Metabolite Profiling of Javanese Ginger Zingiber purpureum and Identification of Antiseizure Metabolites via a Low-Cost Open-Source Zebrafish Bioassay-Guided Isolation.

The rhizomes of Zingiber purpureum, "Bangle", were investigated for its antiseizure properties using a streamlined and cost-effective zebrafish screening strategy and a mouse epilepsy assay. Its hexane extract demonstrated strong antiseizure activity in zebrafish epilepsy assay and was, therefore, selected for bioactivity-guided fractionation. Twelve compounds (1-12) were isolated, and two bioactive phenylbutenoids, trans- (11) and cis-banglene (12), reduced up to 70% of pentylenetetrazole (PTZ)-induced seizures. These compounds showed moderate activity against PTZ-induced seizures in a mouse epilepsy assay. To understand the specificity of Z. purpureum active compounds, its chemical profile was compared to that of Z. officinale. Their composition was assessed by differential metabolite profiling visualized by a molecular network, which revealed only vanillin derivatives and terpenoids as common metabolites and gave a comprehensive view of Z. purpureum composition. This study demonstrates the efficacy of a streamlined zebrafish epilepsy assay, which is therefore suitable for routine screening in phytochemistry laboratories.

Do We have a Satisfactory Cell Viability Assay? Review of the Currently Commercially-Available Assays.

Cell-based assays are an important part of the drug discovery process and clinical research. One of the main hurdles is to design sufficiently robust assays with adequate signal to noise parameters while maintaining the inherent physiology of the cells and not interfering with the pharmacology of target being investigated. A plethora of assays that assess cell viability (or cell heath in general) are commercially available and can be classified under different categories according to their concepts and principle of reactions. The assays are valuable tools, however, suffer from a large number of limitations. Some of these limitations can be procedural or operational, but others can be critical as those related to a poor concept or the lack of proof of concept of an assay, e.g. those relying on differential permeability of dyes in-and-out of viable versus compromised cell membranes. While the assays can differentiate between dead and live cells, most, if not all, of them can just assess the relative performance of cells rather than providing a clear distinction between healthy and dying cells. The possible impact of relatively high molecular weight dyes, used in most of the assay, on cell viability has not been addressed. More innovative assays are needed, and until better alternatives are developed, setup of current cell-based studies and data interpretation should be made with the limitations in mind. Negative and positive control should be considered whenever feasible. Also, researchers should use more than one orthogonal method for better assessment of cell health.

Insignificant medium-term vitamin D status change after 25-hydroxyvitamin D testing in a large managed care population.

OBJECTIVES: To examine the clinical utility of 25-hydroxyvitamin D (25(OH)D) testing in achieving medium-term vitamin D (VD) sufficiency in a managed care population. METHODS: Retrospective study of a continuously-enrolled patient population in a 3-year period between 2011 and 2013. Primary outcome was VD status at ∼1 year after 25(OH)D testing. Patient demographics, comorbidities, medications, and 25(OH)D test results were gathered from relevant databases and multivariate logistic regression analysis used to study the risk factors of persistent VD deficiency or insufficiency. RESULTS: Of 22,784 patients, 7533 (females 69.3%) did 14,563 25(OH)D tests, with an estimated cost of $582,520. Of the 7533 patients, 1126 had another 25(OH)D test at 300-400 days after the first one. Based on the two test results, 234 patients (20.8%) maintained sufficient 25(OH)D levels; 132 (11.7%) turned from VD-sufficient into VD-insufficient or -deficient; 538 (47.8%) remained VD-insufficient or -deficient, and only 222 (19.7%) improved to be VD-sufficient. Overall, only 8.0% more patients were VD-sufficient at ∼1 year after 25(OH)D testing. Only younger age and higher BMI were independent risk factors for persistent low 25(OH)D levels and high-dose VD use was not associated with achieving VD sufficiency. CONCLUSIONS: 25(OH)D testing only benefits a small portion of patients thus lacks clinical utility in achieving VD sufficiency in the medium term but incurs a significant cost. A practical strategy to treat VD deficiency or insufficiency is needed; without it, 25(OH)D testing adds little value to most patients' health and should be used with discretion.

The utility of yeast as a tool for cell-based, target-directed high-throughput screening.

Many Neglected Tropical Diseases (NTDs) have recently been subject of increased focus, particularly with relation to high-throughput screening (HTS) initiatives. These vital endeavours largely rely of two approaches, in vitro target-directed screening using biochemical assays or cell-based screening which takes no account of the target or targets being hit. Despite their successes both of these approaches have limitations; for example, the production of soluble protein and a lack of cellular context or the problems and expense of parasite cell culture. In addition, both can be challenging to miniaturize for ultra (u)HTS and expensive to utilize. Yeast-based systems offer a cost-effective approach to study and screen protein targets in a direct-directed manner within a eukaryotic cellular context. In this review, we examine the utility and limitations of yeast cell-based, target-directed screening. In particular we focus on the currently under-explored possibility of using such formats in uHTS screening campaigns for NTDs.

An improved bioassay facilitates the screening of repellents against cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae).

BACKGROUND: Repellents are a common method for preventing flea bites, making an effective system for flea repellent screening advantageous. We describe an improved technique to facilitate repellent activity screening of numerous plant-based Ctenocephalides felis (cat flea) repellents. RESULTS: Two long strips of filter paper were impregnated with test compounds (dissolved in ethanol) and ethanol only, respectively. After drying, the two filter papers were glued together along the long side and inserted into a glass tube containing non-fed cat fleas. The distribution of cat fleas in each half of the filter paper was recorded after 30 min to calculate repellency. Results showed that the essential oil of Cinnamomum osmophloeum (from leaf), Taiwania cryptomerioides (from heartwood) and Plectranthus amboinicus (from leaf) exhibits repellent activity against cat fleas in a dose dependent manner. Moreover, the repellent activities against cat fleas of 2% trans-cinnamaldehyde (the main constituent of Ci. osmophloeum essential oil) and 0.5% thymol (the main constituent of P. amboinicus essential oil) are 97.6% and 90.6%, and can persist for up to 4 and 8 h, respectively. These results are comparable to those of 15% DEET. CONCLUSION: The proposed screening technique can facilitate the pre-screening of numerous flea repellents for further evaluation on animal or human subjects.

Drug-mediated toxicity: illuminating the 'bad' in the test tube by means of cellular assays?

Health problems are rising worldwide, be it as a consequence of lifestyle and longevity in increasingly affluent societies or due to a sharp rise in bacterial antibiotic resistance. The pharmaceutical industry is caught between high rates of attrition and the rather slow pace of a historically large regulatory system for pharmacological safety. Meanwhile, the past decade has seen a tremendous evolution of the biological toolbox, most notably of cellular assays, stem-cell differentiation and organ-mimicking systems. These systems were readily adapted for lead-compound identification. However, their use as toxicological test systems is lagging behind, not least because of a lack of regulatory acceptance. This review tries to elucidate the scale of the problem and discusses the applicability of the assays currently available, with particular regard to the use of stem cells.

Exploration of novel in vitro assays to study drugs against Trichuris spp.

Though trichuriasis is a significant public health problem, few effective drugs are available underscoring the need for new drug therapies. For the evaluation of trichuricidal activity of test compounds in vitro an accurate, reliable, sensitive, fast and cheap drug sensitivity assay is essential. The aim of the present investigation was to evaluate the performance of different in vitro drug sensitivity assays in comparison to the standard motility assay. Trichuris muris L4 larvae or adult worms were isolated from the intestinal tract from infected female C57BL/10 mice and incubated in the presence of ivermectin, levamisole and nitazoxanide (200, 100 and 50 µg/ml) for 72 h. The health status of the worms was either evaluated microscopically using a motility scale from 0 to 3 (motility assay), by examination of absorbance or emission in response to metabolic activity (MTT (Thiazolyl Blue Tetrazolium Bromide) and Alamar Blue assay), through analysis of absorbance of an enzyme-substrate reaction (acid phosphatase activity assay), by measuring the noise amplitudes (isothermal microcalorimetry and xCELLigence System) or the heat flow (isothermal microcalorimetry) of T. muris. The Alamar Blue assay, xCELLigence and microcalorimetry compared favorably to the standard motility assay. These three assays precisely determined the trichuricidal activity of the three test drugs. The acid phosphatase and the MTT assays showed a poorer performance than the motility assay. In conclusion, the colorimetric Alamar Blue in vitro assay is a good alternative to the motility assay to study drug effects against T. muris L4 and adults, since it is easy to perform, precise and of low cost.

Whole cell strategies based on lux genes for high throughput applications toward new antimicrobials.

The discovery/development of novel drug candidates has witnessed dramatic changes over the last two decades. Old methods to identify lead compounds are not suitable to screen wide libraries generated by combinatorial chemistry techniques. High throughput screening (HTS) has become irreplaceable and hundreds of different approaches have been described. Assays based on purified components are flanked by whole cell-based assays, in which reporter genes are used to monitor, directly or indirectly, the influence of a chemical over the metabolism of living cells. The most convenient and widely used reporters for real-time measurements are luciferases, light emitting enzymes from evolutionarily distant organisms. Autofluorescent proteins have been also extensively employed, but proved to be more suitable for end-point measurements, in situ applications - such as the localization of fusion proteins in specific subcellular compartments - or environmental studies on microbial populations. The trend toward miniaturization and the technical advances in detection and liquid handling systems will allow to reach an ultra high throughput screening (uHTS), with 100,000 of compounds routinely screened each day. Here we show how similar approaches may be applied also to the search for new and potent antimicrobial agents.

Induction of glutathione S-transferase pi as a bioassay for the evaluation of potency of inhibitors of benzo(a)pyrene-induced cancer in a murine model.

There is a growing need for short-term and cost-effective bioassay to assess the efficacy of potential chemo-preventive agents. We report that the induction of glutathione (GSH) S-transferase pi (mGSTP1-1) by a chemo-preventive agent can be used as a reliable marker to assess its efficacy in retarding chemical carcinogenesis induced by benzo(a)pyrene (BP), which is a widespread environmental pollutant and believed to be a risk factor in human chemical carcinogenesis. This conclusion is based on 1) the relative contribution of mGSTP1-1 of the liver and forestomach of female A/J mice in the detoxification of the ultimate carcinogenic metabolite of BP, (+)-anti-7,8-dihydroxy-9, 10-oxy-7,8,9, 10-tetrahydrobenzo(a)pyrene [(+)-anti-BPDE]; and 2) a positive correlation between the induction of hepatic and forestomach mGSTP1-1 by 5 naturally occurring organosulfides (OSCs) from garlic (diallyl sulfide, diallyl disulfide, diallyl trisulfide, dipropyl sulfide and dipropyl disulfide) and their effectiveness in preventing BP-induced forestomach neoplasia in mice. In the liver, the combined contribution of other GSTs in the detoxification of (+)-anti-BPDE was far less than the contribution of mGSTP1-1 alone. Likewise, in the forestomach, the contribution of mGSTP1-1 far exceeded the combined contribution of other GSTs. Studies on the effects of OSCs against BP-induced forestomach neoplasia revealed a good correlation between their chemo-preventive efficacy and their ability to induce mGSTP1-1 expression in the liver (r = -0.89; p < 0.05) as well as in the forestomach (r = -0.97; p < 0.05). Our results suggest that the induction of mGSTP1-1 may be a reliable marker for evaluating the efficacy of potential inhibitors of BP-induced cancer in a murine model.

Effect of dietary iron concentration, age, and length of iron feeding on feed intake and tissue iron concentration of broiler chicks for use as a bioassay of supplemental iron sources.

Three experiments were conducted with day-old chicks to study the effects of dietary Fe concentration and age on Fe accumulation in tissues as an estimate of supplemental Fe bioavailability, and of delaying the time of initial high Fe supplementation up to 7 d of age on feed intake to 3 wk of age. In Experiment 1, chicks were fed a basal corn-soybean meal diet (188 mg/kg Fe, DM basis) or the basal supplemented with 400, 600, or 800 mg/kg added Fe as reagent grade FeSO4.7H2O for either 1, 2, or 3 wk. Dietary Fe depressed (P < 0.001) feed intake and body weight gain, especially at 3 wk. Kidney Fe concentrations increased linearly (P < 0.001) with increasing dietary Fe. Liver Fe concentration also increased linearly, but reached a plateau in birds fed 600 mg/kg Fe. Bone Fe increased linearly (P < 0.05) at 1 wk, but not at 2 or 3 wk. Liver and kidney Fe regressed on daily Fe intake had the best fit to a linear model at 2 wk. In Experiment 2, chicks were fed either a basal diet (320 mg/kg Fe, DM basis) continuously, the basal supplemented with 800 mg/kg added Fe as FeSO4.7H2O continuously, or were started on the control diet and switched to the high Fe diet on Day 3, 5, or 7. Feed intake was lower (P < 0.05) in birds started on Fe on Days 1 or 3, but delaying feeding of high Fe diets until Day 5 resulted in intake at 3 wk similar to that of birds fed the basal diet. In Experiment 3, the basal diet (123 mg/kg Fe) was fed to chicks for 6 d, then experimental diets were fed for 14 d. Diets were the basal or basal supplemented with 400, 600, or 800 mg/kg added Fe as reagent grade or feed grade Fe sulfate or an Fe methionine complex. When estimated from regression of log10 liver Fe concentration on total analyzed dietary Fe concentration, relative bioavailability was set at 100% for reagent grade Fe sulfate, and the feed grade sulfate was 92.3% and Fe methionine was 88.3%. Liver Fe concentrations may be useful criteria for determining Fe bioavailability and 2 wk of feeding was the optimal time required for such a bioassay. Delaying feeding high Fe diets until 5 d of age alleviated the decreased feed intake associated with high Fe diets.

Contribution of "in vitro" assays to preclinical and premarketing testing in immunotoxicology.

An attempt to replace the "in vivo" testing by "in vitro" exposure of cells to drugs is discussed from the point of view of the limitations of these artificial test conditions. The critical reminders are evaluated, the most important ones being the pharmacokinetics, pharmacodynamics and metabolism of drugs in the body what may be a cause of the discrepancy between "in vivo" and "in vitro" exposure assays. Various sensitivity of different cell types and of different phases of the generation cycle represent the crucial importance in the immune response and in the drug-cell interactions. The "in vitro" assays are technically sophisticated and mostly based on the tissue-culture techniques. However, both in immunology and immunotoxicology standardization, validation and usage of rules of Good Laboratory Practice are required. Finally, possibilities and contribution of "in vitro" assays to immunotoxicology are listed. It is apparent that for establishment of common rules these studies require intensive international and interlaboratory cooperation and coordination.