Resazurin Dye Research Articles

Phytochemical, antibacterial and antifungal properties of an aqueous extract of Eucalyptus microcorys leaves

Australia is home to over 800 different species of Eucalyptus and traditionally, many Eucalyptus species have been utilised to heal wounds and treat fungal infections by the Indigenous people of Australia. In view of this, our study was designed to investigate the phytochemical, antibacterial and antifungal properties of crude aqueous extract of E. microcorys leaves. The freeze-dried powdered extract was prepared and the phytochemical profile was studied by analysing the total phenolic content (TPC), total flavonoid content (TFC), proanthocyanidins, antioxidants and saponins. The TPC, TFC and proanthocyanidin values found were: 501.76±14.47mg of gallic acid equivalents per g, 61.53±0.83mg of rutin equivalents per g and 10.76±0.89mg of catechin equivalents per g, respectively. The antioxidant values expressed in mg trolox equivalents per g of extract (mg TE/g) were: ABTS=1073.13±10.73mg TE/g, DPPH=1035.44±65.54mg TE/g and CUPRAC=1524.30±66.43mg TE/g. The powdered extract was also evaluated for activity against three pathogenic bacterial strains (Escherichia coli, Enterobacter aerogenes, Staphylococcus lugdunensis); and three fungal strains (Geotrichum candidum, Aspergillus brasiliensis and Candida albicans) using the disc diffusion method and 96 well plate-based method with resazurin dye. The extract exhibited clear zones of inhibition against the tested bacteria and fungi. Minimum inhibitory concentration (MIC) values were demonstrated to be: A. brasiliensis=2.44μg/mL, G. candidum=4.88μg/mL, S. lugdunensis=78μg/mL, E. coli=156.25μg/mL, E. aerogenes=312.5μg/mL and C. albicans=1250μg/mL. These results reveal the significant potential of E. microcorys as a source of phenolics, antioxidants and antimicrobial agents and also highlight the necessity of further purification and characterisation of solitary bioactive compounds for their prospective applications in food, nutraceutical and pharmaceutical industries.

Antibiofilm activity of Streptomyces toxytricini Fz94 against Candida albicans ATCC 10231

Candida albicans is a significant cause of morbidity and mortality in immunocompromised patients worldwide. Biofilm formation by Candida species is a significant virulence factor for disease pathogenesis. Keeping in view the importance of Streptomyces' metabolites, the present study was initiated during the bioprospecting programme of Egyptian Streptomyces carried by the authors since 2013. Native Streptomyces isolates were recovered from soil samples collected from different governorates. Antifungal activity of forty isolates of Streptomyces were performed against planktonic (free cells) of C. albicans ATCC 10231 and resistant clinical Candida isolates. Streptomyces isolates showed high inhibition activity against free cells of Candida were further assayed against biofilm of C. albicans reference strain. The most active Streptomyces sp. (no.6) was identified phenotypically, biochemically and by using 16S rRNA. The 16S rRNA sequences obtained were compared with those deposited in the GenBank Database and registered with accession number KM052378 as S. toxytricini Fz94. Screening of S. toxytricini Fz94 extract capability in prevention and destruction of C. albicans reference strain biolfilm was assessed by resazurin dye adopted technique. In the pre-exposure scheme, the lowest concentration of 5 gL-1 showed biofilm viability inhibition of 92% after 120 min, while Ketoconazole® gave 90 % inhibition at concentration of 2 gL-1. In post exposure, the concentration of S. toxytricini Fz94 extract 7gL-1 caused 82 % inhibition of biofilms viability after 120 min, while Ketoconazole did not show any destruction capability. The cytotoxicity of S. toxytricini Fz94 crude extract results showed that it was nontoxic at 10 gL-1. S. toxytricini Fz94 is maintained in the Fungarium of Arab Society for Fungal Conservation (ASFC) with accession number FSCU-2017-1110.

Camptothecin Sensitizes Refractory Lung Cancer Cells to Receptor Kinase Inhibitor

BackgroundReceptor tyrosine kinases (RTKs) represent a large class of receptors that are upregulated in many cancers. The Ephrin‐A type‐2 precursor (EphA2) is an important RTK whose overexpression in lung cancer promotes growth, metastasis and acquisition of resistance phenotypes to therapies. Further, EphA2 is a downstream target of the epidermal growth factor receptor (EGFR) which is transcriptionally regulated by the oncogenic SOX2 transcription. This study investigates the effect of combination treatment with camptothecin (CPT) and the RTK inhibitor (RTKI) drug Erlotinib (Erl) in inhibiting survival mechanism in lung cancer cells.MethodsCell viability of H1299 and H1975 was determined using the Resazurin dye assay following single and combination treatments with CPT and Erl for 72 hours. The effect of 48‐hour single and combination CPT (1.0 μM) and Erl (10 μM and 20 μM) treatments on H1299 cell migration, apoptosis, and protein expression were investigated by wound healing assay, acridine orange‐ethidium bromide (AO‐EB) staining, and western blot respectively.ResultsH1299 and H1975 were resistant to Erl treatment (concentrations that killed 50% of cells, IC50 = 291.14±15.84 μM and 92.06±10.06 μM respectively). CPT was relatively more potent in inhibiting H1299 cell proliferation (IC50 = 7.01±0.35 μM). CPT co‐treatment at concentrations of 0.1 μM and 0.5 μM with different concentrations of Erl inhibited H1299 cell proliferation by 2‐(IC50 = 137.12±13.53 μM) and 10‐fold (IC50 = 27.88±2.88 μM) respectively. Combination treatment with CPT and Erl induced apoptotic events and inhibited H1299 cell migration compared to control and single treatment 48 hours after treatment. Additionally, SOX2 and c‐Myc expression as well as EphA2‐mediated cell migration signaling (phospho‐EphA2 Tyr588 and Tyr594 expression) were abrogated.ConclusionCPT is a potent, but highly toxic anticancer agent whose clinical utility is hindered by adverse dose‐dependent hematopoetic and other adverse effects. In this study, however, sub‐toxic doses of CPT was effective in sensitizing highly resistant H1299 cells to Erl through inhibition of cell migration and induction of apoptosis. Combination treatment with low‐dose CPT may be a viable means of sensitizing refractory cancer cells to therapy.Support or Funding InformationDr. Terrick Andey MCPHS UniversityE‐mail address:Terrick.andey@mcphs.edu

Bioassay-guided isolation of active principles from Nigerian medicinal plants identifies new trypanocides with low toxicity and no cross-resistance to diamidines and arsenicals

Ethnopharmacological relevanceLeaves from the plant species studied herein are traditionally used in northern Nigeria against various protozoan infections. However, none of these herbal preparations have been standardized, nor have their toxicity to mammalian cells been investigated. In search of improved and non-toxic active antiprotozoal principles that are not cross-resistant with current anti-parasitics, we here report the results of the in vitro screening of extracts from seven selected medicinal plant species (Centrosema pubescens, Moringa oleifera, Tridax procumbens, Polyalthia longifolia, Newbouldia laevis, Eucalyptus maculate, Jathropha tanjorensis), used traditionally to treat kinetoplastid infections in Nigeria, and the isolation of their bioactive principles. Aim of the studyTo investigate the efficacies of medicinal plant extracts, and of compounds isolated therefrom, against kinetoplastid parasites, assess cross-resistance to existing chemotherapy, and assay their toxicity against mammalian cells in vitro. Material and methodsPlants were extracted with hexane, ethyl acetate and methanol. Active principles were isolated by bioassay-led fractionation, testing for trypanocidal activity, and identified using NMR and mass spectrometry. EC50 values for their activity against wild-type and multi-drug resistant Trypanosoma brucei were obtained using the viability indicator dye resazurin. ResultsSeven medicinal plants were evaluated for activity against selected kinetoplastid parasites. The result shows that crude extracts and isolated active compounds from Polyalthia longifolia and Eucalyptus maculata, in particular, display promising activity against drug-sensitive and multi-drug resistant Trypanosoma brucei. The EC50 value of a clerodane (16α-hydroxy-cleroda-3,13(14)-Z-dien-15,16-olide) isolated from Polyalthia longifolia was as low as 0.38µg/mL, while a triterpenoid (3β,13β-dihydroxy-urs-11-en-28-oic acid) isolated from Eucalyptus maculata displayed an EC50 of 1.58µg/mL. None of the isolated compounds displayed toxicity towards Human Embryonic Kidney cells at concentrations up to 400µg/mL. In addition, the isolated compounds were active against Leishmania mexicana, as well as against T. congolense. ConclusionWe have isolated a clerodane compound from Polyalthia longifolia that shows low toxicity, no cross-resistance with current treatments, and promising activity against both human-infective and veterinary Trypanosoma species.

YM155 induces apoptosis in p53-deficient T-acute lymphoblastic leukemia cells independent of survivin inhibition

T-acute lymphoblastic leukemia (T-ALL) is an aggressive hematological cancer that arises from the malignant transformation of T-cell progenitors. Despite the significant progress in current treatment, challenges remain the lifelong morbidity after current chemotherapy regimens and postrelapse survival. In addition, patients with T-ALL have inferior outcomes compared with those with B-cell precursor; consequently, novel therapeutic approaches are still necessary to improve the outcome in this cohort. YM155 is an imidazolium derivative originally discovered as a suppressant of survivin expression. It has been reported that YM155 has potent antiproliferative activity on a variety of human cancer cell lines; however, its effects in T-ALL cells have been underexplored. The aim of the present study was to examine the effects of YM155 on p53-deficient T-ALL cell lines, JURKAT and CCRF-CEM. Resazurin dye was used to evaluate cell viability. Colony formation was observed in MethoCult methylcellulose medium. Apoptotic cells were detected by flow cytometry (annexin V labeling and TUNEL assay). Cell cycle analysis was carried out by DNA quantification in flow cytometry. DNA damage was assessed using a comet assay and the survivin expression profile was evaluated by real-time PCR and immunoblotting. YM155 treatment decreased cell viability and clonogenicity capacity of T-ALL cells, increased the apoptosis index and DNA damage, and altered the cell cycle dynamic, independent of survivin inhibition. Taken together, the data reinforce that YM155 may be useful as a therapeutic possibility to combat leukemia.

In vitro Sperm Function Tests and Testicular Biometry for Fertility Prediction in Boar

The study was conducted on six porcine males and thirty six semen ejaculates for its fertility assessment. The average length of the left and right testicle of the boars measured by ultrasonography was 9.47 ± 0.73 cm and 9.09 ± 0.65 cm, respectively. The sperm concentration/ml increased significantly as testicular diameter increased in size. The average length of right and left of boar testicle measured by Vernier caliper was 10.4 ± 0.57 cm and average width was 4.3 ± 0.14 cm. The average volume of boar semen was 115.00 ± 11.83 ml with milky colour. Thick consistency was observed in 83.3% semen samples whereas 16.6 % semen samples were having thin consistency. Out of 36 semen ejaculates, 16.6 % semen samples had density of DD where as 83.3 % had a density of DDD. The Mass activity, live percentage, percentage of morphologically abnormal spermatozoa and total sperm concentration in boar semen were 3 ± 0, 75.41 ± 2.07 %, 0 %, and 523 ± 60.07 million/ml, respectively. Mean percentage of hypo osmotic swelling test (HOS-Test) of semen found in the present study was 73.47 ± 2.26. The average time for reduction of resazurin dye from blue to violet was 1.805 ± 0.163 and from violet to pink was 9.944 ± 0.890. None of the sample change colour from pink to white. In the present study 36 ejaculates of boar semen were subjected for TVC of bacteria. In order to differentiate the bacterial species contaminating semen raw semen was placed on different agar plates. The species isolated with higher frequency in boar semen were of Staphylococcus species-83.3% (30 samples) followed by E.coli -63.8% (23 samples). Mean TVC obtained in this study was 45.13 × 10 3 .

Human Brain Malignant Glioma (BMG-1) 3D Aggregate Morphology and Screening for Cytotoxicity and Anti-Proliferative Effects.

Two interesting aspects of cell lines grown in 3 Dimensional (3D) conditions are their distinct morphology and production of extracellular matrix (ECM). Also, it is known that 3D aggregates have different susceptibilities to damage-inducing agents compared to their 2D monolayer counterparts. We describe the effect of ECM on 3D aggregate morphology, the effect of cisplatin, bleomycin, and UV on the 3D aggregates and 2 Dimensional (2D) monolayers of the BMG-1 cell line. We also present a rapid method for analyzing cytotoxicity and anti-proliferative effects of 3D aggregates in 96-well plates. We utilized a single-step protocol using the dye resazurin. BMG-1 cells formed floating aggregates on 1% agarose hydrogels. The extent of ECM formed by them was dependent on number of cells seeded irrespective of the seeding density, which in turn directed the 3D aggregate compactness. The 3D aggregates were less susceptible to cisplatin and UV-induced cytotoxicity compared to 2D counterparts. The IC50 value of cisplatin was elevated at 210 μg/ml for the aggregates compared to 170 μg/ml for the monolayers. Exposure to UV for 0, 10, 20, and 30 min gave inhibition values of 2.98%, 8%, 22.99%, and 31.8% for the aggregates as compared to 3.06%, 7.5%, 39.4%, and 46.7% for the monolayers. While bleomycin-induced effects were unapparent when analyzed by vital staining for the doses used, the rapid, single-step method in 96-well plates was able to provide a dose-response for cytotoxicity and anti-proliferative effects. Also, comparative analysis of results obtained from vital staining and the single-step method demonstrates the reliability of the assay described. J. Cell. Physiol. 232: 685-690, 2017. © 2016 Wiley Periodicals, Inc.

A rapid and miniaturized system using Alamar blue to assess fungal spore viability: implications for biosecurity

Long-lasting viable fungal spores are one of the important aspects in emergence, spread and disease development of pathogenic fungi. We developed a rapid and miniaturized system using Alamar Blue (resazurin dye; 7-hydroxy-3H-phenoxazin-3-one 10-oxide) for assessing fungal spore viability, using the ascomycete Leptosphaeria maculans (causing blackleg disease on canola) as a ‘model pathogen’. The assay is dependent on the metabolic activity of viable fungal spores to convert the dark blue of resazurin (maximum absorbance 605 nm) into the pink colour of resorufin (maximum absorbance 573 nm). The Alamar Blue assay uses an optimised micro-titre based format that was far superior for determining fungal spore viability in comparison with current conventional techniques including trypan blue staining, a TC10 cellometer cell counter, or by assessing germination of the spores under the microscope. This new assay was also more rapid and reproducible than current conventional tests to detect viable spores. Viable spores could be reliably detected within two hours. The successful application of the Alamar Blue assay to measure fungal spore viability in the current study has important benefits for biosecurity operations relating to faster and more reliable confirmation of viability of potential invasive exotic fungal pathogens and in minimising any consequent disease outbreaks. The effectiveness of the Alamar Blue assay was confirmed by successfully determining the relative retention times of viable L. maculans ascospores across a range of different potential spore-carrier materials, including steel, fabric, wood, paper, rubber and leather, over a time period of eight months. To further confirm the wide applicability of the Alamar Blue assay, it was successfully applied to detect viable spores of fungal pathogens of diverse taxonomic groups, including Kabatiella caulivora, Magnaporthe oryzae and Puccinia striiformis f.sp. tritici, and also of the yeast Saccharomyces cerevisiae.

A rapid method to estimate NADH regeneration rates in living cells

We introduce a rapid whole cell assay for the estimation of NADH regeneration rates based on the fluorescent dye resazurin. A co-feed of formate and glucose, known to increase the intrinsic NADH regeneration rate of P. putida KT2440, was chosen as model system for the validation of this assay.

A preferential precursor for photocatalytically active titanium dioxide thin films: Titanium bis-ammonium lactato dihydroxide as an alternative to titanium tetra iso-propoxide

Titanium bis-ammonium lactato dihydroxide (TiBALD) precursor is used in place of the traditional sol–gel precursor, titanium tetra-isopropoxide (TTIP), to determine which precursor produces the most photocatalytically active titanium dioxide thin films. Films were deposited on glass substrates using modified sol–gel routes and the effects of non-ionic surfactant addition with Brij 58 and Brij 98 investigated. The morphology and nanostructure of the films have been assessed using SEM, XRD and Raman spectroscopy. The photocatalytic performance of the films was assessed using resazurin dye, whereby TiBALD precursor was found to produce thin films with enhanced photocatalytic efficiency, with an average rate of degradation for resazurin dye of 9.52×1012of dye molecules reduced per cm−2s−1 compared to 1.76×1012 dye molecules reduced per cm−2s−1 as for TTIP prepared films. A smaller average particle size (10–25nm) was also observed for thin films produced with TiBALD precursor, opening up potential for this precursor to be used for applications where a small TiO2 particle size is required. An overview of the differences between the two precursors for sol–gel deposition of TiO2 thin films is given within this study, including a discussion into the proposed differing decomposition mechanisms that may occur during sol–gel preparation.

Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay.

Most pathogenic bacteria are able to form biofilms during infection, but due to the difficulty of manipulating and assessing biofilms, the vast majority of laboratory work is conducted with planktonic cells. Here, we describe a peg plate biofilm assay as performed with Staphylococcus aureus. Bacterial biofilms are grown on pegs attached to a 96-well microtiter plate lid, washed through gentle submersion in buffer, and placed in a drug challenge plate. After subsequent incubation they are again washed and moved to a final recovery plate, in which the fluorescent dye resazurin serves as a viability indicator. This assay offers greatly increased ease-of-use, reliability, and reproducibility, as well as a wealth of data when conducted as a kinetic read. Moreover, this assay can be adapted to a medium-throughput drug screening approach by which an endpoint fluorescent readout is taken instead, offering a path for drug discovery efforts.

Resazurin-based 96-well plate microdilution method for the determination of minimum inhibitory concentration of biosurfactants.

ObjectivesTo develop and validate a microdilution method for measuring the minimum inhibitory concentration (MIC) of biosurfactants.ResultsA standardized microdilution method including resazurin dye has been developed for measuring the MIC of biosurfactants and its validity was established through the replication of tetracycline and gentamicin MIC determination with standard bacterial strains.ConclusionThis new method allows the generation of accurate MIC measurements, whilst overcoming critical issues related to colour and solubility which may interfere with growth measurements for many types of biosurfactant extracts.

Metabolic activity of sperm cells: correlation with sperm cell concentration, viability and motility in the rabbit

The resazurin reduction test (RRT) is a useful technique to assess the metabolic rate of sperm cells. RRT depends on the ability of metabolically active cells to reduce the non-fluorescent dye resazurin to the fluorescent resorufin. The aim of this study was to develop a vital fluorometric method to evaluate metabolic activity of rabbit sperm cells. Twenty-five rabbit males were included in the study. Viability and morphology, motility and metabolic activity were evaluated using an eosin-nigrosin staining, a computer-assisted semen analysis (CASA) and the RRT, respectively. Spearman rank correlation analysis was used to determine the correlation between RRT and semen parameters. After evaluation, a concentration of 10 × 106 sperm cells/ml was selected for further experiments with RRT. No significant correlation was found between the RRT results and the motility parameters. However, after RRT a significant positive correlation between relative fluorescence units and the percentage of alive spermatozoa (r = 0.62; P = 0.001) and a negative one with the percentage of sperm cells with acrosomic abnormalities (r = -0.45; P < 0.05) were detected. The vital assessment of metabolic rate of sperm cells by RRT could provide more information about semen quality than other routine semen analysis, correlating with sperm viability and acrosome status information.

Chemotherapeutic efficiency of drugs in vitro: Comparison of doxorubicin exposure in 3D and 2D culture matrices

The interest in the use of 3D matrices for in vitro analysis, with a view to increasing the relevance of in vitro studies and reducing the dependence on in vivo studies, has been growing in recent years. Cells grown in a 3D in vitro matrix environment have been reported to exhibit significantly different properties to those in a conventional 2D culture environment. However, comparison of 2D and 3D cell culture models have recently been noted to result in differing responses of cytotoxic assays, without any associated change in viability. The effect was attributed to differing conversion rates and effective concentrations of the resazurin assay in 2D and 3D environments, rather than differences in cellular metabolism. In this study, the efficacy of a chemotherapeutic agent, doxorubicin, is monitored and compared in conventional 2D and 3D collagen gel exposures of immortalized human cervical cells. Viability was monitored with the aid of the Alamar Blue assay and drug internalisation was verified using confocal microscopy. Drug uptake and retention within the collagen matrix was monitored by absorption spectroscopy. The viability studies showed apparent differences between the 2D and 3D culture systems, the differences attributed in part to the physical transition from 2D to a 3D environment causing alterations to dye resazurin uptake and conversion rates. The use of 3D culture matrices has widely been interpreted to result in “reduced” toxicity or cellular “resistance” to the chemotherapeutic agent. The results of this study show that the reduced efficiency of the drug to cells grown in the 3D environment can be accounted for by a sequential reduction of the effective concentration of the test compound and assay. This is due to absorption within the collagen gel inducing a higher uptake of both drug and assay thereby influencing the toxic impact of the drug and conversion rate of resazurin, and. The increased effective surface area of the cell exposed to the drug and assay in the 3D environment. The effect was noted to be higher after shorter exposure periods and should be accounted for in in vitro 2D and 3D culture environment comparisons.

Farnesol increases the susceptibility of Burkholderia pseudomallei biofilm to antimicrobials used to treat melioidosis.

The aim of this study was to analyse the in vitro activity of farnesol alone and combined with the antibacterial drugs amoxicillin, doxycycline, ceftazidime and sulfamethoxazole-trimethoprim against Burkholderia pseudomallei biofilms. Susceptibility was assessed by the broth microdilution test and cell viability was read with the oxidation-reduction indicator dye resazurin. The biofilms were evaluated through three microscopic techniques (optical, confocal and electronic microscopy). The minimum biofilm erradication concentration (MBEC) for farnesol was 75-2400 mmol l(-1). In addition, farnesol significantly reduced the MBEC values for ceftazidime, amoxicillin, doxycycline and sulfamethoxazole-trimethoprim by 256, 16, 4 and 4 times respectively (P < 0·05). Optical, confocal and electronic microscopic analyses of farnesol-treated B. pseudomallei biofilms demonstrated that this compound damages biofilm matrix, probably facilitating antimicrobial penetration in the biofilm structure. This study demonstrated the effectiveness of farnesol against B. pseudomallei biofilms and its potentiating effect on the activity of antibacterial drugs, in particular ceftazidime, amoxicillin, doxycycline and sulfamethoxazole-trimethoprim. The intrinsic antimicrobial resistance of B. pseudomallei is a serious challenge for the treatment of melioidosis. Thus, this paper reports the inhibitory potential of farnesol against B. pseudomallei biofilms, as well as highlights the favourable pharmacological interaction of farnesol with antibiotics tested, not only on cell viability, but also in the structural morphology of biofilms.

Interaction of Chitosan/Zinc Oxide Nanocomposites and their Antibacterial Activities with Escherichia coli

Zinc Oxide nanoparticles (ZNPs) and CS/Zinc oxide nanocomposites (CS/ZnO NCs) were successfully synthesized by wet chemical method. Formation of ZNPs and the interaction of CS/ZnO NCs were characterized by X-ray diffraction (XRD), Zeta potential, and Transmission Electron Microscopy (TEM) techniques. Thermal stability of the prepared samples was characterized by Thermogravimetric Analysis (TGA). TEM images shows the interaction between the ZNPs and CS polymer and the particles has spherical like structure with average crystallite size of 22 nm and is in good agreement with XRD results. The aim of this study was to investigate the attachment of these nanoparticles with Escherichia coli (E. coli) bacterial outermost cell membrane and their mode of action against these bacterial strains by agar well diffusion method and resazurin dye reduction method. Both the test materials synthesized revealed resistivity against E. coli up to 6 mg/mL.

Development and application of a sensitive, phenotypic, high-throughput image-based assay to identify compound activity against Trypanosoma cruzi amastigotes

We have developed a high content 384-well, image-based assay to estimate the effect of compound treatment on Trypanosoma cruzi amastigotes in 3T3 fibroblasts. In the same well, the effect of compound activity on host cells can also be determined, as an initial indicator of cytotoxicity. This assay has been used to identify active compounds from an in-house library of compounds with either known biological activity or that are FDA approved, and separately, from the Medicines for Malaria Venture Malaria Box collection. Active compounds were screened against T. cruzi trypomastigotes, utilising an assay developed with the viability dye resazurin. Twelve compounds with reconfirmed solid sample activity, with IC50 values of less than 10 μM and selectivity indices to T. cruzi amastigotes over 3T3 host cells of between >22 and 319 times were identified from these libraries. As 3T3 cells are contact inhibited, with limited proliferation in the assay, selective compounds of interest were profiled in a separate assay to estimate the viability of compound treated, replicating HEK293 cells. Selective compounds that were not previously reported in the literature were further profiled by extending the incubation time against amastigote infected 3T3 cells to determine if there were residual amastigotes post-treatment, important for the consideration of the exposure time required for further biological characterisation. The assay development process and the suitability of identified compounds as hit molecules for Chagas disease research are discussed.

Synthesis and characterization of silver nanoparticles from Alpinia calcarata by Green approach and its applications in bactericidal and nonlinear optics

Development of green route for the synthesis of nanoparticles with plant extracts plays a very important role in nanotechnology without any toxicity chemicals. Herein we report a new approach to synthesize silver nanoparticles (AgNPs) using aqueous extract of Alpinia calcarata root as a reducing as well as stabilizing agent. The crystal structure and purity of the synthesized AgNPs were studied using Powder X-ray Diffraction analysis. The Surface Plasmon Resonance bands of synthesized silver nanoparticles have been obtained and monitored using UV–Visible spectrum. The morphologies of the AgNPs were analyzed using High resolution transmission electron microscopy (HRTEM). The elements present in the A. calcarata extract were determined by the inductively coupled plasma-optical emission Spectrometry (ICP-OES) and Fourier transform infrared spectroscopy (FTIR). Silver nanoparticles from A. calcarata possess very good antimicrobial activity which was confirmed by resazurin dye reduction assay method and thus it is a potential source of antimicrobial agent. The synthesized Ag nanoparticles exhibit good optical nonlinearity and the nonlinear optical studies have been carried out by Z-scan technique.

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