Real-Time Cytotoxicity Assay for Rapid and Sensitive Detection of Ricin from Complex Matrices

BACKGROUND: In the context of a potential bioterrorist attack sensitive and fast detection of functionally active toxins such as ricin from complex matrices is necessary to be able to start timely countermeasures. One of the functional detection methods currently available for ricin is the endpoint cytotoxicity assay, which suffers from a number of technical deficits. METHODOLOGY/FINDINGS: This work describes a novel online cytotoxicity assay for the detection of active ricin and Ricinus communis agglutinin, that is based on a real-time cell electronic sensing system and impedance measurement. Characteristic growth parameters of Vero cells were monitored online and used as standardized viability control. Upon incubation with toxin the cell status and the cytotoxic effect were visualized using a characteristic cell index-time profile. For ricin, tested in concentrations of 0.06 ng/mL or above, a concentration-dependent decrease of cell index correlating with cytotoxicity was recorded between 3.5 h and 60 h. For ricin, sensitive detection was determined after 24 h, with an IC50 of 0.4 ng/mL (for agglutinin, an IC50 of 30 ng/mL was observed). Using functionally blocking antibodies, the specificity for ricin and agglutinin was shown. For detection from complex matrices, ricin was spiked into several food matrices, and an IC50 ranging from 5.6 to 200 ng/mL was observed. Additionally, the assay proved to be useful in detecting active ricin in environmental sample materials, as shown for organic fertilizer containing R. communis material. CONCLUSIONS/SIGNIFICANCE: The cell-electrode impedance measurement provides a sensitive online detection method for biologically active cytotoxins such as ricin. As the cell status is monitored online, the assay can be standardized more efficiently than previous approaches based on endpoint measurement. More importantly, the real-time cytotoxicity assay provides a fast and easy tool to detect active ricin in complex sample matrices

Inexplicable inefficiency of avian molt? Insights from an opportunistically breeding Arid-zone species, Lichenostomus penicillatus

The majority of bird species studied to date have molt schedules that are not concurrent with other energy demanding life history stages, an outcome assumed to arise from energetic trade-offs. Empirical studies reveal that molt is one of the most energetically demanding and perplexingly inefficient growth processes measured. Furthermore, small birds, which have the highest mass-specific basal metabolic rates (BMRm), have the highest costs of molt per gram of feathers produced. However, many small passerines, including white-plumed honeyeaters (WPHE; Lichenostomus penicillatus), breed in response to resource availability at any time of year, and do so without interrupting their annual molt. We examined the energetic cost of molt in WPHE by quantifying weekly changes in minimum resting metabolic rate (RMRmin) during a natural-molt period in 7 wild-caught birds. We also measured the energetic cost of feather replacement in a second group of WPHEs that we forced to replace an additional 25% of their plumage at the start of their natural molt period. Energy expenditure during natural molt revealed an energy conversion efficiency of just 6.9% (±0.57) close to values reported for similar-sized birds from more predictable north-temperate environments. Maximum increases in RMRmin during the molt of WPHE, at 82% (±5.59) above individual pre-molt levels, were some of the highest yet reported. Yet RMRmin maxima during molt were not coincident with the peak period of feather replacement in naturally molting or plucked birds. Given the tight relationship between molt efficiency and mass-specific metabolic rate in all species studied to date, regardless of life-history pattern (Efficiency (%) = 35.720•10−0.494BMRm; r2 = 0.944; p = <0.0001), there appears to be concomitant physiological costs entrained in the molt period that is not directly due to feather replacement. Despite these high total expenditures, the protracted molt period of WPHE significantly reduces these added costs on a daily basis.

Castor Bean Metabolomics: Current Knowledge and Perspectives Toward Understanding of Plant Plasticity Under Stress Condition

Metabolomics provides vital information for the understanding of biological processes and has been vastly applied in plant studies. Several metabolite-profiling studies have correlated physiological events, such as germination or seedling establishment, with metabolic and molecular changes under different environmental conditions. Castor bean displays high plasticity during initial vegetative growth, which is reflected in the metabolome of the seeds and seedlings. In general, several metabolite-profiling techniques are required to obtain a complete response in terms of metabolism plasticity of the studied biological system. Carbohydrates, amino acids, and organic acids have been measured in castor bean seeds and seedlings by nuclear magnetic resonance, gas chromatography coupled to a quadrupole time of flight mass spectrometry (GC-TOF-MS), as well as by high-performance liquid chromatography (HPLC). Fatty acids and some secondary metabolites have been quantified in castor bean seeds and seedlings by gas chromatography coupled to a triple-axis detector (GC-MS). In this chapter, we initially discuss how metabolomics studies suggested a possible role of gamma-aminobutyric acid (GABA) accumulation during early imbibitions and seedling establishment. Later, we consider a specific metabolic signature of castor bean: a shift in carbon–nitrogen metabolism as its main biochemical response to high temperatures. This metabolic shift is usually associated with adjusted growth, and it is likely involved in maintaining cellular homeostasis under heat stress. The castor bean metabolome has been vastly investigated, especially with regard to its ability to respond to external stimuli. These results might help us understand the molecular requirements for vigorous castor bean seed germination and seedling growth under different environmental conditions