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Adrian Ponce - One of the best experts on this subject based on the ideXlab platform.
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validation of a clostridium endospore Viability Assay and analysis of greenland ices and atacama desert soils
Applied and Environmental Microbiology, 2011Co-Authors: Wanwan Yang, Adrian PonceAbstract:Bacterial endospores are dormant microbial structures that are highly resistant to chemical, physical, and radiation sterilization processes (20, 31). They represent one of the most successful survival strategies of microorganisms and are formed when members of spore-forming genera (e.g., Bacillus and Clostridium) face unfavorable conditions, such as environmental extremes or starvation (19, 34). Once they are formed, endospores can stay dormant for extended periods of time, from thousands (8, 13, 20, 25, 33) to millions (3, 35) of years, although for the more extreme claims of longevity it is difficult to rule out modern contamination (37). Anaerobic spore-forming clostridia include numerous pathogenic species that are dangerous contaminants. For example, Clostridium botulinum and C. perfringens are common food-poisoning agents that produce toxins which cause diseases such as botulism and human necrotic enteritis (9, 17). C. perfringens, C. difficile, and C. tetani are causative agents of gas gangrene, pseudomembranous colitis, and tetanus (9, 30). Some psychrotrophic clostridia are also responsible for the spoilage of chilled vacuum-packed meat (9). In addition, C. perfringens has been used as an indicator of fecal contamination, because it is present in large numbers in human and animal wastes (4, 6). Due to their resistance to various extreme conditions, Clostridium endospores are also employed as biological indicators to monitor the effectiveness of various sterilization processes (11, 12). Currently, the standard method for quantifying viable Clostridium endospores is measuring CFU after heat shock killing of vegetative cells. This method requires several days of incubation and a tedious anaerobic culturing technique, and it is amenable for culturing only fewer than 1% of environmental species (24). Other molecular endospore Viability Assays include ATP Assay (26) and quantitative PCR (qPCR) coupled with propidium monoazide (PMA) (28), which, unlike microscopy-based endospore Viability Assay (micro-EVA), require extensive sample preparation and are labor-intensive. Previously, we described a spectroscopy-based endospore Viability Assay (spectro-EVA) to quantify dipicolinic acid (DPA) released from germinating Clostridium spores in liquid suspension (39). Germination was triggered by various germinants, such as l-alanine/NaHCO3, l-lactate, or d-alanine, which cause the release of approximately 108 molecules of a unique biomarker, DPA, from the spore core. Spectro-EVA is based on the detection of DPA in bulk solution via Tb3+-DPA luminescence spectroscopy, with a limit of detection (LOD) of 1,000 spores/ml. Unfortunately, spectro-EVA has even lower detection limits when environmental extracts are analyzed due to sensitivity to interference from contaminants. Previously, this limitation was overcome for the case of Bacillus endospores by employing a microscopy-based EVA (41), where individual spores are enumerated as they germinate in a microscope field of view. Here we report details of a rapid microscopy-based endospore Viability Assay (micro-EVA) that enables enumeration of single germinating Clostridium endospores on Tb3+- and d-alanine-doped agarose. d-Alanine was used as a germinant, which serves to trigger Clostridium spore germination while inhibiting Bacillus spore germination (2, 14, 38). Germination releases DPA from endospores, and subsequent Tb3+-DPA binding results in green luminescent spots under pulsed UV excitation in a field of view of a time-gated microscope. These were enumerated as germinable Clostridium endospores (GCEs) using time-gated Tb3+-DPA luminescence microscopy (i.e., micro-EVA). A parallel comparison of micro-EVA data with culturing data validated this method. Finally, we compared micro-EVA to culturing methods to quantify GCEs from two Mars analog environments, Greenland ice core and Atacama Desert.
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rapid endospore Viability Assay of clostridium sporogenes spores
International Journal of Food Microbiology, 2009Co-Authors: Wanwan Yang, Adrian PonceAbstract:A rapid Endospore Viability Assay (EVA), previously developed for Bacillus spores, was modified for enumeration of germinable Clostridium sporogenes spores. The EVA is based on the detection of dipicolinic acid (DPA), which is released during stage I germination and quantified by terbium (III) ion Tb-DPA luminescence. Germination of C. sporogenes spores in aqueous suspension was induced by L-alanine and NaHCO_3 addition, and germinable endospore numbers were determined by reference to a standard curve. Determination of the fractions of germinable C. sporogenes spores by EVA and phase-contrast microscopy yielded comparable results of 54.0% ± 2.9% and 59.3% ± 2.6%, respectively, while only 32.3% ± 5.3% of spores produced colonies on reinforced clostridial medium (RCM). Rates of germination were measured as a function of temperature (30 °C–60 °C) using EVA, yielding a linear relationship between the square root of the rate constant and inverse temperature.
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rapid endospore Viability Assay of clostridium sporogenes spores
International Journal of Food Microbiology, 2009Co-Authors: Wanwan Yang, Adrian PonceAbstract:A rapid Endospore Viability Assay (EVA), previously developed for Bacillus spores, was modified for enumeration of germinable Clostridium sporogenes spores. The EVA is based on the detection of dipicolinic acid (DPA), which is released during stage I germination and quantified by terbium (III) ion Tb-DPA luminescence. Germination of C. sporogenes spores in aqueous suspension was induced by L-alanine and NaHCO(3) addition, and germinable endospore numbers were determined by reference to a standard curve. Determination of the fractions of germinable C. sporogenes spores by EVA and phase-contrast microscopy yielded comparable results of 54.0%+/-2.9% and 59.3%+/-2.6%, respectively, while only 32.3%+/-5.3% of spores produced colonies on reinforced clostridial medium (RCM). Rates of germination were measured as a function of temperature (30 degrees C-60 degrees C) using EVA, yielding a linear relationship between the square root of the rate constant and inverse temperature.
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applications of a rapid endospore Viability Assay for monitoring uv inactivation and characterizing arctic ice cores
Applied and Environmental Microbiology, 2006Co-Authors: Hannah S Shafaat, Adrian PonceAbstract:We have developed a rapid endospore Viability Assay (EVA) in which endospore germination serves as an indicator for Viability and applied it to (i) monitor UV inactivation of endospores as a function of dose and (ii) determine the proportion of viable endospores in arctic ice cores (Greenland Ice Sheet Project 2 [GISP2] cores; 94 m). EVA is based on the detection of dipicolinic acid (DPA), which is released from endospores during germination. DPA concentrations were determined using the terbium ion (Tb3+)-DPA luminescence Assay, and germination was induced by l-alanine addition. The concentrations of germinable endospores were determined by comparison to a standard curve. Parallel EVA and phase-contrast microscopy experiments to determine the percentage of germinable spores yielded comparable results (54.3% ± 3.8% and 48.9% ± 4.5%, respectively), while only 27.8% ± 7.6% of spores produced CFU. EVA was applied to monitor the inactivation of spore suspensions as a function of UV dose, yielding reproducible correlations between EVA and CFU inactivation data. The 90% inactivation doses were 2,773 J/m2, 3,947 J/m2, and 1,322 J/m2 for EVA, phase-contrast microscopy, and CFU reduction, respectively. Finally, EVA was applied to quantify germinable and total endospore concentrations in two GISP2 ice cores. The first ice core contained 295 ± 19 germinable spores/ml and 369 ± 36 total spores/ml (i.e., the percentage of germinable endospores was 79.9% ± 9.3%), and the second core contained 131 ± 4 germinable spores/ml and 162 ± 17 total spores/ml (i.e., the percentage of germinable endospores was 80.9% ± 8.8%), whereas only 2 CFU/ml were detected by culturing.
Eiji Tokunaga - One of the best experts on this subject based on the ideXlab platform.
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Noninvasive and Safe Cell Viability Assay for Breast Cancer MCF-7 Cells Using Natural Food Pigment.
Biology, 2020Co-Authors: Kyohei Yamashita, Ryoma Tagawa, Yoshikazu Higami, Eiji TokunagaAbstract:A dye exclusion test (DET) was performed to determine the Viability of human breast cancer cells MCF-7, using natural food pigments as compared with trypan blue (TB), a typical synthetic dye for DET known to exhibit teratogenicity and cytotoxicity. We demonstrated that Monascus pigment (MP) is noninvasive to living cells and can effectively stain only dead cells. This study is the first verification of the applicability of MP to cancer cells. The appropriate MP concentration was 0.4% (0.02% as the concentration of pure MP) and all the dead cells were stained within 10 min. We found that the cell proliferation or the reduced nicotinamide adenine dinucleotide (NADH) activity of living cells was maintained over 48 h. Although 0.1% TB did not show an increase in dead cells, a marked decrease in NADH activity was confirmed. In addition, even when MP coexisted with cisplatin, staining of dead cells was maintained for 47 h, indicating stability to drugs (reagents). The cost of MP is estimated to be about 1/10 of TB. The fact that MP can be used as a cell Viability determination reagent for Euglena and Paramecium, as shown in preceding papers, and also for MCF-7, as shown in this paper, indicates the possibility of application in more cells of different species.
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Noninvasive and safe cell Viability Assay for Paramecium using natural pigment extracted from food.
Scientific reports, 2020Co-Authors: Kyohei Yamashita, Eiji TokunagaAbstract:Noninvasive, safe and cost-effective cell Viability Assay is important in many fields of biological research such as cell culture and counting. We examined ten typical natural pigments extracted from food to find that Monascus pigment (MP) or anthocyanin pigment (AP: purple sweet potato and purple cabbage) with Tris (Trimethylolaminomethane) works as a good indicator of Viability Assay for dye exclusion test (DET) of Paramecium. This was confirmed spectrally by scan-free, non-invasive absorbance spectral imaging A (x, y, λ) microscopy. We developed a new method of cell capture using a metal mesh to confine live Paramecium in a restricted space. This has the advantage that a low-cost and robust capture can be fabricated without using special equipment, compared to a conventional lab-on-a-chip. As a result, MP and AP stained dead cells as quick as methylene blue (MB), a synthetic dye conventionally used in DET within 1 min when treated with microwave and benzalkonium chloride. The natural pigments with Tris had little effect on inhibiting the growth of Paramecium, but MB killed all the cells within 1 h. MP is most useful because it allows non-invasive DET without Tris. This approach provides less invasive and safe DET.
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Noninvasive and safe cell Viability Assay for Euglena gracilis using natural food pigment
PeerJ, 2019Co-Authors: Kyohei Yamashita, Kengo Suzuki, Koji Yamada, Eiji TokunagaAbstract:Noninvasive and safe cell Viability Assay is required in many fields such as regenerative medicine, genetic engineering, single-cell analysis, and microbial food culture. In this case, a safe and inexpensive method which is a small load on cells and the environment is preferable without requiring expensive and space-consuming equipment and a technician to operate. We examined eight typical natural food pigments to find Monascus pigment (MP) or anthocyanin pigment (AP) works as a good Viability indicator of dye exclusion test (DET) for Euglena gracilis which is an edible photosynthetic green microalga. This is the first report using natural food pigments as cell Viability Assay. Euglena gracilis stained by MP or AP can be visually judged with a bright field microscope. This was spectrally confirmed by scan-free, non-invasive absorbance spectral imaging A(x, y, λ) microscopy of single live cells and principal component analysis (PCA). To confirm the ability of staining dead cells and examine the load on the cells, these two natural pigments were compared with trypan blue (TB) and methylene blue (MP), which are synthetic dyes conventionally used for DET. As a result, MP and AP had as good ability of staining dead cells treated with microwave as TB and MB and showed faster and more uniform staining for dead cells in benzalkonium chloride than them. The growth curve and the ratio of dead cells in the culture showed that the synthetic dyes inhibit the growth of E. gracilis, but the natural pigments do not. As the cell density increased, however, AP increased the ratio of stained cells, which was prevented by the addition of glucose. MP can stain dead cells in a shorter time than AP, while AP is more stable in color against long-term irradiation of intense light than MP. Due to the low toxicity of these pigments, Viability of cells in culture can be monitored with them over a long period.
Philippe Buscher - One of the best experts on this subject based on the ideXlab platform.
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Luminescent multiplex Viability Assay for Trypanosoma brucei gambiense.
Parasites & Vectors, 2013Co-Authors: Nick Van Reet, Pati Pyana, Stijn Rogé, Filip Claes, Philippe BuscherAbstract:Background New compounds for the treatment of human African trypanosomiasis (HAT) are urgently required. Trypanosoma brucei (T.b.) gambiense is the leading cause of HAT, yet T.b. gambiense is often not the prime target organism in drug discovery. This may be attributed to the difficulties in handling this subspecies and the lack of an efficient Viability Assay to monitor drug efficacy.
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luminescent multiplex Viability Assay for trypanosoma brucei gambiense
Parasites & Vectors, 2013Co-Authors: Nick Van Reet, Pati Pyana, Stijn Rogé, Filip Claes, Philippe BuscherAbstract:New compounds for the treatment of human African trypanosomiasis (HAT) are urgently required. Trypanosoma brucei (T.b.) gambiense is the leading cause of HAT, yet T.b. gambiense is often not the prime target organism in drug discovery. This may be attributed to the difficulties in handling this subspecies and the lack of an efficient Viability Assay to monitor drug efficacy. In this study, a T.b. gambiense strain, recently isolated in the D.R. Congo, was made bioluminescent by transfection with Renilla luciferase (RLuc) without altering its in vitro and in vivo growth characteristics. A luminescent multiplex Viability Assay (LMVA), based on measurement of the Renilla luciferase activity and the ATP content of the cells within the same experiment, was investigated as an alternative to the standard fluorimetric resazurin Viability Assay for drug sensitivity testing of T.b. gambiense. In a 96-well format, the RLuc transfected strain showed a detection limit of 2 × 104 cells ml-1 for the Renilla luciferase measurement and 5 × 103 cells ml-1 for the ATP measurement. Both Assays of the LMVA showed linearity up to 106 cells ml-1 and correlated well with the cell density during exponential growth of the long slender bloodstream forms. The LMVA was compared to the fluorimetric resazurin Viability Assay for drug sensitivity testing of pentamidine, eflornithine, nifurtimox and melarsoprol with both the wild type and the RLuc transfected population. For each drug, the IC50 value of the RLuc population was similar to that of the wild type when determined with either the fluorimetric resazurin method or the LMVA. For eflornithine, nifurtimox and melarsoprol we found no difference between the IC50 values in both Viability Assays. In contrast, the IC50 value of pentamidine was higher when determined with the fluorimetric resazurin method than in both Assays of the LMVA. LMVA has some advantages for Viability measurement of T.b. gambiense: it requires less incubation time for Viability detection than the fluorimetric resazurin Assay and in LMVA, two sensitive and independent Viability Assays are performed in the same experiment.
Ishac Nazy - One of the best experts on this subject based on the ideXlab platform.
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a platelet Viability Assay pva for the diagnosis of heparin induced thrombocytopenia
Platelets, 2019Co-Authors: Nikola Ivetic, Angela Huynh, John G. Kelton, Donald M Arnold, James W. Smith, Ishac NazyAbstract:AbstractDiagnosing heparin-induced thrombocytopenia (HIT) requires functional Assays measuring platelet activation as they are highly specific and sensitive. A useful functional test for diagnosing...
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performance characteristics of a novel platelet Viability Assay for heparin induced thrombocytopenia
Blood, 2017Co-Authors: Nikola Ivetic, Angela Huynh, John G. Kelton, Donald M Arnold, James W. Smith, Ishac NazyAbstract:Background: Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction that causes platelet activation, leading to thrombocytopenia and a high risk of thrombosis. Platelet activation is induced by antibodies that bind to the complex of platelet factor 4 (PF4), a platelet α-granule protein, and heparin, a common blood anticoagulant. The 14C-serotonin release Assay (SRA) is considered the gold standard functional Assay for HIT testing due to its high sensitivity and specificity. Positive results in the SRA correlate strongly with clinical HIT; however, the SRA also requires radioactive serotonin as an endpoint marker and this limits its availability for use in diagnostic testing. In this study, we developed a platelet Viability Assay (PVA) using Calcein-AM, a fluorescent Viability dye, as a marker to identify platelet-activating antibodies in patients with HIT. Methods: Patient sera were tested in parallel in the SRA and PVA and the results were compared and correlated for sensitivity and specificity. The SRA was performed using 14C-serotonin-labelled donor target platelets (350,000/µL; 75µL per test) incubated with 20µL of test serum and 5µL of heparin (0.1 U/mL).1 Following the reaction (60 minutes, room temperature), 100 µL of 5mM PBS/EDTA was added and the amount of 14C-serotonin released in the supernatant was measured. In our novel PVA, following the initial incubations with serum and heparin, platelets were stained with 2 µl of Calcein-AM (2 µg/mL final concentration) for 30 minutes at 37⁰C. 150 µl of 5mM PBS/EDTA was added and the fluorescence intensity of platelets was measured via flow cytometry (488nm excitation; Cytoflex). Platelet Viability was reported as the percentage of platelets that maintained Calcein-AM fluorescence intensity relative to control. In total, 35 SRA-positive and 43 SRA-negative sera were tested. Results: In the PVA, the percentage of viable platelets were 91±4% for sera that were negative in the SRA (n=43); and 39±14% for sera that were positive in the SRA (n=35) (figure 1). Using a cut off defined as two-standard deviations from the mean of negative sera, sensitivity of the PVA was 100% and specificity was 93%. These results were consistent across different donor platelets. An inverse linear correlation (R=0.93) was observed between platelet Viability in the PVA and serotonin release in the SRA. Conclusions:The PVA can accurately differentiate SRA positive from SRA negative HIT sera. Advantages of the PVA are that the Assay is flow-cytometry based, and uses an inexpensive, easy to handle and a readily available endpoint marker for measuring platelet activation. Further prospective studies are needed to optimize the Assay to develop a useful functional Assay for HIT diagnosis. Disclosures Arnold: Dova: Consultancy; Amgen: Consultancy, Research Funding; Rigel: Consultancy; Bristol Myers Squibb: Research Funding; Novartis: Consultancy, Research Funding; UCB: Consultancy.
Kyohei Yamashita - One of the best experts on this subject based on the ideXlab platform.
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Noninvasive and Safe Cell Viability Assay for Breast Cancer MCF-7 Cells Using Natural Food Pigment.
Biology, 2020Co-Authors: Kyohei Yamashita, Ryoma Tagawa, Yoshikazu Higami, Eiji TokunagaAbstract:A dye exclusion test (DET) was performed to determine the Viability of human breast cancer cells MCF-7, using natural food pigments as compared with trypan blue (TB), a typical synthetic dye for DET known to exhibit teratogenicity and cytotoxicity. We demonstrated that Monascus pigment (MP) is noninvasive to living cells and can effectively stain only dead cells. This study is the first verification of the applicability of MP to cancer cells. The appropriate MP concentration was 0.4% (0.02% as the concentration of pure MP) and all the dead cells were stained within 10 min. We found that the cell proliferation or the reduced nicotinamide adenine dinucleotide (NADH) activity of living cells was maintained over 48 h. Although 0.1% TB did not show an increase in dead cells, a marked decrease in NADH activity was confirmed. In addition, even when MP coexisted with cisplatin, staining of dead cells was maintained for 47 h, indicating stability to drugs (reagents). The cost of MP is estimated to be about 1/10 of TB. The fact that MP can be used as a cell Viability determination reagent for Euglena and Paramecium, as shown in preceding papers, and also for MCF-7, as shown in this paper, indicates the possibility of application in more cells of different species.
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Noninvasive and safe cell Viability Assay for Paramecium using natural pigment extracted from food.
Scientific reports, 2020Co-Authors: Kyohei Yamashita, Eiji TokunagaAbstract:Noninvasive, safe and cost-effective cell Viability Assay is important in many fields of biological research such as cell culture and counting. We examined ten typical natural pigments extracted from food to find that Monascus pigment (MP) or anthocyanin pigment (AP: purple sweet potato and purple cabbage) with Tris (Trimethylolaminomethane) works as a good indicator of Viability Assay for dye exclusion test (DET) of Paramecium. This was confirmed spectrally by scan-free, non-invasive absorbance spectral imaging A (x, y, λ) microscopy. We developed a new method of cell capture using a metal mesh to confine live Paramecium in a restricted space. This has the advantage that a low-cost and robust capture can be fabricated without using special equipment, compared to a conventional lab-on-a-chip. As a result, MP and AP stained dead cells as quick as methylene blue (MB), a synthetic dye conventionally used in DET within 1 min when treated with microwave and benzalkonium chloride. The natural pigments with Tris had little effect on inhibiting the growth of Paramecium, but MB killed all the cells within 1 h. MP is most useful because it allows non-invasive DET without Tris. This approach provides less invasive and safe DET.
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Noninvasive and safe cell Viability Assay for Euglena gracilis using natural food pigment
PeerJ, 2019Co-Authors: Kyohei Yamashita, Kengo Suzuki, Koji Yamada, Eiji TokunagaAbstract:Noninvasive and safe cell Viability Assay is required in many fields such as regenerative medicine, genetic engineering, single-cell analysis, and microbial food culture. In this case, a safe and inexpensive method which is a small load on cells and the environment is preferable without requiring expensive and space-consuming equipment and a technician to operate. We examined eight typical natural food pigments to find Monascus pigment (MP) or anthocyanin pigment (AP) works as a good Viability indicator of dye exclusion test (DET) for Euglena gracilis which is an edible photosynthetic green microalga. This is the first report using natural food pigments as cell Viability Assay. Euglena gracilis stained by MP or AP can be visually judged with a bright field microscope. This was spectrally confirmed by scan-free, non-invasive absorbance spectral imaging A(x, y, λ) microscopy of single live cells and principal component analysis (PCA). To confirm the ability of staining dead cells and examine the load on the cells, these two natural pigments were compared with trypan blue (TB) and methylene blue (MP), which are synthetic dyes conventionally used for DET. As a result, MP and AP had as good ability of staining dead cells treated with microwave as TB and MB and showed faster and more uniform staining for dead cells in benzalkonium chloride than them. The growth curve and the ratio of dead cells in the culture showed that the synthetic dyes inhibit the growth of E. gracilis, but the natural pigments do not. As the cell density increased, however, AP increased the ratio of stained cells, which was prevented by the addition of glucose. MP can stain dead cells in a shorter time than AP, while AP is more stable in color against long-term irradiation of intense light than MP. Due to the low toxicity of these pigments, Viability of cells in culture can be monitored with them over a long period.
