Comparison of the photocatalytic degradation of trypan blue by undoped and silver-doped zinc oxide nanoparticles

Zinc oxide (ZnO) and silver doped zinc oxide (ZnO:Ag) nanoparticles were prepared using nitrates of zinc and silver as oxidizers and ethylene diaminetetraacetic acid (EDTA) as a fuel via low-temperature combustion synthesis (LCS) at 500 degrees C. X-ray diffraction (XRD) pattern indicates the presence of silver in the hexagonal wurtzite structure of ZnO. Fourier transform infrared (FTIR) spectrum indicates the presence of Ag-Zn-O stretching vibration at 510 cm(-1). Transmission electron microscopy (TEM) images shows that the average particle size of ZnO and ZnO:Ag nanoparticles were found to be 58 nm and 52 nm, respectively. X-ray photoelectron spectroscopy (XPS) data clearly indicates the presence of Ag in ZnO crystal lattice. The above characterization techniques indicate that the incorporation of silver affects the structural and optical properties of ZnO nanoparticles. ZnO:Ag nanoparticles exhibited 3% higher photocatalytic efficiency than pure ZnO nanoparticles. ZnO:Ag nanoparticles show better photocatalytic activity for the degradation of trypan blue (TrB) compared to undoped ZnO nanoparticles. (C) 2014 Elsevier Ltd. All rights reserved.

In vivo safety of trypan blue use in vitreoretinal surgery

Purpose: The purpose of this study was to evaluate "in vivo" safety of trypan blue (TB) in patients undergoing TB-assisted internal limiting membrane or epiretinal membrane peeling. Methods: Prospective study including 21 patients (21 eyes) with full-thickness macular hole and/or epiretinal membrane undergoing TB-assisted internal limiting membrane/epiretinal membrane peeling. Main outcome measures included distance visual acuity, near visual acuity, amplitude of P50 and N95 of the pattern electroretinogram, and fundus autofluorescence; these were assessed preoperatively, at 6 months (n = 21) and 12 months (n = 10) postoperatively. Results: There was a statistically significant improvement in distance visual acuity, near visual acuity, P50, and N95 amplitude at 6 months and 12 months postoperatively. The mean logarithm of the minimum angle of resolution distance visual acuity and near visual acuity improved from baseline by 0.31 (SD 0.37) and 0.17 (SD 0.31) at 6 months, respectively, and by 0.4 (SD 0.25) and 0.35 (SD 0.28) at 12 months, respectively. The mean P50 and N95 component amplitudes improved by 28% compared with baseline at 6 months (P50 0.4 [SD 0.8]; N95 0.53 [SD 1.07]) and by 63% at 12 months (P50 0.9 [0.85]; N95 1.04 [1.34]). Autofluorescence did not demonstrate damage to the retinal pigment epithelium attributable to TB. Conclusion: No deleterious effects of TB were observed in this study. Copyright © 2011 Lippincott Williams &Wilkins.

Experimental investigations with trypan blue in connection with the intraspinal treatment of lues nervosa ...

"Stellingen" (2 leaves) laid in.

Staining of the Internal Limiting Membrane with the Use of Heavy Brilliant Blue G

Background: Brilliant blue G (BBG) is frequently used in chromovitrectomy to facilitate internal limiting membrane (ILM) peeling. A study was initiated to evaluate if heavy BBG is safe and effective in staining the ILM. Methods: We studied 30 eyes, 23 with idiopathic macular holes and 7 of patients with diabetic macular edema. Removal of the ILMs was assisted by heavy BBG staining. In cases with histopathological correlation the ILMs were evaluated with hematoxylin and eosin, Masson's trichrome, periodic acid-Schiff and glial fibrillary acidic protein staining. In addition, immunohistochemistry was also performed using specific antibodies for vimentin, neuron-specific enolase, factor VIII and CD68. Using the Image-Pro Plus software of Media Cybernetics Co. we found an average thickness in ILMs. Results: Of the ILM specimens sent, 19/30(63.33%) could not be processed properly because of the limited sample material, recognizing only fragments of dispersed fibrillar material. In macular hole ILMs we found an average thickness of 1.3 +/- 0.65 mu m, and in diabetic macular edema ILMs an average thickness of 6.2 +/- 1.4 mu m. Conclusions: In heavy BBG-assisted ILM peeling we observed no intraoperative or postoperative complications after a mean follow-up of 12 months. Heavy BBG could be an effective and safe vehicle for staining the ILM. Copyright (C) 2012 S. Karger AG, Basel

The manipulation of apoptosis-related genes to generate resistance to Fusarium wilt and water stress in banana

Bananas are susceptible to a diverse range of biotic and abiotic stresses, many of which cause serious production constraints worldwide. One of the most destructive banana diseases is Fusarium wilt caused by the soil-borne fungus, Fusarium oxysporum f. sp. cubense (Foc). No effective control strategy currently exists for this disease which threatens global banana production. Although disease resistance exists in some wild bananas, attempts to introduce resistance into commercially acceptable bananas by conventional breeding have been hampered by low fertility, long generation times and association of poor agronomical traits with resistance genes. With the advent of reliable banana transformation protocols, molecular breeding is now regarded as a viable alternative strategy to generate disease-resistant banana plants. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi. Further, the transgenic plants showed increased resistance to a range of abiotic stresses. In this thesis, the use of anti-apoptosis genes to generate transgenic banana plants with resistance to Fusarium wilt was investigated. Since water stress is an important abiotic constraint to banana production, the resistance of the transgenic plants to water stress was also examined. Embryogenic cell suspensions (ECS) of two commercially important banana cultivars, Grand Naine (GN) and Lady Finger (LF), were transformed using Agrobacterium with the anti-apoptosis genes, Bcl-xL, Bcl-xL G138A, Ced-9 and Bcl- 2 3’ UTR. An interesting, and potentially important, outcome was that the use of anti-apoptosis genes resulted in up to a 50-fold increase in Agrobacterium-mediated transformation efficiency of both LF and GN cells over vector controls. Regenerated plants were subjected to a complete molecular characterisation in order to detect the presence of the transgene (PCR), transcript (RT-PCR) and gene product (Western blot) and to determine the gene copy number (Southern blot). A total of 36 independently-transformed GN lines (8 x Bcl-xL, 5 x Bcl-xL G138A, 15 x Ced-9 and 8 x Bcl-2 3’ UTR) and 41 independently-transformed LF lines (8 x Bcl-xL, 7 x BclxL G138A, 13 x Ced-9 and 13 x Bcl-2 3’ UTR) were identified. The 41 transgenic LF lines were multiplied and clones from each line were acclimatised and grown under glasshouse conditions for 8 weeks to allow monitoring for phenotypic abnormalities. Plants derived from 3 x Bcl-xL, 2 x Ced-9 and 5 x Bcl-2 3’ UTR lines displayed a variety of aberrant phenotypes. However, all but one of these abnormalities were off-types commonly observed in tissue-cultured, non-transgenic banana plants and were therefore unlikely to be transgene-related. Prior to determining the resistance of the transgenic plants to Foc race 1, the apoptotic effects of the fungus on both wild-type and Bcl-2 3’ UTR-transgenic LF banana cells were investigated using rapid in vitro root assays. The results from these assays showed that apoptotic-like cell death was elicited in wild-type banana root cells as early as 6 hours post-exposure to fungal spores. In contrast, these effects were attenuated in the root cells of Bcl-2 3’ UTR-transgenic lines that were exposed to fungal spores. Thirty eight of the 41 transgenic LF lines were subsequently assessed for resistance to Foc race 1 in small-plant glasshouse bioassays. To overcome inconsistencies in rating the internal (vascular discolouration) disease symptoms, a MatLab-based computer program was developed to accurately and reliably assess the level of vascular discolouration in banana corms. Of the transgenic LF banana lines challenged with Foc race 1, 2 x Bcl-xL, 3 x Ced-9, 2 x Bcl-2 3’ UTR and 1 x Bcl-xL G138A-transgenic line were found to show significantly less external and internal symptoms than wild-type LF banana plants used as susceptible controls at 12 weeks post-inoculation. Of these lines, Bcl-2 3’ UTR-transgenic line #6 appeared most resistant, displaying very mild symptoms similar to the wild-type Cavendish banana plants that were included as resistant controls. This line remained resistant for up to 23 weeks post-inoculation. Since anti-apoptosis genes have been shown to confer resistance to various abiotic stresses in other crops, the ability of these genes to confer resistance against water stress in banana was also investigated. Clonal plants derived from each of the 38 transgenic LF banana plants were subjected to water stress for a total of 32 days. Several different lines of transgenic plants transformed with either Bcl-xL, Bcl-xL G138A, Ced-9 or Bcl-2 3’ UTR showed a delay in visual water stress symptoms compared with the wild-type control plants. These plants all began producing new growth from the pseudostem following daily rewatering for one month. In an attempt to determine whether the protective effect of anti-apoptosis genes in transgenic banana plants was linked with reactive oxygen species (ROS)-associated programmed cell death (PCD), the effect of the chloroplast-targeting, ROS-inducing herbicide, Paraquat, on wild-type and transgenic LF was investigated. When leaf discs from wild-type LF banana plants were exposed to 10 ìM Paraquat, complete decolourisation occurred after 48 hours which was confirmed to be associated with cell death and ROS production by trypan blue and 3,3-diaminobenzidine (DAB) staining, respectively. When leaf discs from the transgenic lines were exposed to Paraquat, those derived from some lines showed a delay in decolourisation, suggesting only a weak protective effect from the transgenes. Finally, the protective effect of anti-apoptosis genes against juglone, a ROS-inducing phytotoxin produced by the causal agent of black Sigatoka, Mycosphaerella fijiensis, was investigated. When leaf discs from wild-type LF banana plants were exposed to 25 ppm juglone, complete decolourisation occurred after 48 hours which was again confirmed to be associated with cell death and ROS production by trypan blue and DAB staining, respectively. Further, TdT-mediated dUTP nick-end labelling (TUNEL) assays on these discs suggested that the cell death was apoptotic. When leaf discs from the transgenic lines were exposed to juglone, discs from some lines showed a clear delay in decolourisation, suggesting a protective effect. Whether these plants are resistant to black Sigatoka is unknown and will require future glasshouse and field trials. The work presented in this thesis provides the first report of the use of anti-apoptosis genes as a strategy to confer resistance to Fusarium wilt and water stress in a nongraminaceous monocot, banana. Such a strategy may be exploited to generate resistance to necrotrophic pathogens and abiotic stresses in other economically important crop plants.

Cytotoxicity testing of burn wound dressings, ointments and creams : a method using polycarbonate cell culture inserts on a cell culture system

We have developed a method to test the cytotoxicity of wound dressings, ointments, creams and gels used in our Burn Centre, by placing them on a permeable Nunc Polycarbonate cell culture insert, incubated with a monolayer of cells (HaCaTs and primary human keratinocytes). METHODS: We performed two different methods to determine the relative toxicity to cells. (1) Photo visualisation: The dressings or compounds were positioned on the insert's membrane which was placed onto the monolayer tissue culture plate. After 24 h the surviving adherent cells were stained with Toluidine Blue and photos of the plates were taken. The acellular area of non-adherent dead cells which had been washed off with buffer was measured as a percentage of the total area of the plate. (2) Cell count of surviving cells: After 24 h incubation with the test material, the remaining cells were detached with trypsin, spun down and counted in a Haemocytometer with Trypan Blue, which differentiates between live and dead cells. RESULTS: Seventeen products were tested. The least cytotoxic products were Melolite, White soft Paraffin and Chlorsig1% Ointment. Some cytotoxicity was shown with Jelonet, Mepitel((R)), PolyMem((R)), DuoDerm((R)) and Xeroform. The most cytotoxic products included those which contained silver or Chlorhexidine and Paraffin Cream a moisturizer which contains the preservative Chlorocresol. CONCLUSION: This in vitro cell culture insert method allows testing of agents without direct cell contact. It is easy and quick to perform, and should help the clinician to determine the relative cytotoxicity of various dressings and the optimal dressing for each individual wound.

Cytotoxicity testing of silver-containing burn treatments using primary and immortal skin cells

A novel burn wound hydrogel dressing has been previously developed which is composed of 2-acrylamido-2-methylpropane sulfonic acid sodium salt with silver nanoparticles (silver AMPS). This study compared the cytotoxicity of this dressing to the commercially available silver products; Acticoat™, PolyMem Silver® and Flamazine™ cream. Human keratinocytes (HaCaT and primary HEK) and normal human fibroblasts (NHF) were exposed to dressings incubated on Nunc™ polycarbonate inserts for 24, 48 and 72h. Four different cytotoxicity assays were performed including; Trypan Blue cell count, MTT, Celltiter-Blue™ and Toluidine Blue surface area assays. The results were expressed as relative cell viability compared to an untreated control. The cytotoxic effects of Acticoat™ and Flamazine™ cream were dependent on exposure time and cell type. After 24h exposure, Acticoat™ and Flamazine™ cream were toxic to all tested cell lines. Surprisingly, HaCaTs treated with Acticoat™ and Flamazine™ had an improved ability to survive at 48 and 72h while HEKs and NHFs had no improvement in survival with any treatment. The novel silver hydrogel and PolyMem Silver® showed low cytotoxicity to all tested cell lines at every time interval and these results support the possibility of using the novel silver hydrogel as a burn wound dressing. Researchers who rely on HaCaT cells as an accurate keratinocyte model should be aware that they can respond differently to primary skin cells.

Synthesis of 2-(5-((5-(4-chlorophenyl)furan 2-yl)methylene)-4-oxo-2-thioxo-thiazolidi n-3-yl)acetic acid derivatives and evaluation of their cytotoxicity and induction of apoptosis in human leukemia cells

In order to explore the anticancer effect associated with the thiazolidinone framework, several 2-(5-((5-(4-chlorophenyl)furan-2-yl)methylene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid derivatives 5(a-1) were synthesized. Variation in the functional group at C-terminal of the thiazolidinone led to set of compounds bearing amide moiety. Their chemical structures were confirmed by H-1 NMR, IR and Mass Spectra analysis. These thiazolidinone compounds containing furan moiety exhibits moderate to strong antiproliferative activity in a cell cycle stage-dependent and dose dependent manner in two different human leukemia cell lines. The importance of the electron donating groups on thiazolidinone moiety was confirmed by MTT and Trypan blue assays and it was concluded that the 4th position of the substituted aryl ring plays a dominant role for its anticancer property. Among the synthesized compounds, 5e and 5f have shown potent anticancer activity on both the cell lines tested. To rationalize the role of electron donating group in the induction of cytotoxicity we have chosen two molecules (5e and 5k) having different electron donating group at different positions. LDH assay, Flow cytometric analysis and DNA fragmentation suggest that 5e is more cytotoxic and able to induce the apoptosis. (C) 2009 Elsevier Ltd. All rights reserved.

Synthesis and in vitro cytotoxic evaluation of novel diazaspiro bicyclo hydantoin derivatives in human leukemia cells: A SAR study

To study the structure activity relationship (SAR) on the cytotoxic activity and probe the structural requirement for the potent antitumor activity, a series of novel diazaspiro bicyclo hydantoin derivatives were designed and synthesized. Their structures were confirmed by H-1 NMR, LCMS and IR analyses. The antiproliferative effect of these compounds were determined against human leukemia, K562 (chronic myelogenous leukemia) and CEM (T-cell leukemia) cells using trypan blue and MTT assay, and the SAR associated with the position of N-terminal substituents in diazaspiro bicyclo hydantoin have also been discussed. It has been observed that these compounds displayed strong, moderate and weak cytotoxic activities. Interestingly, compounds having electron withdrawing groups at third and fourth position of the phenyl ring displayed selectively cytotoxic activities to both the cell lines tested with IC50 value lower than 50 mu M. In addition, the cytotoxic activities of the compounds 7(a-o) bearing the substituents at N-3 position of diazaspiro bicyclo hydantoin increases in the order alkene > ester > ether and plays an important role in determining their antitumor activities. The position and number of substituents in benzyl group attached to N-8 of diazaspiro bicyclo hydantoin nucleus interacted selectively with specific targets leading to the difference of biochemical and pharmacological effects.

Detection of Polymyxa graminis in a barley crop in Australia.

Polymyxa graminis was detected in the roots of barley plants from a field near Wondai, Queensland, in 2009. P. graminis was identified by characteristic sporosori in roots stained with trypan blue. The presence of P. graminis f. sp. tepida (which is hosted by wheat and oats as well as barley) in the roots was confirmed by specific PCR tests based on nuclear ribosomal DNA. P. graminis is the vector of several damaging soil-borne virus diseases of cereals in the genera Furovirus, Bymovirus and Pecluvirus. No virus particles were detected in sap extracts from leaves of stunted barley plants with leaf chlorosis and increased tillering. Further work is required to determine the distribution of P. graminis in Australian grain crops and the potential for establishment and spread of the exotic soil-borne viruses that it vectors.

Regulation of oxidative-stress responsive genes by arecoline in human keratinocytes

Background and Objective: Arecoline, an arecanut alkaloid present in the saliva of betel quid chewers, has been implicated in the pathogenesis of a variety of inflammatory oral diseases, including oral submucous fibrosis and periodontitis. To understand the molecular b asis of arecoline action in epithelial changes associated with these diseases, we investigated the effects of arecoline on human keratinocytes with respect to cell growth regulation and the expression of stress-responsive genes.Material and Methods:Human keratinocyte cells (of the HaCaT cell line) were treated with arecoline, following which cell viability was assessed using the Trypan Blue dye-exclusion assay, cell growth and proliferation were analyzed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and 5-bromo-2-deoxyuridine incorporation assays, cell cycle arrest and generation of reactive oxygen species were examined using flow cytometry, and gene expression changes were investigated using the reverse transcription-polymerase chain reaction technique. The role of oxidative stress, muscarinic acetylcholine receptor and mitogen-activated protein kinase (MAPK) pathways were studied using specific inhibitors. Western blot analysis was performed to study p38 MAPK activation.Results:Arecoline induced the generation of reactive oxygen species and cell cycle arrest at the G1/G0 phase in HaCaT cells without affecting the expression of p21/Cip1. Arecoline-induced epithelial cell death at higher concentrations was caused by oxidative trauma without eliciting apoptosis. Sublethal concentrations of arecoline upregulated the expression of the following stress-responsive genes: heme oxygenase-1; ferritin light chain; glucose-6-phosphate dehydrogenase; glutamate-cysteine ligase catalytic subunit; and glutathione reductase.Additionally, there was a dose-dependent induction of interleukin-1alfa mRNA by arecoline via oxidative stress and p38 MAPK activation. Conclusion:our data highlight the role of oxidative stress in arecoline-mediated cell death, gene regulation and inflammatory processes in human keratinocytes.

Synthesis, Characterization and Evaluation of Cytotoxicity of New Aminophosphonic Acid Diesters in Human Leukemia Cells

A series of novel fluoroaminophosphates 4a-4j were synthesized by one-pot method in presence of tetramethylguanidine (TMG) as a catalyst and were characterized by elemental analysis, FTIR, H-1, C-13, P-31, F-19 NMR, and mass spectra. All the title compounds were evaluated forin vitro cytotoxicity against leukemic cell line derived from T-cells of leukemia patient (CEM cells) by Trypan blue exclusion and MTT assays, and these were found to exert concentration dependent cytotoxic effects. Among them 4f, 4g & 4j possessed marked cytotoxicity. 4g (with IC50 value of 6 mu M) had emerged as lead compound.

Synthesis and identification of a new class of (S)-2,6-diamino-4,5,6,7-tetrahydrobenzo[d]thiazole derivatives as potent antileukemic agents

Benzothiazoles are multitarget agents with broad spectrum of biological activity. Among the antitumor agents discovered in recent years, the identification of various 2-(4-aminophenyl) benzothiazoles as potent and selective antitumor drugs against different cancer cell lines has stimulated remarkable interest. Some of the benzothiazoles are known to induce cell cycle arrest, activation of caspases and interaction with DNA molecule. Based on these interesting properties of benzothiazoles and to obtain new biologically active agents, a series of novel 4,5,6,7-tetrahydrobenzo[d]thiazole derivatives 5(a-i) were synthesized and evaluated for their efficacy as antileukemic agents in human leukemia cells (K562 and Reh). The chemical structures of the synthesized compounds were confirmed by H-1 NMR, LCMS and IR analysis. The cytotoxicity of these compounds were determined using trypan blue exclusion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Results showed that, these compounds mediate a significant cytotoxic response to cancer cell lines tested. We found that the compounds having electron withdrawing groups at different positions of the phenyl ring of the thiourea moiety displayed significant cytotoxic effect with IC50 value less than 60 mu M. To rationalize the role of electron withdrawing group in the induction of cytotoxicity, we have chosen molecule 5g (IC50 similar to 15 mu M) which is having chloro substitution at ortho and para positions. Flow cytometric analysis of annexin V-FITC/ propidium iodide (PI) double staining and DNA fragmentation suggest that 5g can induce apoptosis.

Novel rhodanine derivatives induce growth inhibition followed by apoptosis

We have designed and synthesized three novel compounds, 5-isopropylidiene derivatives of 3-dimethyl-2-thio-hydantoin (ITH-1), 3-ethyl-2-thio-2,4-oxazolidinedione (ITO-1), and 5-benzilidene-3-ethyl rhodanine (BTR-1), and have tested their chemotherapeutic properties. Our results showed that all three compounds induced cytotoxicity in a time-and concentration-dependent manner on leukemic cell line, CEM. Among the compounds tested, BTR-1 was 5- to 7-fold more potent than ITH-1 and ITO-1 when compared by trypan blue and MTT assays. IC50 value of BTR-1 was estimated to be <10 mu M. Both cell cycle analysis and tritiated thymidine assays revealed that BTR-1 affects DNA replication by inducing a block at S phase. BTR-1 treatment led to increased level of ROS production and DNA strand breaks suggesting activation of apoptosis for induction of cell death. (C) 2010 Elsevier Ltd. All rights reserved.

Synthesis and Identification of a new class of antileukemic agents containing 2-(arylcarboxamide)-(S)-6-amino-4,5,6,7-tetrahydrobenzod]thiazole

Recently we have reported the effect of (S)-6-aryl urea/thiourea substituted-2-amino-4,5,6,7-tetrahydrobenzod]thiazole derivatives as potent anti-leukemic agents. To elucidate further the Structure Activity Relationship (SAR) studies on the anti-leukemic activity of (S)-2,6-diamino-4,5,6,7 tetrahydrobenzod]thiazole moiety, a series of 2-arlycarboxamide substituted-(S)-6-amino-4,5,6,7-tetrahydrobenzod]thiazole were designed, synthesized and evaluated for their anti-leukemic activity by trypan blue exclusion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), lactate dehydrogenase (LDH) assays and cell cycle analysis. Results suggest that the position, number and bulkiness of the substituent on the phenyl ring of aryl carboxamide moiety at 2nd position of 6-amino-4,5,6,7-tetrhydrobenzod]thiazole play a key role in inhibiting the proliferation of leukemia cells. Compounds with ortho substitution showed poor activity and with meta and para substitution showed good activity. (C) 2010 Elsevier Masson SAS. All rights reserved.