Staining Agent Research Articles

Abstract 5333: TH-302 potentiates the antitumor activity of topotecan in neuroblastoma and rhabdomyosarcoma preclinical models

Abstract Background: There is strong evidence that tumor cells in the hypoxic regions cause drug resistance and tumor relapse. Hypoxia targeting agents may make cytotoxic therapy more effective. In this study, we investigated the efficacy of TH-302, a hypoxia-activated prodrug of bromo-isophosphoramide mustard, both alone and in combination with the cytotoxic agent topotecan, in preclinical models of neuroblastoma (NBL) and rhabdomyosarcoma (RMS). Materials and methods: A panel of four NBL and three RMS cell lines was tested in vitro under normoxic or hypoxic conditions to assess the effect of TH-302 on cell proliferation, both as a single agent and in combination with topotecan. In vivo activity was also evaluated in different xenograft models. TH-302 (50 mg/kg) and topotecan (1 mg/kg) were administered daily for 2 consecutive weeks, as single agents or in combination. Animal survival was studied with an NBL (SK-N-BE(2)) metastatic tumor model. Cleaved caspase-3 and the hypoxia staining agent pimonidazole were used as apoptotic and hypoxic markers in vivo. Results: Under normoxic conditions, all tested lines showed an anti-proliferative response to TH-302 with the IC50s ranging from 4.6 to 52μM. When exposed to hypoxia (1% O2) overnight, a dramatic decrease of IC50 was observed, ranging from 0.07 to 2.4μM. Adding topotecan to the TH-302 treatment significantly increased cytotoxicity in all tested tumor cell lines. In both NBL models (SK-N-BE(2) and CHLA-20), single-agent TH-302 treatment delayed tumor growth. Complete tumor regression was observed in the combined treatment group after a two-week treatment period, but all the regressed tumors relapsed after treatment ended. In the CHLA-20 xenograft model, single-agent topotecan also induced complete tumor regression. However, median time to relapse in the combination group was significantly longer than in the topotecan group (43 days vs. 16 days; p<0.01). In the combination group, all relapsed tumors remained responsive to the drug treatment when re-challenged with the original treatment regimen. In the neuroblastoma metastatic model, all three regimens prolonged animal survival, but the combined treatment group showed better survival than single-agent treatments (p<0.01). In a RMS xenograft model, combined treatment was also more effective than single-agent TH-302 or topotecan. Double immunostaining of apoptotic and hypoxic markers showed that TH-302 mostly targeted tumor cells residing at hypoxic regions while topotecan was more effective on tumor cells outside the hypoxic zones. The combined treatment induced tumor cell apoptosis in both normoxic and hypoxic regions. Conclusions: TH-302 showed antitumor effects in neuroblastoma and rhabdomyosarcoma xenograft models. Compared to single-agent TH-302 or topotecan, the combination treatment significantly improved tumor response, delayed tumor relapse, and enhanced animal survival. Citation Format: Libo Zhang, Bing Wu, Paula Marrano, Paul Thorner, Sylvain Baruchel. TH-302 potentiates the antitumor activity of topotecan in neuroblastoma and rhabdomyosarcoma preclinical models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5333. doi:10.1158/1538-7445.AM2015-5333

Silorane, ormocer, methacrylate and compomer long-term staining susceptibility using ΔE and ΔE 00 colour-difference formulas.

The aim of this study was to evaluate the staining susceptibility of a silorane (Filtek Silorane), an ormocer (Ceram X Duo), a methacrylate (Tetric EvoCeram) and a compomer (Dyract) exposed on the long term to various staining agents by using ΔE and ΔE 00 colour-difference formulas. Thirty-six disc-shaped specimens were made of each of the four chemically different materials, randomly divided in six groups (n=6) and immersed in five staining solutions (red wine, juice, coke, tea and coffee) or stored dry (control) in an incubator at 37°C for 99days. Spectrophotometric measurements by means of a spectrophotometer (Spectroshade Handy Dental, MHT) were repeated over a white (L*=92.6, a*=-1.2, b*=2.9) and black (L*=1.6, a*=1.2, b*=-1.0) background made of plasticized paper, in order to determine the colour changes according to ΔE, ΔE 00 and translucency formulas. Statistical analysis was performed by means of factorial Anova, Fisher's LSD test (post hoc) and a Spearman rank correlation between ΔE and ΔE 00. When analysed over a white background, mean ΔE 00 values were highly significantly different and varied from 0.8 (Ceram X Duo/air) to 20.9 (Ceram X Duo/red wine). When analysed over a black background, mean ΔE 00 values were highly significantly different and varied from 1.0 (Ceram X Duo and Tetric/air) to 25.2 (Ceram X Duo/red wine). Differences in translucency varied from 0.3 (Ceram X Duo/air) to 21.1 (Ceram X Duo/juice). The correlation between ΔE and ΔE 00 over a white background was 0.9928, while over a black background, it was 0.9886.

Multifactorial Optimization of Contrast-Enhanced Nanofocus Computed Tomography for Quantitative Analysis of Neo-Tissue Formation in Tissue Engineering Constructs.

To progress the fields of tissue engineering (TE) and regenerative medicine, development of quantitative methods for non-invasive three dimensional characterization of engineered constructs (i.e. cells/tissue combined with scaffolds) becomes essential. In this study, we have defined the most optimal staining conditions for contrast-enhanced nanofocus computed tomography for three dimensional visualization and quantitative analysis of in vitro engineered neo-tissue (i.e. extracellular matrix containing cells) in perfusion bioreactor-developed Ti6Al4V constructs. A fractional factorial ‘design of experiments’ approach was used to elucidate the influence of the staining time and concentration of two contrast agents (Hexabrix and phosphotungstic acid) and the neo-tissue volume on the image contrast and dataset quality. Additionally, the neo-tissue shrinkage that was induced by phosphotungstic acid staining was quantified to determine the operating window within which this contrast agent can be accurately applied. For Hexabrix the staining concentration was the main parameter influencing image contrast and dataset quality. Using phosphotungstic acid the staining concentration had a significant influence on the image contrast while both staining concentration and neo-tissue volume had an influence on the dataset quality. The use of high concentrations of phosphotungstic acid did however introduce significant shrinkage of the neo-tissue indicating that, despite sub-optimal image contrast, low concentrations of this staining agent should be used to enable quantitative analysis. To conclude, design of experiments allowed us to define the most optimal staining conditions for contrast-enhanced nanofocus computed tomography to be used as a routine screening tool of neo-tissue formation in Ti6Al4V constructs, transforming it into a robust three dimensional quality control methodology.

Insights into the Interaction Mechanism of Ligands with Aβ42 Based on Molecular Dynamics Simulations and Mechanics: Implications of Role of Common Binding Site in Drug Design for Alzheimer's Disease.

Aggregation of β-amyloid (Aβ) into oligomers and further into fibrils is hypothesized to be a key factor in pathology of Alzheimer's disease (AD). In this study, mapping and docking were used to study the binding of ligands to protofibrils. It was followed by molecular simulations to understand the differences in interactions of known therapeutic agents such as curcumin, fluorescence-based amyloid staining agents such as thioflavin T, and diagnostic agents such as florbetapir (AV45), with Aβ protofibrils. We show that therapeutic agents bind to and distort the protofibrils, thus causing destabilization or prevention of oligomerization, in contrast to diagnostic agents which bind to but do not distort such structures. This has implications in the rational design of ligands, both for diagnostics and therapeutics of AD.

Color stability of artificial teeth after exposure to acid and staining agents

Objective: Changes in color of artificial teeth mainly occur due to ingestion of beverages and use of products for cleaning and disinfection. The more aggressive solutions must be identified and the patient provided with explanations in order to avoid or reduce the frequency of their use, to a higher longevity of the denture. The purpose of this study was to evaluate the changes in color of artificial teeth before and after immersion in beverages and disinfectants. Material and Methods: 96 artificial resin teeth were randomly divided into 8 groups. Each group was immersed for 10 min into a test solution (coffee, lemon juice, chlorhexidine gluconate, red wine, cola-based soft drink, vinegar or antiseptic with and without alcohol) and then the specimens were stored in artificial saliva for 23 h and 50 min, completing a period of 24 h. This procedure was performed for 14 consecutive days and after this period the second color measurement was made.The data obtained with the spectrophotometer using the CIEL*a*b* system were statistically analyzed using ANOVA non parametric, Kruskal-Walis and the Dunn test. Results: There were found differences in color variation for each experimental group after the challenge. statistically significant differences were found between the coffee, red wine and lemon juice groups. Conclusion: All the substances changed the color of artificial teeth; coffee was the substance that caused most staining of artificial teeth, altering color and luminosity; the oral antiseptics with and without alcohol promoted whitening of the artificial teeth.Keywords: Artificial tooth; Denture; Oral rehabilitation; Staining.

Effect of Staining Solutions on Color Stability of Silorane & Methacrylate Restorative Material

Color stability throughout the functional lifetime of restorations is important for the durability of treatment and of cosmetic importance. The purpose of this study was to evaluate the discoloration properties of a silorane-based (Filtek P90) and methacrylate-based (Z100) composites upon exposure to different staining solutions that are used on day to day basis (turmeric, tea, coffee, cocoa, lime, yoghurt and distilled water) for different immersion periods (1, 7, 14 and 28 days). The colors of all specimens before and after storage in the solutions were measured by a reflectance spectrophotometer based on CIE Lab system and the color differences were calculated. Data were statistically analyzed by repeated measures of ANOVA and sidak post hoc test (for immersion period); 't' test (for each material) and one way ANOVA (for staining agents). All the staining agents showed significant difference in staining over time in both the materials. However, Z100 showed higher quantum of discoloration at all time periods at each staining agents (p<0.005). In conclusion, the silorane-based resin (Filtek P90) composites exhibited better color stability (less change in ΔE) after exposure to the staining solutions. Among the staining agents cocoa was found to be least staining followed by lime, yoghurt, coffee, tea whereas turmeric discolored the composites to the maximum. Highest discoloration was seen at day 28 in all staining agents. Cocoa and lime discolored to maximum at early stages but remained stable thereafter whereas tea, coffee and turmeric progressively discolored the composite over time.

Influence of whitening and regular dentifrices on orthodontic clear ligature color stability.

This study evaluated the effect of brushing orthodontic clear ligatures with a whitening dentifrice containing a blue pigment (Close Up White Now, Unilever, London, UK) on their color stability, when exposed to a staining agent. Ligatures from 3M Unitek (Monrovia, CA, USA) and Morelli (Sorocaba, SP, Brazil) were tested. Baseline color measurements were performed and nonstained groups (control) were stored in distilled water whereas test groups were exposed for 1 hour daily to red wine. Specimens were brushed daily using regular or whitening dentifrice. Color measurements were repeated after 7, 14, 21, and 28 days using a spectrophotometer based on the CIE L*a*b* system. Decreased luminosity (CIE L*), increased red discoloration (CIE a* axis), and increased yellow discoloration (CIE b* axis) were generally observed for ligatures exposed to the staining agent. Color variation was generally lower in specimens brushed with regular dentifrice, but ligatures brushed with whitening dentifrice were generally less red and less yellow than regular dentifrice. The whitening dentifrice led to blue discoloration trend, with visually detectable differences particularly apparent according to storage condition and ligature brand. The whitening dentifrice containing blue pigment did not improve the ligature color stability, but it decreased yellow discoloration and increased a blue coloration. The use of a whitening dentifrice containing blue pigment during orthodontic treatment might decrease the yellow discoloration of elastic ligatures.

Differential vital staining of normal fibroblasts and melanoma cells by an anionic conjugated polyelectrolyte

Molecular probes for imaging of live cells are of great interest for studying biological and pathological processes. The anionic luminescent conjugated polythiophene (LCP) polythiophene acetic acid (PTAA), has previously been used for vital staining of cultured fibroblasts as well as transformed cells with results indicating differential staining due to cell phenotype. Herein, we investigated the behavior of PTAA in two normal and five transformed cells lines. PTAA fluorescence in normal cells appeared in a peripheral punctated pattern whereas the probe was more concentrated in a one-sided perinuclear localization in the five transformed cell lines. In fibroblasts, PTAA fluorescence was initially associated with fibronectin and after 24 h partially localized to lysosomes. The uptake and intracellular target in malignant melanoma cells was more ambiguous and the intracellular target of PTAA in melanoma cells is still elusive. PTAA was well tolerated by both fibroblasts and melanoma cells, and microscopic analysis as well as viability assays showed no signs of negative influence on growth. Stained cells maintained their proliferation rate for at least 12 generations. Although the probe itself was nontoxic, photoinduced cellular toxicity was observed in both cell lines upon irradiation directly after staining. However, no cytotoxicity was detected when the cells were irradiated 24 h after staining, indicating that the photoinduced toxicity is dependent on the cellular location of the probe. Overall, these studies certified PTAA as a useful agent for vital staining of cells, and that PTAA can potentially be used to study cancer-related biological and pathological processes.

A luminescent poly(amidoamine)-iridium complex as a new singlet-oxygen sensitizer for photodynamic therapy.

A polymer complex (1P) was synthesized by binding bis(cyclometalated) Ir(ppy)2(+) fragments (ppy = 2-phenylpyridyl) to phenanthroline (phen) pendants of a poly(amidoamine) copolymer (PhenISA, in which the phen pendants involved ∼6% of the repeating units). The corresponding molecular complex [Ir(ppy)2(bap)](+) (1M, bap = 4-(butyl-4-amino)-1,10-phenanthroline) was also prepared for comparison. In water solution 1P gives nanoaggregates with a hydrodynamic diameter of 30 nm in which the lipophilic metal centers are presumed to be segregated within polymer tasks to reduce their interaction with water. Such confinement, combined with the dilution of triplet emitters along the polymer chains, led to 1P having a photoluminescence quantum yield greater than that of 1M (0.061 vs 0.034, respectively, in an aerated water solution) with a longer lifetime of the (3)MLCT excited states and a blue-shifted emission (595 nm vs 604 nm, respectively). NMR data supported segregation of the metal centers. Photoreaction of O2 with 1,5-dihydroxynaphthalene showed that 1P is able to sensitize (1)O2 generation but with half the quantum yield of 1M. Cellular uptake experiments showed that both 1M and 1P are efficient cell staining agents endowed with two-photon excitation (TPE) imaging capability. TPE microscopy at 840 nm indicated that both complexes penetrate the cellular membrane of HeLa cells, localizing in the perinuclear region. Cellular photodynamic therapy tests showed that both 1M and 1P are able to induce cell apoptosis upon exposure to Xe lamp irradiation. The fraction of apoptotic cells for 1M was higher than that for 1P (74 and 38%, respectively) 6 h after being irradiated for 5 min, but cells incubated with 1P showed much lower levels of necrosis as well as lower toxicity in the absence of irradiation. More generally, the results indicate that cell damage induced by 1M was avoided by binding the iridium sensitizers to the poly(amidoamine).

Acetic acid as an adjunct vital stain in diagnosis of tobacco-associated oral lesions: A pilot study.

Background:One of the most important risk factors for oral precancer and cancer in India is the use of tobacco. In chronic tobacco users, the mucosa may appear clinically healthy, however, changes are observed histologically. Screening of such tobacco users for an early diagnosis is, therefore, of paramount importance. Several adjunctive diagnostic modalities have been used in the past, but none has been conclusively validated as confirmative and cost-effective screening methodology. The aim of this study was to evaluate the use of 5% acetic acid as a vital staining agent in tobacco-associated oral lesions.Materials and Methods:The study subjects were divided into two groups. Group I (n = 40) subjects with a history of chronic tobacco use and clinically apparent normal mucosa. Group II (n = 40) subjects suspected of having oral cancer, 5% acetic acid was applied to the mucosa/lesions, followed by incisional biopsy for confirmatory diagnosis.Results:The sensitivity and specificity for Groups I and II were 97%, 50% and 95%, 60%, respectively. Positive predictive value (PPV) and negative predictive value (NPV) of Group I were 0.95 and 0.66. Group II showed PPV and NPV of 0.95 and 0.60.Conclusion:The results of this study suggest that acetic acid holds promise for future. Hence, further studies are needed to be undertaken on a large scale to assess its potential as a screening tool for high-risk individuals and oral cancer.

Influência da técnica de acabamento e do tempo de exposição na estabilidade cromática do bis‐acrílico

AimTo study the effect of several polishing techniques and exposure time to a staining agent on the color stability of two bis‐acrylic resins. Methods60 cylindrical specimens of bis‐acrylic were prepared, 30 with Protemp‐4 and 30 with Strutur‐3. Both sides of specimens were cleaned with alcohol and the specimens were allocated to 12 experimental groups (n=5) according to the material and surface treatment performed (without any other treatment, goat hair brushes, Sof‐Lex coarser grit disc followed by goat hair brushes, Sof‐Lex sequence, Sof‐Lex coarser grit disc with Fortify, Sof‐Lex coarser grit disc with Z‐Prime Plus). Sixty minutes after polishing, the baseline colour values (CIE L*a*b*) were measured, and all the specimens were immersed in the staining solution (coffee). After 24hours and 7 days additional measurements were taken. Colour changes were calculated by the formula: ΔE*=[(ΔL*)2+(Δa*)2+(Δb*)2]1/2. All data were statistically analysed by non‐parametric tests according to Kruskal‐Wallis, Mann‐Whitney and Wilcoxon methods (alpha=0.05). ResultsΔE mean values ranged between 4.9 and 11.7 after 24hours of immersion and between 13.3 and 21.4 after 7 days. Increasing the immersion time in the staining solution led to an increase of ΔE values (p<0.001). After 24hours of immersion, there were no statistical differences among materials (p=0.941). After 7 days of immersion, Protemp‐4 obtained lower ΔE (p=0.032) than Structur‐3. The ΔE was influenced by the polishing technique, except for Protemp‐4 after 7 days of immersion (p<0.05). ConclusionAll specimens presented values for ΔE that were considered clinically unacceptable.

Optical properties of some novel 2,5-disubstituted 1,3,4-oxadiazole derivatives and their application as an efficient cell staining azo dyes.

A series of six 2,5-disubstituted 1,3,4-oxadiazole derivatives with various length of conjugation have been synthesized and their optical properties including UV-visible absorption spectra, fluorescence emission spectra, molar absorption co-efficient, Stokes shift and the relative fluorescence quantum yield were measured in a variety of organic solvents. Correlation of the absorption spectra and fluorescence emission response of the 2,5-disubstituted 1,3,4-oxadiazole derivatives with the substituent effect revealed that the optical response can easily be tuned toward red shift by increasing conjugation length. The synthesized compounds were further employed in bioimaging assay in order to investigate their potential as an efficient cell staining agent using L-929 cells under confocal fluorescence microscope which showed scrupulous cell permeability and no toxicity as assessed by MTT assay.

Iodine vapor staining for atomic number contrast in backscattered electron and X-ray imaging.

Iodine imparts strong contrast to objects imaged with electrons and X-rays due to its high atomic number (53), and is widely used in liquid form as a microscopic stain and clinical contrast agent. We have developed a simple technique which exploits elemental iodine's sublimation-deposition state-change equilibrium to vapor stain specimens with iodine gas. Specimens are enclosed in a gas-tight container along with a small mass of solid I2. The bottle is left at ambient laboratory conditions while staining proceeds until empirically determined completion (typically days to weeks). We demonstrate the utility of iodine vapor staining by applying it to resin-embedded tissue blocks and whole locusts and imaging them with backscattered electron scanning electron microscopy (BSE SEM) or X-ray microtomography (XMT). Contrast is comparable to that achieved with liquid staining but without the consequent tissue shrinkage, stain pooling, or uneven coverage artefacts associated with immersing the specimen in iodine solutions. Unmineralized tissue histology can be read in BSE SEM images with good discrimination between tissue components. Organs within the locust head are readily distinguished in XMT images with particularly useful contrast in the chitin exoskeleton, muscle and nerves. Here, we have used iodine vapor staining for two imaging modalities in frequent use in our laboratories and on the specimen types with which we work. It is likely to be equally convenient for a wide range of specimens, and for other modalities which generate contrast from electron- and photon-sample interactions, such as transmission electron microscopy and light microscopy. Microsc. Res. Tech. 77:1044–1051, 2014. © 2014 The Authors. Microscopy Research Technique published by Wiley Periodocals, Inc.

Characterization and Activity of an Immobilized Antimicrobial Peptide Containing Bactericidal PEG-Hydrogel

A single step immobilization-polymerization strategy of a highly active antimicrobial peptide into a soft hydrogel network on a poly(ethylene terephthalate) surface using thiol-ene chemistry is described. The bactericidal hydrogel was molecularly characterized via Coomassie and Lowry assay protein staining agents as well as by X-ray photoelectron spectroscopy. The bactericidal activity was established against Staphylococcus aureus and Staphylococcus epidermidis, two bacterial strains commonly associated with biomaterial infections. To gain further insight into the biological stability, the hydrogels were incubated with human serum prior to activity testing without loss of activity. These studies revealed a promising bactericidal hydrogel with good stability under physiological conditions.

Functional molecular lumino-materials to probe serum albumins: solid phase selective staining through noncovalent fluorescent labeling.

Selective staining of human serum albumin protein in gel electrophoresis over wide range of other protein(s) is extremely important because it contains more than 60% volume of serum fluid in human body. Given the nonexistence of suitable dye materials for selective staining of serum albumins in gel electrophoresis, we report a new class of easy synthesizable and low molecular weight staining agents based on 3-amino-N-alkyl-carbazole scaffold for selective staining of serum albumins in solid phase. A detailed structure-efficiency relationship (SER) study enabled us to develop two such potent functional molecular probes which stain both human and bovine serum albumin selectively in gel electrophoresis in the presence of other proteins and enzymes. The present gel staining process was found to be very simple and less time-consuming as compared to the conventional coomassie blue staining which in turn makes these probes a new class of serum albumin-specific staining materials in proteome research. Moreover, these molecular lumino-materials can detect serum albumins at subnanomolar level in the presence of broad spectrum of other proteins/enzymes in aqueous buffer (99.9% water, pH = 7.3) keeping the protein secondary structure intact. Our experimental and the docking simulation results show that these probes bind preferentially at 'binding site I' of both the serum proteins.

A dataset comprising four micro-computed tomography scans of freshly fixed and museum earthworm specimens.

BackgroundAlthough molecular tools are increasingly employed to decipher invertebrate systematics, earthworm (Annelida: Clitellata: ‘Oligochaeta’) taxonomy is still largely based on conventional dissection, resulting in data that are mostly unsuitable for dissemination through online databases. In order to evaluate if micro-computed tomography (μCT) in combination with soft tissue staining techniques could be used to expand the existing set of tools available for studying internal and external structures of earthworms, μCT scans of freshly fixed and museum specimens were gathered.FindingsScout images revealed full penetration of tissues by the staining agent. The attained isotropic voxel resolutions permit identification of internal and external structures conventionally used in earthworm taxonomy. The μCT projection and reconstruction images have been deposited in the online data repository GigaDB and are publicly available for download.ConclusionsThe dataset presented here shows that earthworms constitute suitable candidates for μCT scanning in combination with soft tissue staining. Not only are the data comparable to results derived from traditional dissection techniques, but due to their digital nature the data also permit computer-based interactive exploration of earthworm morphology and anatomy. The approach pursued here can be applied to freshly fixed as well as museum specimens, which is of particular importance when considering the use of rare or valuable material. Finally, a number of aspects related to the deposition of digital morphological data are briefly discussed.

Cell type related differences in staining with pentameric thiophene derivatives

Fluorescent compounds capable of staining cells selectively without affecting their viability are gaining importance in biology and medicine. Recently, a new family of optical dyes, denoted luminescent conjugated oligothiophenes (LCOs), has emerged as an interesting class of highly emissive molecules for studying various biological phenomena. Properly functionalized LCOs have been utilized for selective identification of disease-associated protein aggregates and for selective detection of distinct cells. Herein, we present data on differential staining of various cell types, including cancer cells. The differential staining observed with newly developed pentameric LCOs is attributed to distinct side chain functionalities along the thiophene backbone. Employing flow cytometry and fluorescence microscopy we examined a library of LCOs for stainability of a variety of cell lines. Among tested dyes we found promising candidates that showed strong or moderate capability to stain cells to different extent, depending on target cells. Hence, LCOs with diverse imidazole motifs along the thiophene backbone were identified as an interesting class of agents for staining of cancer cells, whereas LCOs with other amino acid side chains along the backbone showed a complete lack of staining for the cells included in the study. Furthermore, for p-HTMI,a LCO functionalized with methylated imidazole moieties, the staining was dependent on the p53 status of the cells, indicating that the molecular target for the dye is a cellular component regulated by p53. We foresee that functionalized LCOs will serve as a new class of optical ligands for fluorescent classification of cells and expand the toolbox of reagents for fluorescent live imaging of different cells.

New ruthenium(ii) arene complexes of anthracenyl-appended diazacycloalkanes: effect of ligand intercalation and hydrophobicity on DNA and protein binding and cleavage and cytotoxicity

A series of half-sandwich Ru(II) arene complexes of the type [Ru(η(6)-arene)(L)Cl](PF6) 1-4, where arene is benzene (1, 2) or p-cymene (3, 4) and L is N-methylhomopiperazine (L1) or 1-(anthracen-10-ylmethyl)-4-methylhomopiperazine (L2), has been isolated and characterized by using spectral methods. The X-ray crystal structures of 2, 3 and 4 reveal that the compounds possess a pseudo-octahedral "piano-stool" structure equipped with the arene ligand as the seat and the bidentate ligand and the chloride ion as the legs of the stool. The DNA binding affinity determined using absorption spectral titrations with CT DNA and competitive DNA binding studies varies as 4 > 2 > 3 > 1, depending upon both the arene and diazacycloalkane ligands. Complexes 2 and 4 with higher DNA binding affinities show strong hypochromism (56%) and a large red-shift (2, 10; 4, 11 nm), which reveals that the anthracenyl moiety of the ligand is stacked into the DNA base pairs and that the arene ligand hydrophobicity also dictates the DNA binding affinity. In contrast, the monocationic complexes 1 and 3 are involved in electrostatic binding in the minor groove of DNA. The enhancement in viscosities of CT DNA upon binding to 2 and 4 are higher than those for 1 and 3 supporting the DNA binding modes of interaction inferred. All the complexes cleave DNA effectively even in the absence of an external agent and the cleavage ability is enhanced in the presence of an activator like H2O2. Tryptophan quenching measurements suggest that the protein binding affinity of the complexes varies as 4 > 2 > 3 > 1, which is the same as that for DNA binding and that the fluorescence quenching of BSA occurs through a static mechanism. The positive ΔH(0) and ΔS(0) values for BSA binding of complexes indicate that the interaction between the complexes and BSA is mainly hydrophobic in nature and the energy transfer efficiency has been analysed according to the Förster non-radiative energy transfer theory. The variation in the ability of complexes to cleave BSA in the presence of H2O2, namely, 4 > 2 > 3 > 1, as revealed from SDS-PAGE is consistent with their strong hydrophobic interaction with the protein. The IC50 values of 1-4 (IC50: 1, 28.1; 2, 23.1; 3, 26.2; 4, 16.8 μM at 24 h; IC50: 1, 19.0; 2, 15.9; 3, 18.1; 4, 9.7 μM at 48 h) obtained for MCF 7 breast cancer cells indicate that they have the potency to kill cancer cells in a time dependent manner, which is similar to cisplatin. The anticancer activity of complexes has been studied by employing various biochemical methods involving different staining agents, AO/EB and Hoechst 33258, which reveal that complexes 1-4 establish a specific mode of cell death in MCF 7 breast cancer cells. The comet assay has been employed to determine the extent of DNA fragmentation in cancer cells.

Strophanthus hispidus attenuates the Ischemia-Reperfusion induced myocardial Infarction and reduces mean arterial pressure in renal artery occlusion.

Background:The myocardium is generally injured in the case of reperfusion injury and arterial damage is caused by hypertension. In reference to these statements, the present study was focused. Cardiac glycosides were said to have protective effects against myocardial infarction and hypertension. Strophanthus hispidus was thus incorporated in the study.Objective:The prime objective of the study was to investigate the protective effects of Strophanthus hispidus against ischemia-reperfusion myocardial Infarction and renal artery occluded hypertension in rats.Materials and Methods:The animal model adopted was surgically-induced myocardial ischemia, performed by means of left anterior descending coronary artery occlusion (LAD) for 30 min followed by reperfusion for another 4 h. Infarct size was assessed by using the staining agent TTC (2,3,5-triphenyl tetrazolium chloride). Hypertension was induced by clamping the renal artery with renal bulldog clamp for 4 h.Results:The study was fruitful by the effect of Strophanthus hispidus on infarction size, which got reduced to 27.2 ± 0.5and 20.0 ± 0.2 by 500 mg/Kg and 1000 mg/Kg ethanolic extracts which was remarkably significant when compared with that of the control group 52.8 ± 4.6. The plant extract did reduce heart rate at various time intervals. There was also a protective effect in the case of mean arterial blood pressure were the 500 mg/Kg and 1000 mg/Kg of the plant extract did reduce the hypertension after 60 minutes was 60.0 ± 4.80 and 50.50 ± 6.80.Conclusion:The results suggest that 500 mg/Kg and 100 mg/Kg ethanolic extract of Strophanthus hispidus was found to possess significant cardiac protective and anti-hypertensive activity.

Laser ablation based bioimaging with simultaneous elemental and molecular mass spectrometry: towards spatially resolved speciation analysis

Biological functions of metals are not only specified by the element itself, but also by its chemical form and by its organ, cell and subcellular location. The developed laser ablation based setup enables spatially resolved analysis with simultaneous elemental and molecular mass spectrometry (MS) and promises therefore localization, identification and quantification of metal or heteroelement-containing species in biological samples such as tissue sections. A UV laser ablation (LA) system is hyphenated in parallel both with an elemental and a molecular mass spectrometer via flow splitted transfer lines to simultaneously obtain data from both of the mass spectrometers. Elemental MS was performed using inductively coupled plasma (ICP)-MS, whereas atmospheric pressure chemical ionization (APCI)-MS with an orbitrap mass analyzer was utilized for molecular MS. Simultaneous elemental and molecular MS imaging with high lateral resolution down to 25 µm was presented for the staining agents eosin Y and haematoxylin as well as for the chemotherapy drug cisplatin in thin tissue sections. For molecular MS, target compounds were identified by their exact masses and by characteristic fragment ions. The first simultaneous elemental and molecular MS imaging approach based on laser ablation sampling was introduced for spatially resolved speciation analysis. The combination of the advantages of LA-ICP-MS such as low detection limits and high spatial resolution with information on the chemical identity promises not only localization of metals, but also identification of metal species in biological samples. Therefore, this novel technique opens up new possibilities to address complex challenges in life science research.