An image-based high-content screening assay for compounds targeting intracellular Leishmania donovani amastigotes in human macrophages.

Leishmaniasis is a tropical disease threatening 350 million people from endemic regions. The available drugs for treatment are inadequate, with limitations such as serious side effects, parasite resistance or high cost. Driven by this need for new drugs, we developed a high-content, high-throughput image-based screening assay targeting the intracellular amastigote stage of different species of Leishmania in infected human macrophages. The in vitro infection protocol was adapted to a 384-well-plate format, enabling acquisition of a large amount of readouts by automated confocal microscopy. The reading method was based on DNA staining and required the development of a customized algorithm to analyze the images, which enabled the use of non-modified parasites. The automated analysis generated parameters used to quantify compound activity, including infection ratio as well as the number of intracellular amastigote parasites and yielded cytotoxicity information based on the number of host cells. Comparison of this assay with one that used the promastigote form to screen 26,500 compounds showed that 50% of the hits selected against the intracellular amastigote were not selected in the promastigote screening. These data corroborate the idea that the intracellular amastigote form of the parasite is the most appropriate to be used in primary screening assay for Leishmania.

Nationwide monitoring of mercury in wild and farmed fish from fresh and coastal waters of Korea.

Mercury (Hg) concentrations were monitored in wild and cultured fish collected from fresh and coastal waters in the Korean peninsula from April 2006 to August 2008 nationwide. Total Hg concentrations were reported for 5043 fish samples, including 78 species from 133 locations. Significant interspecies variation was noted in the Hg levels. The average Hg concentration in each fish species ranged from 6.31 µg kg(-1) for mullet (Mugil cephalus) to 200 µg kg(-1) for mandarin fish (Siniperca scherzeri). Among the species collected, the maximum concentration of Hg, 1720 µg kg(-1), was measured in an Amur catfish (Silurus asotus). Only wild freshwater fish exceeded the WHO ingestion standard. Wild freshwater piscivorous fish samples from a large artificial upstream lake contained the highest Hg levels. Hg concentrations were compared between fish groups categorized as wild and farmed fish from freshwater and coastal waters. Although the wild freshwater fish had similar size ranges, their Hg concentrations were higher than those of the other groups. Compared to the feed of farmed marine and freshwater fishes, the prey of wild freshwater fish had a higher Hg concentration, and the total Hg concentrations in freshwater and associated sediment samples were higher than those in coastal water and associated sediment samples. In the freshwater environment, piscivorous fish bioaccumulated two times more Hg than carnivorous and omnivorous fish and four times more than planktivorous fish. The difference in Hg concentrations among trophic groups might have been due to differences in the size of fish, in addition to the variations among different trophic groups. These data will be useful for developing the fish consumption advisory as a management measure to reduce Hg exposure.

Atmospheric transport of water-soluble ions (NO3(-), NH4(+) and nss-SO4(2-)) to the southern East Sea (Sea of Japan).

Atmospheric deposition of different types of aerosols over the southern East Sea has received little attention in terms of seawater biogeochemistry. We investigated the concentrations of water-soluble ions (NO3(-), NH4+ and nss-SO4(2-)) in the aerosols associated with air mass transport patterns arriving at the east coast of Korea, adjacent to the southern East Sea, in order to determine source regions affecting chemical composition of aerosols and to assess the atmospheric pathway as a significant controlling mechanism of the biogeochemistry in this marginal sea. Concentrations of certain elements (Al, Na, Ca, V, Zn and Pb) together with the water-soluble ions were measured in the aerosol samples (n=34) collected during the period March 2002-February 2003. The geometric mean concentrations of the water-soluble ions were NO3(-) 2.98 (0.56-16.22), NH4+ 1.42 (0.37-6.73) and nss-SO4(2-) 2.47 (0.17-17.35) microgm(-3). The backward trajectories revealed that air masses passing slowly over eastern China contributed more to increases in the concentrations of water-soluble ions than those associated with fast-moving northwesterly and maritime winds. Therefore, the correlation between the NH4+ and NO3+ concentrations increased, suggesting that gas-phase NH3 and HNO3 was forming fine-mode NH4NO3. The atmospheric N input accounted for approximately 10% of new production over the southern East Sea on an annual scale, while it accounted for over approximately 25% of new production during the water column stratification seasons (summer and early fall).

Confocal-based method for quantification of diffusion kinetics in microwell plates and its application for identifying a rapid mixing method for high-content/throughput screening.

Rapid mixing in microplates is still an underappreciated challenge in screening assay development, particularly with the use of noncontact nanoliter liquid handlers. In high-content/throughput screening (HC/TS), fast and efficient mixing between compounds and cell culture medium is even more critical as biological kinetics dictates speed of mixing, usually within a few minutes. Moreover, mixing in HC/TS should be gentle enough to avoid any negative disruption in cell layer. Here the authors introduce a method to accurately quantify drop diffusion into a microplate well, independently of buffer, liquid handler, or dispensing protocol. This method was used to determine the effect of various mixing methods on the diffusion of a nanoliter drop of pure DMSO in aqueous buffer in 384-well plates. Rapid plate shaking and additional buffer addition were shown to be the most efficient and effective mixing methods for HC/TS. However, efficient mixing by plate shaking is limited by assay volume. Bulk addition shows fast and efficient mixing, without negative effects on cells. Moreover, this simple, fast, and inexpensive method can be easily adapted on any platform.

Atmospheric metal and phosphorus concentrations, inputs, and their biogeochemical significances in the Japan/East Sea.

Atmospheric metals and phosphorus over the southern Japan/East Sea were investigated in order to evaluate their sources, concentrations and inputs, and to identify their biogeochemical roles in this marginal sea. Aerosols were collected on the east coast of Korea from February 2002 to April 2003 (n=101) as well as at a remote island (Ulleung) and on a ship from February 2002 to June 2003 (n=13). The aerosols were analyzed for Al, Co, Cu, Ni, P, Pb and Zn. Simultaneous collections of aerosols at both coast and offshore were performed, and several high dust aerosols (Al>5 microg m(-3)) were collected at both regions. At the coastal site, both dust mineral and pollutants were transported by westerly winds from the Asian continent, but local emissions were significant (e.g., Cu, Ni, P and Zn) as well during the summer monsoon (May-August). The experimental relationships between the coast and offshore sites were defined. From these relationships, it was possible to obtain the annually averaged atmospheric metal and P concentrations over the southern Japan/East Sea, which has increased by over 2 times for the last decade. Through the estimation of atmospheric metal and phosphorus fluxes and comparisons with inputs from the Tsushima Warm Current, the atmospheric pathway was found to be a significant source for Al, Pb and Zn.