Effect of Acridine Orange and Zoledronic Acid on Bone Metastasis in Renal Cell Carcinoma.

BACKGROUND/AIM: The increasing incidence of renal cell carcinoma (RCC) and its associated bone metastasis pose challenges in surgical interventions, warranting the exploration of novel therapeutic approaches. Therefore, this study aimed to assess the impact of hematogenously administering acridine orange (AO) alone and in combination with zoledronic acid (ZA) on bone metastasis in RCC. MATERIALS AND METHODS: RENCA cells (1.0×106 cells/10 µl) were directly injected into the right femur of male BALB/c mice. The mice were categorized into four groups based on the applied therapeutic intervention and were euthanized after five weeks. Micro-computed tomography was performed to quantify the extent of periosteal reaction, indicative of bone metastasis, along the entire length of the femur. Tumor weight and volume were measured at euthanization. Hematoxylin and eosin staining was used to examine the extent of tumor development in the bone. Apoptotic cell, osteoclast, and vascular endothelial growth factor (VEGF)-positive cell counts were assessed using TdT-mediated dUTP-biotin nick end labeling, tartrate-resistant acid phosphatase staining, and VEGF staining, respectively. RESULTS: The periosteal reaction was significantly reduced in the intervention groups compared to the control group (p<0.05). The apoptotic cell numbers in the intervention groups surpassed that in the control group (p<0.05), whereas those of osteoclasts and VEGF-positive cells in the intervention groups were lower than those in the control group (p<0.05). CONCLUSION: AO hinders bone metastasis progression in RCC, and combination therapy with ZA may be more effective than AO administration alone.

Novel furan chalcone modulates PHD-2 induction to impart antineoplastic effect in mammary gland carcinoma.

Normoxic inactivation of prolyl hydroxylase-2 (PHD-2) in tumour microenvironment paves the way for cancer cells to thrive under the influence of HIF-1α and NF-κB. Henceforth, the present study is aimed to identify small molecule activators of PHD-2. A virtual screening was conducted on a library consisting of 265,242 chemical compounds, with the objective of identifying molecules that exhibit structural similarities to the furan chalcone scaffold. Further, PHD-2 activation potential of screened compound was determined using in vitro 2-oxoglutarate assay. The cytotoxic activity and apoptotic potential of screened compound was determined using various staining techniques, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, 4',6-diamidino-2-phenylindole (DAPI), 1,1',3,3'-tetraethylbenzimi-dazolylcarbocyanine iodide (JC-1), and acridine orange/ethidium bromide (AO/EB), against MCF-7 cells. 7,12-Dimethylbenz[a]anthracene (DMBA) model of mammary gland cancer was used to study the in vivo antineoplastic efficacy of screened compound. [(E)-1-(4-fluorophenyl)-3-(furan-2-yl) prop-2-en-1-one] (BBAP-7) was screened and validated as a PHD-2 activator by an in vitro 2-oxo-glutarate assay. The IC50 of BBAP-7 on MCF-7 cells is 18.84 µM. AO/EB and DAPI staining showed nuclear fragmentation, blebbing and condensation in MCF-7 cells following BBAP-7 treatment. The red-to-green intensity ratio of JC-1 stained MCF-7 cells decreased after BBAP-7 treatment, indicating mitochondrial-mediated apoptosis. DMBA caused mammary gland dysplasia, duct hyperplasia and ductal carcinoma in situ. Carmine staining, histopathology, and scanning electron microscopy demonstrated that BBAP-7, alone or with tirapazamine, restored mammary gland surface morphology and structural integrity. Additionally, BBAP-7 therapy significantly reduced oxidative stress and glycolysis. The findings reveal that BBAP-7 activates PHD-2, making it a promising anticancer drug.

Detection of dsRNA by Acridine Orange Staining.

Acridine orange is a nucleic acid binding dye that emits green fluorescence when bound to double-stranded DNA or RNA and red fluorescence when bound to single-stranded DNA or RNA under ultraviolet light. This unique characterization allows it to be used for distinguishing or visualization of dsRNA. Here, we present a convenient and efficient protocol for detecting dsRNA in polyacrylamide gels.

Hyaluronic acid/silicon nanoparticle scaffold induces proliferation and differentiation of mouse spermatogonial stem cells transplanted to epididymal adipose tissue.

Spermatogonia stem cells (SSCs) are a unique cell population maintaining male spermatogenesis during life, through their potential for proliferation and differentiation. The application of silicon nanoparticles (SNs) and hyaluronic acid (HA) to induce the differentiation of SSCs seems promising. Herein, we investigate the effect of SN and HA scaffolds on the progression of SSCs spermatogenesis in mice. Initially SSCs were isolated from healthy immature mice and cultured on prepared scaffolds (HA, SN, and HA/SN) in a 3D culture system. Then viability of SSCs cultured on scaffolds was examined using MTT assay and Acridine Orange staining. Then SSCs cultured on scaffolds were transplanted into epididymal adipose tissue (EAT) in mature mice and the result was studied by H&E and IHC staining 8 weeks after transplantation. MTT and Acridine Orange analysis revealed that among three different scaffolds HA/SN based scaffold causes considerable toxicity on SSCs (P < 0.05) while H&E staining showed that culture of SSCs on HA, SN, and HA/SN scaffolds has a positive effect on the progression of SSCs spermatogenesis after transplantation into EAT. IHC staining identified TP1, TEKT1, and PLZF as crucial biomarkers in the spermatogenesis development of SSCs transplanted to EAT. According to the presence of these biomarkers in different experimental groups, we found the most spermatogenesis development in SSCs cultured on HA/SN scaffold (PLZF, P < 0.01) (TEKT1, P < 0.01) (TP1, P < 0.001). Our study showed that, although the cytotoxic effect of the HA/SN scaffold decreases the viability rate of SSCs; however, SSCs that survive on HA/SN scaffold showed more ability to progress in spermatogenesis after transplantation into EAT.

The dyeing effect of acridine orange for multiple plasmid systems is sensitive to temperature.

The Goldview dyeing of the natural multiplasmid system of Lactobacillus plantarum PC518 was affected by temperature. The article want to identify the specific molecules that cause temperature sensitivity, then experiment on the universality of temperature sensitivity, and finally preliminarily analyze the influencing factors. At 5°C and 25°C, single pDNA, multiplasmid system, and linear DNA samples were electrophoretic on agarose gel prestained by Goldview 1, 2, 3, and acridine orange (AO), respectively. Eighteen vectors of Escherichia coli and two vectors shortened by cloning were mixed into multiplasmid systems with different member numbers, and then electrophoresis with AO staining was performed within the range of 5°C-45°C, with a linearized multiplasmid system as the control. The lane profiles (peaks) were captured with Image Lab 5.1 software. After electrophoresis, the nine-plasmid-2 system was dyed with AO solutions of different ionic strengths to detect the effect of ionic strength on temperature sensitivity. It was measured that the UV-visible absorption spectra of the nine-plasmid-2 system dissolved in AO solutions with different ionic strengths and pH. Further, a response surface model was constructed using Design-Expert.V8.0.6 software. The electrophoresis result showed that the multiplasmid system from L. plantarum PC518 stained by AO staining showed a weak band at 5°C and five bands at 25°C, which was similar to the result of staining with Goldview 1, 2, and 3. The synthetic nine-plasmid-1 system and nine-plasmid-2 system displayed different band numbers on the electrophoresis gel in the electrophoresis temperature range of 5°C-45°C, namely 3, 4, 6, 4, and 2 bands, as well as 2, 6, 7, 8, and 5 bands. Using the 1× Tris-acetate-EDTA (TAE)-AO solution, the poststaining results of the nine-plasmid-2 system in the temperature range of 5°C-45°C were 4, 6, 9, 9, and 7 bands, respectively. Further, using 5×, 10×, or 25× TAE buffer, the AO poststaining results at 5°C were 4, 2, and 1 bands, respectively. The ultraviolet spectral results from 5°C to 25°C showed that there was a significant difference (3.5 times) in the fluctuation amplitude at the absorption peak of 261.2 nm between 0× and 1-10× TAE-AO solution containing the nine-plasmid-2 system. Specifically, the fluctuation amplitudes of 0×, 1×, 5×, and 10× samples were 0.032, 0.109, 0.112, and 0.110, respectively. At the same time, using 1× and 10× TAE buffer, the AO-stained linear nine-plasmid-2 system remained stable and did not display temperature sensitivity. The response surface models of the AO-stained nine-plasmid-2 system intuitively displayed that the absorbance of the 1× TAE samples increased significantly with increasing temperature compared to the 0× TAE samples, regardless of the pH value. The findings confirmed a temperature-dependent effect in AO staining of natural or synthetic multiplasmid systems, with the optimum staining result occurring at 25°C. Ion strength was a necessary condition for the temperature sensitivity mechanism. This study layed the groundwork for further investigation into the reasons or underlying mechanisms of temperature sensitivity in AO staining of multiplasmid systems.

The Combination of Gold and Silver Food Nanoparticles with Gluten Peptides Alters the Autophagic Pathway in Intestinal Crypt-like Cells.

Metallic nanoparticles (mNPs) are widely used as food additives and can interact with gliadin triggering an immune response, but evaluation of the effects on crypts, hypertrophic in celiac subjects, is still lacking. This study evaluated the effects of gold and silver mNPs in combination with gliadin on crypt-like cells (HIEC-6). Transmission electron microscopy (TEM) was used to evaluate gliadin-mNP aggregates in cells. Western blot and immunofluorescence analysis assessed autophagy-related molecule levels (p62, LC3, beclin-1, EGFR). Lysosome functionality was tested with acridine orange (AO) and Magic Red assays. TEM identified an increase in autophagic vacuoles after exposure to gliadin + mNPs, as also detected by significant increments in LC3-II and p62 expression. Immunofluorescence confirmed the presence of mature autophagosomes, showing LC3 and p62 colocalization, indicating an altered autophagic flux, further assessed with EGFR degradation, AO and Magic Red assays. The results showed a significant reduction in lysosomal enzyme activity and a modest reduction in acidity. Thus, gliadin + mNPs can block the autophagic flux inducing a lysosomal defect. The alteration of this pathway, essential for cell function, can lead to cell damage and death. The potential effects of this copresence in food should be further characterized to avoid a negative impact on celiac disease subjects.

Expulsion of plasmid-mediated antibiotic resistance genes in E. coli by ethidium bromide and acridine orange treatment.

Plasmid borne antibiotics resistance is the global threat to healthcare facilities. Such antibiotics resistance is inherited stably within the same bacterial generations and transmitted horizontally to other species of bacteria. The elimination of such resistance plasmid is of great importance to contain dispersal of antibiotics resistance. E. coli strains were identified, screened for the presence of antibiotics resistance by disc diffusion method, and cured by sub-lethal concentrations of Ethidium bromide and Acridine orange. After curing, again antibiotic resistance was determined. Before and after curing, plasmids were extracted by column spin Kit and subjected to 1% agarose gel electrophoresis and antibiotic resistance genes were identified by PCR. The Ethidium bromide was more effective than Acridine orange in eliminating antibiotics resistance and resistance genes bearing plasmids (4, 5, 6, 8, 9, 10 and <10kb). The most frequently eliminated antibiotic resistance was against Imipenem and Meropenem followed by Cefoperazone-sulbactam, Amikacin and cephalosporins in sequence. The loss of antibiotic resistance was associated with the elimination of plasmid-borne antibiotic resistance genes; bla-TEM, bla-SHV, bla-CTX-M, qnrA, qnrB, qnrC and qnrD. Some E. coli strains did not show the removal of antibiotics resistance and plasmids, suggesting the presence of resistance genes on main chromosome and or non-curable plasmids.

Polystyrene microplastics modulated bdnf expression triggering neurotoxicity via apoptotic pathway in zebrafish embryos.

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.

Incorporation of acridine orange and methylene blue in Langmuir monolayers mimicking releasing nanostructures.

The efficiency of methylene blue (MB) and acridine orange (AO) for photodynamic therapy (PDT) is increased if encapsulated in liposomes. In this paper we determine the molecular-level interactions between MB or AO and mixed monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DPPG) and cholesterol (CHOL) using surface pressure isotherms and polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS). To increase liposome stability, the effects from adding the surfactants Span® 80 and sodium cholate were also studied. Both MB and AO induce an expansion in the mixed monolayer, but this expansion is less significant in the presence of either Span® 80 or sodium cholate. The action of AO and MB occurred via coupling with phosphate groups of DPPC or DPPG. However, the levels of chain ordering and hydration of carbonyl and phosphate in headgroups depended on the photosensitizer and on the presence of Span® 80 or sodium cholate. From the PM-IRRAS spectra, we inferred that incorporation of MB and AO increased hydration of the monolayer headgroup, except for the case of the monolayer containing sodium cholate. This variability in behaviour offers an opportunity to tune the incorporation of AO and MB into liposomes which could be exploited in the release necessary for PDT.

Spectroscopic, calorimetric and cytotoxicity studies on the combined binding of daunorubicin and acridine orange to a DNA tetrahedron.

Chemotherapy-phototherapy (CTPT) combination drugs co-loaded by targeted DNA nanostructures can achieve controlled drug delivery, reduce toxic side effects and overcome multidrug resistance. Herein, we constructed and characterized a DNA tetrahedral nanostructure (MUC1-TD) linked with the targeting aptamer MUC1. The interaction of daunorubicin (DAU)/acridine orange (AO) alone and in combination with MUC1-TD and the influence of the interaction on the cytotoxicity of the drugs were evaluated. Potassium ferrocyanide quenching analysis and DNA melting temperature assays were used to demonstrate the intercalative binding of DAU/AO to MUC1-TD. The interactions of DAU and/or AO with MUC1-TD were analyzed by fluorescence spectroscopy and differential scanning calorimetry. The number of binding sites, binding constant, entropy and enthalpy changes of the binding process were obtained. The binding strength and binding sites of DAU were higher than those of AO. The presence of AO in the ternary system weakened the binding of DAU to MUC1-TD. In vitro cytotoxicity studies demonstrated that the loading of MUC1-TD augmented the inhibitory effects of DAU and AO and the synergistic cytotoxic effects of DAU + AO on MCF-7 cells and MCF-7/ADR cells. Cell uptake studies showed that the loading of MUC1-TD was beneficial in promoting the apoptosis of MCF-7/ADR cells due to its enhanced targeting to the nucleus. This study has important guiding significance for the combined application of DAU and AO co-loaded by DNA nanostructures to overcome multidrug resistance.

Open flow cytometer with the combination of 3D hydrodynamic single cell focusing and confocal laser-induced fluorescence detection.

Flow cytometry is among the most powerful tools for single-cell analysis, while the high cost and mechanical complexity of the commercial instrumentation limit the applications in personalized single-cell analysis. For this issue, we hereby construct an open and low-cost flow cytometer. It is highly compact to integrate the functions of (1) single cell aligning by a lab-made modularized 3D hydrodynamic focusing device, and (2) fluorescence detection of the single cells by a confocal laser-induced fluorescence (LIF) detector. The ceiling cost of the entire hardware for the LIF detection unit and 3D focusing device is $ 3200 and $ 400 respectively. A sheath flow velocity of 150 µL/min produces a focused sample stream of 17.6 µm × 14.6 µm at sample flow of 2 µL/min, based on the LIF response frequency and the laser beam spot diameter. The assay performance of the flow cytometer was evaluated by characterizing fluorescent microparticles and acridine orange (AO) stained HepG2 cells, producing throughputs of 40.5/s and 6.2/s respectively. Favorable assay precision and accuracy were demonstrated by the agreement of frequency histogram with imaging analysis, and good Gaussian-like distributions of fluorescent microparticles and AO-stained HepG2 cells. Practically, the flow cytometer was successfully applied for the evaluation of ROS generation in single HepG2 cells.

Phototoxic Potential of Different DNA Intercalators for Skin Cancer Therapy: In Vitro Screening.

Photodynamic therapy is a minimally invasive procedure used in the treatment of several diseases, including some types of cancer. It is based on photosensitizer molecules, which, in the presence of oxygen and light, lead to the formation of reactive oxygen species (ROS) and consequent cell death. The selection of the photosensitizer molecule is important for the therapy efficiency; therefore, many molecules such as dyes, natural products and metallic complexes have been investigated regarding their photosensitizing potential. In this work, the phototoxic potential of the DNA-intercalating molecules-the dyes methylene blue (MB), acridine orange (AO) and gentian violet (GV); the natural products curcumin (CUR), quercetin (QT) and epigallocatechin gallate (EGCG); and the chelating compounds neocuproine (NEO), 1,10-phenanthroline (PHE) and 2,2'-bipyridyl (BIPY)-were analyzed. The cytotoxicity of these chemicals was tested in vitro in non-cancer keratinocytes (HaCaT) and squamous cell carcinoma (MET1) cell lines. A phototoxicity assay and the detection of intracellular ROS were performed in MET1 cells. Results revealed that the IC50 values of the dyes and curcumin in MET1 cells were lower than 30 µM, while the values for the natural products QT and EGCG and the chelating agents BIPY and PHE were higher than 100 µM. The IC50 of MB and AO was greatly affected by irradiation when submitted to 640 nm and 457 nm light sources, respectively. ROS detection was more evident for cells treated with AO at low concentrations. In studies with the melanoma cell line WM983b, cells were more resistant to MB and AO and presented slightly higher IC50 values, in line with the results of the phototoxicity assays. This study reveals that many molecules can act as photosensitizers, but the effect depends on the cell line and the concentration of the chemical. Finally, significant photosensitizing activity of acridine orange at low concentrations and moderate light doses was demonstrated.

Insights into the Formation of Intermolecular Complexes of Fluorescent Probe 10-N-Nonyl Acridine Orange with Cardiolipin and Phosphatidylglycerol in Bacterial Plasma Membrane by Molecular Modeling.

In this article, we used molecular dynamics (MD), one of the most common methods for simulations of membranes, to study the interaction of fluorescent membranotropic biological probe 10-N-nonyl acridine orange (NAO) with the bilayer, mimicking a plasma membrane of Gram-negative bacteria. Fluorescent probes serve as an effective tool to study the localization of different components in biological membranes. Revealing the molecular details of their interaction with membrane phospholipids is important both for the interpretation of experimental results and future design of lipid-specific stains. By means of coarse-grained (CG) MD, we studied the interactions of NAO with a model membrane, imitating the plasma membrane of Gram-negative bacteria. In our simulations, we detected different NAO forms: monomers, dimers, and stacks. NAO dimers had the central cardiolipin (CL) molecule in a sandwich-like structure. The stacks were formed by NAO molecules interlayered with anionic lipids, predominantly CL. Use of the CG approach allowed to confirm the ability of NAO to bind to both major negatively charged phospholipids, phosphatidylglycerol (PG) and CL, and to shed light on the exact structure of previously proposed NAO-lipid complexes. Thus, CG modeling can be useful for the development of new effective and highly specific molecular probes.

Targeted delivery of nuclear targeting probe for bladder cancer using cyclic pentapeptide c(RGDfK) and acridine orange

Purpose Both cyclic pentapeptide c(RGDfK) and acridine orange (AO) exhibit antitumor effects and cell permeability. This study aimed to evaluate the nuclear targeting efficiency and safety of the nuclear targeting probe for bladder cancer (BCa) synthesized by c(RGDfK) and AO. Tethods The nuclear targeting probe AO-(cRGDfK)2 was synthesized from AO hydrochloride, azided c(RGDfK), and a near-infrared skeleton synthesized via click chemistry reactions. The effect of the AO-(cRGDfK)2 probe on cell viability was assessed in BCa 5637 cells. The tumor cell targeting efficacy of the AO-(cRGDfK)2 probe was evaluated in BCa cells in vitro and in tumor-bearing mice in vivo. Nuclear-specific accumulation of fluorescence probe in BCa tumor cells was evaluated using laser scanning confocal microscopy (LSCM). Hematoxylin and eosin staining was performed to detect histopathological changes in the spleen, heart, liver, and kidney. Results The AO-(cRGDfK)2 probe did not cause a significant reduction in cell viability. LSCM analysis showed that AO-(cRGDfK)2 exhibited nuclear-specific ambulation in BCa cells and was not accumulated in 293T cells. Also, this probe efficiently targeted tumor cells in the serum and urine samples. In vivo imaging system of tumor-bearing mice showed that ~ 80% percent of fluorescence signal was accumulated in the tumor sites. The probe did not change histopathology in the heart, liver, spleen, and kidney in tumor-bearing mice after the 21-day treatment. Conclusions The AO-(cRGDfK)2 probe exhibited nuclear-specific accumulation in BCa cells without cytotoxicity, which provides an innovative alternative to improve anticancer therapy for BCa (AU)

F4-Neuroprostane Effects on Human Sperm.

Swim-up selected human sperm were incubated with 7 ng F4-neuroprostanes (F4-NeuroPs) for 2 and 4 h. Sperm motility and membrane mitochondrial potential (MMP) were evaluated. The percentage of reacted acrosome was assessed by pisum sativum agglutinin (PSA). Chromatin integrity was detected using the acridine orange (AO) assay and localization of the ryanodine receptor was performed by immunofluorescence analysis. Sperm progressive motility (p = 0.02) and the percentage of sperm showing a strong MMP signal (p = 0.012) significantly increased after 2 h F4-NeuroP incubation compared to control samples. The AO assay did not show differences in the percentage of sperm with dsDNA between treated or control samples. Meanwhile, a significantly higher number of sperm with reacted acrosomes was highlighted by PSA localization after 4 h F4-NeuroP incubation. Finally, using an anti-ryanodine antibody, the immunofluorescence signal was differentially distributed at 2 and 4 h: a strong signal was evident in the midpiece and postacrosomal sheath (70% of sperm) at 2 h, whereas a dotted one appeared at 4 h (53% of sperm). A defined concentration of F4-NeuroPs in seminal fluid may induce sperm capacitation via channel ions present in sperm cells, representing an aid during in vitro sperm preparation that may increase the positive outcome of assisted fertilization.

Watermelon rinds as cost-efficient adsorbent for acridine orange: a response surface methodological approach.

In the current investigation, watermelon rinds (WMR) have been utilized as an eco-friendly and cost-efficient adsorbent for acridine orange (AO) from contaminated water samples. Adsorption of AO onto raw (RWM) and thermally treated rinds (TTWM250 and TTWM500) has been studied. The adsorption efficiency of the three adsorbents was evaluated by measuring the % removal (%R) of AO and the adsorption capacity (qe, mg/g). Dependent variables (%R and qe) were optimized as a function of four factors: pH, sorbent dosage (AD), the concentration of AO (DC), and contact time (ST). Box-Behnken (BB) design has been utilized to obtain the optimum adsorption conditions. Prepared adsorbents have been characterized using scanning electron microscopy (SEM), Fourier-transform infrared (FT-IR), and Raman spectroscopies. The surface area of RWM, TTWM250, and TTWM500, as per the Brunauer-Emmett-Teller (BET) analysis, was 2.66, 2.93, and 5.03 m2/g, respectively. Equilibrium investigations suggest that Freundlich model was perfectly fit for adsorption of AO onto TTWM500. Maximum adsorption capacity (qmax) of 69.44 mg/g was obtained using the Langmuir equation. Adsorption kinetics could be best described by the pseudo-second-order (PSO) model. The multi-cycle sorption-desorption study showed that TTWM500 could be regenerated with the adsorption efficiency being preserved up to 87% after six cycles.

Targeted delivery of nuclear targeting probe for bladder cancer using cyclic pentapeptide c(RGDfK) and acridine orange.

PURPOSE: Both cyclic pentapeptide c(RGDfK) and acridine orange (AO) exhibit antitumor effects and cell permeability. This study aimed to evaluate the nuclear targeting efficiency and safety of the nuclear targeting probe for bladder cancer (BCa) synthesized by c(RGDfK) and AO. METHODS: The nuclear targeting probe AO-(cRGDfK)2 was synthesized from AO hydrochloride, azided c(RGDfK), and a near-infrared skeleton synthesized via click chemistry reactions. The effect of the AO-(cRGDfK)2 probe on cell viability was assessed in BCa 5637 cells. The tumor cell targeting efficacy of the AO-(cRGDfK)2 probe was evaluated in BCa cells in vitro and in tumor-bearing mice in vivo. Nuclear-specific accumulation of fluorescence probe in BCa tumor cells was evaluated using laser scanning confocal microscopy (LSCM). Hematoxylin and eosin staining was performed to detect histopathological changes in the spleen, heart, liver, and kidney. RESULTS: The AO-(cRGDfK)2 probe did not cause a significant reduction in cell viability. LSCM analysis showed that AO-(cRGDfK)2 exhibited nuclear-specific ambulation in BCa cells and was not accumulated in 293T cells. Also, this probe efficiently targeted tumor cells in the serum and urine samples. In vivo imaging system of tumor-bearing mice showed that ~ 80% percent of fluorescence signal was accumulated in the tumor sites. The probe did not change histopathology in the heart, liver, spleen, and kidney in tumor-bearing mice after the 21-day treatment. CONCLUSIONS: The AO-(cRGDfK)2 probe exhibited nuclear-specific accumulation in BCa cells without cytotoxicity, which provides an innovative alternative to improve anticancer therapy for BCa.

Single-cell microglial transcriptomics during demyelination defines a microglial state required for lytic carcass clearance.

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.

Ultrastructural changes induced by açaí (Euterpe oleracea Mart) in MCF-7 breast cancer cell line.

Açaí (Euterpe oleracea Mart) is an Amazon plant with many biological properties. Previous report of this group evidenced autophagy induction after treatment with açaí seed extract in MCF-7 breast cancer cell lines by acridine orange assay. The aim of this study was to evaluate the ultrastructural changes induced by açaí seed extract in MCF-7 breast cancer cell lines. First, MCF- 7 breast cancer cell line viability was evaluated by MTT assay. Acridine orange assay showed increase in the acidic compartments, suggesting autophagolysosome formation. These cells were treated with 25 µg/ml for 24 h and evaluated by transmission electron microscopy (MET). This analysis showed that açaí seed extract induced autophagy, confirmed by autophagolysosome formation. Furthermore, açaí seed extract increased the number of mitochondria, suggesting the enrollment of reactive oxygen species in autophagy.

Aggregation of molecules is controlled in microdroplets.

Molecular de-aggregation was observed at the air/water interface of aqueous microdroplets. We probed this phenomenon using dyes such as Rhodamine 6G (R6G), Rhodamine B, acridine orange, and fluorescein, which show aggregation-induced shift in fluorescence. The fluorescence micrographs of microdroplets derived from the aqueous solutions of these dyes show that they are monomeric at the air/water interface, but highly aggregated at the core. We propose that rapid evaporation of the solvent influences the de-aggregation of molecules at the air-water interface of the microdroplets.