Fig. 1. Labeling of the liposome. (A)Outline of the method. Cells were rapidly frozen, freeze-fractured, and evaporated with carbon (C) and platinum/carbon (Pt/C) in vacuum. The replica of the split membrane was digested with SDS to remove noncast molecules and labeled by GST-PH. Both the cytoplasmic and exoplasmic halves of the membrane were examined. (B) Labeling of small unilamellar liposome replicas. Freeze-fracture replicas of liposomes containing 95 mol % of phosphatidylcholine (PC) and 5 mol % of phosphatidylinositol or a phosphoinositide were labeled. Only liposomes containing PI(4,5)P 2 were labeled intensely by GST-PH. A PH mutant, GST-PH (K30N, K32N), which does not bind PI(4,5)P 2, showed little labeling in the PI(4,5)P 2-containing liposome. (C) Quantification of the GST-PH labeling in the liposomes. The number of gold particles per 1 m 2 of the liposome surface is shown (blue). The labeling on the convex (green) and concave (yellow) surfaces showed equivalent results.

Brooks, Bryan W., Susan V. James, Theodore W. Valenti, Jr., Fabiola Urena‐Boeck, Carlos Serrano, Jason P. Berninger, Leslie Schwierzke, Laura D. Mydlarz, James P. Grover, and Daniel L. Roelke, 2010. Comparative Toxicity of Prymnesium parvum in Inland Waters. Journal of the American Water Resources Association (JAWRA) 46(1):45‐62. DOI: 10.1111/j.1752‐1688.2009.00390.x Abstract:  Numerous studies have examined the impacts of Prymnesium parvum to aquatic life, but the majority of information available is from experiments or field studies performed at salinities of marine and coastal ecosystems. Ambient toxicity of P. parvum has been characterized with in vitro and in vivo models because reliable quantitation of P. parvum toxins in environmental matrices is often precluded by a lack of available analytical standards. Hemolytic activity and fish mortality assays have been used most frequently to characterize toxic conditions; however, relatively few in vivo studies employed standardized methods. Because the relative sensitivities of different taxa to P. parvum toxins in inland waters were undefined, we assessed the comparative toxicity of P. parvum filtrate from a laboratory study (20°C, 12:12 light:dark cycle, f/8 media, 2.4 psu) to several common standardized in vitro and in vivo models. After exposure to cell‐free filtrate hemolytic activity (1 h EC50 = 13,712 cells/ml) and juvenile fish (Pimephales promelas) survival (48 h LC50 = 21,754 cells/ml) were the most sensitive assay responses examined, followed by rotifer (Brachionus calyciflorus) population growth rate [48 h no observable adverse effect levels (NOAEL) = 19,072 cells/ml] and cladoceran (Daphnia magna) reproduction (10‐day NOAEL = 47,680 cells/ml). Green algae (Pseudokirchneriella subcapitata) growth (96 h) was not adversely affected but was instead significantly stimulated by P. parvum toxins. We further propose an initial species sensitivity distribution approach for P. parvum, which may be used to support future environmental management decisions. Our findings from these laboratory studies indicate that although fish kills are increasingly associated with P. parvum blooms occurring in inland waters, further study is required to define the influences of toxin sensitivities of phytoplankton, zooplankton, and fish communities on P. parvum bloom initiation and termination.

Power transformations are commonly used in image processing techniques to manipulate image contrast. Many analytical results, including chromatograms, are essentially presented as images, often to convey qualitative information. Power transformations have remarkable effects on the appearance of the image, in chromatography, for example, increasing apparent resolution between peaks by the factor √n and apparent column efficiency (plate counts) by a factor of n for an nth-power transform. The profile of a Gaussian peak is not qualitatively changed, but the peak becomes narrower, whereas for an exponentially tailing peak, asymmetry at the 10% peak height level changes markedly. Using several examples we show that power transforms also increase signal-to-noise ratio and make it easier to discern an event of detection. However, they may not improve the limit of detection. Power responses are intrinsic to some detection schemes, and in others they are imbedded in instrument firmware to increase apparent linear range that the casual user may not be aware of. The consequences are demonstrated and discussed.

Erythrocyte lysis and fish mortality assays, in combination with high performance liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis, were investigated for bioassay-guided fractionation of cultured golden alga (Prymnesium parvum). Intracellular constituents from isolated cell pellets and extracellular supernatant growth medium were fractionated by a variety of common separation modes, including reversed phase and normal phase solid phase extraction step fractionation procedures. For reversed phase fractionation of extracellular growth medium, one fraction was obtained that displayed hemolytic activity and adversely affected fish survival. Effective dose concentrations for this sample were similar in both assays and the LC-ESI-MS analysis of the fraction showed a number of mass spectral signals which were distinct to this fraction. Fractions obtained from separation of an ethanol extract of the lyophilized cell pellet provided one sample that was highly hemolytic, but not toxic to fish. Discrepancies such as this, along with notable fish behavioral responses from other nonhemolytic cell pellet fractions, problems with the use of unbonded silica gel for fractionation, and misleading mass spectral signatures are interesting in the context of our current understanding of P. parvum toxicity and remain to be investigated further. This work provides an account of ongoing research aimed toward comprehensive elucidation of toxic constituents produced by golden alga for the purpose of providing a better understanding and means to potentially remediate the ecological impact of this harmful bloom organism.(KEY TERMS: erythrocyte lysis; fish mortality; liquid chromatography; electrospray ionization; mass spectrometry; prymnesin.)

In this study, two novel ionic liquid matrices (ILMs), N,N-diisopropylethylammonium 3-oxocoumarate and N,N-diisopropylethylammonium dihydroxymonooxoacetophenoate, were tested for the structural elucidation of recently developed aliphatic biodegradable polymers by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The polymers, formed by a condensation reaction of three components, citric acid, octane diol, and an amino acid, are fluorescent, but the exact mechanism behind their luminescent properties has not been fully elucidated. In the original studies, which introduced the polymer class (J. Yang et al., Proc. Natl. Acad. Sci. USA 2009, 106, 10086-10091), a hyper-conjugated cyclic structure was proposed as the source for the photoluminescent behavior. With the use of the two new ILMs, we present evidence that supports the presence of the proposed cyclization product. In addition, the new ILMs, when compared with a previously established ILM, N,N-diisopropylethylammonium α-cyano-3-hydroxycinnimate, provided similar signal intensities and maintained similar spectral profiles. This research also established that the new ILMs provided good spot-to-spot reproducibility and high ionization efficiency compared with corresponding crystalline matrix preparations. Many polymer features revealed through the use of the ILMs could not be observed with crystalline matrices. Ultimately, the new ILMs highlighted the composition of the synthetic polymers, as well as the loss of water that was expected for the formation of the proposed cyclic structure on the polymer backbone.