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A ubiquitous presence of microplastics and nanoplastics created a new toxicological research area arising concept of "plastic rivers". But, the precise molecular mechanisms by which its exposure affects developmental neurotoxicity are poorly understood. Hence, in the present investigation, healthy zebrafish embryos were exposed to different concentrations of 500 nm polystyrene microplastics (0.1 ppm, 1 ppm and 10 ppm) to assess the neurotoxicity and the underlying biomolecular mechanism. On the last day of exposure, behaviour, accumulation, embryotoxicity, acridine orange staining, antioxidant enzyme assay, acetylcholinesterase assay, nitric oxide (NO) estimation, along with neurotransmitter (serotonin, dopamine) quantification and gene expression using qRT-PCR (bdnf, p53, bcl-2, caspase-3, caspase-9) were performed. As a result, we found that zebrafish embryos ingest and bioaccumulate microplastic without causing any morphological changes and lethality. The survival and hatching rates of the embryos were also unaffected but the swimming behaviour patterns were found to be altered. Further, acridine orange staining exhibited more apoptosis in treated groups with increased p53, caspase-3, caspase-9 and decreased bcl-2 gene expression. Moreover, polystyrene microplastics exposure resulted in reduced acetylcholinesterase activity leading to elevated NO concentration along with altered serotonin and dopamine levels and subsequently leading to down-regulated bdnf gene expression and modulated downstream apoptotic signalling, confirming the neurotoxicity potential of microplastics causing neuronal dysfunction. This study also compared the binding affinities between styrene and human proteins (Bdnf, p53 and Bcl-2) using bioinformatics methods, and docking results showed negative binding energy resulting in high binding affinities of Bcl-2 then p53 and Bdnf with styrene., Competing Interests: Declaration of competing interest The authors show no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Microglia regulate the response to injury and disease in the brain and spinal cord. In white matter diseases microglia may cause demyelination. However, how microglia respond and regulate demyelination is not fully understood., Methods: To understand how microglia respond during demyelination, we fed mice cuprizone-a potent demyelinating agent-and assessed the dynamics of genetically fate-mapped microglia. We then used single-cell RNA sequencing to identify and track the microglial subpopulations that arise during demyelination. To understand how microglia contribute to the clearance of dead oligodendrocytes, we ablated microglia starting at the peak of cuprizone-induced cell death and used the viability dye acridine orange to monitor apoptotic and lytic cell morphologies after microglial ablation. Lastly, we treated serum-free primary microglial cultures to model distinct aspects of cuprizone-induced demyelination and assessed the response., Results: The cuprizone diet generated a robust microglial response by week 4 of the diet. Single-cell RNA sequencing at this time point revealed the presence of several cuprizone-associated microglia (CAM) clusters. These clusters expressed a transcriptomic signature indicative of cytokine regulation and reactive oxygen species production with altered lysosomal and metabolic changes consistent with ongoing phagocytosis. Using acridine orange to monitor apoptotic and lytic cell death after microglial ablation, we found that microglia preferentially phagocytose lytic carcasses. In culture, microglia exposed to lytic carcasses partially recapitulated the CAM state, suggesting that phagocytosis contributes to this distinct microglial state during cuprizone demyelination., Conclusions: Microglia serve multiple roles during demyelination, yet their transcriptomic state resembles other neurodegenerative conditions. The phagocytosis of cellular debris is likely a universal cause for a common neurodegenerative microglial state., (© 2022. The Author(s).)

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: Frameshift mutations in LRPAP1 are responsible for autosomal recessive high myopia in human beings but its underlying mechanism remains elusive. This study aims to investigate the effect of LRPAP1 defect on ocular refractive development and its involved mechanism., Methods: A lrpap1 mutant zebrafish line with homozygous frameshift mutation was generated by CRISPR/Cas9 technology and confirmed by Sanger sequencing. The ocular refractive phenotype was analyzed by calculating the relative refractive error (RRE) with vivo photography and histological analysis at different development stages, together with examining ocular structure change via transmission electron microscopy. Further, RNA sequencing and bioinformatics analysis were performed. The potentially involved signaling pathway as well as the interacted protein were investigated in vivo., Results: The lrpap1 homozygous mutant zebrafish line showed myopic phenotype. Specifically, the mutant lines showed larger eye axial length-to-body length in one-month old individuals and a myopic shift with an RRE that changed after two months. Collagen fibers became thinning and disordered in the sclera. Further, RNA sequencing and bioinformatics analysis indicated that apoptosis signaling was activated in mutant line; this was further confirmed by acridine orange and TUNEL staining. Moreover, the expression of TGF-β protein was elevated in the mutant lines. Finally, the treatment of wild-type embryos with a TGF-β agonist aggravated the degree of eyeball apoptosis; conversely, the use of a TGF-β inhibitor mitigated apoptosis in mutant embryos., Conclusion: The study provides functional evidence of a link between lrpap1 and myopia, suggesting that lrpap1 deficiency could lead to myopia through TGF-β-induced apoptosis signaling. Video abstract., (© 2022. The Author(s).)

Bisphenol A (BPA) is one of the world's most widely used plasticizer, and its hazardous impacts have been well studied. However, few studies focused on the effects of parental long-term BPA exposure on the bone development of offspring. In the present study, the bone development of offspring was studied following long-term exposure of parental zebrafish to environmentally relevant 15 and 225 µg/L BPA. The results showed that BPA increased the mortality and deformity rate of offspring and caused craniofacial deformities characterized by changes in various cartilage angles and lengths. The alizarin red and calcein staining showed that BPA could delay bone mineralization and reduce bone mass accumulation. The results of acridine orange staining indicated that BPA induced apoptosis of the skull. The degree of harm of BPA presented a dose-dependent pattern. The results of the comparative transcriptome showed that there were 380 different expression genes (DEGs) in the 15 µg/L BPA group, and 645 DEGs in the 225 µg/L BPA group. MAPK/Wnt/FoxO signaling pathway-related genes were significantly down-regulated in the BPA-exposed groups. The present study demonstrates that long-term parental BPA exposure would severely affect cartilage development and bone mineralization of fish offspring, and MAPK/Wnt/FoxO signaling pathways may be involved in this process., 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 © 2022. Published by Elsevier Inc.)

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.)

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.)

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.)

Nonyl acridine orange (NAO) is a lipophilic and positively charged molecule widely used as a mitochondrial fluorescent probe. NAO is cytotoxic at micromolar concentration and might be potentially used as a mitochondria-targeted drug for cancer therapy. However, the use of NAO under in vivo conditions would be compromised by the unspecific interactions with off-target cells and negatively charged proteins present in the bloodstream. To tackle this limitation, we have synthesized NAO analogues carrying an imidazole group for their specific binding to nitrilotriacetic (NTA) functionalized gold nanorods (AuNRs). We demonstrate that AuNRs provide 10 4 binding sites and a controlled delivery under acidic conditions. Upon incubation with mouse embryonic fibroblasts, the endosomal acidic environment releases the NAO analogues from AuNRs, as visualized through the staining of the mitochondrial network. The addition of the monoclonal antibody Cetuximab to the conjugates enhanced their uptake within lung cancer cells and the conjugates were cytotoxic at subnanomolar concentrations ( c 50 ≈ 0.06 nM). Moreover, the specific interactions of Cetuximab with the epidermal growth factor receptor (EGFR) provided a specific targeting of EGFR-expressing lung cancer cells. After intravenous administration in patient-derived xenografts (PDX) mouse models, the conjugates reduced the progression of EGFR-positive tumors. Overall, the NAO-AuNRs provide a promising strategy to realize membrane mitochondria-targeted conjugates for lung cancer therapy.

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.)

Acridine orange (AO), a basic carcinogenic fluorochrome dye, is used in the industry for staining. In this study, Gram-positive bacteria, Bacillus cereus M 1 16 (MTCC 5521) dry biomass was tested as an eco-friendly, easily available, and cheap biosorbent for the AO dye removal. We obtained optimum biosorption of AO at a biomass concentration of 0.25 g/L and initial dye concentrations of 50-400 mg/L at neutral to basic pH within the 300 min contact time. Kinetics analysis of the biosorption process was best fitted with the pseudo-second-order reaction type. We also performed the isotherm analysis to predict the nature of the reaction taking place, which was found to follow the Redlich Peterson isotherm model with high determination coefficients. The maximum sorption capacity was 210.46 mg/g of dry biomass. The differential FTIR spectroscopic analysis of pristine and AO-treated Bacillus cereus M 1 16 cells suggested the potential involvement of carbonyl, hydroxyl, and amine groups in the biosorption process. Also, the scanning electron microscopy of the cells after AO removal confirmed a gross surface alteration compared to the untreated cells. Furthermore, Response Surface Model (RSM) analysis with the three-way ANOVA test confirms statistically significant interactions between the dye concentration, pH, and temperature with the biosorption capacity (p < 0.001). Hence, the dry biomass of Bacillus cereus M 1 16 was found to be an effective bio-remedial for the AO removal., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Described is the new cytometric approach do detect either stimulation or a collapse of lysosomal proton pump (lysosomes rupture) combined with activation of transglutaminase 2 (TG2) during induction of apoptosis. Apoptosis of human lymphoblastoid TK6 cells was induced by combination of 2-deoxyglucose with the isoquinoline alkaloid berberine, by DNA topoisomerase I inhibitor camptothecin, its analog topotecan, topoisomerase II inhibitors etoposide or mitoxantrone, as well as by the cytotoxic anticancer ribonuclease ranpirnase (onconase). Activity of the proton pump of lysosomes was assessed by measuring entrapment and accumulation of the basic fluorochrome acridine orange (AO) resulting in its metachromatic red luminescence (F >640 ) within these organelles. Activation of TG2 was detected in the same cell subpopulation by the evidence of crosslinking of cytoplasmic proteins revealed by the increased intensity of the side light scatter (SSC) as well as following cell lysis by detergent, by its red fluorescence after staining by sulforhodamine 101. Because at low AO concentration nuclear DNA of the lysed cells was stoichiometrically stained green (F 530 ) its quantity provided information on effects of the drug treatments on cell cycle in relation to activation of TG2. The data reveal that activation of lysosomal proton pump was evident in subpopulations of cells treated with 2-deoxyglucose plus berberine, topotecan, etoposide and mitoxantrone but not with ranpirnase. The collapse of lysosomal proton pump possibly reporting rupture of these organelles was observed in definite cell subpopulations after treatment with each of the studied drugs. Because regardless of the inducer of apoptosis TG2 activation invariably was correlated with lysosomes rupture it is likely that it was triggered by calcium ions or protons released from the ruptured lysosomes. This new methodological approach offers the means to investigate mechanisms and factors affecting autophagic lysosomes proton pump activity vis-à-vis TG2 activation that are common in several pathological states. © 2019 International Society for Advancement of Cytometry., (© 2018 International Society for Advancement of Cytometry.)

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.)

Organotin compounds, being biologically active, affect a variety of cellular functions due to their ability to accumulate in and penetrate biological membranes. These compounds influence the distribution of electrostatic charges, alter organization, disrupt molecular dynamics and change mechanical properties of biological membranes. It was found that the membrane/water partition coefficient equals 4, a value significantly higher than octanol/water partition coefficient. In addition, the effect of di- and tri-phenyltin chlorides on the mechanics of model lipid membranes was measured for the first time. It has been determined that phenyltins affect the global model lipid bilayer properties by reducing the membrane expansion modulus, when measured using micromanipulation technique, and elevating the bending rigidity coefficient of the lipid bilayer, as determined with the flickering noise spectroscopy. In addition, the elevated water permeability shows that phenyltins also cause the local defects formation in the lipid bilayer, i.e. lipid pores. These data shows that phenyltins may interfere indirectly with variety cellular processes by altering non-specifically the entire cellular membrane system. Accordingly, when phenyltins are added to macrophages in culture, they inflict massive alterations of cell morphology and interfere with membrane-associated processes, as visualized using fluorescence labelling of selected subcellular compartments., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.)

Na+/H+ exchange is essential for survival of all organisms, having a role in the regulation of the intracellular Na+ concentration, pH and cell volume. Furthermore, Na+/H+ exchangers were shown to be involved in the virulence of the bacterium Yersinia pestis, indicating they might be potential targets for novel antibiotic treatments. The model system for Na+/H+ exchangers is the NhaA transporter from Escherichia coli, EcNhaA. Therefore, the general transport mechanism of NhaA exchangers is currently well characterized. However, much less is known about NhaB exchangers, with only a limited number of studies available. The pathogen Klebsiella pneumoniae, which is a major source of nosocomial infection, possesses three electrogenic Na+/H+ exchangers, KpNhaA1, KpNhaA2 and KpNhaB, none of which have been previously investigated. Our aim in this study was to functionally characterize KpNhaB using solid supported membrane-based electrophysiology as the main investigation technique, and thus provide the first electrophysiological investigation of an NhaB Na+/H+ exchanger. We found that NhaB can be described by the same competition-based mechanism that was shown to be valid for electrogenic NhaA and NapA, and for electroneutral NhaP Na+/H+ exchangers. For comparison we also characterized the activity of KpNhaA1 and KpNhaA2 and found that the three exchangers have complementary activity profiles, which is likely a survival advantage for K. pneumoniae when faced with environments of different salinity and pH. This underlines their importance as potential antibiotic drug targets.

Whole-cell High-Throughput Screening (HTS) is a key tool for the discovery of much needed malaria transmission blocking drugs. Discrepancies in the reported outcomes from various HTS Plasmodium falciparum gametocytocidal assays hinder the direct comparison of data and ultimately the interpretation of the transmission blocking potential of hits. To dissect the underlying determinants of such discrepancies and assess the impact that assay-specific factors have on transmission-blocking predictivity, a 39-compound subset from the Medicines for Malaria Venture Malaria Box was tested in parallel against three distinct mature stage gametocytocidal assays, under strictly controlled parasitological, chemical, temporal and analytical conditions resembling the standard membrane feeding assay (SMFA). Apart from a few assay-specific outliers, which highlighted the value of utilizing multiple complementary approaches, good agreement was observed (average ΔpIC 50 of 0.12 ± 0.01). Longer compound incubation times improved the ability of the least sensitive assay to detect actives by 2-fold. Finally, combining the number of actives identified by any single assay with those obtained at longer incubation times yielded greatly improved outcomes and agreement with SMFA. Screening compounds using extended incubation times and using multiple in vitro assay technologies are valid approaches for the efficient identification of biologically relevant malaria transmission blocking hits.

Cellular injury and death are ubiquitous features of disease, yet tools to detect them are limited and insensitive to subtle pathological changes. Acridine orange (AO), a nucleic acid dye with unique spectral properties, enables real-time measurement of RNA and DNA as proxies for cell viability during exposure to various noxious stimuli. This tool illuminates spectral signatures unique to various modes of cell death, such as cells undergoing apoptosis versus necrosis/necroptosis. This new approach also shows that cellular RNA decreases during necrotic, necroptotic, and apoptotic cell death caused by demyelinating, ischemic, and traumatic injuries, implying its involvement in a wide spectrum of tissue pathologies. Furthermore, cells with pathologically low levels of cytoplasmic RNA are detected earlier and in higher numbers than with standard markers including TdT-mediated dUTP biotin nick-end labeling and cleaved caspase 3 immunofluorescence. Our technique highlights AO-labeled cytoplasmic RNA as an important early marker of cellular injury and a sensitive indicator of various modes of cell death in a range of experimental models., (© 2017 Plemel et al.)

The aim of this study was to evaluate the anticancer activity of bioinspired silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) against mouse myoblast cancer cells (C 2 C 12 ). Both AgNPs and AuNPs were biologically synthesized using Spinacia oleracea Linn., aqueous leaves extract. UV-Vis. spectrophotometer, high resolution-transmission electron microscopy (HR-TEM), field emission-scanning electron microscopy (FE-SEM) and X-ray diffraction (XRD) studies supported the successful synthesis of AgNPs and AuNPs. Both these NPs have shown cytotoxicity against C 2 C 12 cells even at very low concentration (5μg/mL). Acridine orange/Ethidium bromide (AO/EB) dual staining confirmed the apoptotic morphological features. The levels of caspase enzymes (caspase-3 and caspase-7) were significantly up-regulated in NPs treated myoblast cells than the plant extract. Furthermore, in zebrafish embryo toxicity study, AgNPs showed 100% mortality at 3μg/mL concentration while AuNPs exhibited the same at much higher concentration (300mg/mL). Taken together, these results provide a preliminary guidance for the development of biomaterials based drugs to fight against the fatal diseases for example cancer., (Copyright © 2016 Elsevier B.V. All rights reserved.)

There exists a broad range of techniques that can be used to classify and count white blood cells in a point-of-care (POC) three-part leukocyte differential test. Improvements in lenses, light sources, and cameras for image-based POC systems have renewed interest in acridine orange (AO) as a contrast agent, whereby subpopulations of leukocytes can be differentiated by colorimetric analysis of AO fluorescence emission. We evaluated the effect on test accuracy using different AO staining and postprocessing methods in the context of an image-based POC colorimetric cell classification scheme. Thirty blood specimens were measured for percent cell counts using our POC system and a conventional hematology analyzer for comparison. Controlling the AO concentration used during whole-blood staining, the incubation time with AO, and the colorimetric ratios among the three population of leukocytes yielded a percent deviation of 0.706%, ? 1.534 % , and ? 0.645 % for the lymphocytes, monocytes, and granulocytes, respectively. Overall, we demonstrated that a redshift in AO fluorescence was observed at elevated AO concentrations, which lead to reproducible inaccuracy of cell counts. This study demonstrates there is a need for a strict control of the AO staining and postprocessing methods to improve test accuracy in these POC systems.

Acridine Orange is a cell-permeable green fluorophore that can be protonated and trapped in acidic vesicular organelles (AVOs). Its metachromatic shift to red fluorescence is concentration-dependent and, therefore, Acridine Orange fluoresces red in AVOs, such as autolysosomes. This makes Acridine Orange staining a quick, accessible and reliable method to assess the volume of AVOs, which increases upon autophagy induction. Here, we describe a ratiometric analysis of autophagy using Acridine Orange, considering the red-to-green fluorescence intensity ratio (R/GFIR) to quantify flow cytometry and fluorescence microscopy data of Acridine-Orange-stained cells. This method measured with accuracy the increase in autophagy induced by starvation or rapamycin, and the reduction in autophagy produced by bafilomycin A1 or the knockdown of Beclin1 or ATG7. Results obtained with Acridine Orange, considering R/GFIR, correlated with the conversion of the unlipidated form of LC3 (LC3-I) into the lipidated form (LC3-II), SQSTM1 degradation and GFP-LC3 puncta formation, thus validating this assay to be used as an initial and quantitative method for evaluating the late step of autophagy in individual cells, complementing other methods., (© 2016. Published by The Company of Biologists Ltd.)

Pancreatic cancer is only the 12th most common cancer, but the fourth leading cause of cancer-related deaths in the world. This is due to late prognosis, poor response to chemotherapy and early metastases. Natural prodrugs may play an important role in the treatment of pancreatic cancer. The main aim of this study was to determine the cytotoxicity of five natural prodrugs, namely harpagoside, hyperoside, hypoxoside, oleuropein and polydatin, by investigating apoptosis and autophagy as possible mechanism/s of action. Hypoxoside and hyperoside have shown selective cytotoxicity at IC50 values of ∼25 and 50μM against INS-1 and MIA PaCa-2 pancreatic cancer cells, respectively. Hypoxoside and hyperoside induced G2/M phase arrest and caspase-3 activation in INS-1 and MIA PaCa-2 cells, respectively. Hoechst/phalloidin staining confirmed morphological changes, including condensed chromatin multinucleation, membrane blebbing and loss of cytoskeletal arrangement in INS-1 and MIA PaCa-2 cells. Acridine orange staining was absent in INS-1 (hypoxoside) and MIA PaCa-2 (hyperoside) treated cells, whereas LC3B expression was not significantly increased. INS-1 and MIA PaCa-2 treated cells favour the cell death pathway, apoptosis, over the cell survival pathway, autophagy., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

The interaction of acridine orange (AOH(+)) with calf thymus DNA (ct-DNA) under different dye-DNA conditions has been investigated in detail using multispectroscopic techniques, unraveling a number of hitherto unexplored intricacies of dye-DNA binding. The observed results intriguingly show contrasting binding features when low (2.4 μM) and significantly high (23 μM) dye concentrations are used. It is conclusively inferred from absorption, steady-state fluorescence, circular dichroism, fluorescence decay and anisotropy decay studies that at low [DNA] to [dye] ratio, especially with higher dye concentration, dimeric AOH(+) predominantly binds externally to DNA surfaces through electrostatic interactions. At sufficiently high [DNA] to [dye] ratios, however, the interaction intriguingly changes to monomeric AOH(+) bound to DNA, predominantly in the intercalative mode between DNA base pairs, with partly an electrostatic binding on DNA surfaces. With very low initial dye concentration, monomeric (AOH(+)) mostly binds to DNA through intercalative and electrostatic modes for most DNA to dye ratios. The present study demonstrates a systematic correlation of the striking changes in the photophysical properties of the dye upon multimode binding with DNA. The observed results are of great significance in understanding the fundamental insights of dye/drug binding to DNA hosts, of use in the design of effective therapeutic agents.

The loading of chemotherapics into smart nanocarriers that simultaneously possess more than one useful property for specifically targeting a tumor site improves their therapeutic effectiveness, reducing their side effects. Hence, we proposed a combined approach for the treatment of human breast cancer (BC) consisting of the co-encapsulation of doxorubicin and curcumin or doxorubicin and quercetin into multifunctional niosomes, which results in prolonged blood circulation and an ability to spontaneously accumulate at the tumor site (passive target) and to recognize and bind the tumor cells through dual ligand-receptor interactions (active target). The drug-loaded vesicles showed high stability and good capability of loading doxorubicin and antioxidants alone or in combination. Their diameter was around 400 nm. The drugs released from the vesicles were found to be controlled and sustained for over 24 h, with a strong dependence on the co-presence of the loaded molecules. Transferrin and/or folic acid were conjugated on the external surface of the niosomes as ligands, considerably improving the cellular uptake into MCF-7 and MDA-MB-231 malignant cells when compared with the uptake of nonconjugated samples. In vitro evaluation of anticancer activity demonstrated the strong potential of niosomes loaded with a doxorubicin/curcumin combination as useful devices in breast tumor treatment. These features hold great promise for the development of multifunctional devices that combine several advantages such as biocompatibility, stealth properties, loading capability, and active targeting, moving toward the development of more specific and efficient carriers for personalized tumoral therapy.

Fe3O4 nanoparticles were grown in situ on carbon nanotubes (CNTs) by a solvothermal method. The Fe3O4/CNTs composites were characterised by the Brunauer-Emmett-Teller method and transmission electron microscopy. The results indicated that the Fe3O4 nanoparticles were uniformly deposited on CNTs, and the average diameter was approximately 7.0 nm. The Fe3O4/CNTs were applied as an enzyme mimetic to decompose Orange II, and the decomposing conditions were optimised. At 500 mg L(-1) of Fe3O4/CNTs in the presence of 15.0 mmol L(-1) of H2O2, at 30°C, it degraded 94.0% of Orange II (0.25 mmol L(-1), pH = 3.5), showing higher catalytic activity than pure Fe3O4 nanoparticles. The high activity was attributed to the uniform Fe3O4 nanoparticles growing on the side walls of the CNTs and the synergetic effect between Fe3O4 and CNTs. The Fe3O4/CNTs maintained their activity at temperatures as high as 65°C. The Fe3O4/CNTs presented high reusability and stability even after eight uses. These data proved that the Fe3O4/CNTs-catalysed degradation is a promising technique for wastewater treatment. Fe3O4 nanoparticles were grown in situ on carbon nanotubes (CNTs) by a solvothermal method. The Fe3O4/CNTs was applied as a mimetic enzyme to decompose Orange II. The Fe3O4/CNTs were collected after the reaction by applying an external magnetic field and can use repeatedly.

Colonization of wood blocks by brown and white rot fungi rapidly resulted in detectable wood oxidation, as shown by a reduced phloroglucinol response, a loss of autofluorescence, and acridine orange (AO) staining. This last approach is shown to provide a novel method for identifying wood oxidation. When lignin was mildly oxidized, the association between AO and lignin was reduced such that stained wood sections emitted less green light during fluorescence microscopy. This change was detectable after less than a week, an interval that past work has shown to be too short for significant delignification of wood. Although fungal hyphae were observed in only a few wood lumina, oxidation was widespread, appearing relatively uniform over regions several hundred micrometers from the hyphae. This observation suggests that both classes of fungi release low molecular weight mild oxidants during the first few days of colonization.

We show here that crotamine, a polypeptide from the South American rattlesnake venom with cell penetrating and selective anti-fungal and anti-tumoral properties, presents a potent anti-plasmodial activity in culture. Crotamine inhibits the development of the Plasmodium falciparum parasites in a dose-dependent manner [IC50 value of 1.87 μM], and confocal microscopy analysis showed a selective internalization of fluorescent-labeled crotamine into P. falciparum infected erythrocytes, with no detectable fluorescence in uninfected healthy erythrocytes. In addition, similarly to the crotamine cytotoxic effects, the mechanism underlying the anti-plasmodial activity may involve the disruption of parasite acidic compartments H(+) homeostasis. In fact, crotamine promoted a reduction of parasites organelle fluorescence loaded with the lysosomotropic fluorochrome acridine orange, in the same way as previously observed mammalian tumoral cells. Taken together, we show for the first time crotamine not only compromised the metabolism of the P. falciparum, but this toxin also inhibited the parasite growth. Therefore, we suggest this snake polypeptide as a promising lead molecule for the development of potential new molecules, namely peptidomimetics, with selectivity for infected erythrocytes and ability to inhibit the malaria infection by its natural affinity for acid vesicles., (Copyright © 2016 Elsevier Inc. All rights reserved.)

While opto-genetics has proven to have tremendous value in revealing the functions of the macromolecular machinery in cells, it is not amenable to exploration of small molecules such as phospholipids (PLs). Here, we describe a redox opto-lipidomics approach based on a combination of high affinity light-sensitive ligands to specific PLs in mitochondria with LC-MS based redox lipidomics/bioinformatics analysis for the characterization of pro-apoptotic lipid signals. We identified the formation of mono-oxygenated derivatives of C18:2-containing cardiolipins (CLs) in mitochondria after the exposure of 10-nonylacridine orange bromide (NAO)-loaded cells to light. We ascertained that these signals emerge as an immediate opto-lipidomics response, but they decay long before the commencement of apoptotic cell death. We found that a protonophoric uncoupler caused depolarization of mitochondria and prevented the mitochondrial accumulation of NAO, inhibited the formation of C18:2-CL oxidation product,s and protected cells from death. Redox opto-lipidomics extends the power of opto-biologic protocols to studies of small PL molecules resilient to opto-genetic manipulations.

Pressure microinjection can be used to introduce fluorescent dyes and labeled macromolecules into single cells. The method allows measuring transport activity of macromolecules such as proteins and RNA molecules within and between cells. Routinely, plant mesophyll cells are injected with fluorescent dextran molecules of specific sizes to measure an increase of the size exclusion limit of plasmodesmata in the presence of a co-injected or expressed protein. The mobility of a macromolecule can also be addressed directly by injecting a recombinant protein that itself is labeled with fluorescent dye and following its transport to neighboring cells. This chapter describes a pressure microinjection protocol successfully applied to Nicotiana leaves. This protocol requires basic skills and experience in handling a microscope equipped with an imaging system, a micromanipulator, and a microinjection system attached to an upright microscope. Using this equipment, a trained person can inject approximately 10-20 mesophyll cells per hour.

We sequenced the complete 7118 bp circular plasmid pColE3-CA38 (pColE3) from Escherichia coli, located the previously identified colicin components together with two new ORFs that have homology to mobilization and transfer proteins, and found that pColE3 is highly similar to a plasmid present in enterohemorrhagic E. coli O111. We also found that unusual aspects of the plasmid include the inability to be completely digested with restriction endonucleases and asymmetric Phred DNA sequencing quality scores, with significantly lower scores in the forward direction relative to the colicin and immunity proteins consistent with plus (+) strand DNA. Comparing the A260 with picogreen double-stranded DNA (dsDNA) fluorescence and oligreen single-stranded DNA (ssDNA) fluorescence as well as metachromatic staining by acridine orange, we found that the undigested pColE3 DNA stains preferentially as ssDNA and that it coexists with dsDNA. We also identified ssDNA in pColE5 and pColE9 but not in pColE1. Colicin plasmids producing ssDNA may represent a new subclass of rolling-circle replication plasmids and add to the known similarities between colicins and filamentous phage., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Acidic vesicles can be imaged and tracked in live cells after staining with several low molecular weight fluorescent probes, or with fluorescently labeled proteins. Three fluorescent dyes, acridine orange, LysoTracker Red DND-99, and quinacrine, were evaluated as acidic vesicle tracers for confocal fluorescence imaging and quantitative analysis. The stability of fluorescent signals, achievable image contrast, and phototoxicity were taken into consideration. The three tested tracers exhibit different advantages and pose different problems in imaging experiments. Acridine orange makes it possible to distinguish acidic vesicles with different internal pH but is fairly phototoxic and can cause spectacular bursts of the dye-loaded vesicles. LysoTracker Red is less phototoxic but its rapid photobleaching limits the range of useful applications considerably. We demonstrate that quinacrine is most suitable for long-term imaging when a high number of frames is required. This capacity made it possible to trace acidic vesicles for several hours, during a process of drug-induced apoptosis. An ability to record the behavior of acidic vesicles over such long periods opens a possibility to study processes like autophagy or long-term effects of drugs on endocytosis and exocytosis., (© 2014 International Society for Advancement of Cytometry.)

We previously demonstrated that cyanobacteria-derived microcystin-leucine-arginine (MCLR) is able to induce developing toxicity, such as malformation, growth delay and also decreased heart rates in zebrafish embryos. However, the molecular mechanisms by which MCLR induces its toxicity during the development of zebrafish remain largely unknown. Here, we evaluate the role of apoptosis in MCLR-induced developmental toxicity. Zebrafish embryos were exposed to various concentrations of MCLR (0, 0.2, 0.5, 2, and 5.0 mg L(-1)) for 96 h, at which time reactive oxygen species (ROS) was significantly induced in the 2 and 5.0 mg L(-1) MCLR exposure groups. Acridine orange (AO) staining and terminal deoxynucleotide transferase-mediated deoxy-UTP nick end labelling (TUNEL) assay showed that MCLR exposure resulted in cell apoptosis. To test the apoptotic pathway, the expression pattern of several apoptotic-related genes was examined for the level of enzyme activity, gene and protein expression, respectively. The overall results demonstrate that MCLR induced ROS which consequently triggered apoptosis in the heart of developing zebrafish embryos. Our results also indicate that the p53-Bax-Bcl-2 pathway and the caspase-dependent apoptotic pathway play major roles in MCLR-induced apoptosis in the developing embryos., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Xylose fermentation is necessary for the bioconversion of lignocellulose to ethanol as fuel, but wild-type Saccharomyces cerevisiae strains cannot fully metabolize xylose. Several efforts have been made to obtain microbial strains with enhanced xylose fermentation. However, xylose fermentation remains a serious challenge because of the complexity of lignocellulosic biomass hydrolysates. Genome shuffling has been widely used for the rapid improvement of industrially important microbial strains. After two rounds of genome shuffling, a genetically stable, high-ethanol-producing strain was obtained. Designated as TJ2-3, this strain could ferment xylose and produce 1.5 times more ethanol than wild-type Pichia stipitis after fermentation for 96 h. The acridine orange and propidium iodide uptake assays showed that the maintenance of yeast cell membrane integrity is important for ethanol fermentation. This study highlights the importance of genome shuffling in P. stipitis as an effective method for enhancing the productivity of industrial strains., (© 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Candida albicans is a common fungal pathogen that causes systemic and superficial infections in most immunocompromised patients. Fluconazole, a synthetic triazole antifungal agent, is the most prescribed drug used in treating this pathogen. But because of its poor solubilization in water and the emergence of resistant strains against this antimycotic drug, we aimed at devising a unique microemulsion drug delivery system for fluconazole against candidiasis. A clear oil-in-water microemulsion system, consisting of clove oil as oil phase, Tween 20 as surfactant, and water as aqueous phase was developed using a ternary phase diagram. Physicochemical characterization was done to understand the internal physicochemical state. The bulk drug, fluconazole, that measured several microns in length was reduced to a 10-65 nm range with no means of high-energy methods as confirmed by transmission electron microscopy. The very small and uniform spherical structure of the drug-loaded microemulsion system could be of high impact to the biological system as the efficacy of fluconazole is greatly improved when compared with its conventional bulk form. The optimized microemulsion exhibited significantly higher antifungal activity at a minimum concentration (8 µg/ml) of fluconazole as examined by fluorescence and scanning electron microscopy. Thus, our report discloses an excellent oral drug delivery system., (© 2013 International Union of Biochemistry and Molecular Biology, Inc.)

Environmental exposure to nanomaterials is inevitable as nanomaterials become part of our daily life, and as a result, nanotoxicity research is gaining attention. However, most investigators focus on the evaluation of overall toxicity instead of a certain organism system. In this regard, the evaluation of cardiovascular effects of silica nanoparticles was preformed in vitro and in vivo. It's worth noting that silica nanoparticles induced cytotoxicity as well as oxidative stress and apoptosis. ROS and apoptosis were considered as major factor to endothelial cells dysfunction, involved in several molecular mechanisms of cardiovascular diseases. In vivo study, mortality, malformation, heart rate and whole-embryo cellular death were measured in zebrafish embryos. Results showed that silica nanoparticles induced pericardia toxicity and caused bradycardia. We also examined the expression of cardiovascular-related proteins in embryos by western blot analysis. Silica nanoparticles inhibited the expression of p-VEGFR2 and p-ERK1/2 as well as the downregulation of MEF2C and NKX2.5, revealed that silica nanoparticles could inhibit the angiogenesis and disturb the heart formation and development. In summary, our results suggest that exposure to silica nanoparticles is a possible risk factor to cardiovascular system., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Breast cancer tissue contains a small population of cells that have the ability to self-renew; these cells are known as cancer stem-like cells (CSCs). We have recently shown that autophagy is essential for the tumorigenicity of these CSCs. Salinomycin (Sal), a K (+) /H (+) ionophore, has recently been shown to be at least 100 times more effective than paclitaxel in reducing the proportion of breast CSCs. However, its mechanisms of action are still unclear. We show here that Sal blocked both autophagy flux and lysosomal proteolytic activity in both CSCs and non-CSCs derived from breast cancer cells. GFP-LC3 staining combined with fluorescent dextran uptake and LysoTracker-Red staining showed that autophagosome/lysosome fusion was not altered by Sal treatment. Acridine orange staining provided evidence that lysosomes display the characteristics of acidic compartments in Sal-treated cells. However, tandem mCherry-GFP-LC3 assay indicated that the degradation of mCherry-GFP-LC3 is blocked by Sal. Furthermore, the protein degradation activity of lysosomes was inhibited, as demonstrated by the rate of long-lived protein degradation, DQ-BSA assay and measurement of cathepsin activity. Our data indicated that Sal has a relatively greater suppressant effect on autophagic flux in the ALDH (+) population in HMLER cells than in the ALDH (-) population; moreover, this differential effect on autophagic flux correlated with an increase in apoptosis in the ALDH (+) population. ATG7 depletion accelerated the proapoptotic capacity of Sal in the ALDH (+) population. Our findings provide new insights into how the autophagy-lysosomal pathway contributes to the ability of Sal to target CSCs in vitro.

The surface properties of biomaterials, such as wettability, polar group distribution, and topography, play important roles in the behavior of cell adhesion and proliferation. Gaseous plasma discharges are among the most common means to modify the surface of a polymer without affecting its properties. Herein, we describe the surface modification of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) films using atmospheric pressure plasma processing through exposure to a dielectric barrier discharge (DBD). After treatment the film surface showed significant changes from hydrophobic to hydrophilic as the water contact angle decreasing from 95° to 37°. All plasma-treated films developed more hydrophilic surfaces compared to untreated films, although the reasons for the change in the surface properties of PS and PMMA differed, that is, the PS showed chemical changes and in the case of PMMA they were topographical. Excellent adhesion and cell proliferation were observed in all films. In vitro studies employing flow cytometry showed that the proliferation of L929 cells was higher in the film formed by a 1:1 mixture of PS/PMMA, which is consistent with the results of a previous study. These findings suggest better adhesion of L929 onto the 1:1 PS/PMMA modified film, indicating that this system is a new candidate biomaterial for tissue engineering., (Copyright © 2012 Elsevier B.V. All rights reserved.)

This study was conducted to investigate the modulating effects of green tea polyphenols on genotoxic damage and apoptotic activity induced by hexavalent chromium [Cr (VI)] in CD-1 mice. Animals were divided into the following groups: (i) injected with vehicle; (ii) treated with green tea polyphenols (30 mg/kg) via gavage; (iii) injected with CrO3 (20 mg/kg) intraperitoneally; (iv) treated with green tea polyphenols in addition to CrO3. Genotoxic damage was evaluated by examining micronucleated polychromatic erythrocytes (MN-PCEs) obtained from peripheral blood at 0, 24, 48, and 72 h after treatment. Induction of apoptosis and cell viability were assessed by differential acridine orange/ethidium bromide (AO/EB) staining. Treatment of green tea polyphenols led to no significant changes in the MN-PCEs. However, CrO3 treatment significantly increased MN-PCEs at 24 and 48 h after injection. Green tea polyphenols treatment prior to CrO3 injection led to a decrease in MN-PCEs compared to the group treated with CrO3 only. The average of apoptotic cells was increased at 48 h after treatment compared to control mice, suggesting that apoptosis could contribute to eliminate the DNA damaged cells induced by Cr (VI). Our findings support the proposed protective effects of green tea polyphenols against the genotoxic damage induced by Cr (VI).

We present the new fixation method for RHA (resurfacing hip arthroplasty) endoprostheses by means of the biomimetic multispiked connecting scaffold (MSC-Scaffold). Such connecting scaffold can generate new type of RHA endoprostheses, that is stemless and fixed entirely without cement. The preprototypes of this MSC-Scaffold were manufactured with modern additive laser additive technology (SLM). The pilot surgical implantations in animal model (two laboratory swine) of MSC-Scaffold preprototypes have showed after two months neither implant loosening, migration, and nor other early complications. From the results of performed histopathological evaluation of the periscaffold spikes bone tissue and 10-day culture of human osteoblasts (NHOst) we can conclude that (1) the scaffolding effect was obtained and (2) to improve the osseointegration of the scaffold spikes, their material surface should be physicochemically modified (e.g., with hydroxyapatite). Some histopathological findings in the periscaffold domain near the MSC-Scaffold spikes bases (fibrous connective tissue and metallic particles near the MSC-Scaffold spikes bases edges) prompt considering the necessity to optimize the design of the MSC-Scaffold in the regions of its interspike space near the spikes bases edges, to provide more room for new bone formation in this region and for indispensable post-processing (glass pearl blasting) after the SLM manufacturing.

Acetylcholine-binding protein is a water-soluble homologue of the extracellular ligand-binding domain of cys-loop receptors. It is used as a structurally accessible prototype for studying ligand binding to these pharmaceutically important pentameric ion channels, in particular to nicotinic acetylcholine receptors, due to conserved binding site residues present at the interface between two subunits. Here we report that an aromatic conjugated small molecule binds acetylcholine-binding protein in an ordered π-π stack of three identical molecules per binding site, two parallel and one antiparallel. Acetylcholine-binding protein stabilizes the assembly of the stack by aromatic contacts. Thanks to the plasticity of its ligand-binding site, acetylcholine-binding protein can accommodate the formation of aromatic stacks of different size by simple loop repositioning and minimal adjustment of the interactions. This type of supramolecular binding provides a novel paradigm in drug design.

The interaction of luteolin with calf thymus deoxyribonucleic acid (ctDNA) under physiological conditions (Tris-HCl buffer solutions, pH 7.4) was studied by UV-Vis spectroscopy, fluorescence spectroscopy and viscosity measurement method, respectively. The results indicated that a complex of luteolin with ctDNA can be formed. Spectroscopic techniques together with viscosity determination provided evidences of intercalation mode of binding for the interaction between luteolin and ctDNA. The binding constant of luteolin to DNA calculated based on UV-Vis spectroscopy data was found to be 4.52×10(4)L mol(-1) at 310 K. The thermodynamic parameters of the complex were calculated by a double reciprocal method: Δ(r)H(m)(s)=-8.9×10(3)J mol(-1),Δ(r)S(m)(s)=60.5 JK(-1)mol(-1) and Δ(r)G(m)(s)=-2.76×10(4)J mol(-1) (310 K). The interacting forces between luteolin and DNA mainly included hydrophobic interactions and hydrogen bonds. The acridine orange displacement studies revealed that luteolin had significant effect for acridine orange bounded on DNA, which was indicative of intercalation binding., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Climate change, world population growth, and poverty have led to an increase in the incidence of leptospirosis. Leptospirosis is caused by pathogenic spirochaete bacteria that belong to the genus Leptospira. The bacteria are maintained in the renal tubules of the reservoir hosts (typically a rodent), then shed into the environment via the urine. Water is key for environmental survival and transmission, as leptospires can survive for several weeks in a moist environment. Therefore, environmental epidemiological studies are needed to study the contamination of environmental water sources. However, few such studies have been performed using cultivation of the isolates and PCR assays. But, leptospira cultivation can be easily contaminated by other organisms and takes usually several weeks. Moreover, PCR is a complex and costly analysis for the underdeveloped countries that have the highest incidence of leptospirosis. In this study, we describe two modifications of a fluorescence microscopy assay based on immuno-magnetic separation (IMS) to detect leptospires in environmental water samples that mainly differ in fluorescent dye staining. The first type uses acridine orange fluorescent dye staining combined with multiplexed IMS for sample screening. The detection limit ranged from 10(2) to 10(3) organisms per mL and largely depended on the capture efficiency (CE) of the immuno-magnetic particles. The second type uses serogroup-specific immuno-particles and direct fluorescence antibody staining (DFA) to detect leptospires; the detection limit of this second assay was approximately 10(1) cells per mL. Both assay types were applied to natural and experimentally infected water samples, which were also analysed with DFM and real-time PCR. Our data show that the fluorescent microscopy immunoassay successfully identified experimental leptospire contamination and was as sensitive as PCR. This modified immune-fluorescence assay may therefore enable epidemiological studies of leptospirosis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Aim: Development of a non-toxic, fluorescent-based, agar system for the screening of overexpressed bacterial efflux pump systems with common, inexpensive UV accessories., Materials and Methods: Wild type Gram-negative and positive bacteria expressing intrinsic efflux pumps and their progeny that overexpress a specific efflux pump were selected for evaluation of efflux pump activity in a Mueller-Hinton agar, containing increasing concentrations of the non-toxic fluorescent chromophore acridine orange (AO). The method is based on the same principle as the first-generation ethidium bromide method, according to which the concentration of the fluorescent dye that first produces fluorescence of the overlying bacterial colony represents the maximum concentration of the dye that the bacterium can extrude. The higher the concentration needed to produce fluorescence, the greater the ability of the bacterial efflux pump to extrude the dye., Results: Progeny of Escherichia coli, Salmonella enterica serovar Enteritidis and Staphylococcus aureus that over-expressed a given efflux pump fluoresced (i.e. accumulated AO) at concentrations of AO that were much greater than the ones required for the emission of fluorescence by their corresponding wild-type counterpart which expressed an intrinsic efflux pump., Conclusion: The AO agar method readily identifies strains of Gram-negative and Gram-positive bacteria that overexpress efflux pump systems compared to their wild-type progeny.

We report data demonstrating that zebrafish embryos irradiated by α-particles can release a stress signal into the water, which can be communicated to the unirradiated zebrafish embryos sharing the same water medium, and then these unirradiated zebrafish embryos can release a feedback stress signal back to the irradiated embryos. The effects of radiation on the whole embryos were studied through quantification of apoptotic signals at 24 h post fertilization through staining with the vital dye acridine orange, followed by counting the stained cells under a microscope. We refer to this phenomenon as the "rescue effect", where the unirradiated embryos successfully helped the irradiated embryos mitigate the radiation induced DNA damages. The results showed that the number of apoptotic signals in the irradiated embryos was smaller when they were partnered with bystander unirradiated embryos in the same medium. The results also showed significantly fewer apoptotic signals in the irradiated embryos when the population of bystander embryos increased from 10 to 30, while keeping the population of irradiated embryos at 10. These data suggest that the stress communicated between the unirradiated zebrafish embryos and the irradiated embryos sharing the same medium will help "rescue" the irradiated embryos, and that the strength of the rescue effect depends on the number of rescuing bystander unirradiated embryos.

Pentoxifylline (PTX), a methylxanthine derivative is a non-steroidal immunomodulating agent with unique hemorheologic properties. It is used in the treatment of intermittent claudication as it increases the amount of oxygen reaching tissues by increasing the flexibility of red blood cells. Recently, it has also shown to exhibit anti-metastatic and anti-angiogenic activities in B16F10 melanoma cells both in vitro as well as in vivo. As per the reports, the choice of drug in the treatment of breast cancer is paclitaxel, but the major limitation is its toxicity. However, the effects of PTX on metastatic processes in breast cancer are not currently known. Therefore, in this study, we have examined the effect of PTX in MDA-MB-231 human breast cancer cells. The MTT assay showed dose- and time-dependent decreases in cellular proliferation. The non-toxic concentration of PTX selected were 1, 2.5 and 5 mM for 24 h. PTX induced a G0-G1 cell-cycle arrest leading to apoptosis. Further, it affected adhesion to both the matrigel and collagen type-IV in a time- and dose-dependent manner. The PTX impeded the migration of MDA-MB-231 cells and also decreased the activities of both MMP-2 and MMP-9. Thus, PTX at non-toxic doses affected cellular proliferation, adhesion, migration and invasion. These results demonstrate its anti-metastatic effect on MDA-MB-231 cells, and further studies need to be carried out to understand the mechanism of action.

Auger-emitting radionuclides such as (99m)Tc have been the focus of recent studies aiming at finding more selective therapeutic approaches. To explore the potential usefulness of (99m)Tc as an Auger emitter, we have synthesized and biologically evaluated novel multifunctional structures comprising (1) a pyrazolyl-diamine framework bearing a set of donor atoms to stabilize the [M(CO)(3)](+) (M is Re, (99m)Tc) core; (2) a DNA intercalating moiety of the acridine orange type to ensure close proximity of the radionuclide to DNA and to follow the internalization and subcellular trafficking of the compounds by confocal fluorescence microscopy; and (3) a bombesin (BBN) analogue of the type X-BBN[7-14] (where X is SGS, GGG) to provide specificity towards cells expressing the gastrin releasing peptide receptor (GRPr). Of the evaluated (99m)Tc complexes, Tc ( 3 ) containing the GGG-BBN[7-14] peptide showed the highest cellular internalization in GRPr-positive PC3 human prostate tumor cells, presenting a remarkably high nuclear uptake in the same cell line. Live-cell confocal imaging microscopy studies with the congener Re complex, Re ( 3 ), showed a considerable accumulation of fluorescence in the nucleus, with kinetics of uptake similar to that exhibited by Tc ( 3 ). Together, these data show that the acridine orange intercalator and the metal fragment are colocalized in the nucleus, which indicates that they remain connected despite the lysosomal degradation of Tc ( 3 )/Re ( 3 ). These compounds are the first examples of (99m)Tc bioconjugates that combine specific cell targeting with nuclear internalization, a crucial issue to explore use of (99m)Tc in Auger therapy.

The fate of xenobiotics in living organisms is determined by their in vivo absorption, distribution, metabolism and excretion. A convenient and scalable animal model of these biological processes is thus highly beneficial in understanding the effects of xenobiotics. Here we present a silkworm model to investigate the molecular properties-directed absorption, distribution and excretion of fluorescent compounds as model xenobiotics through introducing the compounds into the silkworm's diet and monitoring the resulting color and fluorescence in the silkworm's body. The efficient uptake of xenobiotics into silk has been further studied through quantitative analysis of the intrinsically colored and highly luminescent silk secreted by silkworm. Our findings provide first-hand insights to better understand the molecular properties that allow specific materials to be incorporated into silk while it is being produced in the silk gland. The use of resulting luminescent silk as scaffold for tissue engineering application has been demonstrated to clearly reveal the interaction of silk with cells. Furthermore, this new development also paves a way to produce various functional silk embedded with stimuli-sensitive dyes or drugs as novel biomaterials for in vivo applications., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Inhibition of the gastric H,K-ATPase by the potassium-competitive acid blocker (P-CAB) 1-[5-(2-fluorophenyl)-1-(pyridin-3-ylsulfonyl)-1H-pyrrol-3-yl]-N-methylmethanamine (TAK-438), is strictly K(+)-competitive with a K(i) of 10 nM at pH 7. In contrast to previous P-CABs, this structure has a point positive charge (pK(a) 9.06) allowing for greater accumulation in parietal cells compared with previous P-CABs [e.g., (8-benzyloxy-2-methyl-imidazo(1,2-a)pyridin-3-yl)acetonitrile (SCH28080), pK(a) 5.6]. The dissociation rate of the compound from the isolated ATPase is slower than other P-CABs, with the t(1/2) being 7.5 h in 20 mM KCl at pH 7. The stoichiometry of binding of TAK-438 to the H,K-ATPase is 2.2 nmol/mg in the presence of Mg-ATP, vanadate, or MgP(i). However, TAK-438 also binds enzyme at 1.3 nmol/mg in the absence of Mg(2+). Modeling of the H,K-ATPase to the homologous Na,K-ATPase predicts a close approach and hydrogen bonding between the positively charged N-methylamino group and the negatively charged Glu795 in the K(+)-binding site in contrast to the planar diffuse positive charge of previous P-CABs. This probably accounts for the slow dissociation and high affinity. The model also predicts hydrogen bonding between the hydroxyl of Tyr799 and the oxygens of the sulfonyl group of TAK-438. A Tyr799Phe mutation resulted in a 3-fold increase of the dissociation rate, showing that this hydrogen bonding also contributes to the slow dissociation rate. Hence, this K(+)-competitive inhibitor of the gastric H,K-ATPase should provide longer-lasting inhibition of gastric acid secretion compared with previous drugs of this class.

Recent studies have demonstrated that vegetable rich diets have protective effects on the occurrence and prognosis of various cancers. In addition to dietary intakes, ascorbic acid and β-carotene are also taken as supplements. The aim of this study was to assess effects of ascorbic acid, β-carotene and their combinations on human hepatocellular carcinoma cell line HepG2. Ascorbic acid and β-carotene were applied to cells as plasma peak concentrations (70 and 8 μM, respectively) and their half concentrations (35 and 4 μM, respectively) for 24 and 48 h. Genotoxic and cytotoxic effects of ascorbic acid and β-carotene were evaluated by alkali single cell gel electrophoresis (SCGE), acridine orange/ethidium bromide staining patterns of cells (apoptosis and necrosis) and lipid peroxidation (thiobarbituric acid reactive substances, TBARS). Results of the SCGE demonstrated that both ascorbic acid and β-carotene caused DNA damage on HepG2 which were also concordant to increased apoptosis and necrosis of cells. Increased TBARS values also demonstrated increased lipid peroxidation in these cells. Results of the present study demonstrates that when dietary intakes of ascorbic acid and β-carotene and their relevant achievable plasma level concentrations were considered, both ascorbic acid and β-carotene induce genotoxic and cytotoxic damage on HepG2 together with increased oxidative damage in contrast to their protective effect on healthy cells. This may be correlated to oxidative status and balance of ROS in hepatocellular carcinoma cells.

In vitro mutagen binding ability of human Lactobacillus rhamnosus 231 (Lr 231) was evaluated against acridine orange (AO), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 2-amino-3, 8-dimethylimidazo-[4,5-f]-quinoxaline (MeIQx) and 4-nitro-o-phenylenediamine (NPD). Binding of AO by Lr 231 is due to adsorption, thereby leading to removal of mutagen in solution and is instantaneous, pH- and concentration-dependent. Whereas, binding of MNNG and MeIQx by Lr 231 results into biotransformation leading to detoxification with subsequent loss of mutagenicity as determined by spectral analysis, thin layer chromatography and Ames test. Binding of mutagen by Lr 231 was dependent on culture age and optimum binding of AO, MNNG and MeIQx was observed to occur with 24 h old culture. Cells of Lr 231 were subjected to different chemical treatments prior to binding studies. Results indicated cell wall component such as cell wall polysaccharide, peptidoglycan, carbohydrates and proteins plays an important role in adsorption of AO, also involving hydrophilic and ionic interactions. Binding, biotransformation and detoxification of MNNG and MeIQx by Lr 231 was dependent on cell surface characteristics mainly involving carbohydrates, proteins, teichoic acid/lipoteichoic acid, hydrophobic interaction and presence of thiol group. L. rhamnosus 231 bound MNNG instantaneously. More than 96 (p < 0.01) and 70% (p < 0.05) cells remained viable after mutagen binding and various pretreatments respectively. This study shows Lr 231 exhibits ability to bind and detoxify potent mutagens, and this property can be useful in formulating fermented foods for removal of potent mutagens., (Copyright © 2011 Elsevier Ltd. All rights reserved.)