Resazurin Dye Research Articles

Rapid, Semiquantitative Assay To Discriminate among Compounds with Activity against Replicating or Nonreplicating Mycobacterium tuberculosis.

The search for drugs that can kill replicating and nonreplicating Mycobacterium tuberculosis faces practical bottlenecks. Measurement of CFU and discrimination of bacteriostatic from bactericidal activity are costly in compounds, supplies, labor, and time. Testing compounds against M. tuberculosis under conditions that prevent the replication of M. tuberculosis often involves a second phase of the test in which conditions are altered to permit the replication of bacteria that survived the first phase. False-positive determinations of activity against nonreplicating M. tuberculosis may arise from carryover of compounds from the nonreplicating stage of the assay that act in the replicating stage. We mitigate these problems by carrying out a 96-well microplate liquid MIC assay and then transferring an aliquot of each well to a second set of plates in which each well contains agar supplemented with activated charcoal. After 7 to 10 days—about 2 weeks sooner than required to count CFU—fluorometry reveals whether M. tuberculosis bacilli in each well have replicated extensively enough to reduce a resazurin dye added for the final hour. This charcoal agar resazurin assay (CARA) distinguishes between bacterial biomasses in any two wells that differ by 2 to 3 log10 CFU. The CARA thus serves as a pretest and semiquantitative surrogate for longer, more laborious, and expensive CFU-based assays, helps distinguish bactericidal from bacteriostatic activity, and identifies compounds that are active under replicating conditions, nonreplicating conditions, or both. Results for 14 antimycobacterial compounds, including tuberculosis (TB) drugs, revealed that PA-824 (pretomanid) and TMC207 (bedaquiline) are largely bacteriostatic.

Abstract 2690: DCC-2618 is a potent inhibitor of wild-type and mutant KIT, including refractory Exon 17 D816 KIT mutations, and exhibits efficacy in refractory GIST and AML xenograft models

Abstract Introduction: KIT kinase mutations are causative of a number of human cancers, including gastrointestinal stromal tumors (GIST), systemic mastocytosis (SM), mast cell leukemia (MCL), and subtypes of melanoma and acute myeloid leukemia (AML). DCC-2618 is a robust Type II switch pocket control inhibitor which potently inhibits exon 17 KIT mutations that are resistant to conventional TKIs. Experimental procedures: DCC-2618 was tested for inhibition of KIT isoforms using a standard PK/LDH coupled spectrophotometric assay. CHO cells were transiently transfected to express mutant KIT or PDGFRα constructs. Transfected cells were treated with a range of DCC-2618 and levels of phosphorylated KIT or PDGFRα in cell lysates were determined by ELISA or western blot. Cell proliferation of several cell lines was measured using the fluorescent dye resazurin. Experiments were performed in triplicate. In vivo xenograft models were performed at Molecular Imaging, Inc. (Ann Arbor, MI) or Molecular Response, LLC (San Diego, CA). Summary of results: DCC-2618 inhibited various forms of KIT with nanomolar potency: WT (IC50 4 nM), V654A (8 nM), T670I (18 nM), D816H (5 nM), D816V (14 nM). In CHO cells transiently transfected with both single and double (primary/secondary) KIT mutants, DCC-2618 robustly inhibited exon 17, exon 9/13, exon 9/14, and exon 9/17 KIT mutants, as well as exon 11/17 KIT mutants, including exon 17 D816V, D816G, D820A, D820E, D820Y, N822K, N822Y, N822H, and Y823D primary or secondary mutations. DCC-2618 inhibited wild type KIT phosphorylation in the MO7e cell line (IC50 36 nM). DCC-2618 potently inhibited KIT activation in human GIST cell lines, including GIST T1 (exon 11 deletion, IC50 2 nM), GIST 430 (exon 11 deletion/exon 13 V654A, IC50 7 nM), and GIST 48 (exon 11 V560D/exon 17 D820A, IC50 53 nM). In the murine mastocytosis P815 cell line expressing the exon 17 D816Y mutation, DCC-2618 potently inhibited cell proliferation (IC50 2 nM). In vivo, DCC-2618 administration at 50 mg/kg afforded an ED90 for inhibition of KIT phosphorylation in the GIST T1 xenograft model, corresponding to an EC90 concentration of ∼ 470 ng/mL. When give twice daily, this oral dose resulted in almost complete tumor stasis. This dose of DCC-2618 produced tumor regressions in a patient derived xenograft (PDX) GIST expressing KIT exon 11 delW557K558/exon 17 Y823D, and also in a KIT exon 17 N822K AML xenograft model. Conclusion: DCC-2618 is a potent inhibitor of singly and doubly mutated KIT characterized by primary exon 9 or exon 11 mutations paired with secondary mutations in exons 13, 14 or 17. DCC-2618 inhibits exon 17 mutations, including the D816V mutation refractory to currently marketed KIT inhibitors. DCC-2618 has the potential to treat KIT mutant-driven cancers including GIST, systemic mastocytosis, AML, or melanoma. DCC-2618 has been selected for formal IND-enabling clinical development. Citation Format: Bryan D. Smith, Molly M. Hood, Scott C. Wise, Michael D. Kaufman, Wei-Ping Lu, Thomas Rutkoski, Daniel L. Flynn, Michael C. Heinrich. DCC-2618 is a potent inhibitor of wild-type and mutant KIT, including refractory Exon 17 D816 KIT mutations, and exhibits efficacy in refractory GIST and AML xenograft 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 2690. doi:10.1158/1538-7445.AM2015-2690

Abstract 790: Altiratinib is a potent inhibitor of TRK kinases and is efficacious in TRK-fusion driven cancer models

Abstract Introduction: TRK kinases have been implicated in a variety of cancers, wherein TRK gene fusions, amplifications and mutations have been shown to drive tumor growth. TRK kinases have also been shown to play key roles in metastasis. Recently, large-scale sequencing efforts have identified TRK fusions at a low but significant frequency across all major cancers. Altiratinib is a potent single-digit nanomolar inhibitor of TRK, MET, TIE2, and VEGFR2 kinases. Altiratinib inhibits TRKA, TRKB, and TRKC phosphorylation in both WT TRK and TRK-fusion cell lines and inhibits cell proliferation in these cell lines. In vivo, altiratinib suppressed TRK phosphorylation for >18 hr after a single oral dose and showed significant inhibition of tumor growth in TRKA and TRKC-fusion xenograft efficacy studies. Altiratinib is currently in a Phase 1 clinical trial for patients with advanced solid tumors. Experimental procedures: Altiratinib was tested for inhibition of various recombinant kinases using a standard PK/LDH coupled spectrophotometric continuous assay. In cell assays, cells were treated with a dose response of compound. Levels of phosphorylated TRK in cell lysates were determined Western blot. Cell proliferation was measured using the fluorescent dye resazurin. Experiments were performed in triplicate. In vivo xenograft models were performed at Molecular Imaging, Inc. (Ann Arbor, MI). Summary of results: Altiratinib potently inhibited TRK kinase activity in biochemical assays (IC50 values of 0.9 nM, 4.6 nM, and 0.8 nM for TRKA, B, and C, respectively). Altiratinib inhibited phosphorylation of TRKA in the TPM3-TRKA fusion cell line KM-12 (IC50 = 1.4 nM) and of TRKC in ETV6-TRKC transformed NIH-3T3 cells (IC50 = 0.5 nM). Altiratinib inhibited NGF-stimulated phosphorylation of wild-type TRKA in K562 (IC50 = 0.7 nM) and SK-N-SH cells (IC50 = 1.2 nM) and BDNF-stimulated phosphorylation of TRKB in ATRA-transformed SK-N-SH cells (IC50 = 0.24 nM). In compound washout experiments, altiratinib inhibited TRKA phosphorylation in KM-12 cells for > 24 hr after compound washout, due to its durable Type II switch pocket binding mode to the kinase. In cell lines driven by TRK fusions, altiratinib also inhibited cell proliferation (KM-12 IC50 = 3.8 nM; NIH-3T3 ETV6/TRKC IC50 = 0.5 nM). In vivo, altiratinib inhibited phosphorylation of TRK and downstream targets in the signaling pathway for >12 hr after a single oral dose. Altiratinib treatment led to tumor regression in TRK-fusion xenograft efficacy studies. Conclusions: Altiratinib exhibits potent inhibition of oncogenic TRK fusions in vitro and in vivo. Combined with its inhibition of the tumor and microenvironment targets MET, TIE2, and VEGFR2 kinases, altiratinib provides the potential to treat cancers driven by TRK fusions. Altiratinib is currently in a Phase 1 clinical trial in patients with solid tumors. Citation Format: Bryan D. Smith, Cynthia B. Leary, Benjamin A. Turner, Michael D. Kaufman, Scott C. Wise, Maria E. R. Garcia-Rendueles, James A. Fagin, Daniel L. Flynn. Altiratinib is a potent inhibitor of TRK kinases and is efficacious in TRK-fusion driven cancer 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 790. doi:10.1158/1538-7445.AM2015-790

Real-time monitoring of the metabolic activity of periodontopathic bacteria

Bacterial metabolic activity is associated with the onset and progression mechanisms of oral biofilm-mediated disease; however, at present no method to monitor bacterial metabolism exists, especially for periodontopathic bacteria. Therefore, we aimed to establish a novel method for monitoring the metabolic activity of periodontopathic bacteria, Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi) and Fusobacterium nucleatum (Fn), as well as Streptococcus mutans (Sm) for comparison. The method is based on the dye resazurin, which is converted to the fluorescent molecule resorufin by reducing molecules derived from bacterial metabolism. Additionally, the effects of antimicrobial substances on bacterial metabolic activity were evaluated using this method. When bacterial suspensions were incubated with tryptone, glutamate, aspartate or glucose in the presence of resazurin, the fluorescence intensity increased over time by these bacterial metabolic reactions, indicating that this method can be used to monitor the metabolic activity of periodontopathic bacteria. Chlorhexidine showed the 50% inhibitory concentration (IC50) of 15–49μg/ml for tryptone metabolism by Pg, Pi, and Fn, and 7.1–18μg/ml for glucose metabolism by Pi and Sm. The IC50s for cetylpyridinium chloride and sodium dodecyl sulfate were 0.8–2.1 and 28–44μg/ml, respectively for all bacteria examined. Fluoride had no effect except the IC50 of 640μg/ml for Sm, while minocycline hydrochloride had no effect on any of the bacteria. The present study established the method for real-time monitoring of the metabolic activity of periodontopathic bacteria, and the method might be useful for evaluating the effects of antimicrobial substances on the bacterial metabolic activity.

407. Sensitivity and Reproducibility of a Metabolic Assay for Quality Control of Cell Therapy Lots

Determining cell viability via dye exclusion assays (ie; Trypan Blue, TB) is routinely used in lot release testing of cell therapy products. However, such assays provide a limited snapshot of viability and may not be adequate for monitoring the overall quality of cells intended for clinical use, where injection of nonviable cells could lead to safety issues. TB assay may show high viability at thaw, but could fail to capture cryopreservation-driven apoptosis seen after 24 hours. We developed a custom assay based upon resazurin dye conversion to assess changes in cell metabolic activity after cryopreservation.

Abstract 111: Identification of the agent causing Kawasaki Disease via throat swab cultures

Kawasaki disease (KD) is a paediatric inflammatory disease and is the most common cause of acquired heart disease in children in developed countries. The cause of KD is unknown but epidemiological data indicate that it is most likely to be an infectious agent. The clinical features of KD are consistent with either bacterial or fungal infection rather than a virus, as KD is associated with an intense inflammatory process. It has been suggested that the oral and respiratory mucosa are the primary site of infection as the initial infection in KD patients is likely to be in the mucosal surfaces of the respiratory track with prominent lymphadenopathy and oral mucous membrane inflammation. This study examines whether the agent causing KD induces disease through production of novel toxins, or inflammation inducing antigens in the supernatants of cultured bacteria isolated from KD patients and healthy controls. Throat swabs were collected from children with KD (n=14), other diseases (n=10) and healthy children (n=8), and were inoculated into growth medium, incubated and then centrifuged. The supernatants were collected and toxicity was measured by microscopic observation as well as by resazurin dye reduction of cells after growth in the presence of the KD or control supernatant. An effect of the toxins in activating inflammation was also tested by the addition of the supernatants to healthy donor peripheral blood mononuclear cells (PBMCs) and measurement of interleukin 6 production by Enzyme linked immune assay (ELISA). There was no significant difference in the reduction of cells after growth between control and KD supernatants. No difference in the production of IL-6 was observed between the three groups. Further analysis and larger sample size are required to establish whether novel toxins are present in cultured supernatants of KD patients.

Cell viability assessment using the Alamar blue assay: A comparison of 2D and 3D cell culture models

Comparisons of 2D and 3D cell culture models in literature have indicated differences in cellular morphology and metabolism, commonly attributed the better representation of in vivo conditions of the latter cell culture environment. Thus, interest in the use of 3D collagen gels for in vitro analysis has been growing. Although comparative studies to date have indicated an enhanced resistance of cells on collagen matrices against different toxicants, in the present study it is demonstrated that non-adapted protocols can lead to misinterpretation of results obtained from classical colorimetric dye-based cytotoxic assays. Using the well established Alamar blue assay, the study demonstrates how the transfer from 2D substrates to 3D collagen matrices can affect the uptake of the resazurin itself, affecting the outcome of the assay. Using flow cytometry, it is demonstrated that the cell viability is unaffected when cells are grown on collagen matrices, thus the difference seen in the fluorescence is a result of a dilution of the resazurin dye in the collagen matrix, and an increased uptake rate due to the larger cell surface exposed to the surrounding environment, facilitating more effective diffusion through the cellular membrane. The results are supported by a rate equation based simulation, verifying that differing uptake kinetics can result in apparently different cell viability. Finally, this work highlights the feasibility to apply classical dye-based assays on collagen based 3D cell culture models. However, the diffusion and bioavailability of test substances in 3D matrices used in in vitro toxicological assays must be considered and adaption of the protocols is necessary for direct comparison with the traditional 2D models. Moreover, the observations made based on the resazurin dye can be applied to drugs or nanoparticles which freely diffuse through the collagen matrices, thus affecting the effective concentration exposed to the cells.

Study on inorganic oxidants assisted sonocatalytic degradation of Resazurin dye in presence of β-SnWO4 nanoparticles

This work presents about the study of inorganic oxidants assisted sonocatalytic degradation of Resazurin (RZ) dye in the presence of β-SnWO4 nanoparticles. The catalysts were synthesized via a surfactant free sonochemical method. Physical properties of the as-prepared β-SnWO4 nanoparticles were analyzed by XRD and FE-SEM. The dispersion property and particle size were analyzed by Zeta potential and Particle size analyzer. The degradation of RZ was analyzed in the presence and absence of common oxidants such as PMS (peroxymonosulfate) and H2O2 (hydrogen peroxide). The result shows that the RZ was first mineralized to Resorufin and it was degraded to simple molecule in the presence of β-SnWO4. 75% of degradation efficiency was achieved in presence of β-SnWO4, while in addition of H2O2 and PMS, the efficiency was increased up to 93% and 85% respectively. Our results will pave a new opening in degradation of poisonous dye using inorganic common oxidants.

HISTONE DEACETYLASE INHIBITOR PCI-24781 SHOWS ANTIPROLIFERATIVE EFFECTS AGAINST PEDIATRIC GLIOBLASTOMA CELLS

BACKGROUND: Glioblastoma (GBM) is one of the most aggressive tumors affecting the central nervous system (CNS). Despite combined aggressive treatment schedules, the prognosis of children with GBM remains very poor. Epigenetic mechanisms may interfere with the process of carcinogenesis, and the acetylation of DNA can modulate the expression of genes related to the development and progression of malignancies. Clinical studies have shown that histone deacetylases inhibitors (HDACi are clinically active and well tolerated in the treatment of several tumors. Therefore, this study aims to evaluate the therapeutic potential of HDACi PCI-24781 in pediatric GBM lines, SF188 and KNS42 by proliferation assay. METHODS: Pediatric GMB cell-lines were cultured in HAM-F10 supplemented with 10% fetal bovine serum, at 37°C in a humidified 5% CO2 incubator. Cells were seeded in 96-well plates at 2x103 per well. After 24h, cells were treated with PCI-24781 at different concentrations (0.5, 1, 2, 4, 8 and 16 µM for 24, 48, 72 and 96h). Culture medium was removed and replaced with culture medium containing 5 µL of Resazurin dye in each well. Cells were incubated for 6h at 37°C, and the product was measured at 570 nm and 595 nm using a microplate reader. Statistical analysis was performed by One- and Two-way ANOVA and Bonferoni post-hoc. RESULTS: Both cell-lines were sensitive to PCI-24781 treatment, demonstrating an inhibition of proliferation (P < 0.05). For the SF188 cell-line, the strongest effect was observed at the doses of 4 µM, 8 µM and 16 µM at 96h (growth inhibition of 93%). The KNS42 cell-line showed a time-dependent antiproliferative pattern; the best effect was observed at 96h, when growth inhibition was of approximately 84%. It was observed time effect difference of proliferation between 24h and 48h (P < 0.05), but not between 48 h and 72 h. CONCLUSIONS: These primary data demonstrates that PCI-24781 can induce the inhibition of cell proliferation in pediatric GBM cells. Further experiments will be performed to assess the ability of this HDACi in modulating the cellular response in combinatin to ionizing radiation. Financial Support: FAPESP (process no. 2013/15891-8). SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

N-doped TiO2 visible light photocatalyst films via a sol–gel route using TMEDA as the nitrogen source

Abstract N-doped TiO 2 thin films were successfully prepared using N,N,N′,N′-tetramethylethane-1,2-diamine (TMEDA) via a single step sol–gel synthesis. Characterisation by X-ray diffraction and Raman spectroscopy confirmed that the anatase crystal type of TiO 2 was present in all samples. UV/Vis spectroscopy was used to determine the absorbance and band-gap of the materials. Stearic acid and resazurin dye Photo-degradation measurements were used to show the enhanced photocatalytic properties of the materials. Both of these methods are well known for the assessment of photocatalytic films for self cleaning applications with stearic acid being the preferred model for bacteria. The N-doped films showed superhydrophilicity under filtered white light conditions, whereas the undoped films did not show any photo-induced superhydrophilicity. The N-doped TiO 2 films show interstitial nitrogen content rather than substitutional and a substantial red-shift of the band-gap. They were shown to be superior visible light photocatalysts when compared to similar non-doped films. The nature of the chelating ligand, TMEDA, has a profound effect on the oxidation state of the Ti centres, with low concentrations of TMEDA promoting the formation of Ti 3+ centres giving a superior photocatalyst. This method is to the best of our knowledge the easiest, reliable and most reproducible one step route to achieve N-doped TiO 2 films for photocatalytic applications. These N-doped TiO 2 films could have potential applications; as self-cleaning surfaces in healthcare, water sterilisation, and solar energy harvesting.

Stability andin vitroantimicrobial efficacy of a nanopropolis formulation intended for intramammary treatment of bovine mastitis

II SIMPÓSIO DE QUALIDADE DE LEITE O II Simpósio de Qualidade de Leite aconteceu nos dias 05, 06 e 07 de setembro de 2014, no Centro de Convenções da Faculdade de Ciências Agrárias e Veterinárias, Unesp, Câmpus de Jaboticabal, SP. Foi um evento organizado por pós-graduandos do Departamento de Medicina Veterinária Preventiva, sob a supervisão do Prof. Dr . Antonio Nader Filho (homenageado do V Congresso Brasileiro de Qualidade de Leite, 2013, pelos importantes trabalhos desenvolvidos na área). Tendo em vista o sucesso obtido na edição anterior, o II Simpósio de Qualidade de Leite apresentou uma nova programação, com palestras que abordaram assuntos atuais relacionados à cadeia do leite no Brasil aliados à presença de palestrantes renomados. Temas como fraudes e adulteração do leite; resíduos químicos; programas de autocontrole e boas práticas de fabricação; zoonoses veiculadas pelo leite; tratamento da mastite; manejo alimentar e ambiental do gado de leite; aproveitamento de dejetos; a importância da extensão rural como uma aliada; o controle estratégico de parasitas e análises sobre CBT e CCS, e uma mesa redonda para discutir a Instrução Normativa nº 62, do Ministério da Agricultura, Pecuária e Abastecimento, foram alguns dos principais assuntos abordados durante o evento. O público esperado envolveu profissionais de indústrias de laticínios, criadores de gado de leite, estudantes de graduação e de pós-graduação da área de qualidade de leite, pesquisadores e demais profissionais do segmento envolvidos com a pecuária leiteira. Outro aspecto inovador do Simpósio foi a apresentação de resumos expandidos de trabalhos científicos e de extensão rural relacionados ao tema de qualidade do leite. Os resumos estão publicados na Revista Brasileira de Higiene e Sanidade Animal , no formato de resumo expandido, o que permite maior visibilidade e divulgação das pesquisas relacionadas ao tema. Dessa forma, a Comissão Organizadora do II Simpósio de Qualidade de Leite, agradece a todos que de alguma forma colaboraram para o sucesso dessa edição. Em especial, agradecemos nossos patrocinadores, pelo apoio essencial ao evento e aos palestrantes, que foram de suma importância para o enriquecimento e sucesso do Simpósio. Muito obrigado a todos pela participação! Comissão Organizadora II Simpósio de Qualidade de Leite URL do Portal: http://www.higieneanimal.ufc.br/seer/index.php/index/index 2014 setembro; 8(5 Supl 1): p. 01- 552

High throughput and miniaturised systems for biodegradability assessments

The society demands safer products with a better ecological profile. Regulatory criteria have been developed to prevent risks for human health and the environment, for example, within the framework of the European regulation REACH (Regulation (EC) No 1907, 2006). This has driven industry to consider the development of high throughput screening methodologies for assessing chemical biodegradability. These new screening methodologies must be scalable for miniaturisation, reproducible and as reliable as existing procedures for enhanced biodegradability assessment. Here, we evaluate two alternative systems that can be scaled for high throughput screening and conveniently miniaturised to limit costs in comparison with traditional testing. These systems are based on two dyes as follows: an invasive fluorescent dyes that serves as a cellular activity marker (a resazurin-like dye reagent) and a noninvasive fluorescent oxygen optosensor dye (an optical sensor). The advantages and limitations of these platforms for biodegradability assessment are presented. Our results confirm the feasibility of these systems for evaluating and screening chemicals for ready biodegradability. The optosensor is a miniaturised version of a component already used in traditional ready biodegradability testing, whereas the resazurin dye offers an interesting new screening mechanism for chemical concentrations greater than 10 mg/l that are not amenable to traditional closed bottle tests. The use of these approaches allows generalisation of high throughput screening methodologies to meet the need of developing new compounds with a favourable ecological profile and also assessment for regulatory purpose.

On the Influence of DC Electric Fields on the Aerosol Assisted Chemical Vapor Deposition Growth of Photoactive Titanium Dioxide Thin Films

Titanium dioxide thin films were deposited on fluorine doped tin oxide glass substrate from the electric field assisted aerosol chemical vapor deposition (EACVD) reaction of titanium isopropoxide (TTIP, Ti(OC3H7)4) in toluene on glass substrates at a temperature of 450 °C. DC electric fields were generated by applying a potential difference between the electrodes of the transparent coated oxide coated glass substrates during the deposition. The deposited films were characterized using scanning electron microscopy, X-ray diffraction, atomic force microscopy, Raman spectroscopy, and UV-vis spectroscopy. The photoactivity and hydrophilicity of the deposited films were also analyzed using a dye-ink test and water-contact angle measurements. The characterization work revealed that the incorporation of DC electric fields produced significant reproducible changes in the film microstructure, preferred crystallographic orientation, roughness, and film thickness. Photocatalytic activity was calculated from the half-time (t1/2) or time taken to degrade 50% of the initial resazurin dye concentration. A large improvement in photocatalytic activity was observed for films deposited using an electric field with a strong orientation in the (004) direction (t1/2 17 min) as compared to a film deposited with no electric field (t1/2 40 min).

English

Biological silver nanoparticles were successfully synthesized from a simple green and natural route using the extract of&nbsp;Allium cepa&nbsp;(onion) with the use of silver nitrate as precursor and chemically synthesized using silver nitrate and tri sodium citrate. Nanoparticle synthesis was proven under UV-Visible absorption spectroscopy. Toxicity of sliver nanoparticles was tested using ToxTrak test, in which, fresh overnight broths of&nbsp;Bacillus subtilis&nbsp;and resazurin dye were used to calculate percentage inhibition (PI).&nbsp;PI is only a relative measure and since there is toxic substances that increase respiration, to give result to a negative number.&nbsp;The PI of both chemically and biologically synthesized silver nanoparticles was compared in order to evaluate toxic effect value. The toxic effect value, PI of chemically synthesized silver nanoparticles is much greater (85.45%) than the&nbsp;biologically synthesized sliver nanoparticles from onion (51.39%). These observation shows that the bacteria&nbsp;B. subtilis&nbsp;killed by chemically synthesized silver nanoparticles are more as compare to biologically&nbsp;synthesized sliver nanoparticle. &nbsp; Key words:&nbsp;Silver nanoparticles,&nbsp;Allium cepa, ToxTrak toxicity test, resazurin dye, ultraviolet spectroscopy,&nbsp;Bacillus subtilis&nbsp;and percentage inhibition (PI).

Genotoxicity Testing of Two Anticaking Agents: Sodium and Potassium Ferrocyanide in vitro

Anticaking agents are mainly used in food to prevent agglomeration in certain solids, permitting a free-flowing condition. Sodium and potassium ferrocyanide are among the popular anticaking agents which are used in table salt. The present study focuses on the genotoxic effect of sodium and potassium ferrocyanide on human lymphocyte. Human lymphocytes were exposed to the sodium and potassium ferrocyanide at concentrations ranging from 0 to 10 mM for 3 h at 37oC. Cytotoxicity was evaluated by trypan blue dye exclusion test and resazurin test was carried for cell viability. To assess the extent of DNA damage comet assay was performed. The results show that potassium ferrocyanide was cytotoxic and genotoxic at the concentrations tested, whereas, sodium ferrocyanide was non genotoxic.

Resazurin reduction based colorimetric antibiogram in microfluidic plastic chip

Abstract When drug-resistant bacteria pose a threat, a simple and inexpensive method for identifying their presence and quickly determining the right antibiotic for treatment, is critical. A simple optical detection technique combined with microfluidic technology will have great potential for configuring a portable antibiogram device. We report a colorimetric antibiogram method, based on viability-dependent resazurin dye reduction (intense blue colour changing to pink and leuco). The antibiogram protocol was developed initially in a 96-well plate format. This was tested using 20 clinical bacterial isolates against the minimum inhibitory concentration (MIC) of four antibiotics after 6 h of incubation. The antibiogram results obtained colorimetrically or visually were matched and confirmed with those of the liquid turbidity method (LTM). This antibiogram protocol was then transferred to a Poly(methyl methacrylate) (PMMA) plastic microfluidic chip containing 40 micro wells (8 rows and 5 columns, 20 μl volume per well). These microfluidic units can serve as disposable devices for portable instrumentation. The plastic microfluidic chip antibiogram showed 98% match with what was obtained in the 96-well plate method. The microfluidic chip offers a novel, compact and inexpensive platform technology for testing/screening of drug-resistant bacteria, and determining their antibiogram. This approach will lead the way for developing portable and low-cost point-of-care antibiotic susceptibility testing instrumentation for the future.

A Rapid Resazurin-Based Microtiter Assay to Evaluate QoI Sensitivity for Alternaria alternata Isolates and Their Molecular Characterization.

Chemical management of Alternaria brown spot of citrus is based upon the timely application of site-specific fungicides, many of which are vulnerable to the development of fungicide resistance. A rapid microtiter bioassay based on the colorimetric changes of resazurin (RZ) dye was developed to evaluate the sensitivity of Alternaria alternata to quinone outside inhibitor (QoI) fungicides. Four liquid media (complete medium, minimal medium, potato dextrose broth, and yeast peptone dextrose broth), five conidia concentrations (from 101 to 105 conidia/ ml), and five RZ concentrations (10, 20, 30, 40, and 50 μM) were evaluated. Complete medium at 105 conidia/ml and 40 μM RZ were identified as optimal for measuring RZ reduction. The effective concentration of two QoI fungicides (azoxystrobin and pyraclostrobin) needed to reduce RZ by 50% (EC50) was calculated and compared with those obtained from conidia germination tests on fungicide-amended media. Concordant EC50 values were observed (R2 = 0.923; P < 0.0001) from both methods. Resistant phenotypes were further characterized by the partial sequencing of the cytochrome b gene. Genetic variability associated with the presence or absence of two introns was observed among isolates. The identified resistant isolates had the amino acid substitution G143A, typical of QoI resistance in other fungi.

Analysis of cell viability using time-dependent increase in fluorescence intensity

We report the use of resazurin (AlamarBlue) dye in a robust assay for cell viability of primary cells. Human mononuclear cells were used here for immunological studies, but the method can be applied to monitor reduction potential of any living cell. Reduction of AlamarBlue dye is widely used in several commercial assays of cell viability. Although it is fast and easy with immortal cell lines, the method is impractical for the primary cells due to their slower metabolic activity. We propose that the viability of human primary cells can be determined with AlamarBlue by monitoring the increase in fluorescence intensity in a matter of a few hours. In the presence of AlamarBlue, the dynamic increase in cellular reduction capacity is linear for several hours or, alternatively, the assay can be repeated to monitor the viability at any time point of cell culture. In addition to testing cellular growth rates and cytotoxicity, the application can be used to compare sample quality of cells that have been frozen or represent a pool of multiple donors. This application of the AlamarBlue cell viability assay is simple, rapid, and cost-effective, and therefore it is also well suited for high-throughput studies.

Production of Predominantly Anatase Thin Films on Various Grades of Steel and Other Metallic Substrates From TiCl4 and Ethyl Acetate by Atmospheric Pressure CVD

AbstractTiO2 thin films are deposited by atmospheric pressure (AP)CVD of TiCl4 and ethyl acetate on various substrates heated to 500 °C. The substrates investigated are four different grades of stainless steel (304, 304L, 316, and 316L), titanium metal, gold‐coated titanium metal, and gold‐coated stainless steel (316 grade). The TiO2 films are analyzed by Raman spectroscopy (RS) to determine the percentage of anatase present. Predominantly anatase films are produced on all the substrates, demonstrating the versatility of this method. Scanning electron microscopy (SEM) images reveal a marked difference in particle size between regions of high anatase content and lower anatase content, indicative of a difference in growth rate between anatase and rutile particles. Selected samples are shown to be photocatalytically active by degradation of resazurin dye by UV light. The rate of degradation is significantly faster than on the self‐cleaning Saint Gobain Bioclean™.

Diameter dependent photocatalytic activity of ZnO nanowires grown by vapor transport technique

Here a systematic study on diameter dependence of photocatalytic properties of ZnO nanowires is presented. The photocatalytic activity of ZnO nanowires of various diameters ranging from 30 to 300nm were studied by degradation of Resazurin dye in aqueous phase under UV light. The photocatalytic activity of ZnO nanowires were found to increase anomalously for nanowires of diameters <90nm. The increased photocatalytic activity of thinner nanostructures is due to higher surface to volume ratio. This resulted in increased surface activity because of lowering of activation energy (20–2kJ for diameter 300 and 30nm, respectively).