG cell line, it had equal apoptotic effect on B104-1-1 cell line with MA; and (3) MDMA induced more potent mitochondrial toxicity, stronger necrotic, apoptotic, and autophagic effects than MA in B104-1-1 cell line than U-87 MG cell line., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Bone marrow-derived mesenchymal stem cells (BMSCs) and bone morphogenetic protein-2 (BMP-2) have been studied for bone repair because they have regenerative potential to differentiate into osteoblasts. The development of injectable and in situ three-dimensional (3D) scaffolds to proliferate and differentiate BMSCs and deliver BMP-2 is a crucial technology in BMSC-based tissue engineering., Methods: The proliferation of mouse BMSCs (mBMSCs) in collagen/poly-γ-glutamic acid (Col/γ-PGA) hydrogel was evaluated using LIVE/DEAD and acridine orange and propidium iodide assays. In vitro osteogenic differentiation and the gene expression level of Col/γ-PGA(mBMSC/BMP-2) were assessed by alizarin red S staining and quantitative reverse-transcription polymerase chain reaction. The bone regeneration effect of Col/γ-PGA(mBMSC/BMP-2) was evaluated in a mouse calvarial bone defect model. The cranial bones of the mice were monitored by micro-computed tomography and histological analysis., Results: The developed Col/γ-PGA hydrogel showed low viscosity below ambient temperature, while it provided a high elastic modulus and viscous modulus at body temperature. After gelation, the Col/γ-PGA hydrogel showed a 3D and interconnected porous structure, which helped the effective proliferation of BMSCs with BMP-2. The Col/γ-PGA (mBMSC/BMP-2) expressed more osteogenic genes and showed effective orthotopic bone formation in a mouse model with a critical-sized bone defect in only 3-4 weeks., Conclusion: The Col/γ-PGA(mBMSC/BMP-2) hydrogel was suggested to be a promising platform by combining collagen as a major component of the extracellular matrix and γ-PGA as a viscosity reducer for easy handling at room temperature in BMSC-based bone tissue engineering scaffolds., (© 2022. Korean Tissue Engineering and Regenerative Medicine Society.)

Injury to the intestinal epithelial cells and loss of the intestinal barrier are critical to heatstroke. To reveal the mechanism through which heatstroke leads to intestinal epithelial injury, the relationship between reactive oxygen species (ROS), c-Jun NH2-terminal kinase (JNK), and lysosomes were studied in intestinal epithelial cells subjected to heat stress. Cells of heat stress groups were incubated at 43 °C for 1 h, then incubated at 37 °C as indicated. Control group cells were incubated at 37 °C. Cell-counting kit-8 assay was used to assess cell viability. Cells were labeled with 2'-7'dichlorofluorescin diacetate and acridine orange (AO) staining, respectively, the total ROS and AO were detected by confocal laser scanning microscopy and flow cytometry. Apoptosis was analyzed by flow cytometry using annexin V-fluorescein isothiocyanate/prodium iodide staining, the expressions of mitogen-activated protein kinases were detected by western blotting. Heat stress induced apoptosis and inhibited cell viability, the production of ROS, and lysosomal injury in IEC-6 cells. After pretreatment with the lysosomal cathepsin inhibitor E64, the JNK inhibitor SP600125, or the ROS scavenger NAC, the effect of heat stress on apoptosis or lysosomal injury was significantly attenuated. In conclusion, heat stress induced apoptosis, lysosomal injury, and the accumulation of ROS in IEC-6 cells; mechanistically, this occurred through the ROS-induced activation of JNK signaling, which mediated the lysosomal injury and ultimately apoptosis., Competing Interests: Declaration of competing interest None., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Candida albicans is a common human fungal pathogen that colonizes mucosa and develops biofilm in the oral cavity that causes oral candidiasis. It has been reported that cytochrome P450 enzyme (CYP51), a vital part of the ergosterol synthesis cascade, is associated with Candida infections and its biofilm formation. Thidiazuron, a phenyl-urea cytokinin, exhibits anti-senescence and elicitor activity against fungal infection in plants. However, how Thidiazuron impacts C. albicans biofilm formation is still uncertain. Here, we aimed to investigate the effects of a Thidiazuron against the growth and biofilm formation properties of C. albicans using in silico and in vitro experimental approaches. A preliminary molecular docking study revealed potential interaction between Thidiazuron and amino acid residues of CYP51. Further in vitro antifungal susceptibility test, scanning electron microscopy (SEM) and time kill analysis revealed the anti-fungal activity of Thidiazuron in both dose and time-dependent manner. Crystal violet staining, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay revealed 50% inhibition in C. albicans biofilm by Thidiazuron at concentrations 11 and 19 µM respectively. Acridine orange staining assay visually confirmed the biofilm inhibitory potential of Thidiazuron. The gene expression study showed that Thidiazuron treatment down regulated the expression of genes involved in ergosterol synthesis (ERG3, ERG11, ERG25), cell adhesion (ASL3, EAP1), and hyphae development (EFG1, HWP1, SAP5) in C. albicans. Wherease, the expression of negative transcription regulator of hyphae (NRG1) was upregulated (5.7-fold) by Thidiazuron treatment. Collectively, our data suggest that Thidiazuron is a robust antifungal compound and an outstanding biofilm inhibitor, which may promise further therapeutic development due to CYP51 binding and inhibition of ergosterol formation against C. albicans., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Rhein is an anthraquinone found in Rheum palmatum, used in Chinese medicine. Due to potential anticancer properties, the study assessed its effect on the lysosomal compartment, which indirectly influences cell death. The experiment was performed on HeLa cells by treating them with rhein at concentrations of 100-300 µM. LC3-II protein and caspase 3/7 activity, level of apoptosis, the concentration of reactive oxide species (ROS), and mitochondrial potential (Δψm) were evaluated by the cytometric method. To evaluate the permeability of the lysosomal membrane (LMP), staining with acridine orange and the assessment of activity of cathepsin D and L in the lysosomal and extralysosomal fractions were used. Cell viability was assessed by -(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT) and neutral red (NR) assays. Changes in cells were also demonstrated at the level of electron, optical, confocal, and fluorescence microscopy. Inhibition of autophagy was done using chloroquine. Rhein-induced degradation processes were confirmed by an increase in the number of primary lysosomes, autophagosomes, and autolysosomes. At high concentrations, rhein caused the generation of ROS, which induced LMP expressed by quenching of acridine orange fluorescence. These results correlated with a reduction of lysosomes, as visualized in graphical modeling, with the decreased uptake of NR by lysosomes, and increased activity of cathepsin D and L in the extralysosomal fraction. The studies also showed an increase in the activity of caspase 3/7 and a decrease in the expression of Bcl-2 protein, indicative of rhein-stimulated apoptosis. At the same time, we demonstrated that preincubation of cells with chloroquine inhibited rhein-induced autophagy and contributed to increased cytotoxicity to HeLa cells. Rhein also induced DNA damage and led to cycle arrest in the S phase. Our results indicate that rhein, by inducing changes in the lysosomal compartment, indirectly affects apoptosis of HeLa cells and in combination with autophagy inhibitors may be an effective form of anticancer therapy., (© 2022 Wiley Periodicals LLC.)

Our study was aimed to investigate the effects of lgals3a (Gal-3 encoding gene) on the development of zebrafish embryo and its underlying mechanisms. Morpholino (MO) technology was used to inhibit the expression of zebrafish lgals3a, and the effect of lgals3a gene knockdown on zebrafish embryo development and the number of monocyte macrophages was observed. Effect of lgals3a-e3i3-MO on apoptosis of zebrafish was detected by acridine orange staining. In addition, the mRNA expression levels of Wnt/β-catenin signaling pathway-related genes were detected by RT-qPCR. Compared with control-MO group, the zebrafish embryos injected with lgals3a-e3i3-MO had obvious defects in the head, eyes, and tail, and pericardial edema. Lgals3a-e3i3-MO significantly reduced the number of mononuclear macrophages in zebrafish embryos compared with the control-MO group. The results of acridine orange staining showed that compared with the control-MO group, lgals3a-e3i3-MO promoted cardiomyocyte apoptosis in zebrafish. Furthermore, lgals3a-e3i3-MO significantly up-regulated the expression of dkk1b, wnt9a, lrp5, fzd7a, β-catenin, Gsk-3β, mycn, myca in the Wnt/β-catenin pathway, and decreased the expression of lef1. These results indicate that lgals3a-e3i3-MO inhibits zebrafish embryo development, reduces the number of mononuclear macrophages, activates Wnt/β-catenin signaling pathway and promotes cardiomyocyte apoptosis., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Combinatorial therapies based on the simultaneous administration of multiple drugs can lead to synergistic effects, increasing the efficacy of the cancer therapy. However, it is crucial to develop new delivery systems that can increase the drugs' therapeutic selectivity and efficacy. Gold core silica shell (AuMSS) nanoparticles present physicochemical properties that allow their simultaneous application as drug delivery and imaging agents. Herein, poly(ethylene glycol) was modified with 4-methoxybenzamide and 3-(triethoxysilyl)propyl isocyanate (TPANIS) to create a novel surface functionalization capable of improving the colloidal stability and specificity of AuMSS nanospheres towards cancer cells. Moreover, a dual drug combination based on Doxorubicin (DOX) and Acridine orange (AO) was characterized and administered using the AuMSS-TPANIS nanospheres. The obtained results show that the DOX:AO drug combination can mediate a synergistic therapeutic effect in both HeLa and MCF-7 cells, particularly at the 2:1, 1:1, and 1:2 ratios. Additionally, the TPANIS functionalization increased the AuMSS nanospheres colloidal stability and selectivity towards MCF-7 cancer cells (overexpressing sigma receptors). Such also resulted in an enhanced cytotoxic effect against MCF-7 cells when administering the DOX:AO drug combination with the AuMSS-TPANIS nanospheres. Overall, the obtained results confirm the therapeutic potential of the DOX:AO drug combination as well as the targeting capacity of AuMSS-TPANIS, supporting its application in the cancer-targeted combinatorial chemotherapy., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Aging of red blood cells (RBCs) is associated with alteration in a wide range of RBC features, occurring each on its own timescale. A number of these changes are interrelated and initiate a cascade of biochemical and structural transformations, including band-3 clustering and phosphatidylserine (PS) externalization. Using specific band-3 clustering agents (acridine orange (AO) and ZnCl 2 ), we examined whether treatment of RBCs with these agents may affects PS externalization and whether this process is Ca 2+ -dependent. RBCs were isolated from the blood of eight healthy donors upon obtaining their informed consent. The suspension was supplemented with increasing concentrations of AO or ZnCl 2 (from 0.5 to 2.0 mM) and incubated at 25 °C for 60 min. To detect PS at the RBC surface, we used allophycocyanin-conjugated recombinant human Annexin V. We demonstrated, that treatment of RBCs with both clustering agents caused an elevation in the percent of cells positively labeled by Annexin-V (RBC PS ), and that this value was not dependent on the presence of calcium in the buffer: RBCs treated with AO in the presence of either EDTA, EGTA or calcium exhibited similar percentage of RBC PS . Moreover, the active influx of Zn 2+ into RBCs induced by their co-incubation with both ZnCl 2 and A23187 did not increase the percent of RBC PS as compared to RBCs incubated with ZnCl 2 alone. Taken together, these results demonstrate that the band-3 clustering agents (AO or ZnCl 2 ) induce PS externalization in a Ca 2+ independent manner, and we hereby suggest a possible scenario for this phenomenon., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Monoamine oxidase (MAO) is an important drug target as the MAO isoforms play key roles in neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease, as well as in neuropsychiatric diseases such as depression. Methylene blue is an inhibitor of MAO-A, while azure B, the major metabolite of methylene blue, and various other structural analogues retain the ability to inhibit MAO-A. Based on this, the present study evaluated 22 dyes, many of which are structurally related to methylene blue, as potential inhibitors of human MAO-A and MAO-B. The results highlighted three dye compounds as good potency competitive and reversible MAO inhibitors, and which exhibit higher MAO inhibition than methylene blue: acridine orange, oxazine 170 and Darrow red. Acridine orange was found to be a MAO-A specific inhibitor (IC 50  = 0.017 μM), whereas oxazine 170 is a MAO-B specific inhibitor (IC 50  = 0.0065 μM). Darrow red was found to be a non-specific MAO inhibitor (MAO-A, IC 50  = 0.059 μM; MAO-B, IC 50  = 0.065 μM). These compounds may be advanced for further testing and preclinical development, or be used as possible lead compounds for the future design of MAO inhibitors., (© 2019 John Wiley & Sons A/S.)

The susceptibility of DNA in situ to denaturation is modulated by its interactions with histone and nonhistone proteins, as well as with other chromatin components related to the maintenance of the 3D nuclear structure. Measurement of DNA proclivity to denature by cytometry provides insight into chromatin structure and thus can be used to recognize cells in different phases of the cell cycle, including mitosis, quiescence (G 0 ), and apoptosis, as well as to identify the effects of drugs that modify chromatin structure. Particularly useful is the method's ability to detect chromatin changes in sperm cells related to DNA fragmentation and infertility. This article presents a flow cytometric procedure for assessing DNA denaturation based on application of the metachromatic property of acridine orange (AO) to differentially stain single- versus double-stranded DNA. This approach circumvents limitations of biochemical methods of examining DNA denaturation, in particular the fact that the latter destroy higher orders of chromatin structure and that, being applied to bulk cell populations, they cannot detect heterogeneity of individual cells. Because the metachromatic properties of AO have also found application in other cytometric procedures, such as differential staining of RNA versus DNA and assessment of lysosomal proton pump including autophagy, to avert confusion between these approaches and the use of this dye in the DNA denaturation assay, these AO applications are briefly outlined in this unit as well. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Differential staining of single- versus double-stranded DNA with acridine orange., (© 2019 John Wiley & Sons, Inc.)

Musculoskeletal sarcomas are rare and aggressive human malignancies affecting bones and soft tissues with severe consequences, in terms of both morbidity and mortality. An innovative technique that combines photodynamic surgery (PDS) and therapy (PDT) with acridine orange has been recently suggested, showing promising results. However, due to the low incidence of sarcoma in humans, this procedure has been attempted only in pilot studies and stronger evidence is needed. Naturally occurring tumors in cats are well-established and advantageous models for human cancers. Feline injection-site sarcoma (FISS) shares with human musculoskeletal sarcomas a mesenchymal origin and an aggressive behavior with a high relapse rate. Furthermore, wide surgical excision is not always possible due to the size and site of development. We assessed the feasibility and the effectiveness of PDS and PDT with acridine orange to prevent FISS recurrence by treating a short case series of cats. For PDS, the surgical field was irrigated with an acridine orange solution and exposed to UV light to enlighten the residual tumor tissue, and the resultant fluorescent areas were trimmed. For PDT, before wound closure, the field was again irrigated with acridine orange solution and exposed to visible light to get the antitumoral cytocidal effect. The procedure was easy to perform and well tolerated, we did not observe any major complications, and all the surgical resection margins were free of disease. Finally, at follow-up, all treated patients did not show evidence of tumor recurrence and had a significantly higher event-free survival rate in respect to a control group treated only by surgery. In conclusion, by this study we demonstrated that, in FISS, PDS and PDT with acridine orange may improve local tumor control, granting a better outcome, and we laid the foundation to validate its effectiveness for the treatment of human musculoskeletal sarcomas.

The increasing and indiscriminate use of pesticides may lead to the intoxication and contamination of the environment and foods. In addition, pesticides can cause fungal resistance promoting the selection of resistant phytopathogenic fungi. This is a problem in the agricultural and human health areas, which leads to a need for developing new methodologies to address this problem. Photodynamic inactivation is a promising strategy involving the association of a photosensitizer (PS), light, and molecular oxygen to inhibit the growth of microorganisms. In this work, the PS acridine orange (AO) was deposited using the spray layer-by-layer technique. The effectiveness of the method was evaluated by the analysis of the growth evolution of the colonies as a function of the amount of PS layers applied in field in the presence of sunlight. Image processing and analysis of the fractal theory were used to evaluate the growth of the colonies. The results revealed that AO is a candidate PS for use in field. It was possible to observe the reduction of the growth dynamics of the colonies with the increase of the number of PS layers. The parameters related to the fractality of the system were described by mathematical models of the fractal growth of interfaces. The knowledge of these parameters can help to identify new strategies for the control of phytopathogenic microorganisms that directly affect agricultural production., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We present a new technique for obtaining simultaneous multimodal quantitative phase and fluorescence microscopy of biological cells, providing both quantitative phase imaging and molecular specificity using a single camera. Our system is based on an interferometric multiplexing module, externally positioned at the exit of an optical microscope. In contrast to previous approaches, the presented technique allows conventional fluorescence imaging, rather than interferometric off-axis fluorescence imaging. We demonstrate the presented technique for imaging fluorescent beads and live biological cells.

KRAS is often found mutated in lethal cancers and should be an important target for anticancer drug development. However, no effective inhibitor has been reported so far, prompting the scientific community to describe the RAS proteins as nearly "undruggable". Recent approaches developed to modulate KRAS protein expression comprises the targeting of G-quadruplex (G4) structures formed within the nuclease hypersensitive element of KRAS promoter region, by designing small and specific ligands to stabilize the tertiary fold and reduce gene expression. In this work, we report in vitro and in silico studies of novel acridine orange (AO) derivatives (C 3 -C 8 ), developed as G4 stabilizing agents. The results show that the ligands bind with high affinity and stabilize KRAS22-RT G4 with modest specificity over duplex DNA. The most promising ligand C 8 stabilizes the structure by ≈ 40 °C. Molecular docking using NMR-derived distance restraints reveal atomic details about the ligand structural features in the interaction with KRAS22-RT G4. In vitro studies with HeLa cells show that the ligands are cytotoxic with IC50 values between 0.9 μM and 5.7 μM. Moreover, the ligands tend to localize in the nucleus as shown by confocal fluorescence microscopy. Overall, these results show that the reported AO ligands display favourable properties as G4 ligands and this study provides structural detail for the development of lead KRAS G4 ligands., (Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Human bladder cancer (BC) cells exhibit a high basal level of autophagic activity with accumulation of acridine-orange(AO)-stained acidic vesicular organelles. The rapid AO relocalization was observed in treated BC cells under blue-light emission. To investigate the cytotoxic effects of AO on human BC cell lines under blue-light exposure, human immortalized uroepithelial (SV-Huc-1) and BC cell lines (5637 and T24) were treated with indicated concentrations of AO or blue-light exposure alone and in combination. The cell viability was then determined using WST-1, time-lapse imaging with a Cytosmart System and continuous quantification with a multi-mode image-based reader. Treatment of AO or blue-light exposure alone did not cause a significant loss of viability in BC cells. However, AO exhibited a dose-dependent increment of cytotoxicity toward BC cells under blue-light exposure. Furthermore, the tumor formation of BC cells with treatment was significantly reduced when evaluated in a mouse xenograft model. The photodamage caused by AO was nearly neglected in SV-Huc-1 cells, suggesting a differential effect of this treatment between cancer and normal cells. In summary, AO, as a photosensitizer, disrupts acidic organelles and induces cancer cell death in BC cells under blue-light irradiation. Our findings may serve as a novel therapeutic strategy against human BC.

Candida albicans is responsible for many of the infections affecting immunocompromised individuals. Although most C. albicans are susceptible to antifungal drugs, uncontrolled use of these drugs has promoted the development of resistance to current antifungals. The clinical implication of resistant strains has led to the search for safer and more effective drugs as well as alternative approaches, such as controlled drug release using liposomes and photodynamic inactivation (PDI), to eliminate pathogens by combining light and photosensitizers. In this study, we used layer-by-layer (LBL) assembly to immobilize triclosan and acridine orange encapsulated in liposomes and investigated the possibility of controlled release using light. Experiments were carried out to examine the susceptibility of C. albicans to PDI. The effects of laser irradiation were investigated by fluorescence microscopy, atomic force microscopy, and release kinetics. Liposomes were successfully prepared and immobilized using the self-assembly LBL technique. Triclosan was released more quickly when the LBL film was irradiated. The release rate was approximately 40% higher in irradiated films (fluence of 15J/cm 2 ) than in non-irradiated films. The results of the susceptibility experiments and surface morphological analysis indicated that C. albicans cell death is caused by photodynamic inactivation. Liposomes containing triclosan and acridine orange may be useful for inactivating C. albicans using light. Our results lay the foundation for the development of new clinical strategies to control resistant strains., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Pectinases, produced by microorganisms, have wide range application in food industry, textile processing, paper making, coffee and tea fermentation, etc. It accounts for 10% of the global industrial enzymes produced. The most important and widely used commercial pectinase polygalacturonase is produced by alkalophilic strains of Bacillus sp. and Streptomyces sp. Here, we explored 29 bacterial strains isolated from rotten mango samples for polygalacturonase production and selected 16 strains through preliminary screening by well-plate method for enzyme activity. The maximum zone of inhibition of pectin was observed up to 28 mm in diameter but one strain ZM11 was exhibiting no activity. Quantitative dinitrisalicylic acid (DNS) assay for polygalacturonase enzyme was also performed for the selected bacterial isolates. All the strains bestowed significant enzyme activity with the highest activity of 2.4 U/µL exhibited by strain ZM3 (P ≤0.05). Characterization of the isolates was performed using different biochemical tests which also confirmed the isolates as members of the genus Bacillus. Mutation was induced to the selected strains by UV light and acridine orange for different periods of time. Qualitative and quantitative assays of the mutant bacterial isolates showed that the enzyme activity increased to 4.62 U/µL which clearly indicated that induced mutation enhanced the ability of Bacillus strains to produce more polygalacturonase enzyme up to 3-fold as compared to the wild strains (P ≤0.05). Molecular characterization by 16S rRNA sequences further confirmed that the bacterial isolates belong to Bacillus subtilis and B. amyloliquefaciens.

Singlet oxygen plays a role in cellular stress either by providing direct toxicity or through signaling to initiate death programs. It was therefore of interest to examine cell death, as occurs in Arabidopsis, due to differentially localized singlet oxygen photosensitizers. The photosensitizers rose bengal (RB) and acridine orange (AO) were localized to the plasmalemma and vacuole, respectively. Their photoactivation led to cell death as measured by ion leakage. Cell death could be inhibited by the singlet oxygen scavenger histidine in treatments with AO but not with RB In the case of AO treatment, the vacuolar membrane was observed to disintegrate. Concomitantly, a complex was formed between a vacuolar cell-death protease, RESPONSIVE TO DESSICATION-21 and its cognate cytoplasmic protease inhibitor ATSERPIN1. In the case of RB treatment, the tonoplast remained intact and no complex was formed. Over-expression of AtSerpin1 repressed cell death, only under AO photodynamic treatment. Interestingly, acute water stress showed accumulation of singlet oxygen as determined by fluorescence of Singlet Oxygen Sensor Green, by electron paramagnetic resonance spectroscopy and the induction of singlet oxygen marker genes. Cell death by acute water stress was inhibited by the singlet oxygen scavenger histidine and was accompanied by vacuolar collapse and the appearance of serpin-protease complex. Over-expression of AtSerpin1 also attenuated cell death under this mode of cell stress. Thus, acute water stress damage shows parallels to vacuole-mediated cell death where the generation of singlet oxygen may play a role., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Phototoxicity consists in the capability of certain innocuous molecules to become toxic when subjected to suitable illumination. In order to discover new photoactive drugs or characterize phototoxic pollutants, it would be advantageous to use simple biological tests of phototoxicy. In this work, we present a pilot screening of 37 dyes to test for phototoxic effects in the roundworm Caenorhabditis elegans. Populations of this nematode were treated with different dyes, and subsequently exposed to 30 min of white light. Behavioral outcomes were quantified by recording the global motility using an infrared tracking device (WMicrotracker). Of the tested compounds, 17 dyes were classified as photoactive, being phloxine B, primuline, eosin Y, acridine orange and rose Bengal the most phototoxic. To assess photoactivity after uptake, compounds were retested after washing them out of the medium before light irradiation. Dye uptake into the worms was also analyzed by staining or fluorescence. All the positive drugs were incorporated by animals and produced phototoxic effects after washing. We also tested the stress response being triggered by the treatments through reporter strains. Endoplasmic reticulum stress response (hsp-4::GFP strain) was activated by 22% of phototoxic dyes, and mitochondrial stress response (hsp-6::GFP strain) was induced by 16% of phototoxic dyes. These results point to a phototoxic perturbation of the protein functionality and an oxidative stress similar to that reported in cell cultures. Our work shows for the first time the feasibility of C. elegans for running phototoxic screenings and underscores its application on photoactive drugs and environmental pollutants assessment.

Acridine orange (AO) is an antimalarial drug that accumulates into acidic cellular compartments. Lysosomes are quite acidic in cancer cells, and on this basis we have demonstrated that photoactivated AO is selectively toxic in sarcomas. However, photodynamic therapy is only locally effective, and cannot be used to eradicate systemic residual disease. In this study, we have evaluated the activity of non-photoactivated AO on sensitive and chemoresistant osteosarcoma (OS) cells to be considered for the systemic delivery. Since lysosomes are even more acidic in chemoresistant cells (MDR), we found that AO accumulation was significantly higher in the lysosomes of MDR in respect to parental cells, and in both cell types, therapeutic doses of AO significantly inhibited cell growth. However, the level of growth inhibition was inversely related to the level of lysosomal uptake of AO, suggesting that the main target of this agent is indeed extralysosomal. A significant reduction of intracellular ATP content and of the expression of mitochondrial complex III suggests a mitochondrial targeting. Notably, MDR cells showed a lower mitochondrial activity. Finally, the combined treatment of AO with the anticancer agent doxorubicin (DXR) significantly increased chemotoxicity by promoting DXR mitochondrial targeting, as revealed by the further reduction in ATP intracellular content. In conclusion, AO is able to effectively target both sensitive and resistant OS cells through mitotoxicity.

Aim: We undertook studies to determine the lethal dose 50 (LD50) of acridine orange (AO) using mice in order to confirm the safety of intravenous administration of AO., Materials and Methods: We used 40 mice and AO was administered once intravenously. General behavior and mortality were continuously observed for 14 days. At the end of the experiment, all animals were sacrificed for subsequent studies., Results: The LD50 for AO in male and female mice was determined to be 32 mg/kg and 36 mg/kg, respectively. Histopathological abnormalities were observed in only one mouse which died three days after the administration of AO. The other nine mice which died immediately after the administration of AO had no pathological findings in major organs., Conclusion: The clinical use of AO can be kept at 1.0 mg/kg or below and, therefore, intravenous administration of AO might be safe for use as cancer therapy., (Copyright © 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

It was found in our previous study that berberine (BBR) and fluconazole (FLC) used concomitantly exhibited a synergism against FLC-resistant Candida albicans in vitro. The aim of the present study was to clarify how BBR and FLC worked synergistically and the underlying mechanism. Antifungal time-kill curves indicated that the synergistic effect of the two drugs was BBR dose dependent rather than FLC dose dependent. In addition, we found that BBR accumulated in C. albicans cells, especially in the nucleus, and resulted in cell cycle arrest and significant change in the transcription of cell cycle-related genes. Besides BBR, other DNA intercalators, including methylene blue, sanguinarine, and acridine orange, were all found to synergize with FLC against FLC-resistant C. albicans. Detection of intracellular BBR accumulation by fluorescence measurement showed that FLC played a role in increasing intracellular BBR concentration, probably due to its effect in disrupting the fungal cell membrane. Similar to the case with FLC, other antifungal agents acting on the cell membrane were able to synergize with BBR. Interestingly, we found that the efflux of intracellular BBR was FLC independent but strongly glucose dependent and associated with the drug efflux pump Cdr2p. These results suggest that BBR plays a major antifungal role in the synergism of FLC and BBR, while FLC plays a role in increasing the intracellular BBR concentration.

Oogenesis involves a sequence of cellular divisions and developmental changes leading to the formation of oocytes, whose role in development is to transfer genomic information to the next generation. During this process, the gene expression pattern changes considerably concomitant with genome remodeling, while genomic information is maintained. The development of the gonad in zebrafish is unique in that it goes through an initial ovarian phase and subsequently into either ovarian or testicular phases. How the germ cells choose to commit to an oogenic fate and enter meiosis or alternatively not to enter meiosis and commit to a spermatogenetic fate remains a key question in development. Lack of suitable markers has hampered the understanding of the principles controlling sex differentiation in zebrafish. The current study was aimed at finding substantive cytochemical markers to identify specific oocyte stages primarily focusing on the DNA and RNA component of cells, using fluorescent dyes: acridine orange and propidium iodide. The pattern of synthesis and appearance of nucleoli was stage specific and may be used to identify stages of oogenesis. A distinguishing and possibly diagnostic feature of the staining pattern observed was the low level of chromatin staining compared to other cellular structures. This may be related to the more diffuse state of chromatin that occurs prior to thickening of chromosomes from the pachytene stage onwards. Although the fluorescent dyes may be useful in determining the localization of nucleic acids in tissue sections, it was not possible to quantify the relative contribution of the DNA and RNA components of specific stages of oocyte growth., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Although combination of surgery with chemotherapy has noticeably improved the survival rate of osteosarcoma patients, the application of anticancer drugs is still associated with significant adverse reactions, for instance acquisition of drug-resistant phenotypes, necessitating the development of new chemotherapeutical agents. Therefore, the aim of this study was to research, if taurine chloramine (NCT) induces apoptosis in the osteosarcoma cell lines HOS, MG-63, and SAOS-2. Proliferation of osteosarcoma cells was detected with the "EZ4U Cell Proliferation and Cyotoxicity Assay" showing a time- and dose-dependent cytotoxic effect of NCT on these cell lines. After 3 h of incubation all cell lines showed significantly less cells at 5.5 mM NCT solutions, after 6 h at concentrations of 1.1 and 2.2 mM. Acridine-orange fluorescence nuclear staining showed characteristic features of apoptosis. DNA fragmentation was detected via ELISA, showing significant results for HOS and MG-63 after 6 h at an NCT concentration of 3.3 mM. Results of JC-1 mitochondrial FACS analysis presented a significant increase in apoptotic cells after 6 h at 3.3 mM for the tested cell lines. Summarized, the results of this study indicate that NCT is a promising agent in osteosarcoma therapy., (Copyright © 2012 Orthopaedic Research Society.)

Chromaffin cell catecholamines are released when specialized secretory vesicles undergo exocytotic membrane fusion. Evidence indicates that vesicle supply and fusion are controlled by the activity of the cortical F-actin-myosin II network. To study in detail cell cortex and vesicle interactions, we use fluorescent labeling with GFP-lifeact and acidotropic dyes in confocal and evanescent wave microscopy. These techniques provide structural details and dynamic images of chromaffin granules caged in a complex cortical structure. Both the movement of cortical structures and granule motion appear to be linked, and this motion can be restricted by the myosin II-specific inhibitor, blebbistatin, and the F-actin stabilizer, jasplakinolide. These treatments also affect the position of the vesicles in relation to the plasma membrane, increasing the distance between them and the fusion sites. Consequently, we observed slower single vesicle fusion kinetics in treated cells after neutralization of acridine orange-loaded granules during exocytosis. Increasing the distance between the granules and the fusion sites appears to be linked to the retraction of the F-actin cytoskeleton when treated with jasplakinolide. Thus, F-actin-myosin II inhibitors appear to slow granule fusion kinetics by altering the position of vesicles after relaxation of the cortical network.

For rapid, intra-operative pathological margin assessment to guide staged cancer excisions, multimodal confocal mosaic scan image wide surgical margins (approximately 1 cm) with sub-cellular resolution and mimic the appearance of conventional hematoxylin and eosin histopathology (H&E). The goal of this work is to combine three confocal imaging modes: acridine orange fluorescence (AO) for labeling nuclei, eosin fluorescence (Eo) for labeling cytoplasm, and endogenous reflectance (R) for marking collagen and keratin. Absorption contrast is achieved by alternating the excitation wavelength: 488 nm (AO fluorescence) and 532 nm (Eo fluorescence). Superposition and false-coloring of these modes mimics H&E, enabling detection of cutaneous squamous cell carcinomas (SCC). The sum of mosaic Eo+R is false-colored pink to mimic the appearance of eosin, while the AO mosaic is false-colored purple to mimic the appearance of hematoxylin in H&E. In this study, mosaics of 10 Mohs surgical excisions containing invasive SCC, and five containing only normal tissue were subdivided for digital presentation equivalent to 4 × histology. Of the total 50 SCC and 25 normal sub-mosaics presented, two reviewers made two and three type-2 errors (false positives), respectively. Limitations to precisely mimic H&E included occasional elastin staining by AO. These results suggest that confocal mosaics may effectively guide staged SCC excisions in skin and other tissues.

Placental malaria is a common clinical complication during pregnancy and is associated with abortion, premature delivery, intrauterine growth retardation and low birth weight. The present study was designed to delineate the underlying mechanism of placental pathology during malarial infection with special reference to oxidative stress and apoptosis. Experimentally, pregnant BALB/c mice were infected with Plasmodium berghei infected red blood cells on gestation day 10. The presence of malarial infection in placenta was confirmed by histopathological studies. It was observation that infected placenta had plugged placental sinusoids with parasitized red blood cells and malarial pigments. Interestingly, we found significant increase in the level of malondialdehyde, the index of oxidative stress and decreased activity of catalase, the antioxidant in infected placenta. Furthermore, in infected placenta the oxidative stress mediated apoptosis was determined by DNA fragmentation assay, ethidium bromide/acridine orange staining and caspase activity. It was observed that oxidative stress begin after second day of malarial infection. Interestingly, it was observed that there was down regulation of anti-apoptotic protein Bcl-2 and up regulation of pro-apoptotic protein Bax in infected placenta, suggesting the involvement of mitochondrial pathway of apoptosis which was further confirmed by activation of caspase 9. However, no change in the expression of Fas gene and caspase 8 activity, indicated the absence of death receptor pathway. Thus, it can be concluded that the placental pathology during malarial infection is mediated by mitochondrial pathway of apoptosis occurring due to augmented lipid peroxidation which may in turn jeopardise the materno-fetal relationship.

During the past 20 years, we have found that acridine orange (AO) selectively accumulates in musculoskeletal sarcomas in vivo or exerts selective cytocidal effects against sarcoma cells in vitro after illumination of the tumor cells with visible light or irradiation of the cells with low-dose X-rays. Based on the data obtained from basic research, we have employed reduction surgery followed by photo- or radiodynamic therapy using AO (AO-PDT & RDT) in 71 patients with musculoskeletal sarcomas, in an attempt to maintain excellent limb function in the patients. We have obtained good local control rates and remarkably better limb functions with this approach as compared to the results obtained with the conventional wide resection surgery. Our basic research demonstrated that AO accumulates densely in intracellular acidic vesicles, especially lysosomes, in an acidity-dependent manner. In cancer cells that proliferate under hypoxic conditions or with Warburg's effect, active glycolysis produces an enormous number of protons, which are released by the cells via proton pumps into the extracellular fluid or lysosomes to maintain a neutral pH of the cytosolic fluid. Cancer cells contain many strongly acidic lysosomes of large sizes; therefore, AO shows marked and prolonged accumulation in the acidic lysosomes of cancer cells. Photon energy excites the AO resulting in the production of activated oxygen species, which oxidize the fatty acids of the lysosomal membrane, resulting in the leakage of lysosomal enzymes and protons, followed by apoptosis of the cancer cells. Based on these observations, we conclude that AO-PDT & RDT target acidic vesicles, especially the lysosomes, in cancer cells, to exhibit selective anti-cancer cell activity. Therefore, it is suggested that AO excited by photon energy has excellent potential as an anticancer "Magic Bullet".

Although the survival of patients with osteosarcoma has improved following development of chemotherapy and surgery, the presence of pulmonary metastases indicate a poor prognosis. We developed photodynamic and radiodynamic therapies with acridine orange (AO-PDT and AO-RDT) for minimally invasive surgery to treat musculoskeletal sarcomas and reported a good clinical outcome of local control and limb function. We investigated the effect of AO-PDT using flash-wave light (FWL) on pulmonary metastasis of mouse osteosarcoma. In in vitro and in vivo studies, AO alone and AO-PDT significantly inhibited cell invasion and the growth of pulmonary metastases from primary mouse osteosarcoma. AO may have a specific metastasis-inhibitory effect, different from the effect of AO-PDT. The fluorovisualization effect on pulmonary metastases following intravenous AO administration showed that pulmonary metastases localized on the lung surface were recognized as brilliant green lesions. In conclusion, AO-PDT using FWL inhibited cell invasion and pulmonary metastases in mouse osteosarcoma; therefore, this treatment modality might be applicable for treating pulmonary metastasis from malignant musculoskeletal tumors in humans.

Recent study revealed that most of cancer cells form acidic environment and have many, large size acidic organelle, especially lysosomes due to cancer specific proton dynamics induced by active aerobic glycolysis without TCA cycle in the mitochondoria. We have developed a new cancer therapy with acridine orange photodynamics which targeted on such cancer acidity and treated patients with malignant bone and soft tissue tumors. In this paper, we describe mechanism and clinical outcome of the therapy.

There is performed a comparative analysis of action of four acridine derivatives and of one xanthene derivative (pyronine G) on activity of liver monoamine oxidase (MAO) of two species of poikilothermal freshwater animals: a representative of amphibians--the common frog Rana temnporaria and a representative of the order Salmonidae--the European whitefish Coregonus lavaretus. The studied synthetic hexamerous tricyclic compounds show the irreversible character of inhibition of intermediate potency towards the enzyme from both biological sources. There are obtained qualitative and quantitative differences in the reactional ability and selectivity of action of the studied inhibitors for liver MAO of frog and whitefish. The obtained data of the inhibitory analysis with use of specific substrates are an indirect proof for the existence in liver of the studies frog species of two molecular forms, whereas in the whitefish liver--single molecular MAO form.

Tyrosinase (TYR: EC 1.14.18.1) was covalently modified onto the surface of a cyanuric chloride-activated carbon felt (CF) from the mixed buffer solution of TYR and acridine orange (AO). The resulting TYR-immobilized CF (TYR/AO-CF) was used as a working electrode unit of an electrochemical flow-through detector for mono- and di-phenolic compounds (i.e., p-chlorophenol (p-CP), p-cresol, phenol, and catechol), which detects the reduction current of enzymatically produced o-quinones at -0.05 V (vs. Ag/AgCl). The presence of AO (0.2 mM) in TYR solution during the enzyme immobilization step was significantly effective for the signal enhancements especially for p-CP, and the cathodic peak currents of p-CP by the TYR/AO-CF-based detector were much larger than those by the TYR-CF-based detector prepared from TYR solution without AO. The oxymetry with Clark-type oxygen electrode revealed that monophenolase activity of free TYR in 1 mM phosphate buffer (pH 7.0) was greatly enhanced in the presence of AO (0.2 mM), whereas diphenolase activity was not so much influenced. Furthermore, the comparison of cyclic voltammograms of TYR/AO-CF and TYR-CF in air-saturated phosphate buffer containing each substrate revealed that the electrochemical reduction rate of p-chloro-o-benzoquinone at TYR/AO-CF was faster than that at TYR-CF. In addition, the electrochemical impedance spectroscopy revealed that the structural properties of immobilized TYR on the CF would be influenced by AO. Some kinds of interaction of AO with TYR would affect the enzymatic kinetics and the structural properties of the immobilized TYR, leading to the signal enhancement of the TYR-CF-based flow biosensor especially for monophenolic compounds.

The interaction of acridine orange with blood albumins and tissue cells from different organs of white mouse has been studied by the spectral luminescence method. It was shown that acridine orange, by penetrating cells or orangelles, is able to intercalate between base pairs in the DNA molecule. It was found that the application of acridine orange as a fluorescent probe can influence the metabolic activity of organs.

Cucurbiturils (CBs) of the appropriate size (CB[7] and CB[8]) form strong guest-host complexes in phosphate buffer solution (PBS) with acridine orange (AO) and pyronine Y (PYY) with 1 : 1 and 2 : 1 stoichiometries for CB[7] and CB[8] complexes, respectively. Binding constants in the range 0.87-1.60 x 10(6) M(-1) and 5.2-6.3 x 10(13) M(-2) were determined by titration with fluorescence spectroscopy for 1 : 1 and 2 : 1 complexes, respectively. These binding constants in PBS and the eight-fold excess of CBs minimize the presence of free dye in solution and also stabilize the host-guest complex in the culture medium. Images showing that the CB complexes can cross the cell membrane of 3T3 cells have been acquired using fluorescence microscopy. Given the current importance of supramolecular CB complexes and the search for new drug delivery systems, the present findings open avenues for the use of CBs as nanocapsules to transport drugs into the cells.

One of the early events of the DNA damage response (DDR), particularly if the damage involves induction of DNA double-strand breaks, is remodeling of chromatin structure characterized by its relaxation (decondensation). The relaxation increases accessibility of the damaged DNA sites to the repair machinery. We present here a simple cytometric approach to detect chromatin relaxation based on the analysis of the proclivity of DNA in situ to undergo denaturation after treatment with acid. DNA denaturation is probed by the metachromatic fluorochrome acridine orange (AO) which differentially stains single-stranded (denatured) DNA by fluorescing red and the double-stranded DNA by emitting green fluorescence. DNA damage was induced in both human leukemic TK6 cells and mitogen-stimulated human peripheral blood lymphocytes by exposure to UV light or by treatment with H(2)O(2). Chromatin relaxation was revealed by diminished susceptibility of DNA to denaturation, likely reflecting decreased DNA torsional stress, seen as soon as 10 min after subjecting cells to UV or H(2)O(2). While cells in all phases of the cell cycle showed a comparable extent of chromatin relaxation upon UV or H(2)O(2) exposure, H2AX was phosphorylated on Ser139 predominantly in S-phase cells. The data are consistent with the notion that chromatin relaxation is global, affects all cells with damaged DNA, and is a prerequisite to the subsequent steps of DDR that can be selective to cells in a particular phase of the cell cycle. The method offers a rapid and simple means of detecting genotoxic insult on cells.

TRPA1, a Ca(2+)-permeable cation channel that is expressed in sensory neurones, is involved in the perception of chemical irritants and mechanical hyperalgesia. TRPA1 is activated by either covalent or reversible binding of various chemical compounds, including allylisothiocyanate or acrolein, and is further sensitised by increases in the intracellular Ca(2+) concentration. We here demonstrate that TRPA1 confers a sensitivity towards near ultraviolet (UVA) light, which rapidly causes Ca(2+) entry. In electrophysiological recordings in whole cell and inside out modes, exposure to UVA light activated typical TRPA1 currents in a wavelength-dependent and membrane-delimited manner. In the presence of the photosensitising agents acridine orange (100 nM) or hypericin (10 nM), the sensitivity of light-induced TRPA1 activation was increased and extended towards the visible spectrum. Since extracellular application of hydrogen peroxide mimicked the effect of UVA irradiation and since dithiothreitol partly reversed the activation by UVA exposure, we conclude that reactive oxygen species may mediate the light-induced activation of TRPA1. Accordingly, hydrogen peroxide induced a TRPA1 activation with a membrane-delimited mode of action that was attenuated by dithiothreitol. Intracellular but not extracellular application of FeSO(4), which catalyses the formation of highly reactive hydroxyl radicals potentiated the hydrogen peroxide-stimulated TRPA1 activation. We conclude that, via generation of reactive oxygen species, light-induced TRPA1 activation provides an additional mode of activation, which renders TRPA1 a likely molecular candidate in processes leading to painful or burning sensations during photodynamic therapy or upon local application of hydrogen peroxide.

Nano-hydroxyapatite (nano-HAP) may be a better candidate for an apatite substitute of bone in biomedical applications than micro-sized hydroxyapatite (m-HAP). However, size control is always difficult when synthesizing well-defined nano-HAP particles. In this study, nano-HAP particles with diameters of approximately 20nm (np20) and approximately 80nm (np80) were synthesized and characterized. The size effects of these nano-HAPs and m-HAP were studied on human osteoblast-like MG-63 cells in vitro. Our results demonstrate that both cell proliferation and cell apoptosis are related to the size of the HAP particles. Np20 has the best effect on promotion of cell growth and inhibition of cell apoptosis. This work provides an interesting view of the role of nano-HAPs as ideal biomedical materials in future clinical applications.

The aim of this study was to determine the extent of DNA fragmentation and the presence of denatured single-stranded or normal double-stranded DNA in spermatozoa with large nuclear vacuoles (LNV) selected by high magnification. Fresh semen samples from 30 patients were prepared by discontinuous isolate concentration gradient. Spermatozoa with normal nucleus (NN) and LNV were selected at x8400 magnification and placed on different slides. DNA fragmentation was determined by TUNEL assay. Denatured and double-stranded DNA was identified by the acridine orange fluorescence method. DNA fragmentation in spermatozoa with LNV (29.1%) was significantly higher (P < 0.001) than in spermatozoa with NN (15.9%). Therefore, cleavage of genomic DNA in low molecular weight DNA fragments (mono- and oligonucleosomes), and single-strand breaks (nicks) in high molecular weight DNA occur more frequently in spermatozoa with LNV. Similarly, the percentage of denatured-stranded DNA in spermatozoa with LNV (67.9%) was significantly higher (P < 0.0001) than in spermatozoa with NN (33.1%). The high level of denatured DNA in spermatozoa with LNV suggests precocious decondensation and disaggregation of sperm chromatin fibres. The results show an association between LNV and DNA damage in spermatozoa, and support the routine morphological selection and injection of motile spermatozoa at high magnification for ICSI.

To understand the environmental impacts of carbon nanotubes (CNTs), the toxicity study was carried out with water-soluble multi-walled carbon nanotubes (MWCNTs) using zebrafish (Danio rerio) as a model system. Zebrafish embryos were treated with different concentrations of MWCNTs. Teratogenic effects were evaluated through changes in embryonic development after 24 hours post-fertilization (hpf), 48 hpf and 72 hpf. No observable effect concentration (NOEC) was at 40 microg/mL. Lowest effect concentration (LOEC) of MWCNTs which caused significant phenotypic defects in zebrafish embryo was 60 microg/mL. At concentration above 60 microg/mL, slimy mucus like coating was observed around the embryo. At high concentrations, MWCNTs was found to be involved in the apoptosis, delayed hatching and formation of abnormal spinal chords. Thus, the toxicity of multi-walled carbon nanotubes is concentration dependent.

The temporal organization of the synthesis of RNA and antibodies in immunocompetent cells has been investigated by fluorecsent spectroscopy. Two fluorochromes: acridine orange and 1-anilino-8-naphthalenesulfonate were used. Temporal changes in the mean values of the characteristic parameters alpha' and beta in blood lymphocytes stained with acridine orange and 1-anilino-8-naphthalenesulfonate in the immune reaction to the introduction of ovalbumin were obtained. It was shown that the maximal increase in RNA and protein synthesis in blood lymphocytes by a factor of 6-8 occurs after the fourth immunization. The simultaneous increase in RNA and protein synthesis in cells was indicative of cell activation. A general response of immunocompetent blood cells to the introduction of the antigen was observed. The results confirm the idea about the cooperative interaction between different lymphocyte subpopulations during the synthesis of antibodies.

10-N-Nonyl acridine orange (NAO) is used as a mitochondrial probe because of its high affinity for cardiolipin (CL). Targeting of NAO may also depend on mitochondrial membrane potential. As the nonyl group has been considered essential for targeting, a systematic study of alkyl chain length was undertaken; three analogues (10-methyl-, 10-hexyl-, and 10-hexadecyl-acridine orange) were synthesized and their properties studied in phospholipid monolayers and breast cancer cells. The shortest and longest alkyl chains reduced targeting, whereas the hexyl group was superior to the nonyl group, allowing very clear and specific targeting to mitochondria at concentrations of 20-100 nM, where no evidence of toxicity was apparent. Additional studies in wild-type and cardiolipin-deficient yeast cells suggested that cellular binding was not absolutely dependent upon cardiolipin.

The redox environment of the cell is currently thought to be extremely important to control either apoptosis or autophagy. This study reported that reactive oxygen species (ROS) and nitric oxide (NO) generations were induced by evodiamine time-dependently; while they acted in synergy to trigger mitochondria-dependent apoptosis by induction of mitochondrial membrane permeabilization (MMP) through increasing the Bax/Bcl-2 or Bcl-x(L) ratio. Autophagy was also stimulated by evodiamine, as demonstrated by the positive autophagosome-specific dye monodansylcadaverine (MDC) staining as well as the expressions of autophagy-related proteins, Beclin 1 and LC3. Pre-treatment with 3-MA, the specific inhibitor for autophagy, dose-dependently decreased cell viability, indicating a survival function of autophagy. Importantly, autophagy was found to be promoted or inhibited by ROS/NO in response to the severity of oxidative stress. These findings could help shed light on the complex regulation of intracellular redox status on the balance of autophagy and apoptosis in anti-cancer therapies.

While lysosomal disruption seems to be a late step of necrosis, a moderate lysosomal destabilization has been suggested to participate early in the apoptotic cascade. The origin of lysosomal dysfunction and its precise role in apoptosis or apoptosis-like process still needs to be clarified, especially upon carcinogen exposure. In this study, we focused on the implication of lysosomes in cell death induced by the prototype carcinogen benzo[a]pyrene (B[a]P; 50 nM) in rat hepatic epithelial F258 cells. We first demonstrated that B[a]P affected lysosomal morphology (increase in size) and pH (alkalinization), and that these changes were involved in caspase-3 activation and cell death. Subsequently, we showed that lysosomal modifications were partly dependent on mitochondrial dysfunction, and that lysosomes together with mitochondria participate in B[a]P-induced oxidative stress. Using two iron chelators (desferrioxamine and deferiprone) and siRNA targeting the lysosomal iron-binding protease lactoferrin, we further demonstrated that both lysosomal iron content and lactoferrin were required for caspase-3 activation and apoptosis-like cell death.

Ofloxacin (15 microg/mL) and acridine orange (5 microg/mL) induce mutagenicity by different mechanisms in the photosynthetic flagellate Euglena gracilis. The present study examined whether Pycnogenol (PYC; 5-100 microg/mL) or Ginkgo biloba extract (EGb 761; 5-100 microg/mL) could protect against the mutagenic effects of each of the mutagens and the potential mechanisms underlying such protection. The highest concentration of PYC and EGb 761 effectively reduced the mutagenic activity of both ofloxacin and acridine orange by more than 99% (p < 0.001). Using luminol-dependent photochemical methodology it was demonstrated that EGb 761 and PYC were effective antioxidants. In addition, as determined by spectrophotometry, PYC and EGb 761 bound acridine orange. Both PYC and EGb 761 have been shown to produce dual antimutagenic effects, as evidenced by both antioxidant and physicochemical properties. The findings suggest that EGb 761 and PYC would thus be suitable for future study, not only as antioxidants, but also as antimutagenic agents.

Ultraviolet-irradiation (UV), ethyl methane sulfonate (EMS) and acridine orange (AO) were used to induce citric acid overproduction mutations in Aspergillus niger UMIP 2564. Among 15, eight of the mutant derivatives, were improved with respect to citric acid production from sucrose in batch cultures. Maximum product yield (60.25%) was recorded by W5, a stable UV mutant, with approximately 3.2-fold increase when compared to the parental wild type strain. In terms of the kinetic parameters for batch fermentation processes, the mutation doubled the specific substrate uptake rate and achieved 4.5- and 7.5-fold improvements in citric acid productivity and specific productivity, respectively. For reduction of the fermentation medium cost, corn steep liquor and calcium phosphate pre-treated beet molasses were successfully used as substituents of nitrogen and carbon sources in the growth medium, respectively. These medium substitutions resulted in a W5 citric acid fermentation culture with a product yield of 74.56%.