Characterization of Bitter Taste Receptor-Dependent Autophagy in Oral Epithelial Cells.

Microbial dysbiosis is an important trigger in the development of oral diseases. Oral keratinocytes or gingival epithelial cells (GECs) offer protection against various microbial insults. Recent studies suggest that GECs expressed higher level of bitter taste receptor 14 (T2R14) compared to other taste receptors and toll-like receptors and act as innate immune sentinels. Macroautophagy or autophagy is a cellular conserved process involved in the regulation of host innate immune responses against microbial infection. Here, we describe a robust method for evaluation of T2R14-dependent autophagy flux in GECs. Autophagy flux was detected using Western blot analysis in GECs and further was confirmed using Acridine Orange-dependent flow cytometry analysis.

Assessment of Autophagy in Leishmania Parasites.

Leishmaniasis is a neglected tropical disease caused by numerous species of Leishmania parasites, including Leishmania major. The parasite is transmitted by several species of sandfly vectors and infects myeloid cells leading to a myriad of inflammatory responses, immune dysregulations, and disease manifestations. Every cell undergoes autophagy, a self-regulated degradative process that permits the cells to recycle damaged or worn-out organelles in order to maintain cellular health and homeostasis. Studies have shown that Leishmania modulates their host cell autophagic machinery and there are indications that the parasite-specific autophagic processes may be valuable for parasite virulence and survival. However, the role of autophagy in Leishmania is inconclusive because of the limited tools available to study the Leishmania-specific autophagic machinery. Here, we describe methods to study and definitively confirm autophagy in Leishmania major. Transmission electron microscopy (TEM) allowed us to visualize Leishmania autophagosomes, especially those containing damaged mitochondrial content, as well as dividing mitochondria with ongoing fusion/fission processes. Flow cytometry enabled us to identify the amount of acridine orange dye accumulating in the acidic vacuolar compartments in Leishmania major by detecting fluorescence in the red laser when autophagic inhibitors or enhancers were included. These methods will advance studies that aim to understand autophagic regulation in Leishmania parasites that could provide insights into developing improved therapeutic targets against leishmaniasis.

Bitter Taste Receptor T2R14 and Autophagy Flux in Gingival Epithelial Cells.

Macroautophagy (hereafter autophagy) is a lysosomal degradation pathway that functions in nutrient recycling and as a mechanism of innate immunity. Previously, we reported a novel host-bacteria interaction between cariogenic S. mutans and bitter taste receptor (T2R14) in gingival epithelial cells (GECs), leading to an innate immune response. Further, S. mutans might be using the host immune system to inhibit other Gram-positive bacteria, such as S. aureus. To determine whether these bacteria exploit the autophagic machinery of GEC, it is first necessary to evaluate the role of T2R14 in modulating autophagic flux. So far, the role of T2R14 in the regulation of autophagy is not well characterized. Therefore, in this study, for the first time, we report that T2R14 downregulates autophagy flux in GECs, and T2R14 knockout increases acidic vacuoles. However, the treatments of GEC WT with a T2R14 agonist and antagonist did not lead to a significant change in acidic vacuole formation. Transmission electron microscopy morphometric results also suggested an increased number of autophagic vesicles in T2R14-knockout GEC. Further, our results suggest that S. mutans competence stimulating peptide CSP-1 showed robust intracellular calcium release and this effect is both T2R14- and autophagy protein 7-dependent. In this study, we provide the first evidence that T2R14 modulates autophagy flux in GEC. The results of the current study could help in identifying the impact of T2R in regulation of the immuno-microenvironment of GEC and subsequently oral health.

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.

RNA-based liposomes for oral cancer: From biophysical characterization to biological evaluation.

Oral cancer incidence and mortality are increasing over time. The most common therapies for oral cancers are surgery and radiotherapy, either used alone or combined, and immunotherapy can be also an option. Although there are several therapeutic options, none of them are completely effective, and in addition, there are numerous associated side effects. To overcome these limitations, researchers have been trying to reduce these drawbacks by using drug delivery systems that carry drugs for specific delivery to cancer cells. For that purpose, RNA-coated liposomes to selectively deliver the ligands C8 (acridine orange derivative) and dexamethasone to oral cancer cells were produced, characterized, and biologically evaluated. Firstly, the RNA structure and binding interaction with ligands (C8 and dexamethasone) were evaluated by circular dichroism (CD), thermal difference spectroscopy (TDS), nuclear magnetic resonance (NMR) and fluorescence titrations. The biophysical assays evidenced the formation of an RNA hairpin and duplex structure. Moreover, steady-state and time-resolved fluorescence intensity and anisotropy experiments show that C8 forms a complex with RNA and adopts an open conformation upon RNA binding. Then, RNA-coated liposomes were characterized by dynamic light scattering, and diameters near 160 nm were observed. Time-resolved anisotropy measurements of C8 loaded in RNA-functionalized liposomes indicate the co-existence of free C8 in solution (inside the liposome) and C8 bound to RNA at the external liposome surface. The RNA-functionalized liposomes loaded with C8 or dexamethasone mediated a significant reduction in the cell viability of malignant UPCI-SCC-154 cells while maintaining viable non-malignant NHDF cells. Additionally, the liposomes were able to internalize the cells, with higher uptake by the malignant cell line. Overall, the results obtained in this work can contribute to the development of new drug delivery systems based on RNA-coated liposomes.

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.

Practical Characterization Strategies for Comparison, Qualification, and Selection of Cell Viability Detection Methods for Cellular Therapeutic Product Development and Manufacturing.

Cellular therapy development and manufacturing has focused on providing novel therapeutic cell-based products for various diseases. The International Organization for Standardization (ISO) has provided guidance on critical quality attributes (CQAs) that shall be considered when testing and releasing cellular therapeutic products. Cell count and viability measurements are two of the CQAs that are determined during development, manufacturing, testing, and product release. The ISO Cell Counting Standard Part 1 and 2 addressed the needs for improving the quality of cell counting results. However, there is currently no guidance on the qualification and selection of a fit-for-purpose cell viability detection method. In this work, we present strategies for the characterization and comparison of AO/PI and AO/DAPI staining methods using the heat-killed (HK) and low temperature/nutrient-deprived (LT/ND) cell death models to evaluate the comparability of cell viability measurements and identify potential causes of differences. We compared the AO/PI and AO/DAPI staining methods using HK and LT/ND-generated dead cells, investigated the staining time effects on cell viability measurements, and determined their viability linearity with different mixtures of live and dead cells. Furthermore, we validated AO/PI and AO/DAPI cell viability measurement with a long-term cell proliferation assay. Finally, we demonstrate a practical example of cell viability measurement comparison using AO/PI and AO/DAPI on antibiotic-selected transduced Jurkat and THP-1 cells to select a fit-for-purpose method for functional genomics screening. The proposed strategies may potentially enable scientists to properly characterize, compare, and select cell viability detection methods that are critical for cellular therapeutic product development and manufacturing.

Myeloprotection with activated carbon in doxorubicin-treated rats.

Chemotherapy can often cause a variety of side effects including bone marrow (BM) suppression, termed as myelosuppression. Accordingly, facile and effective management of chemotherapy-induced myelosuppression is currently a pivotal task for experimental pathologists and oncologists. Here, we chose to use activated carbon (AC) with an extensive surface area for studying its possible protective effectiveness with respect to BM in doxorubicin (DOX)-treated rats. Spherical AC with an extended surface area up to 4490 m2/g was prepared for per os (p/o) delivery, whereas for intraperitoneal (i/p) delivery we used the powdered form of AC that was derived from the aforementioned spherical AC. During the monthly treatment of animals with AC and DOX these two components were delivered alternately (not in the same day). After treatment, BM cells were isolated from femurs of sacrificed animals, stained with acridine orange (AO) and analyzed by flow cytometry. Regardless of the route of AC delivery (p/o or i/p), apparent myeloprotection with a possible regenerative effect was observed in animals that received DOX, as evidenced by recovery of the populations of total nucleated cells (TNC) and polychromatic (immature) erythrocytes accompanied by a considerable reduction of the number of apoptotic/dead cells among TNC (≤2.0%). Moreover, as a result of AC administrations, there was a significant increase of AO green and far-red fluorescence intensities in the population of TNC, which is suggestive of the ongoing quantitative and conformational changes in DNA and RNA associated with cell recovery and proliferation. Thus, AC preparations under the present experimental conditions can effectively tackle DOX-induced myelosuppression via mechanisms not necessarily associated with adsorptive detoxification.

Real-Time Monitoring of Lysosomal Membrane Permeabilization Using Acridine Orange.

Loss of lysosomal membrane integrity results in leakage of lysosomal hydrolases to the cytosol which might harm cell function and induce cell death. Destabilization of lysosomes often precede apoptotic or necrotic cell death and occur during both physiological and pathological conditions. The weak base acridine orange readily enters cells and accumulates in the acidic environment of lysosomes. Vital staining with acridine orange is a well-proven technique to observe lysosomal destabilization using fluorescence microscopy and flow cytometry. These analyses are, however, time consuming and only adapted for discrete time points, which make them unsuitable for large-scale approaches. Therefore, we have developed a time-saving, high-throughput microplate reader-based method to follow destabilization of the lysosomal membrane in real-time using acridine orange. This protocol can easily be adopted for patient samples since the number of cells per sample is low and the time for analysis is short.

Fluorescence Microscopy: Determination of Meropenem Activity against Klebsiella pneumoniae.

The development and implementation of diagnostic methods that allow rapid assessment of antibiotic activity against pathogenic microorganisms is an important step towards antibiotic therapy optimization and increase in the likelihood of successful treatment outcome. To determine whether fluorescence microscopy with acridine orange can be used for rapid assessment (≤8 h) of the meropenem activity against Klebsiella pneumoniae, six isolates including three OXA-48-carbapenemase-producers were exposed to meropenem at different levels of its concentration (0.5 × MIC, 1 × MIC, 8 or 16 µg/mL) and the changes in the viable counts within 24 h were evaluated using fluorescence microscopy and a control culture method. The approach was to capture the regrowth of bacteria as early as possible. Within the first 8 h fluorescence microscopy allowed to categorize 5 out of 6 K. pneumoniae strains by their meropenem susceptibility (based on the MIC breakpoint of 8 mg/L), but meropenem activity against three isolates, two of which were OXA-48-producers, could not be accurately determined at 8 h. The method proposed in our study requires improvement in terms of accelerating the bacterial growth and regrowth for early meropenem MIC determination. Volume-dependent elevation in meropenem MICs against OXA-48-producers was found and this phenomenon should be studied further.

Isolation and evaluation of erythroid progenitors in the livers of larval, froglet, and adult Xenopus tropicalis.

Xenopus liver maintains erythropoietic activity from the larval to the adult stage. During metamorphosis, thyroid hormone mediates apoptosis of larval-type erythroid progenitors and proliferation of adult-type erythroid progenitors, and a globin switch occurs during this time. In addition, the whole-body mass and the liver also change; however, whether there is a change in the absolute number of erythroid progenitors is unclear. To isolate and evaluate erythroid progenitors in the Xenopus liver, we developed monoclonal ER9 antibodies against the erythropoietin receptor (EPOR) of Xenopus. ER9 recognized erythrocytes, but not white blood cells or thrombocytes. The specificity of ER9 for EPOR manifested as its inhibitory effect on the proliferation of a Xenopus EPOR-expressing cell line. Furthermore, ER9 recognition was consistent with epor gene expression. ER9 staining with Acridine orange (AO) allowed erythrocyte fractionation through fluorescence-activated cell sorting. The ER9+ and AO-red (AOr)high fractions were highly enriched in erythroid progenitors and primarily localized to the liver. The method developed using ER9 and AO was also applied to larvae and froglets with different progenitor populations from adult frogs. The liver to body weight and the number of ER9+ AOrhigh cells per unit body weight were significantly higher in adults than in larvae and froglets, and the number of ER9+ AOrhigh cells per unit liver weight was the highest in froglets. Collectively, our results show increased erythropoiesis in the froglet liver and demonstrate growth-dependent changes in erythropoiesis patterns in specific organs of Xenopus.

Ultrasonication-Tailored Graphene Oxide of Varying Sizes in Multiple-Equilibrium-Route-Enhanced Adsorption for Aqueous Removal of Acridine Orange.

Graphene oxide (GO) has shown remarkable performance in the multiple-equilibrium-route adsorption (MER) process, which is characterized by further activation of GO through an in-situ reduction process based on single-equilibrium-route adsorption (SER), generating new adsorption sites and achieving an adsorption capacity increase. However, the effect of GO on MER adsorption in lateral size and thickness is still unclear. Here, GO sheets were sonicated for different lengths of time, and the adsorption of MER and SER was investigated at three temperatures to remove the typical cationic dye, acridine orange (AO). After sonication, we found that freshly prepared GO was greatly reduced in lateral size and thickness. In about 30 min, the thickness of GO decreased dramatically from several atomic layers to fewer atomic layers to a single atomic layer, which was completely stripped off; after that, the monolayer lateral size reduction dominated until it remained constant. Surface functional sites, such as hydroxyl groups, showed little change in the experiments. However, GO mainly reduces the C=O and C-O bonds in MER, except for the conjugated carbon backbone (C-C). The SER adsorption kinetics of all temperatures fitted the pseudo-first-order and pseudo-second-order models, yet room temperature preferred the latter. An overall adsorption enhancement appeared as sonication time, but the equilibrium capacity of SER GO generally increased with thickness and decreased with the single-layer lateral size, while MER GO conversed concerning the thickness. The escalated temperature facilitated the exfoliation of GO regarding the adsorption mechanism. Thus, the isotherm behaviors of the SER GO changed from the Freundlich model to Langmuir as size and temperature changed, while the MER GO were all of the Freundlich. A record capacity of ~4.3 g of AO per gram of GO was obtained from the MER adsorption with a sixty-minute ultrasonicated GO at 313.15 K. This work promises a cornerstone for MER adsorption with GO as an adsorbent.

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.

Haemato-biochemical, mutagenic, and histopathological changes in Oreochromis niloticus exposed to BTX.

The study of the DNA damage response in erythrocytes after exposure to volatile organic compounds (VOCs) can present evidence for its potential effect as genotoxic- biomarkers for environmental pollution. Although VOCs are dangerous pollutants, still little is known about hemotoxic, cytotoxic, and genotoxic effects of such pollutants on fish. We optimized an assay method for apoptosis and DNA damage in erythrocytes of adult tilapia fish after 15 days exposure to benzene (0.762 ng/L), toluene (26.614 ng/L), and xylene (89.403 ng/L). The highest level of apoptosis and DNA damage were recorded in benzene-exposed fish, as was the highest level of histopathological alterations in gills, liver, and kidney. The imbalance of the antioxidants profile explained the stress-case reported in exposed fish. These results suggest that hemotoxic, cytotoxic, genotoxic, and tissue damage were recorded after exposure to BTX in Oreochromis niloticus.

Mutation and apoptosis are well-coordinated for protecting against DNA damage-inducing toxicity in Drosophila.

BACKGROUND: Apoptotic cell death is an important survival system for multicellular organisms because it removes damaged cells. Mutation is also a survival method for dealing with damaged cells in multicellular and also unicellular organisms, when DNA lesions are not removed. However, to the best of our knowledge, no reports have comprehensively explored the direct relationship between apoptosis and somatic cell mutations induced by various mutagenic factors. RESULTS: Mutation was examined by the wing-spot test, which is used to detect somatic cell mutations, including chromosomal recombination. Apoptosis was observed in the wing discs by acridine orange staining in situ. After treatment with chemical mutagens, ultraviolet light (UV), and X-ray, both the apoptotic frequency and mutagenic activity increased in a dose-dependent manner at non-toxic doses. When we used DNA repair-deficient Drosophila strains, the correlation coefficient of the relationship between apoptosis and mutagenicity, differed from that of the wild-type. To explore how apoptosis affects the behavior of mutated cells, we determined the spot size, i.e., the number of mutated cells in a spot. In parallel with an increase in apoptosis, the spot size increased with MNU or X-ray treatment dose-dependently; however, this increase was not seen with UV irradiation. In addition, BrdU incorporation, an indicator of cell proliferation, in the wing discs was suppressed at 6 h, with peak at 12 h post-treatment with X-ray, and that it started to increase again at 24 h; however, this was not seen with UV irradiation. CONCLUSION: Damage-induced apoptosis and mutation might be coordinated with each other, and the frequency of apoptosis and mutagenicity are balanced depending on the type of DNA damage. From the data of the spot size and BrdU incorporation, it is possible that mutated cells replace apoptotic cells due to their high frequency of cell division, resulting in enlargement of the spot size after MNU or X-ray treatment. We consider that the induction of mutation, apoptosis, and/or cell growth varies in multi-cellular organisms depending on the type of the mutagens, and that their balance and coordination have an important function to counter DNA damage for the survival of the organism.

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.

Entrapment of Acridine Orange in Metakaolin-Based Geopolymer: A Feasibility Study.

Few studies have explored the immobilization of organic macromolecules within the geopolymer matrix, and some have found their chemical instability in the highly alkaline geopolymerization media. The present work reports on the feasibility of encapsulating the potentially toxic acridine orange (AO) dye in a metakaolin based geopolymer while maintaining its structural integrity. The proper structural, chemical, and mechanical stabilities of the final products were ascertained using Fourier-transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), thermogravimetric (TGA/DTG), and mechanical analyses, whereas the dye integrity and its stability inside the geopolymer were investigated by the UV-Vis analysis. In addition, the antimicrobial activity was investigated. The FT-IR and XRD analyses confirmed the geopolymerization occurrence, whereas the TGA/DTG and mechanical (compressive and flexural) strength revealed that the addition of 0.31% (AO mg/ sodium silicate L) of AO to the fresh paste did not affect the thermal stability and the mechanical properties (above 6 MPa in flexural strength and above 20 MPa for compressive strength) of the hardened product. UV-Vis spectroscopy revealed that the dye did not undergo chemical degradation nor was it released from the geopolymer matrix. The results reported herein provide a useful approach for the safe removal of toxic macromolecules by means of encapsulation within the geopolymer matrix.

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.

Ras-transfected human mammary tumour cells are resistant to photodynamic therapy by mechanisms related to cell adhesion.

AIMS: Photodynamic therapy (PDT) is a treatment modality for several cancers involving the administration of a tumour-localising photosensitiser (PS) and its subsequent activation by light, resulting in tumour damage. Ras oncogenes have been strongly associated with chemo- and radio-resistance. Based on the described roles of adhesion and cell morphology on drug resistance, we studied if the differences in shape, cell-extracellular matrix and cell-cell adhesion induced by Ras transfection, play a role in the resistance to PDT. MATERIALS AND METHODS: We employed the human normal breast HB4a cells transfected with H-RAS and a panel of five PSs. KEY FINDINGS: We found that resistance to PDT of the HB4a-Ras cells employing all the PSs, increased between 1.3 and 2.5-fold as compared to the parental cells. There was no correlation between resistance and intracellular PS levels or PS intracellular localisation. Even when Ras-transfected cells present lower adherence to the ECM proteins, this does not make them more sensitive to PDT or chemotherapy. On the contrary, a marked gain of resistance to PDT was observed in floating cells as compared to adhesive cells, accounting for the higher ability conferred by Ras to survive in conditions of decreased cell-extracellular matrix interactions. HB4a-Ras cells displayed disorganisation of actin fibres, mislocalised E-cadherin and vinculin and lower expression of E-cadherin and ß1-integrin as compared to HB4a cells. SIGNIFICANCE: Knowledge of the mechanisms of resistance to photodamage in Ras-overexpressing cells may lead to the optimization of the combination of PDT with other treatments.

Determination of Barr bodies in Transgender Patients in India - A comparative study.

Sex determination in forensic medicine is considered one of the first and foremost steps in personal identification. The need for identifying the exact sex of the individual arises when deciding whether a person can exercise certain civil rights reserved for one particular sex, for competing in sex-specific athletic and sports events, legitimacy, divorce, paternity disputes and also to some criminal offenses. Nuclear sexing by Barr body examination can be done using buccal smears to establish the sex of the individual when routine methods fail to disclose the exact gender of the individual. Aim: To determine and compare the Barr bodies present in exfoliated buccal epithelial cells in males, females and transgender populations using light and fluorescence microscopy. Materials and Methods: A total of 90 patients were recruited for the study. Group I consisted of 30 female patients. Group II consisted of 30 male patients and group III consisted of 30 transgender patients. The buccal mucosa was then scraped using a wooden spatula and the cells obtained were fixed in 95% ethanol. Two smears per individual were made and stained. One smear was stained with papanicolaou (PAP) stain and the other with Acridine orange and viewed under light microscopy and fluorescent microscopy, respectively. Results: When PAP stained slides were examined, the percentage of Barr-bodies in females ranged from 3% to 5% and in males it was 0% and in transgenders, it ranged from 0% to 5%. In Acridine orange stained smears, the percentage of Barr bodies in females ranged from 1% to 3% and in males it was 0% and in transgenders, it was 0%. Kruskal-Wallis test to study the relation of Barr body percentage in females, males and transgender subjects demonstrated significant differences between the groups (P < 0.001). Wilcoxon signed rank test was done for pairwise comparison, which showed that the distribution of percentage of positive cells in females are statistically significant from males and transgenders (P < 0. 001). Conclusion: Nuclear sexing using Barr bodies offers a simple yet effective method for determining the sex of transgender patients which could help them in understanding their gender identity better and diagnose any underlying chromosomal aberration.